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1. This study examined whether pretreatment of rabbits with infusions of prostaglandin E1 (PGE1) or prostaglandin E0 (PGE0) (which were terminated prior to the onset of ischaemia) reduce myocardial infarct size arising from coronary artery occlusion (60 min) and reperfusion (120 min). In addition, we investigated whether the observed cardioprotective effects of these two prostaglandins were due to the activation of ATP-sensitive potassium (KATP) channels. 2. In the anaesthetized rabbit, infarct size (expressed as a percentage of the area at risk) after 60 min of coronary artery occlusion followed by 2 h of reperfusion was 59 +/- 4% (n = 10). PGE1 or PGE0 treatment (1.0 micrograms kg-1 min-1), administered as 1 h pretreatments (0.05 ml min-1, i.v.), significantly reduced infarct size to 44 +/- 6% (n = 6) or 42 +/- 1% (n = 6), respectively. PGE1 or PGE0 pretreatment resulted in a significant reduction in mean arterial blood pressure, which returned to baseline within 15 min of discontinuation of the infusion (i.e. prior to LAL ligation). 3. The reduction in infarct size afforded by PGE1 was abolished by pretreatment of rabbits with the KATP channel blockers, glibenclamide (60 +/- 4%; n = 8) or 5-hydroxydecanoate (58 +/- 6%; n = 6). Similarly, glibenclamide also largely attenuated the reduction in infarct size afforded by PGE0 (52 +/- 3%; n = 8). 4. We propose that a 1 h pretreatment of PGE1 or PGE0 reduces infarct size by activating protein kinase C resulting in the opening of KATP channels.

1. Endothelin-1 (ET-1) produces vasoconstriction, via activation of ETA and ETB receptors on vascular smooth muscle, and vasodilatation via ETB receptors on endothelial cells. Here we have used the ETA receptor-selective antagonist, BQ-123, the ETB receptor-selective antagonist, BQ-788 and the ETA/ETB receptor non-selective antagonist, PD 145065, to study the role of these receptors in mediating the haemodynamic changes induced by an infusion of ET-1 to the anesthetized ganglion-blocked rat. 2. Infusion of ET-1 (10 pmol kg-1 min-1) increased the mean arterial pressure (MAP) by 57.5 +/- 5.1 mmHg over 70 min. This pressor response was reduced by about 50% by coinfusion of BQ-123 (10 mmol kg-1 min-1), but was unaffected by either BQ-788 (10 nmol kg-1 min-1) or PD 145065 (10 nmol kg-1 min-1). 3. After infusion of ET-1 for 70 min the cardiac output had fallen from 102.6 +/- 11.3 to 55.7 +/- 7.6 ml min-1 and the total peripheral resistance had increased from 3.24 +/- 0.6 to 10.0 +/- 0.8 mmHg ml-1 min-1 (per 100g body weight). BQ-123 decreased the magnitudes of these changes whereas BQ-788 potentiated them. PD 145065 was without effect. 4. ET-1 increased the vascular resistances of all the organs studied except the brain and stomach. These changes were attenuated by BQ-123 in the kidneys, skin, adrenal glands and caecum and potentiated by BQ-788 in the kidneys, small intestine, large intestine and mesentery. PD 145065 had little effect on the individual tissues. 5. Thus, BQ-123, a selective ETA receptor antagonist, inhibits the pressor and vascular constrictor effects of ET-1 more actively than PD 145065. As BQ-788 potentiates some of the vasoconstrictor effects of ET-1 and increases the effects of ET-1 on total peripheral resistance, the predominant role of ETB receptors in the rat circulation is to limit the pressor effects of ET-1.

1. The induction of cyclo-oxygenase-2 (COX-2) afforded by bacterial lipopolysaccharide (LPS, endotoxin) in bovine aortic endothelial cells (BAEC) is mediated by tyrosine kinase. LPS also causes the generation of several cytokines including interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). This study investigates whether endogenous IL-1 beta, TNF-alpha, EGF or PDGF contribute to the induction of COX-2 elicited by LPS in BAEC and if their action is due to activation of tyrosine kinase. Furthermore, we have studied the induction of COX-2 by exogenous cytokines. 2. Accumulation of 6-oxo-prostaglandin (PG) F1 alpha in cultures of BAEC was measured by radioimmunoassay at 24 h after addition of either LPS (1 microgram ml-1) alone or LPS together with a polyclonal antibody to one of the various cytokines. In experiments designed to measure 'COX activity', 6-oxo-PGF1 alpha generated by BAEC activated with recombinant human IL-1 beta, TNF-alpha, EGF or PDGF for 12 h was measured after incubation of washed cells with exogenous arachidonic acid (30 microM for 15 min). Western blot analysis determined the expression of COX-2 protein in BAEC. 3. The accumulation of 6-oxo-PGF1 alpha caused by LPS in BAEC was attenuated by co-incubation with one of the polyclonal antibodies, anti-IL-1 beta, anti-TNF-alpha, anti-EGF, anti-PDGF or with the IL-1 receptor antagonist, in a dose-dependent manner. Exogenous IL-1 beta, TNF-alpha or EGF also caused an increase in COX activity, while PDGF was ineffective. The increase in COX activity elicited by IL-1,beta(10 ng ml-1), TNF-alpha (100 ng ml-1) or EGF (1000 ng ml-1) in BAEC was attenuated by erbstatin (0.005 to 5 microg ml-1), as was the expression of COX-2 protein measured by Western blot analysis.4. PDGF (10 ng ml-1) significantly augmented the rise in COX activity and COX-2 protein caused by shorter incubation of BAEC with LPS (1 microg ml-1 for 3 h). Combination of PDGF (10 ng ml-1) with a low concentration of IL-l beta (1 ng ml-1) for 12 h, also increased 'COX activity', but combination of PDGF and TNF-alpha (10 ng ml-1) did not show any increased activity.5. These results suggest that (i) the induction of COX activity and COX-2 protein elicited by LPS in BAEC is mediated by TNF-alpha with lesser contributions from PDGF, EGF or IL-1 beta; (ii) exogenous IL-1 beta,TNF-alpha or EGF alone induce COX-2 activity and protein in BAEC; (iii) PDGF synergizes with IL-1 beta,but not TNF-alpha, to cause expression of COX-2; and (iv) the induction of COX-2 protein and activity caused by these cytokines involves the activation of tyrosine kinase.

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.

The release of nitric oxide (NO) by NG-hydroxy-L-arginine (L-OHArg) from endothelial cells (ECs) grown in culture was assessed by two bioassays. The first was a cascade bioassay system, in which the effluent from a column of ECs grown on beads superfused a cascade of isolated endothelium-denuded rabbit aortic strips. Exclusion of calcium from the Krebs' buffer perfusing the ECs (but not from the cascade) diminished the relaxant effects of L-OHArg. Growing the ECs in the presence of dexamethasone did not affect the relaxations of the bioassay cascade induced by L-OHArg. However, these relaxations were potentiated by superoxide dismutase (SOD) and inhibited by haemoglobin, but not inhibited by NG-nitro-L-arginine methyl ester (NO2 ArgMeE). The second bioassay was a transfer assay, in which accumulation of cGMP was measured in detector, RFL-6 fibroblast cells. The accumulation of cGMP induced by the basal release of NO from ECs was increased by L-OHArg and by SOD. However, L-OHArg, unlike SOD, did not scaven...

Compared with endothelin-1 (ET-1), big endothelin-1 (big ET-1) is only weakly active on isolated vascular smooth muscle preparations. However, on systemic administration high doses of big ET-1 (1 nmol.kg-1) are approximately equipotent to ET-1, indicating the existence of an endothelin converting enzyme in the circulation that rapidly converts big ET-1 to ET-1. In this study arterial blood levels of big ET-1 and ET-1 immunoreactivity were measured after bolus i.v. administration of big ET-1 (1 or 3 nmol.kg-1) or ET-1 (1 nmol.kg-1) in anaesthetised male Wistar rats. In addition, the effect of phosphoramidon (10 mg.kg-1) on the pressor response to big ET-1 and its disappearance rate from the circulation were examined. After big ET-1 injection, circulating ET-1 concentrations did not exceed 2% of the big ET-1 level. Phosphoramidon reduced the pressor response to big ET-1 by 93%, but did not alter its rate of clearance from the circulation. Thus exogenous big ET-1 is converted locally in the vasculature and its disappearance from the circulation is not dependent on conversion to ET-1.

Purpose: To determine the effects of the nitric oxide synthase inhibitor, nitro-L-arginine on energy metabolism in transplantable and spontaneous murine tumors. Methods and Materials: The responses of the transplantable murine tumor SCCVII/Ha and a range of spontaneously arising murine mammary adenocarcinomas to 10 mg/kg IV nitro-L-arginine were examined using in vivo 31 P magnetic resonance spectroscopy (MRS). The influence of Hypnorm/Hypnovel anesthesia on the response to nitro-L-arginine was also determined in the SCCVII/Ha tumors. Data were expressed as changes in the inorganic phosphate peak area relative to the sum of all peak areas from the 31 P MR spectrum, or Pi/total. Results: Nitro-L-arginine at 10 mg/kg IV increased Pi/total 2–3-fold in the SCCVII/Ha tumors for at least 2 h after administration, in both anesthetized and nonanesthetized mice, consistent with increased tumor hypoxia. Similar increases in Pi/total were observed after 10 mg/kg IV nitro-L-arginine in 13 spontaneous murine tumors from three different mouse strains, where anesthetic was used. Conclusion: The results indicate that tumor metabolism may be modified by an inhibitor of nitric oxide synthesis, that this modification occurs in both transplantable and spontaneous murine tumors and is not affected by anesthetic.

1. We have characterized the receptors mediating contractions to endothelin-1 (ET-1) or IRL 1620, an ETB receptor selective agonist, in isolated strips of tissue prepared from different parts of the guinea-pig airways. We used as antagonists BQ-123 and FR139317 (ETA receptor-selective) and PD 145065 (ETA/ETB receptor non-selective). 2. ET-1 and IRL 1620 (10(-10) M to 10(-6) M) caused similar concentration-dependent contractions of strips of guinea-pig trachea and upper bronchus. In the guinea-pig trachea without epithelium or lung parenchyma, IRL 1620 was less potent than ET-1. 3. In the trachea, contraction to ET-1 (< 10(-8) M) was preceded by a transient relaxation which was inhibited by BQ-123 (10(-5) M) or FR 139317 (10(-5) M) or by the removal of the epithelium. The concentration-response curve to ET-1 in the trachea was shifted to the right by PD 145065 (10(-5) M to 10(-4) M). PD 145065 (10(-4) M) also inhibited the response to ET-1 (3 x 10(-7) M) by 55%. Contractions induced by IRL 1620 were not affected by BQ-123 (10(-6) M) or FR139317 (10(-6) M) but were significantly attenuated by 10(-5) M of either antagonist. PD 145065 at 10(-6) M strongly attenuated and at 10(-5) M abolished contractions induced by IRL 1620. 4. In the trachea, removal of the epithelium potentiated the effects of both agonists. BQ-123 (10-5 M)had no effect on contractions of the trachea without epithelium induced by ET-1, but FR139317 (10-5 M)caused a significant inhibition. PD 145065 (10-5 M to 10-4 M) caused a shift to the right of the ET-1 concentration-response curve without affecting the contractile effect at 3 x 10-7 M. All three antagonists inhibited contractions induced by IRL 1620.5. In the upper bronchus, BQ-123 (10-5 M) did not affect contractions induced by ET-1, whileFR139317 (10-5 M) attenuated (20-26%) only contractions induced by 1-3 x 10-7 M ET-1. PD 145065(10-5 M to 10-4 M) caused a shift to the right of the ET-1 concentration-response curve. The contractions induced by IRL 1620 were inhibited by BQ-123 or FR139317 (10-5M to 10-4 M). PD 145065(10-6 M) strongly inhibited contractions induced by IRL 1620 and PD 145065 (10-5 M) totally abolished them.6. The contractile action of ET-1 in the lung parenchyma was significantly and similarly attenuated by BQ-123 (10-5 M) or indomethacin (10-5 M), while FRI39317 (10-5 M) was less effective. PD 145065(10-6 to 10-5 M) inhibited contractions to ET-1. IRL 1620, which is less potent than ET-1 in this preparation, was antagonized by PD 145065 (10-5 to 10-6 M) but unaffected by BQ-123 (10-6 M to10-5M) or FR139317 (10-6 M).7. Thus, ETB receptors mediate contractions to ET-1 in all four guinea-pig airway preparations. In addition, contractions to ET-1 in the trachea and lung parenchyma are mediated in part by ETA receptors. In the latter tissue, these ETA receptors mediate contraction through the release of cyclooxygenase metabolites. Similarly, ETA receptors located on the epithelial cells also mediate the release of prostanoids in the trachea with epithelium but they are responsible for transient relaxations. Interestingly,contractions induced by IRL 1620 were more susceptible to inhibition by the different antagonists,most probably because it binds to the endothelin receptors in a reversible manner. High concentrations(10-5 M) of ETA-selective antagonists also inhibit responses to IRL 1620, most probably by an effect at ETB receptors in both the trachea and the upper bronchus.

1. To characterize the receptor subtype(s) mediating the renal vasoconstrictor effects of the endothelin (ET) and sarafotoxin (SX) peptides in the isolated perfused kidney of the rat, we have examined the effects of endothelin-1 (ET-1), sarafotoxin 6b (SX6b) and sarafotoxin 6c (SX6c) as agonists, BQ-123 and FR 139317 as selective ETA receptor antagonists, and PD 145065 as a non-selective (ETA and ETB) receptor antagonist. We have also compared in the anaesthetized rat the systemic pressor and renal vasoconstrictor effects of ET-1 and SX6c alone or after pretreatment with PD 145065. 2. In the isolated perfused kidney, ET-1, SX6b and SX6c all gave similar concentration-dependent increases in perfusion pressure. The ETA receptor selective antagonists, BQ-123 and FR 139317, both partially blocked the increase in perfusion pressure induced by ET-1. In contrast, PD 145065 completely blocked the increase in perfusion pressure caused by ET-1. 3. Indomethacin (10 microM) had no effect on the ET-1-induced increases in perfusion pressure but significantly reduced the vasoconstriction induced by low concentrations of SX6c, without affecting responses to high concentrations. In the anaesthetized rat, indomethacin (5 mg kg-1) did not modify the systemic pressor or renal vasoconstrictor effects of ET-1 or SX6c. 4. In anaesthetized rats, bolus intravenous injections of ET-1 or SX6c (0.1, 0.25, 0.5 or 1.0 nmol kg-1) produced initial transient depressor responses followed by sustained and dose-dependent increases in mean arterial pressure (MAP). Both peptides caused an equipotent fall in renal blood flow (RBF).PD 145065 (5 mg kg-1) partially antagonized the systemic pressor effects of ET-1 and SX6c but completely blocked the fall in RBF and rise in renal vascular resistance (RVR) induced by ET-1 and SX6c. PD 145065 also antagonized the transient depressor effect following the bolus administration of either ET-1 or SX6c.5. These results indicate that ET/SX induced renal vasoconstriction is mediated via ETA and ETB-like receptors with ETB receptors having a predominant role in vivo. This may be of therapeutic relevance for an ETA receptor-selective antagonist may offer only limited protection against the deleterious renal effects of endogenous ETs.

The vascular endothelium plays a vital role in the control of the circulation. It metabolizes various vasoactive substances, coverts angiotensin I to angiotensin II and secretes the potent vasodilators prostacyclin and EDRF(NO) and the vasoconstrictor peptide endothelin-1. The balance between these mediators determines the responses of the cardiovascular system in diseases such as hypertension, atherosclerosis and myocardial infarction.

This study investigates the role of endogenous platelet-activating factor (PAF) in the production of nitric oxide (NO) by constitutive and inducible isoforms of NO synthase (NOS) in endotoxin shock. In anesthetized rats, 3 hours of endotoxemia resulted in a fall in mean arterial blood pressure (MAP) from 127 +/- 5 (control) to 61 +/- 7 mm Hg and a reduction of the pressor responses to norepinephrine (NE, 1 microgram.kg-1) from 33 +/- 3 (control) to 17 +/- 2 mm Hg. Endotoxemia for 3 hours also resulted in a significant reduction in the contractile effects of NE (10(-8) to 10(-6) mol/L) in thoracic aortas ex vivo. This hyporeactivity to NE was due to an enhanced formation of NO, for it was restored by the NOS inhibitor NG-nitro-L-arginine methyl ester. Animals pretreated with the PAF receptor antagonist WEB 2086 maintained higher MAP (MAP at 180 minutes, 98 +/- 6 mm Hg) and exhibited more pronounced pressor responses to NE at 180 minutes after LPS injection. Moreover, WEB 2086 attenuated by 58% the lipopolysaccharide (LPS)-induced hyporeactivity of the rat aortic rings ex vivo. At 3 hours after LPS injection, calcium-independent NOS activity was induced in the lung. The activity of inducible NOS was significantly lower (by 31%) in lungs of rats pretreated with WEB 2086. The hypothesis that WEB 2086 attenuates the induction of NOS in vivo was substantiated in vitro by the finding that pretreatment with WEB 2086 for 30 minutes inhibited the LPS-stimulated NO production in cultured murine macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.

1. To characterize the receptors mediating the effects of the endothelin/sarafotoxin family of peptides on the responses to electrical stimulation of the rat vas deferens (RVD) and guinea-pig ileum (GPI) we have used endothelin-1 (ET-1), ET-3, sarafotoxin 6b (SX6b) and SX6c as agonists and the endothelin-receptor antagonists BQ-123 (ETA receptor selective) and PD 142893 (non-selective). 2. In the RVD, ET-1 and SX6b increased the twitches induced by field stimulation starting at a threshold concentration of 10(-10) M while the threshold concentration for ET-3 was 3 x 10(-9) M. SX6c (up to 3 x 10(-8) M) did not potentiate the twitches. SX6b produced significantly (P0.05) greater potentiations than ET-1 at concentrations of 3 x 10(-9) M and higher, and 10(-7) M ET-3 also produced a significantly greater effect than ET-1 at the same concentration. Thus, at threshold the rank order of peptides was ET-1 = SX6bET-3SX6c, and at concentrations of 3 x 10(-8) M and higher, SX6bET-3ET-1SX6c. 3. In the presence of BQ-123 or PD 142893 (10(-5) M) the threshold concentrations for ET-1 to augment the twitches were increased 30 fold. In the same conditions neither SX6b nor ET-3 potentiated the responses. The relative activities of the endothelin/sarafotoxin peptides and the effectiveness of the endothelin receptor antagonists are consistent with postjunctional ETA receptors mediating these effects. 4. In the transmurally stimulated GPI the endothelin/sarafotoxin peptides produced two effects; an increase in the basal tension of the tissues and an inhibition of the twitch responses. To increase the basal tension the peptides had the order of potency ET-1SX6bET-3 = SX6c. These direct effects of ET-1 or SX6b were strongly antagonized (100 fold) by either BQ-123 (10-5M) or PD 142893(10-5 M). Thus, ETA receptors mediate contractions of the GPI induced by these peptides.5. The endothelin/sarafotoxin peptides were approximately equipotent at depressing twitches of the GPI in response to transmural stimulation (EC50s, 4 x 10-11 to 1.5 x 10-10 M). The depressions induced byET-1 were unaffected by either BQ-123 (10-5 M) or PD 142893 (10-5 M). BQ-123 produced a small(three fold) antagonism of the inhibitory effects of ET-3 or SX6c. These results indicate that a receptor of the ETB type mediates the inhibitory effects of the endothelin/sarafotoxin peptides on neurotransmission in the GPI.6. Thus, both ETA receptors and ETB receptors mediate the effects of the endothelin/sarafotoxinpeptides on neurotransmission.

1. The effects of FR139317 (ETA antagonist) or PD145065 (non-selective ETA/ETB antagonist) on endothelin-1 (ET-1)-induced changes in blood pressure and inhibition of ex vivo platelet aggregation were investigated in the anaesthetized rabbit. 2. ET-1 (1 nmol kg-1, i.a. bolus) caused a sustained increase in mean arterial pressure (MAP) (peak increase 47 +/- 5 mmHg, n = 8). Intravenous infusion of FR139317 at 0.2 (n = 4) or 0.6 mg kg-1 min-1 (n = 4) inhibited the ET-1 pressor response by 83 or 89%, respectively. Infusion of PD145065 at 0.2 (n = 4) or 0.6 mg kg-1 min-1 (n = 4) inhibited the ET-1-induced increase in MAP by 79 or 75%, respectively. 3. The transient depressor response (-16 +/- 3 mmHg) which preceded the rise in blood pressure induced by ET-1 (1 nmol kg-1, i.a., n = 8) was enhanced by an intravenous infusion of FR139317 (0.6 mg kg-1 min-1) to -35 +/- 5 mmHg (P0.05, n = 4). This enhancement was abolished by indomethacin (5 mg kg-1, i.v.) pretreatment (-17 +/- 1 mmHg, n = 4). PD145065 (0.2 mg kg-1 min-1, i.v.) attenuated the ET-1-induced fall in blood pressure to -9 +/- 1 mmHg (n = 4), while a higher dose of this antagonist (0.6 mg kg-1 min-1, i.v.) completely abolished the ET-1-mediated depressor response. 4. ET-1 (1 nmol kg-1, n = 8) inhibited ex vivo platelet aggregation by 96% at 5 min after injection of the peptide. FR139317 (0.2 or 0.6 mg kg-1 min-1, i.v.) or PD145065 (0.2mg kg-1 min-1, i.v.) did not affect the inhibition of ex vivo platelet aggregation in response to ET-1. In contrast, intravenous infusion of PD145065 (0.6 mg kg-1 min-1) abolished the anti-aggregatory effects of ET-1.5. Thus, FR139317 inhibits the pressor, but not the depressor actions of ET-1 and has no effect on the ET-l-induced inhibition of ex vivo platelet aggregation. In contrast, PD145065 antagonizes the pressor and depressor responses to ET-1 and abolishes the anti-aggregatory effects of the peptide.6. These results strongly suggest that ET-1-induced vasoconstriction in the anaesthetized rabbit is primarily mediated via the ETA receptor while the depressor and antiaggregatory actions of ET-1 are due to activation of the ETB receptor.

1. The role of an enhanced formation of nitric oxide (NO) and the relative importance of the constitutive and inducible NO synthase (NOS) for the development of immediate (within 60 min) and delayed (at 180 min) vascular hyporeactivity to noradrenaline was investigated in a model of circulatory shock induced by endotoxin (lipopolysaccharide; LPS) in the rat. 2. Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate. In addition, the calcium-dependent and calcium-independent NOS activity was measured ex vivo by the conversion of [3H]-arginine to [3H]-citrulline in homogenates from several organs obtained from vehicle- and LPS-treated rats. 3. E. coli LPS (10 mg kg-1, i.v. bolus) caused a rapid (within 5 min) and sustained fall in MAP. At 30 and 60 min after LPS, pressor responses to noradrenaline (0.3, 1 or 3 micrograms kg-1, i.v.) were significantly reduced. The pressor responses were restored by NG-nitro-L-arginine methyl ester (L-NAME, 1 mg kg-1, i.v. at 60 min), a potent inhibitor of NO synthesis. In contrast, L-NAME did not potentiate the noradrenaline-induced pressor responses in control animals. 4. Dexamethasone (3 mg kg-1, i.v., 60 min prior to LPS), a potent inhibitor of the induction of NOS, did not alter initial MAP or pressor responses to noradrenaline in control rats, but significantly attenuated the LPS-induced fall in MAP at 15 to 60 min after LPS. Dexamethasone did not influence the development of the LPS-induced immediate (within 60 min) hyporeactivity to noradrenaline. However,dexamethasone pretreatment prevented the hypotension and vascular hyporeactivity at 180 min.5. At 60 min after LPS a moderate increase in the activity of a calcium-independent (inducible) NOS activity was detected in the aorta, but not in any of the other tissues studied. However, at 180 min after LPS, a significant NOS induction was observed in the lung, liver, spleen, mesentery, heart and aorta.This NOS induction was substantially prevented by pretreatment with dexamethasone.6. These results suggest that the immediate hypotension and vascular hyporeactivity to noradrenaline in endotoxin shock is caused by an enhanced formation of NO due to activation of the constitutive enzyme. The delayed hypotension and vascular hyporeactivity, however, is due to enhanced NO formation by the LPS-induced enzyme.

1. Using endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6b (SX6b) and sarafotoxin 6c (SX6c) as agonists and BQ-123 as a selective ETA receptor antagonist, we have examined the endothelin receptor subtypes mediating the systemic pressor and renal vasoconstrictor effects of the ET/SX family of peptides. 2. In anaesthetized rats, bolus intravenous injections of ET-1, ET-3, SX6b or SX6c (0.1, 0.25 and 0.50 nmol kg-1) produced initial transient depressor responses followed by sustained and dose-dependent increases in mean arterial pressure (MAP) with the following rank order of potency: SX6b > ET-1 >> SX6c > ET-3. In contrast, in the renal vasculature these peptides caused equipotent dose-dependent falls in renal blood flow (RBF) (ET-1 = ET-3 = SX6b = SX6c). 3. BQ-123 (1 mg kg-1, i.v. bolus) significantly reduced the systemic pressor effects of all the peptides but was largely ineffective against the renal vasoconstrictions. 4. These results indicate that although the systemic pressor effects of the ET/SX peptides are mediated via ETA receptors, the vasoconstriction in the kidney in vivo may be mediated predominantly via ETB-like receptors. This may be of therapeutic relevance, for an ETA-receptor-selective antagonist could offer only poor protection of the renal circulation from the deleterious effects of endogenously produced members of this peptide family.

1. Exposure of smooth muscle cells (SMC) to glyceryl trinitrate (GTN, 75-600 microM) for 30 min led to a concentration-dependent increase in nitrite (NO2-), one of the breakdown products of nitric oxide (NO). This was not affected by 30 min pretreatment of the cells with 0.5 mM of sulphobromophthalein (SBP) an inhibitor of glutathione-S-transferase (GST), by metyrapone or SKF-525A inhibitors of cytochrome P450. These experiments were confirmed by organ bath studies using rabbit aortic strips denuded of endothelium and contracted with phenylephrine. Thus, a 30 min incubation of the strips with 0.5 mM SPB, metyrapone or SKF-525A did not affect the relaxations in response to GTN (10(-10)-10(-6) M). 2. Potentiation of the anti-platelet effect of GTN (44 microM) by endothelial cells (EC, 40 x 10(3) cells) was not affected by prior incubation of EC with SBP, metyrapone or SKF-525A (all at 0.5 mM). 3. Potentiation of the antiplatelet activity of GTN (11-352 microM) by small numbers of SMC (24 x 10(3) cells) or EC (40 x 10(3) cells) treated with indomethacin (10 microM) was attenuated when the SMC or EC were treated in culture with a high concentration of GTN (600 microM) for 18 h beforehand (referred to as 'tolerant' cells). In addition, tolerant SMC produced far less NO2- than non-tolerant SMC. 4. Exposure of non-tolerant SMC or EC (10(5) cells) to GTN (200 microM) for 3 min increased (3-4 fold) the levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP). This increase was much less (< I fold) in the tolerant SMC or EC (105 cells). The basal levels of cyclic GMP were similar in normal or tolerant SMC or EC. Sodium nitroprusside (80 JAM) or atrial natriuretic factor (ANF, I0- M) increased the levels of cyclic GMP in normal or tolerant SMC or EC to the same extent.5 The anti-platelet effects of GTN (44 JM) were potentiated by the sulphydryl donor N-acetylcysteine(NAC, 0.5mM). Incubation of GTN (150-1200fJM) for 30min with NAC (0.1-1mM) led to aconcentration-dependent increase in N02- formation. The reduced ability of tolerant SMC or EC to potentiate the anti-platelet activity of GTN was restored by NAC (0.5 mM). These anti-aggregatory effects were abolished by concurrent co-incubation with oxyhaemoglobin (10 JM) indicating that they were due to NO release.6 Thus, in SMC or EC, metabolism of GTN to NO does not depend on glutathione-S-transferase or the cytochrome P450 system. Furthermore, when compared to normal cells, tolerant SMC or EC metabolize GTN to NO less effectively as assessed by the reduced capacity to potentiate the antiplatelet effects of GTN, to release NO2- and to increase the level of cyclic GMP. This decrease in NO formation shows that tolerance to GTN is mainly due to impaired biotransformation of GTN to NO. NAC, by directly forming NO from GTN, compensates for this failing mechanism.

Endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6b (SX6b), and sarafotoxin 6C (SX6c) were used as agonists, and BQ-123 (ETA-selective) and PD 142893 (ET receptor-nonselective) were used as antagonists to characterize the receptors mediating the effects of the ET/SX peptides on a variety of isolated smooth-muscle preparations. Contractions of the rat thoracic aorta, rat isolated perfused mesentery, and guinea pig ileum and potentiation of twitch of the rat vas deferens were mediated by ETA receptors in that they showed the order of potency ET-1 = SX6bET-3SX6C. These effects were antagonized by BQ-123 or PD 142893. Contractions of the rabbit pulmonary artery and rat stomach strip, inhibition of twitches in the guinea pig ileum, and vasodilatations of the rat isolated perfused mesentery showed the order of potency ET-1 = SX6b = ET-3 = SX6c and were unaffected by BQ-123, suggesting involvement of ETB receptors. However, in these tissues, PD 142893 antagonized only dilatations of the rat mesentery to ET-1 but not any of the other effects of ET-1. Thus, we suggest that there are three types of endothelin receptors: ETA and two subtypes of ETB.

Using selective endothelin (ET) receptor antagonists, we investigated which ET receptor subtypes mediate the changes in blood pressure and hematocrit produced by intraarterial injection of ET-1 in the anesthetized rabbit. In addition, the receptor through which ET-1 stimulates the release of prostacyclin (PGI2) and, hence, inhibits ex vivo platelet aggregation, was identified. FR 139317 (ETA antagonist, 0.6 mg kg-1 min-1 preceded by a loading dose of 3 mg kg-1 i.v.) and PD 145065 (nonselective ETA/ETB antagonist, 0.6 mg kg-1 min-1 preceded by a loading dose of 3 mg kg-1 i.v.) attenuated the ET-1 (1 nmol kg-1 i.a.)-induced rise in mean arterial pressure (MAP) by 89% and 75%, respectively. In contrast to FR 139317, PD 145065 also abolished the initial, transient depressor response brought about by ET-1. ET-1 caused a significant increase in hematocrit 15 min after its injection. PD 145065 caused a significantly greater inhibition of this hemoconcentration than FR 139317. ET-1 inhibited ex vivo platelet aggregation by 96%, measured 5 min after injection of the peptide. PD 145065, but not FR 139317, abolished the antiaggregatory effects of ET-1. Thus, the ET-1-induced vasoconstriction in the anesthetized rabbit is predominantly mediated via the ETA receptor, whereas the depressor and antiaggregatory actions of ET-1 are caused by activation of the ETB receptor. Moreover, activation of both receptor subtypes by ET-1 accounts for the increase in hematocrit produced by ET-1 in vivo.

We investigated the effects of NG-hydroxy-L-arginine (L-HOArg) and hydroxyguanidine (HOG) on the synthesis and vasorelaxant activity of endothelium-derived relaxing factor (NO) released from the rabbit aortic endothelium. Both L-HOArg (10 microM) and HOG (10 microM) equally potentiated the vasorelaxant activity of NO released by Ach (0.1 or 0.3 microM) from the luminally perfused rabbit aorta and bioassayed using the superfused strips of the endothelium-denuded rabbit aorta. This potentiation was caused by the generation of a more stable vasodilator during the chemical reaction of L-HOArg or HOG with NO and it was abolished by NG-nitro-L-arginine methyl ester (L-NO2Arg, 10 microM). In contrast, in organ baths, L-HOArg (10 microM) or HOG (10 microM) did not affect the relaxations of intact rabbit aortic rings induced by Ach (0.01-1 microM). At concentrations higher than 10 microM, both L-HOArg and HOG were endothelium-independent vasorelaxants. However, L-HOArg (100 microM) prevented the inhibition by L-NO2Arg (10 microM) of Ach-induced relaxations of bathed aortic rings which indicates that L-HOArg is still a substrate for the NO synthase in the endothelium of the rabbit aorta.

1. We have investigated the effects of L-hydroxy-L-arginine (L-HOArg), an intermediate in the biosynthesis of nitric oxide (NO) from L-arginine (L-Arg), on the haemodynamic effects (systemic blood pressure and renal blood flow) of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in the anaesthetized rat. 2. L-Arg or L-HOArg (3 mg kg-1 min-1), but not D-arginine (D-Arg) or NG-hydroxy-D-arginine (D-HOArg), elicited a slight but significant increase in total renal blood flow (RBF) of 11 +/- 2% and 11 +/- 1%. Since mean arterial blood pressure (MAP) did not change this dose of L-Arg or L-HOArg resulted in a reduced renal vascular resistance (RVR) of the same magnitude. 3. Bolus injections of L-NAME, at 0.3 or 1 mg kg-1 i.v., produced a significant fall in RBF of 11 +/- 2% and 32 +/- 5% and an increase in MAP of 7 +/- 3 mmHg and 22 +/- 5 mmHg, respectively. Consequently, RVR was elevated by 21 +/- 5% and 52 +/- 10%. 4. L-Arg or L-HOArg (3 mg kg-1 min-1) reduced the L-NAME-induced (0.3 or 1 mg kg-1) falls in RBF and increases in RVR by more than 65%. Neither D-Arg nor D-HOArg (3 mg kg-1 min-1) had any significant effect on the changes in RBF or RVR induced by L-NAME. 5. L-Arg or L-HOArg (3 mg kg-' min-') attenuated the pressor effect of L-NAME (3 mg kg-') by 73% and 64%, respectively, while neither the D-isomer of arginine nor hydroxyarginine had any effect.6. These results demonstrate that L-HOArg antagonizes the haemodynamic effects of NO-biosynthesis inhibition in vivo, thus supporting the hypothesis that L-HOArg is an intermediate in the formation of NO from L-Arg.

The metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) was studied in the mouse macrophage cell line J774 and in the human monocytic cell line U937 in the absence or presence of Escherichia coli lipopolysaccharide (LPS). Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in J774 cells. In addition, NO produced from GTN by cells or by cellular fractions was measured as nitrite (NO − 2 ) one of its breakdown products. J774 cells (1.25 × 10 5 cells) treated with indomethacin (10 μM) enhanced the platelet inhibitory activity of GTN (22-352 μM) but not that of sodium nitroprusside (4 μM). This effect was abrogated by co-incubation with oxyhaemoglobin (oxyHb, 10 μM) indicating release of NO from GTN. U937 cells (up to 60 × 10 5 ) did not metabolize GTN to NO. LPS (0.5 μg/mL for 18 hr) enhanced at least 2-fold the capacity of J774 cells but not that of U937 cells to form NO from GTN and this enhancement was attenuated when cycloheximide (10 μg/mL) was incubated together with LPS. In the absence of LPS stimulation, cycloheximide had no effect. Furthermore, when incubated with GTN (200 μM), J774 cells treated with LPS released more NO from GTN as indicated by a 3-fold greater increase in their level of cGMP which was prevented by oxyHb (10 μM). Incubation of J774 cells with GTN (75–600 μM) for 30 min led to a concentration-dependent increase in NO − 2 which was substantially reduced when the cells were boiled. The microsomal fraction was more potent than the cytosol in producing NO − 2 from GTN (1.2–2.4 mM). Release of NO − 2 from GTN by J774 cells was not affected by treating the cells with the NO synthase inhibitor, N G -monomethyl- l -arginine (MeArg, 300 μM). In J774 cells made tolerant to GTN, potentiation of the anti-platelet effects of GTN (11–352 μM) and release of NO − 2 from GTN was reduced. Thus, J774 cells but not U937 cells convert GTN to NO. This enzymic pathway (present mainly in the microsomal fraction of the J774 cells) is induced by LPS and is not regulated by endogenous NO released from L-Arg by the enzyme NO synthase. Furthermore, when compared to normal cells, tolerant J774 cells metabolize GTN to NO less effectively as assessed by a reduced capacity to potentiate the anti-platelet effect of GTN and to release NO − 2 . The presence of this enzyme system and its induction by LPS in cells other than smooth muscle cells and endothelial cells has therapeutic implications.

1. Endothelin-1 (ET-1), endothelin-3 (ET-3) and noradrenaline (NA) were administered as intra-arterial bolus injections into the isolated, blood-perfused spleen of the dog to assess agonist properties and relative molar potencies on the vascular and capsular smooth muscle. 2. An initial small vasodilatation was observed occasionally at low doses (1.0-10 pmol) of ET-1. 3. ET-1, ET-3 and NA all caused graded increases in splenic arterial vascular resistance. The molar ED50 for the splenic vasoconstrictor response to ET-1 was significantly less (P less than 0.001) than that to ET-3; both peptides were significantly more potent as vasoconstrictor agents than NA. The maximum increase in splenic arterial vascular resistance was not significantly different for either ET-1, ET-3 or NA. 4. The time course of the splenic vasoconstrictor response to ET-1 was significantly (P less than 0.01) longer than that to equieffective doses of ET-3 or NA. 5. The splenic vasoconstrictor responses to ET-1 and ET-3 were accompanied by reductions in spleen volume. The rank order of molar potency in causing splenic capsular contraction was ET-1 greater than ET-3 greater than NA. The maximum reduction in splenic volume was significantly greater for NA than for either ET-1 or ET-3. The two peptides (ET-1, ET-3) were equiefficacious in contracting splenic capsular smooth muscle. 6. The high molar potency of ET-1 as a splenic arterial vasoconstrictor, over 1,700 times more potent than NA, suggests that it may play an important local role in the control of splenic haemodynamics.

Here, we demonstrate that the metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) occurs not only in bovine aortic smooth muscle cells (SMCs) but also in endothelial cells (ECs) and that this biotransformation is enhanced by pretreatment with Escherichia coli lipopolysaccharide (LPS). Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in SMCs or ECs. In addition, NO produced from GTN by cells was measured as nitrite (NO2-), one of its breakdown products. Indomethacin (10 microM)-treated SMCs or ECs enhanced the platelet inhibitory activity of GTN. This effect was abrogated by coincubation with oxyhemoglobin (oxyHb; 10 microM), indicating release of NO from GTN. LPS (0.5 microgram/ml; 18 h) enhanced at least 2- to 3-fold the capacity of SMCs or ECs to form NO from GTN, and this enhancement was attenuated when cycloheximide (10 micrograms/ml) was incubated together with LPS. Furthermore, when incubated with GTN (200 microM) SMCs or ECs treated with LPS (0.5 microgram/ml; 18 h) released more NO from GTN than nontreated cells as indicated by a much higher (8- to 9-fold) increase in the levels of cGMP. Exposure of SMCs to GTN (600 microM) for 30 min led to an increase in the levels of NO2- dependent on cell numbers, which was enhanced when SMCs were treated with LPS. Incubation of nontreated or LPS-treated cells with NG-monomethyl-L-arginine (300 microM; 60 min) did not influence the metabolism of GTN to NO. SMCs failed to enhance the antiplatelet activity of sodium nitroprusside. Anesthetized rats treated with an intraperitoneal injection of LPS (20 mg/kg) 18 h beforehand showed enhanced hypotensive responses to GTN (0.25-1 mg/kg). These effects were blocked by methylene blue (10 mg/kg) but not by indomethacin (3 mg/kg). LPS did not alter the hypotensive responses induced by phentolamine, verapamil, or SIN-1. Thus, both in vitro and in vivo, LPS induces the enzyme(s) metabolizing GTN to NO.

1. Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 microM) promoted a cell number-related inhibition of platelet aggregation induced by thrombin (40 mu ml-1). This inhibition was not attributable to products of the cyclo-oxygenase pathway for the SMC were also treated with indomethacin (10 microM). 2. The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 micrograms ml-1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 micrograms ml-1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non-treated cells. 3. The inhibition of platelet aggregation obtained with non-treated or LPS-treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml-1) and ablated by oxyhaemoglobin (OxyHb, 10 microM). Preincubation of the SMC with NG-monomethyl-L-arginine (L-NMMA, 30-300 microM) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with L-arginine (L-Arg, 100 microM) but not with D-arginine (D-Arg, 100 microM). 4. Exposure of the non-treated SMC (5 x 10(5) cells) to stirring (1000 r.p.m., 37 degrees C) for 10 min led to a significant increase in their levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) but not adenosine 3':5'-cyclic monophosphate (cyclic AMP). L-NMMA (300 microM) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells.5. These findings support the idea that non-treated or LPS-treated cultured SMC can produce an NO-like factor. Production by the latter requires protein synthesis as evidenced by blockade with cycloheximide. This NO-like factor may play a role in the auto-regulation of smooth muscle cell reactivity through a cyclic GMP-dependent mechanism.

Serosal mast cells (MC) from 6 month old spontaneously hypertensive rats (SHR) were compared to MC from 6 month old Wistar Kyoto rats (WKYR) for their ability to release nitric oxide (NO). The relationship between histamine release and NO-like activity from these cells was also investigated. MC from SHR released less NO-like factor than MC from WKYR as assessed by the use of platelet aggregation and soluble guanylate cyclase activation as bioassays for NO. Sodium nitroprusside elevated the concentrations of cGMP to a similar extent in MC from SHR or WKYR. No changes in the levels of cAMP were observed. The release of histamine from MC induced by compound 48/80 or the calcium ionophore A23187 was greater in MC from SHR than in MC from WKYR. Thus, MC from SHR show a decreased production of NO-like activity which is reflected by a decreased ability to inhibit platelet aggregation. The decreased production of cGMP in the MC leads to an increased stimulated release of histamine.

1. Release of endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2) from bovine cultured aortic endothelial cells (EC) was measured by bioassay and radioimmunoassay, respectively. 2. Bradykinin (BK, 3-30 pmol), adenosine diphosphate (ADP, 2-6 nmol) or the sodium ionophore monensin (40-100 nmol) injected through a column of EC released EDRF. L-Arginine free base (FB; 10-20 mumol) or D-arginine FB (10-20 mumol) injected through the column of EC released similar amounts of EDRF and also caused an increase in pH of the Krebs solution perfusing the EC from 7.5-8.0 to 8.6-9.5. Sodium carbonate (Na2CO3) an alkaline buffer which caused the same changes in the pH of the Krebs solution also induced the same release of EDRF. The hydrochloride salts of L- or D-arginine did not cause either release of EDRF when injected through the column of EC or increases in the pH of the Krebs solution. 3. Inhibitors of either diacylglycerol lipase (RHC 80267) or kinase (R59022) inhibited the release of EDRF induced by BK or ADP but potentiated the release induced by L-arginine FB, monensin (40-100 nmol) or alkaline buffer (Na2CO3). R59022 and RHC 80267 infused through the EC increased the basal release of EDRF. 4. When calcium chloride was omitted from the Krebs solution the release of EDRF induced by alkaline buffer (Na2CO3; pH 8.6-9.5) or L-arginine FB (10-20 mumol) was selectively inhibited when compared to that induced by BK (3-30 pmol) or ADP (2-6 nmol). This inhibition was reversed when calcium (2.5 mM) was restored. 5. NG-monomethyl-L-arginine (NMMA; 30 microM) inhibited release of EDRF induced by BK (10-30 pmol) or alkaline buffers (Na2CO3 or D-arginine FB; pH 8.6-9.5). This inhibition was partially reversed by L- but not D-arginine FB or HCl (30-100 microM). 6. Prostacyclin was released when BK (10 pmol), ADP (2 nmol) or arachidonic acid (30 nmol) were injected through the column of EC. However, monensin (40 nmol) or alkaline buffers (pH 8.6-9.5) did not release detectable amounts of PGI2 as measured by radioimmunoassay for 6-oxo-prostaglandin F1 alpha. 7. Thus alkalinisation of the external bathing solution can release EDRF from cultured EC by a mechanism which does not involve receptor activation and which depends on the presence of extracellular calcium.

The osteoclast is unique in its ability to resorb bone, and excessive osteoclastic activity has been implicated in osteoporosis, Paget disease of bone, rheumatoid arthritis, and the growth of metastases in bone. The activity of this cell is controlled by the main circulating inhibitor, calcitonin, in association with locally produced modulators. We show that nitric oxide (NO) may be an important member of the latter group. NO is produced by the vascular endothelium and nervous system and is involved in both neurotransmission and the regulation of blood pressure. However, our results show that the autocoid is also a potent inhibitor of osteoclast function. NO (30 microM) produced a decrease to approximately 50% of the original osteoclast spread area. Similar effects were also produced by 3-morpholinosydnonimine or sodium nitroprusside, reagents that spontaneously release NO. These shape changes were associated with a reduction of bone resorption after a 24-hr incubation of isolated osteoclasts on devitalized bone slices. NO is thought to act by stimulating guanylate cyclase, with a consequent increase in cyclic GMP, but a different mode of action is likely in the osteoclast since dibutyryl or 8-bromo cyclic GMP have no effect. It should be noted that calcitonin can produce similar changes in shape and activity but is associated with an increase in osteoclast intracellular calcium and cessation of membrane movement; neither of these is produced by NO, suggesting that its mode of action is different. The abundance of NO-producing endothelial cells in bone marrow and their proximity to osteoclasts suggests that marrow endothelial cells may play a physiological role in the regulation of osteoclastic activity.

1. In the present study the role of endogenous nitric oxide (NO) was investigated, in the regulation of renal cortical blood flow (RCBF) in vivo in anaesthetized rats under conditions in which prostacyclin involvement had been eliminated. 2. Infusions of the NO synthesis inhibitor NG-monomethyl-L-arginine (MeArg) at 1 or 3 mg kg-1 min-1, i.v., produced significant decreases in RCBF of 29 +/- 7% and 35 +/- 5%, respectively. These effects were reversed by co-infusion of a 3 fold excess of L-arginine (L-Arg). 3. Similarly, intravenous infusion of N omega-nitro-L-arginine methyl ester (NO2Arg) at 30 or 300 micrograms kg-1 min-1 attenuated RCBF by 21 +/- 4% or 53 +/- 4%, respectively, and these effects were reversed by L-Arg (3 or 10 mg kg-1 min-1, i.v.). Most importantly, a low dose of NO2Arg (30 micrograms kg-1 min-1, i.v.), while having no pressor effect, considerably reduced RCBF, indicating that basal release of NO is important for the maintenance of renal cortical blood flow. 4. MeArg (3 mg kg-1 min-1, i.v.) or NO2Arg (300 micrograms kg-1 min-1, i.v.) inhibited endothelium-dependent acetylcholine (ACh, 10 micrograms kg-1 min-1, i.v. for 3 min) increases in RCBF in an L-Arg reversible manner, but did not affect endothelium-independent (dopamine 10 micrograms kg-1 min-1, i.v., for 3 min) increases. Endothelin- 1 (1 nmol kg-1, i.v.), when given as a control for the vasoconstrictor effects of MeArg and NO2Arg, produced a slight inhibition of the ACh-induced increase in RCBF, but this effect was significantly weaker than that produced by MeArg or NO2Arg. 5. Our findings suggest that MeArg and NO2Arg inhibit basal and ACh-stimulated release of NO in the renal cortical vasculature. Thus, endogenous NO formation may play an important role in the local regulation of renal cortical blood flow.

The neuropeptide FMRFamide (L-Phe-L-Met-L-Arg-L-Phe-NH2) increases mean arterial blood pressure (MABP) and heart rate (HR) in the anaesthetised rat at concentrations ranging from 10-1000 micrograms/kg. Here, we demonstrate that peptides containing L-arginyl-L-phenylalanine (L-Arg-L-Phe), the C-terminal sequence of FMRFamide, mimic its haemodynamic effects. L-Arg-L-Phe was approximately 4 fold more potent in increasing MABP and HR than FMRFamide. In 40 different peptides investigated, the following order of potency of the effective compounds was established: L-Arg-L-Phe-L-Ala = L-Arg-L-Phe greater than FMRFamide greater than L-Met-L-Arg-L-Phe = L-Arg-L-Trp greater than L-Arg-L-Tyr greater than D-Arg-L-Phe = L-Arg-L-Phe-OMe greater than L-Arg-L-Leu = L-Arg-L-Ile greater than L-Lys-L-Phe greater than L-Arg-L-Met. L-Arg-L-Phe or FMRFamide did not cause any pressor response in pithed rats, indicating a central mechanism of action. In anaesthetised rats, intravenous injections of FMRFamide or L-Arg-L-Phe (100 micrograms/kg) were associated with a 2-3 fold increase in plasma noradrenaline levels, whereas plasma adrenaline levels remained unchanged. Thus, L-Arg-L-Phe may represent the active principle of FMRFamide acting by a central mechanism involving the release of noradrenaline from sympathetic nerve terminals.

The stimulation of gastric-acid secretion by pentagastrin, a synthetic analogue of the endogenous peptide gastrin, is associated with an increased blood flow to the stomach mucosa, commonly referred to as functional hyperaemia. There are at least two potent vasodilator substances, the local release of which from endothelial cells could contribute to this hyperaemia, endothelium-derived relaxing factor (EDRF) and prostacyclin. EDRF has been identified as nitric oxide, released enzymatically from the guanidino group of L-arginine. In the present studies, the involvement of prostacyclin in the pentagastrin-induced increase in stomach blood flow was eliminated by using the cyclo-oxygenase inhibitor indomethacin. Thus this work was designed to elucidate the participation of EDRF/NO in the pentagastrin-induced hyperaemia and not its relative importance to prostacyclin. The increase in blood flow to the gastric mucosa in response to pentagastrin was measured by laser Doppler flowmetry in situ . Inhibition of EDRF/NO biosynthesis with the L-arginine analogues N G -monomethyl-L-arginine (MeArg) or N ω -nitro-Larginine (NO 2 Arg) significantly attenuated (by more than 80%) the increase in mucosal blood flow in response to pentagastrin. However, infusions of the natural substrate L-arginine reversed the inhibitor effect of MeArg on pentagastrin-induced increase in mucosal blood flow. Local intra-arterial injections of the endothelium-independent vasodilator glyceryl trinitrate produced a dose-related increase in blood flow to the rat stomach mucosa that was unaffected by infusion of MeArg. Thus, in the absence of prostacyclin, EDRF/NO participates in the pentagastrin-induced increase in blood flow to the rat stomach mucosa.

Endothelin (ET)-1 (0.1–1 nmol/kg), ET-2 (0.1–1 nmol/kg) or ET-3 (0.3–3 nmol/kg) dose dependently inhibited platelet aggregation induced by adenosine di-phosphate (ADP) ex vivo in anaesthetised rabbits, while having no effect on aggregations induced by ADP, collagen or arachidonic acid in vitro. This anti-aggregatory effect of the peptides is most likely due to the release of prostacyclin into the circulation, for the inhibition was abolished by an injection of indomethacin (5 mg/ml). All three peptides produced a significant, bi-phasic reduction of the euglobulin clot lysis time. This ET-induced enhancement of plasma fibrinolytic activity was associated with a release of tissue plasminogen activator into the circulation. ET-1 or ET-2 caused a transient decrease in left ventricular systolic pressure (LVSP) followed by a prolonged pressor response. However, ET-3, while inducing a similar transient fall in LVSP caused a second, more prolonged, decrease in LVSP. The haemodynamic responses to all three peptides were modulated by the release of prostanoids, as evidenced by the elevation of the pressure responses by indomethacin.

1. A non-invasive technique for the scintigraphic determination of 111indium-labelled platelet aggregation stimulated with submaximal doses of adenosine diphosphate (ADP, 56 micrograms kg-1 i.v.), collagen (100 micrograms kg-1 i.v.), platelet-activating factor (PAF, 0.1 microgram kg-1 i.v.) or thrombin (18 iu kg-1 i.v.) was used to investigate the platelet-inhibitory effects of endothelin 1 (ET-1) in anaesthetized rabbits in vivo. 2. ET-1 (1 nmol kg-1 i.v.) inhibited ADP-stimulated platelet aggregation in vivo; a maximum inhibition of 78% of the control value was reached at 3 min, with 45% inhibition at 15 min, and a return to control values at 30 min after injection of the peptide. 3. ET-1 (1 nmol kg-1 i.v.) inhibited in vivo platelet aggregation in response to collagen or PAF by 86% and 52%, respectively, but had no effect on thrombin-induced platelet aggregation. 4. Indomethacin (5 mg kg-1 i.v.) abolished the ET-1-induced inhibition of ADP-stimulated platelet aggregation and significantly potentiated and prolonged the pressor response brought about by ET-1. 5. In conclusion, the data demonstrate that ET-1 potently inhibits platelet aggregation in the anaesthetized rabbit in vivo by releasing a hypotensive and anti-aggregatory cyclo-oxygenase product, presumably prostacyclin, into the circulation.

Elevated plasma levels of endothelin-1 (ET-1) in patients with septic shock have implicated ET-1 in the vascular hypoperfusion and organ dysfunction, including renal failure, that occur in this condition. Administration of endotoxin to rats induces renal dysfunction characterized by increased blood urea nitrogen (BUN) and plasma creatinine (Cr) levels. Here, we have found that neither SB 209670 (an ETA and ETB receptor antagonist) nor an affinity-purified ET antibody blocked the endotoxin-induced changes in renal function, suggesting that ET-1 is not involved in the pathophysiology of endotoxin-induced renal dysfunction.

Just over 100 years ago Felix Hoffman synthesised acetylsalicylic acid, which was marketed as Aspirin by the Bayer Company. It is now selling more than 45,000 tonnes per year. The story of the aspirin-like drugs [1] is a fascinating one, in which our discovery that they inhibit the formation of prostaglandins (PGs) [2] was a key stepping stone in its development. The enzyme involved is cyclooxygenase (COX) and we now know that there are at least two isozymes, COX-1 and COX-2. Now we have selective COX-2 inhibitors, which have the promise of being excellent anti-inflammatory drugs without the side effects on the stomach [3].

Prostaglandins formed by cyclooxygenase-1 (COX-1) or COX-2 produce hyperalgesia in sensory nerve endings. To assess the relative roles of the two enzymes in pain processing, we compared responses of COX-1- or COX-2-deficient homozygous and heterozygous mice with wild-type controls in the hot plate and stretching tests for analgesia. Preliminary observational studies determined that there were no differences in gross parameters of behavior between the different groups. Surprisingly, on the hot plate (55°C), the COX-1-deficient heterozygous groups showed less nociception, because mean reaction time was longer than that for controls. All other groups showed similar reaction times. In the stretching test, there was less nociception in COX-1-null and COX-1-deficient heterozygotes and also, unexpectedly, in female COX-2-deficient heterozygotes, as shown by a decreased number of writhes. Measurements of mRNA levels by reverse transcription–PCR demonstrated a compensatory increase of COX-1 mRNA in spinal cords of COX-2-null mice but no increase in COX-2 mRNA in spinal cords of COX-1-null animals. Thus, compensation for the absence of COX-1 may not involve increased expression of COX-2, whereas up-regulation of COX-1 in the spinal cord may compensate for the absence of COX-2. The longer reaction times on the hot plate of COX-1-deficient heterozygotes are difficult to explain, because nonsteroid anti-inflammatory drugs have no analgesic action in this test. Reduction in the number of writhes of the COX-1-null and COX-1-deficient heterozygotes may be due to low levels of COX-1 at the site of stimulation with acetic acid. Thus, prostaglandins made by COX-1 mainly are involved in pain transmission in the stretching test in both male and female mice, whereas those made by COX-2 also may play a role in the stretching response in female mice.

Rat serosal mast cells were evaluated for their capacity to generate a nitric oxide-like factor by two bioassays: inhibition of platelet aggregation and stimulation of mast cell guanylate cyclase. Incubation of mast cells with human washed platelets, both treated with indomethacin, inhibited thrombin-induced platelet aggregation which was potentiated by superoxide dismutase and reversed by oxyhaemoglobin. When mast cells alone were stirred at 1000 rpm, a time dependent increase in the levels of their cGMP but not cAMP was observed. Preincubation of mast cells with NG-monomethyl-L-arginine significantly enhanced E. coli lipopolysaccharide-evoked histamine release. Our results show that mast cell histamine release can be modulated by an intrinsically generated nitric oxide-like factor.

1 Isolated rings of hamster aorta produced an unstable substance which inhibited platelet aggregation in vitro and had the same characteristics as prostacyclin. 2 Prostacyclin inhibited adenosine diphosphate (ADP)-induced aggregation of hamster platelets in vitro. 3 The effects of prostacyclin on ADP-induced platelet thrombi in the microcirculation of the hamster cheek pouch were studied with a television microscope. 4 Prostacyclin caused a dose-dependent increase in the time of iontophoretic application of ADP which was required to induce platelet thrombi formation and embolization in venules (30 to 40 micron diameter). 5 Prostacyclin caused a dose-dependent reduction in the total time during which ADP-induced thrombi were observed following local electrical damage to arterioles (40 to 80 micron diameter). 6 Thrombus formation in venules and arterioles was abolished by 500 ng/ml prostacyclin in the Krebs solution superfusing the hamster cheek pouch. 7 Prostacyclin was approximately twenty times more potent than prostaglandin E1 in preventing thrombus formation in the microcirculation.

1. This study investigates the effects of the non-selective ETA/ETB receptor antagonist, SB 209670, on systemic haemodynamics, renal function, liver function, acid-base balance and survival in a rat model of endotoxic shock. 2. Injection of E. coli lipopolysaccharide (LPS, 10 mg kg-1, i.v.) resulted in increases in the serum levels of tumour necrosis factor-alpha (TNF-alpha, maximum 60 min after LPS), endothelin-1, (ET-1; maximum 120 min after LPS), and interferon-gamma (IFN-gamma, maximum 180 min after LPS). 3. Injection of LPS also resulted in a fall in blood pressure from 113 +/- 3 mmHg (time = 0) to 84 +/- 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the vasoconstrictor responses elicited by noradrenaline (NA, 1 microgram kg-1, i.v.). Pretreatment of rats with a continuous infusion of SB 209670 (3 mg kg-1, i.v. bolus + 100 micrograms kg-1, i.v. infusion commencing 15 min prior to LPS) significantly augmented the hypotension as well as the vascular hyporeactivity to NA caused by endotoxaemia. 4. Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v. bolus given 15 min prior to LPS) or infusion of SB 209670 (bolus dose and infusion as above) resulted in a reduction in 6 h-survival from 71% (control) to 30% and 13%, respectively. 5. Endotoxaemia for 4 h resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), but not in the serum levels of bilirubin, GPT and GOT (indicators of liver dysfunction and/or hepatocellular injury). Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v. bolus 15 min prior to LPS) significantly augmented the serum levels of creatinine, bilirubin, GPT and GOT caused by endotoxin. In addition, endotoxaemia caused, within 15 min, an acute metabolic acidosis (falls in pH, HCO3- and base excess) which was compensated by hyperventilation (fall in PaCO2). Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v. bolus) significantly augmented the metabolic acidosis caused by LPS. 6. Thus, the non-selective ETA/ETB receptor antagonist, SB 209670, augments the degree of (i) hypotension, (ii) vascular hyporeactivity to noradrenaline, (iii) renal dysfunction and (iv) metabolic acidosis caused by endotoxin in the anaesthetized rat. In contrast to rats treated with LPS alone, LPS-rats treated with SB 209670 exhibited liver dysfunction and hepatocellular injury. We propose that the release of endogenous ET-1 serves to maintain blood pressure and subsequently organ perfusion in septic shock.

1. Inflammatory disease states predispose to myocardial infarction. Here we have investigated the effects of a systemic inflammatory response syndrome, i.e. lipopolysaccharide (LPS)-induced circulatory shock in rats, on coronary vascular tone. 2. Anaesthetized rats were given LPS (10 mg kg-1, i.v.) and the hearts excised 180 min later for isolated perfusion at constant flow by the Langendorff technique. Once the ex vivo perfusion was started, the perfusion pressure strongly increased in these hearts compared to hearts from control rats (130 +/- 3 vs. 49 +/- 3 mmHg after 10 min). This increase in coronary resistance was not associated with a reduction in endothelial cell function, for the vasodilator responses to bradykinin were unchanged. 3. When hearts were removed 30 min after the injection of LPS, the LPS-induced rise in perfusion pressure was delayed. No changes in perfusion pressure were seen when the hearts were removed 15 min after the injection of LPS. Pre-treatment with cycloheximide or an anti-tumour necrosis factor-alpha (TNF-alpha) antibody or continuous infusion in vivo and in vitro of the endothelin ETA receptor selective antagonist FR 139317, greatly decreased the increase in coronary vascular resistance induced by LPS. 4. These data suggest that TNF-alpha may induce the release of endothelin-1 (ET-1) and that this mediates at least part of the coronary vasoconstriction. This hypothesis is supported by the demonstration that LPS administration increased the circulating levels of both TNF-alpha and ET-1. 5. We conclude, therefore, that in inflammatory disease states, such as LPS-induced septic shock, there is the sequential release of TNF-alpha and endothelin-1 which leads to an increase in coronary vascular tone and so a predisposition to myocardial ischaemia. Inactivation of TNF-alpha by an antibody as well as ETA receptor blockade by a selective antagonist may effectively interfere with this pathway.

1. This study investigates the effects of two structurally different antagonists of platelet-activating factor (PAF), BN52021 and WEB2086, on the circulatory and renal failure elicited by lipoteichoic acid (LTA) from Staphylococcus aureus (an organism without endotoxin) in anaesthetized rats. 2. Administration of LTA (10 mg kg-1, i.v.) caused hypotension and vascular hyporeactivity to noradrenaline (1 microgram kg-1, i.v.) WEB2086 (5 mg kg-1, i.v., 20 min before and 150 min after LTA) inhibited the delayed fall in mean arterial blood pressure (at 300 min: 99 +/- 6 mmHg vs. 75 +/- 6 mmHg, P < 0.01) and prevented the decrease in pressor response to noradrenaline (at 300 min: 36 +/- 5 mmHg min vs. 17 +/- 5 mmHg min, P < 0.01). Surprisingly, BN52021 (20 mg kg-1, i.v., 20 min before and 150 min after LTA) neither prevented the hypotension (74 +/- 6 mmHg) nor the vascular hyporeactivity (21 +/- 5 mmHg min). However, BN52021 inhibited the hypotension to injections of PAF as well as the circulatory failure elicited by lipopolysaccharides (10 mg kg-1, i.v.). 3. LTA caused an increase in plasma concentration of creatinine from 39 +/- 5 microM (sham-operated) to 70 +/- 8 microM and urea from 4.7 +/- 0.1 to 13.1 +/- 1.6 mM. The renal failure elicited by LTA was significantly inhibited by WEB2086 (creatinine: 45 +/- 4 microM and urea: 5.7 +/- 0.7 mM), but not by BN52021. 4. The induction of nitric oxide synthase activity in lungs by LTA was attenuated by WEB2086 from 98 +/- 17 to 40 +/- 15 pmol L-citrulline 30 min-1 mg-1 protein (P < 0.01), but not by BN52021 (148 +/- 21 pmol L-citrulline 30 min-1 mg-1 protein). Similarly, WEB2086, but not BN52021, inhibited the increase in plasma nitrite concentration associated with the delayed circulatory failure caused by LTA. The release of tumour necrosis factor-alpha (TNF-alpha) after injection of LTA was not attenuated by WEB2086. 5. The induction of nitrite release by cultured macrophages activated with LTA (10 micrograms ml-1 for 24 h) was inhibited by 74 +/- 4% by WEB2086 (3 x 10(-4) M), but not by BN52021, indicating that only WEB2086 acts on intracellular PAF receptors. 6. Thus, the intracellular release of PAF contributes to the circulatory and renal failure and induction of nitric oxide synthase elicited by LTA in anaesthetized rats. The difference between the two structurally different PAF antagonists in our septic shock models using either LTA or lipopolysaccharide (LPS), shows the importance of models for Gram-positive sepsis in the elucidation of the pathophysiology of septic shock and for the evaluation of potential drugs.

1. We have investigated whether (i) endotoxaemia caused by E. coli lipopolysaccharide in the anaesthetized rat causes a multiple organ dysfunction syndrome (MODS; e.g. circulatory failure, renal failure, liver failure), and (ii) an enhanced formation of nitric oxide (NO) due to induction of inducible NO synthase (iNOS) contributes to the MODS. In addition, this study elucidates the beneficial and adverse effects of aminoethyl-isothiourea (AE-ITU), a relatively selective inhibitor of iNOS activity, and NG-methyl-L-arginine (L-NMMA), a non-selective inhibitor of NOS activity on the MODS caused by endotoxaemia. 2. In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 117 +/- 3 mmHg (time 0) to 97 +/- 4 mmHg at 2 h (P < 0.05, n = 15) and 84 +/- 4 mmHg at 6 h (P < 0.05, n = 15). The pressor effect of noradrenaline (NA, 1 micrograms kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS-rats with AE-ITU (1 mg kg-1, i.v. plus 1 mg kg-1 h-1 starting at 2 h after LPS) caused only a transient rise in MAP, but significantly attenuated the delayed vascular hyporeactivity seen in LPS-rats. Infusion of L-NMMA (3 mg kg-1, i.v. plus 3 mg kg-1 h-1) caused a rapid and sustained rise in MAP and attenuated the delayed vascular hyporeactivity to NA. Neither AE-ITU nor L-NMMA had any effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 3. Endotoxaemia for 6 h was associated with a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT), gamma-glutamyl-transferase (gamma GT), and bilirubin, and hence, liver dysfunction. Treatment of LPS-rats with AE-ITU significantly attenuated this liver dysfunction (rise in GOT, GPT, gamma GT and bilirubin) (P < 0.05, n = 10). In contrast, L-NMMA reduced the increase in the serum levels of gamma GT and bilirubin, but not in GOT and GPT (n = 5). Injection of LPS also caused a time-dependent, but rapid (almost maximal at 2 h), increase in the serum levels of urea and creatinine, and hence, renal dysfunction. This renal dysfunction was not affected by either AE-ITU (n = 10) or L-NMMA (n = 5). In rats infused with saline rather than LPS, neither AE-ITU (n = 4) nor L-NMMA (n = 4) had any significant effect on the serum levels of GOT, GPT, gamma GT, bilirubin, creatinine or urea. 4. Endotoxaemia for 6 h resulted in a 4.5 fold rise in the serum levels of nitrite (9.13 +/- 0.77 microM, P < 0.01, n = 15), which was significantly reduced by treatment with AE-ITU (6.32 +/- 0.48 microM, P < 0.05, n = 10) or L-NMMA (5.10 +/- 0.40 microM, P < 0.05, n = 5). In addition, endotoxaemia for 6 h was also associated with a significant increase in iNOS activity in lung and liver homogenates, which was significantly reduced in lung or liver homogenates obtained from LPS-rats treated with either AE-ITU or L-NMMA. 5. Thus, AE-ITU or L-NMMA (i) inhibits iNOS activity in LPS-rats without causing a significant increase in MAP in rats infused with saline and, hence inhibition of endothelial NOS activity, and (ii) attenuates the delayed circulatory failure as well as the liver dysfunction caused by endotoxaemia in the rat. Thus, an enhanced formation of NO may contribute to the development of liver failure in endotoxic shock.

Here we demonstrate that perfused hearts removed from polyarthritic rats develop a pronounced coronary vasoconstriction ex vivo. This vasoconstriction is almost entirely blocked by in vivo pretreatment of the rats with the endothelin receptor antagonist, SB 209670. Thus, inflammatory states may be associated with an increased activity of the endothelin system, leading to vascular dysfunction and vasoconstriction.

1. We have investigated the effects of aminoguanidine, a relatively selective inhibitor of the cytokine-inducible isoform of nitric oxide synthase (iNOS), on the delayed circulatory failure, vascular hyporeactivity to vasoconstrictor agents, and iNOS activity in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide; LPS). In addition, we have evaluated the effect of aminoguanidine on the 24 h survival rate in a murine model of endotoxaemia. 2. Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg-1, i.v.) resulted in a fall in MAP from 115 +/- 4 mmHg (time 0, control) to 79 +/- 9 mmHg at 180 min (P0.05, n = 10). The pressor effect of noradrenaline (NA, 1 microgram kg-1, i.v.) was also significantly reduced at 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with aminoguanidine (15 mg kg-1, i.v., 20 min prior to LPS injection) maintained a significantly higher MAP (at 180 min, 102 +/- 3 mmHg, n = 10, P0.05) when compared to rats given only LPS (LPS-rats). Cumulative administration of aminoguanidine (15 mg kg-1 and 45 mg kg-1) given 180 min after LPS caused a dose-related increase in MAP and reversed the hypotension. Aminoguanidine also significantly alleviated the reduction of the pressor response to NA: indeed, at 180 min, the pressor response returned to normal in aminoguanidine pretreated LPS-rats. 3. Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractile responses elicited by NA (10-9- 10-6 M). Pretreatment with aminoguanidine (15 mg kg- 1, i.v.,at 20 min prior to LPS) significantly prevented this LPS-induced hyporeactivity to NA ex vivo.4. Endotoxaemia for 180 min resulted in a significant increase in iNOS activity in the lung from 0.6 +/- 0.2 pmol mg-1 min-1 (control, n = 4) to 4.8 +/- 0.3 pmol mg-1 min-1 (P0.05, n = 6). In LPS-rats treated with aminoguanidine, iNOS activity in the lung was attenuated by 44+/- 5% (n = 6, P0.05).Moreover, when added in vitro to lung homogenates obtained from LPS-rats, aminoguanidine and N omega-nitro-L-arginine methyl ester (L-NAME; 10-8 to 10-3 M) caused a concentration-dependent inhibition of iNOS activity (n = 3-6, IC50: 30 +/- 12 and 11 +/- 6pEM, respectively P0.05). In contrast,aminoguanidine was a less potent inhibitor than L-NAME of the constitutive nitric oxide synthase in rat brain homogenates (n = 3-6, IC50 is 140 +/- 10 and 0.6 +/- 0.1 I1M, respectively, P0.05). In addition, the inhibitory effect of aminoguanidine on iNOS activity showed a slower onset than that of L-NAME(maximal inhibition at 90 min and 30 min, respectively).5. Treatment of conscious Swiss albino (T/O) mice with a high dose of endotoxin (60 mg kg-1, i.p.)resulted in a survival rate of only 8% at 24 h (n = 12). However, therapeutic application of aminoguanidine (15 mg kg-1, i.p. at 2 h and 6 h after LPS) increased the 24 h survival rate to 75%(n = 8), whereas L-NAME (3 mg kg-1, i.p. at 2 h and 6 h after LPS) did not affect the survival rate(11%, n=9).6 Thus, aminoguanidine inhibits iNOS activity and attenuates the delayed circulatory failure caused by endotoxic shock in the rat and improves survival in a murine model of endotoxaemia. Aminoguanidine,or novel, more potent selective inhibitors of iNOS may be useful in the therapy of septic shock.

A single addition of 3 x I0-7 M ET-1, ET-2 or ET-3 produced contractions that reached a steady state in 28.2 +/- 4.2, 21.1 +/- 1.3 and 24.0 +/- 3.8 min, respectively and took 2.7 +/- 0.4, 2.1 + 0.1 and 1.6 +/- 0.1 min to reach half of this steady-state response.4. Contractions induced by 3 x I0-7 M big ET-11-38 or big ET-11- 39 reached a plateau in 38.5 +/- 3.6 and 35.6 +/- 3.3 min, respectively, and half of these responses were attained in 12.0 +/- 2.5 and 7.1 +/- 1.1 min.Thus, these contractions developed more slowly than those induced by ET-1. Contractions induced by 3 x 10-7 M big ET-21-38 were also much slower to develop than those to ET-2, for these took 49 +/- 2 min to reach plateau and 19.4 +/- 2.1 min to attain half that response. Contractions induced by 3 x 10-7 M big ET-31-41 amide took 50.2 +/- 3.7 min to reach a plateau and 27.3 +/- 3.0 min to reach half of this response.5. Phosphoramidon (0.1, 1 and 3 x 10-4 M) inhibited contractions induced by big ET-11.39. For instance,the contractions induced by 3 x 10-7 M big ET-11-39 were inhibited by 10-4 M or 3 x 10-4 M of phosphoramidon by 62.8 +/- 6.7% or 74.5 +/- 4.6%, respectively. Similarly, contractions induced by ET-21-38 were inhibited by 91.3 +/- 5.4% and the small response induced by big ET-3l-4l amide was abolished by 3 x 10-4M phosphoramidon. Conversely, the neutral endopeptidase (EC 24.11) inhibitor DL-thiorphan(3 x 10-4 M) had no effect. Captopril (10-5 M), pepstatin A (10-5 M), phenylmethylsulphonylfluoride(PMSF, 10-3 M), aprotinin (10-5 M), E-64 (10-5 M), cystatin (10-6 M), leupeptin (10-4 M),chymostatin (10-4 M), or bestatin (10-5 M) did not inhibit but rather increased to a similar, but small degree the contractions induced by 3 to 30 x 10-9 M big ET-11-39. Only captopril (10-5 M) or leupeptin(10-4 M) increased the contraction induced by 3 x 10-7 M big ET-11-39. Phosphoramidon (10-4 M),pepstatin (10-5 M) or PMSF (10-3 M) did not affect contractions induced by ET-1.6. Removal of the epithelium increased by 70% the size of the contraction induced by 5 microM histamine(1.08 +/- 0.05 g; n = 160 to 1.84 +/- 0.14 g; n = 12) but did not affect, in absolute terms, the contraction induced by ET-1 (as a % of the response to histamine, these responses were, of course, apparently depressed). Epithelium removal did, however, increase the size of the contractions induced by 3 to 30 x 10-9 M big ET-1 -39 which was very similar to the effect of the protease inhibitors.7. In competition binding studies on membranes prepared from the guinea-pig gallbladder, 10-11 MET-1 inhibited by 76.9 +/- 3.1% the binding of [125]-ET-I while porcine big ET-11-39 caused no inhibition(0.7 +/- 3.0; n = 3). ET-1 (10-6 M) inhibited binding by 95.7 =/- 1.1% (n = 3) while at this much higher concentration, big ET-11-39 inhibited binding by only 16.8 +/- 4.2% (n = 3). This clearly suggests that big ET-11-39 does not bind directly to ET receptors.8. Thus, a phosphoramidon-sensitive endothelin-converting enzyme (ECE), different from neutral endopeptidase (NEP; EC 24.11) and not located on the epithelium, converts big ET-1 into ET-1 in the gallbladder of the guinea-pig. This ECE appears to act preferentially on big ET-1 or big ET-2 over bigET-3.

Treatment of rats with cytokines has been associated with an increase in the circulating levels of endothelin 1 (ET-1). Here we show that administration of tumor necrosis factor alpha (TNF-alpha; 4 micrograms.kg-1) to anesthetized rats caused within 15 min a strong elevation in the circulating levels of ET-1. This was associated with a striking coronary vasoconstriction in hearts from these animals when they were removed and perfused in vitro by the Langendorff technique. This vasoconstriction was largely overcome by treatment with either the endothelin type A (ETA) receptor antagonist FR 139317 or antibody against ET-1. Furthermore, it was mimicked by in vivo exposure to exogenous ET-1. Endogenously produced TNF-alpha may also cause such a coronary vasoconstriction, for treatment with interleukin 2 (600 micrograms.kg-1) produced an increase in coronary perfusion pressure that correlated with the increases in circulating TNF-alpha. This coronary vasoconstriction was substantially reversed by treatment either with antibody against TNF-alpha or with FR 139317. We suggest, therefore, that cytokine-driven changes in the production of ET-1 are key events in the development of vascular pathologies.

Human and porcine big ET-1 and big ET-2 are similarly potent in contracting parenchymal strips of the guinea-pig lung while big ET-3 is inactive,suggesting that the endothelin-converting enzyme (ECE) which converts big ET-3 is not present and that at least two distinct ECE activities exist, one selective for big ET-1 and big ET-2 and one for big ET-3. Metalloendoprotease inhibitors (phosphoramidon and DL-thiorphan), but not captopril, inhibited the contractions elicited by human big ET-1 and big ET-2 but DL-thiorphan was less active, suggesting that a non-selective enzymatic process is involved in conversion of big ET-1 and big ET-2 in addition to a phosphoramidon-sensitive ECE. Big ET-1 and big ET-2 induced much higher contractions than their corresponding mature peptides. Both metalloendoprotease inhibitors, but not captopril, similarly potentiated contractions induced by ET-1, ET-2 or ET-3 to the level of those evoked by big ET-1 and big ET-2, indicating that only mature ET isopeptides and not their precursors are suceptible to degradation by metalloendoproteases.

Endotoxin causes the expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2. We have compared the ability of endotoxin to increase the activities of these enzymes in bovine aortic endothelial cells and the macrophage cell line (J774.2). Endotoxin (1 microgram ml-1; for 24 h) caused a time-dependent increase in the accumulation of cyclooxygenase metabolites from endogenous arachidonic acid, in both cell types. Cyclooxygenase activity towards exogenous arachidonic acid (30 microM; for 15 min) was also increased in both cell types. Endothelial cells and macrophages also contained comparable amounts of cyclooxygenase-2 protein after incubation with endotoxin for 24 h which was prevented by pretreatment with cycloheximide (10 micrograms ml-1; 30 min prior to endotoxin). Endotoxin for 24 h caused a time-dependent increase in nitrite accumulation in macrophages, but not in endothelial cells. Thus, endotoxin increased cyclooxygenase activity and induced cyclooxygenase-2 protein in endothelial cells and macrophages. Endotoxin also increased NO synthase activity in macrophages, but not in endothelial cells.

1. Cyclo-oxygenase metabolizes arachidonic acid to prostaglandin H2 (PGH2) and exists in at least two isoforms. Cyclo-oxygenase-1 (COX-1) is expressed constitutively whereas COX-2 is induced by lipopolysaccharide (LPS) and some cytokines in vitro and at the site of inflammation in vivo. Epithelial cells may be an important source of prostaglandins in the airways and we have, therefore, investigated the expression of COX-1 or COX-2 isoforms in primary cultures of human airway epithelial cells or in a human pulmonary epithelial cell line (A549). 2. COX-1 or COX-2 protein was measured by western blot analysis using specific antibodies to COX-2 and selective antibodies to COX-1. The activity of COX was assessed by the conversion of either endogenous or exogenous arachidonic acid to four metabolites, PGE2, PGF2 alpha, thromboxane B2 or 6-oxo PGF1 alpha measured by radioimmunoassay. Thus, COX-1 or COX-2 activity was measured under two conditions; initially the accumulation of the COX metabolites formed from endogenous arachidonic acid was measured after 24 h. In other experiments designed to measure COX activity directly, cells were treated with cytokines for 12h before fresh culture medium was added containing exogenous arachidonic acid (30 microM) for 15 min after which COX metabolites were measured. 3. Untreated primary cells or A549 cells contained low amounts of COX-1 or COX-2 protein. Bacterial LPS (1 micro g ml-1 for 24 h) induced COX-2 protein in the primary cells, a process which was enhanced by interferon-gamma, with no further increase in the presence of a mixture of cytokines (interleukin-1 beta, tumour necrosis factor-alpha and interferon-gamma, 10 ng ml-1 for all). In contrast, A549 cells contained only low levels of COX-2 protein after exposure to LPS or LPS plus interferon-y, but contained large amounts of COX-2 protein after exposure to the mixture of cytokines.4. Untreated human pulmonary primary cells or A549 cells released low levels of all COX metabolites measured over a 24 h incubation period. This release was enhanced by treatment of either cell type with the mixture of cytokines (interleukin-1 beta , tumour necrosis factors- and interferon-gamma, 10 ng ml-1 for all).PGE2 was the principal COX metabolite released by cytokine-activated epithelial cells. The release of PGE2 induced by cytokines occurred after a lag period of more than 6 h.5. The glucocorticosteroid, dexamethasone (1 micro M; 30 min prior to cytokines) completely suppressed the cytokine-induced expression of COX-2 protein and activity in both primary cells and A549 cells.6. In experiments where COX-2 activity was supported by endogenous stores of arachidonic acid,treatment of A549 cells with interleukin-l beta but not tumour necrosis factor a or interferon-gamma alone caused a similar release of PGE 2 to that seen when the cytokines were given in combination. However, both interleukin-l beta and necrosis factor- alone produced similar increases in COX-2 activity (measured in the presence of exogenous arachidonic acid) as seen when the mixture of interleukin-l beta, tumour necrosis factor- alpha and interferon-gamma were used to stimulate the cells.7. These findings show that COX-2 expression correlates with the exaggerated release of prostaglandins from cytokine-activated human pulmonary epithelial cells and that the induction of the enzyme is suppressed by a glucocorticosteroid. These findings may be relevant to inflammatory diseases of the lung, such as asthma.

1 We have characterized the receptors mediating contractions induced by endothelin-1 (ET-1), ET-2, ET-3 and the ETB-selective receptor agonists, sarafotoxin 6c (SX6c), IRL 1620, BQ-3020, [Ala1,3,11,15]ET-1 and ET (16–21) in strips of the isolated gall bladder of the guinea-pig (GPGB). We used as antagonists BQ-123 (ETA receptor selective) and PD 145065 (ETA/ETB receptor non-selective). 2 ET-1, ET-2 and ET-3 (10−10 M to 3 × 10−7 M) caused similar slowly-developing concentration-dependent contractions of the GPGB. Contractile effects induced by ET-1, ET-2 or ET-3 (at 3 × 10−7 M) were also similar (230 ± 25, 241 ± 7 and 287 ± 37% of that to histamine at 5 × 10−6 M, n = 7, 6, 12, respectively). However, the threshold concentration for ET-1 or ET-2 was 10−1 M whereas it was 3 × 10−9 M for ET-3. 3 SX6c (10−10 M to 3 × 10−7 M) also caused slowly-developing concentration-dependent contractions at a threshold concentration of 10−1 M (n = 16). However, the contraction caused by SX6c at 3 × 10−7 M was 116 ± 9% of that to histamine at 5 × 10−6 M, which was half of that induced by the same concentration of the ET isopeptides. The contraction induced by IRL 1620 at 3 × 10−7 M (n = 9) was 43 ± 9% of that to histamine at 5 × 10−6 M, which was one fifth of that produced by the same concentration of ET-1. Contractions induced by BQ-3020 or [Ala1,3,11,15]ET-1 at 3 × 10−7 M were even less than those produced by IRL 1620. ET (16–21) was inactive up to 10−5 M. Addition of a concentration of 3 × 10−7 M of ET-1 to tissues with developed contractions induced by the bolus addition of 3 × 10−7 M SX6c caused a further contraction of the GPGB to the level observed with ET-1 alone at 3 × 10−7 M (n = 8). 4 BQ-123 (10−5 M) did not affect the concentration-response curve to ET-1 and the contraction induced by 3 × 10−7 M was also not affected (n = 5; 239 ± 19% of histamine at 5 × 10−6 M). PD 145065 (10−5 M) shifted the ET-1 concentration-response curve to the right and the contraction induced by ET-1 at 3 × 10−7 M was inhibited by 15% (n = 6; NS). A higher concentration of BQ-123 (10−4 M) caused a significant shift to the right of the ET-1 concentration-response curve similar to that caused by PD 145065 (10−5 M) and caused a 24% (n = 6; NS) inhibition of the contractions induced by ET-1 at 3 × 10−7 M. PD 145065 (10−4 M) abolished contractions induced by ET-1 (up to 10−7 M) and inhibited the response to ET-1 at 3 × 10−7 M by 52% (n = 4; P < 0.05). 5 Contractions induced by ET-3 were more sensitive to inhibition by the antagonists. BQ-123 (10−6, 10−5 or 10−4 M) inhibited responses to 3 × 10−7 M ET-3 by 66, 71 and 83%, respectively (n = 5, 5, 3; P < 0.05). PD 145065 (10−6, 10−5 or 10−4 M) attenuated more strongly than did BQ-123 the contractions induced by ET-3. For instance, the contractions caused by ET-3 at 3 × 10−7 M were decreased by 73 and 80% (n = 5, 5; P < 0.05) in the presence of PD 145065 (10−6 or 10−5 M, respectively). PD 145065 (10−4 M) completely abolished contractions to ET-3 (n = 4; up to 3 × 10−7 M). 6 Contractions induced by SX6c, especially those observed at concentrations lower than 10−8 M, were attenuated by BQ-123 (up to 10−4 M). PD 145065 (10−5 M) shifted to the right the concentration-response curve to SX6c and inhibited by 38% (P < 0.05) the contractions induced by 3 × 10−7 M. However, the contractions induced by a bolus addition of a high concentration of SX6c (3 × 10−7 M) and the subsequent addition of an identical concentration of ET-1 on top of SX6c were not affected by BQ-123 (10−6 or 10−5 M). 7 These results suggest that ETB receptors are involved in the contractions induced by endothelins in the GPGB. However, SX6c and other selective ETB agonists produced only half or less than half of the contractile response induced by non-selective agonists. In addition, the responses to ET-1 but not to ET-3, were insensitive to the antagonist action of BQ-123 at 10−5 M whereas BQ-123 or PD 145065 at 10−5 M strongly antagonized contractions induced by ET-3. Finally, BQ-123 at 10−4 M inhibited contractions to ET-1 and SX6c. Thus, within the GPGB there may well be additional ET receptor(s) not conforming to the established ETA/ETB receptor subtype classification, as well as ETB receptors.