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Cell death is a common feature observed in neurodegenerative disorders, and is often associated with calpain activation and overproduction of reactive oxygen species (ROS). This study investigated the use of calpain inhibitors and antioxidants in combination to protect cells against necrosis. Maitotoxin (MTX), which induces a massive influx of calcium, was used to provoke neuronal cell death. This toxin increased, in a concentration-dependent manner, both calpain activity and ROS formation. Calpain inhibitors or antioxidants inhibited MTX-induced necrosis only marginally (below 20%), whereas their association protected against cell death by 40-66% in a synergistic manner. BN 82204, which possesses both calpain-cathepsin L inhibitory and antioxidant properties, and its acetylated pro-drug BN 82270, totally protected cells at 100 microm. The pro-drug BN 82270, which had better cell penetration, was twice as effective as the active principle BN 82204 in protecting glioma C6 or neuroblastoma SHSY5Y cells against death. These results suggest the potential therapeutic relevance of using a single molecule with multiple activities (cysteine protease inhibitor/antioxidant), and warrant further in vivo investigations in models of neuronal disorders.

There is substantial evidence that excitotoxicity and oxidative damage may contribute to Huntington's disease (HD) pathogenesis. We examined whether the novel anti-oxidant compound BN82451 exerts neuroprotective effects in the R6/2 transgenic mouse model of HD. Oral administration of BN82451 significantly improved motor performance and improved survival by 15%. Oral administration of BN82451 significantly reduced gross brain atrophy, neuronal atrophy and the number of neuronal intranuclear inclusions at 90 days of age. These findings provide evidence that novel anti-oxidants such as BN82451 may be useful for treating HD.

Nitric oxide (NO) and reactive oxygen species are both implicated in neuronal death due to cerebral ischemia. BN 80933, an original compound associating an inhibitor of neuronal NO synthase with an antioxidant, has been shown to reduce functional and histological damage in rat submitted to cerebral ischemia. The aim of the present study was to confirm these results in mice and to further examine the effects of BN 80933 on inflammatory response, including blood-brain barrier (BBB) disruption, brain edema, and neutrophil infiltration after transient middle cerebral artery occlusion (MCAO). Intravenous administration of BN 80933 at 3 and 10 mg/kg 3 h after MCAO significantly reduced by 26 to 36% the infarct volume evaluated 24 and 48 h after ischemia, and improved the neurological score. Furthermore, BN 80933 at both dosages decreased by 42 to 75% the extravasation of Evans blue in brain parenchyma observed 24 h after ischemia. This reduction in BBB disruption was associated with decreased brain edema as demonstrated by the 37% reduction in brain water content induced by BN 80933 at 3 mg/kg 24 h after MCAO. Neutrophil infiltration in brain parenchyma, evaluated by the myeloperoxidase activity, was also reduced by 45 to 56% in animals treated with BN 80933 at 3 and 10 mg/kg. Together, these results extend the protective capacity of BN 80933 against brain ischemic injury and confirm that BN 80933 represents a promising treatment for stroke.

Both nitric oxide synthase (NOS) inhibitors and free radical scavengers have been shown to protect brain tissue in ischemia-reperfusion injury. Nitric oxide and superoxide anion act via distinct mechanisms and react together to form the highly deleterious peroxynitrite. Therefore the authors examined the effects and the interaction between the NOS inhibitor, NG nitro-L-arginine (LNA) and the antioxidant/superoxide scavenger, di-tert-butyl-hydroxybenzoic acid (DtBHB) in the rat submitted to 2 hours of middle cerebral artery occlusion. Posttreatment was initiated 4 hours after the onset of ischemia and infarct volume was measured at 48 hours. The dose-related effect of LNA resulted in a bell-shaped curve: 15, 56, 65, and 33% reduction of total infarct for 0.03, 0.1, 0.3, and 1 mg/kg (intravenously [IV]) respectively and 11% increase in infarct volume for 3 mg/kg (IV). Whereas DtBHB (20 mg/kg; intraperitoneally [IP]) was ineffective, the dose of 60 mg/kg produced 65% protection in infarct volume. The combination of a subthreshold dose of LNA (0.03 mg/kg; IV) and DtBHB (20 mg/kg; IP) resulted in significant reduction (49%) in infarct volume. These results show that LNA and DtBHB act synergistically to provide a consistent neuroprotection against ischemic injury when administered 4 hours after ischemia. This suggests that nitric oxide and free radicals are involved and interact in synergy in ischemia-reperfusion injury.

This study examines the ability of different nitric oxide synthase (NOS) inhibitors and NO donors to inhibit the endothelium-dependent relaxation of the rat aorta and the NANC relaxation of the rat gastric fundus.NG-Nitro- l -arginine,N-monomethyl- l -arginine, andS-methyl- l -thiocitrulline elicite comparable potency in the aorta and in the fundus. However, 1-(2-trifluoromethyl)imidazole (TRIM), unlike 7-nitroindazole, is more potent on the fundus than on the aorta, showing that TRIM elicits a selective functional inhibition of the neural NOS isoform. (1H)-(1,2,4)Oxadiazole(4,3-a)quinoxalin-1-one, a selective inhibitor of soluble guanylyl cyclase, inhibits the dilator response in both tissues and the cyclic GMP mimetic, 8-Br-cGMP, is 16 times more potent for inducing relaxation in the gastric fundus than in the aorta. However, methylene blue and LY-83583, two other inhibitors of soluble guanylyl cyclase and superoxide anion-generating agents, are at least 100 times less potent on fundus strips than on aortic rings. The data suggest that once released into the extracellular space, NO is more susceptible to inactivation by superoxide anions in the vascular tissue than in the gastric fundus. Thus, the study shows that selective inhibition of NO in a target tissue may be reached not only at the NOS isoform level but also by the manipulation of the NO pathway.

Endothelin receptors were characterized in rat prostate and potential modification of these receptors was investigated in prostatic hypertrophy induced by testosterone. Both ET(A) and ET(B) endothelin receptor mRNA were detected in rat prostate, whereas binding experiments show the presence of only ET(A) receptors. Testosterone administration produced a 75% increase in prostate weight. Although the density of prostatic endothelin receptors was decreased from 348 +/- 75.0 fmol/mg protein in control rats to 252 +/- 39.9 fmol/mg protein in testosterone-treated animals, the total amount of receptors per prostate was unchanged. The steady-state level of ET(A)- and ET(B)-receptor mRNA was not altered by testosterone treatment. These results suggest that endothelin receptors are not affected in prostatic hypertrophy induced by testosterone.

Endothelin-1 (Et1), like angiotensin II, is implicated in postnatal maturation and development. The present study was designed to identify Et1 receptors and subtype Et1 receptors present in rat kidney between 1 and 30 days of postnatal life. On day 1, high-affinity and high-density Et1 binding sites were identified in rat kidney. The dissociation constant and maximum binding for ET1 to membranes from whole kidney were 0.073 +/- 0.05 nM and 1,345.9 +/- 73 fmol/mg protein, respectively. On day 30, affinity and receptor density were markedly decreased. The dissociation constant and maximum binding were 0.147 +/- 0.021 nM (P0.01) and 633.2 +/- 56.4 fmol/mg protein (P0.001), respectively. Using BQ 123 (EtA-selective antagonist) and sarafotoxin S6c (EtB-selective agonist), the two Et1 receptor subtypes EtA and EtB were identified in 1- and 30-day-old rat kidney. BQ 123 selectively recognized EtA receptors with high affinity (2.9 +/- 0.44 on day 1 and 4.0 +/- 0.5 nM on day 30) and sarafotoxin S6c bound with higher affinity EtB receptors (0.871 +/- 0.14 on day 1 and 0.717 +/- 0.12 nM on day 30). Between birth and day 30, the EtA binding capacity was decreased (304 +/- 27 vs. 752 +/- 202 fmol/mg protein, P0.05), whereas EtB binding was not affected (514 +/- 87 vs. 656 +/- 171 fmol/mg protein, NS). The decrease in the total number of Et1 receptors during the 1st month of life may be due to the concomitant decrease in the number of EtA receptors. Increased Et1 receptor density in early postnatal life suggests an influence of Et1 on immature kidney circulation and/or kidney growth.

The synthetic polyribonucleotide poly(A).poly(U) induces 2',5'-oligoadenylate synthetase activity in the murine macrophage cell line J774A1. The possible role of several cytokines involved in macrophage activation (i.e., IL-1, IL-6, TNF, and IFN) was examined in the present study. It was first demonstrated that among the anticytokine antibodies, only monoclonal antibodies directed against IL-6 inhibited the induction of 2',5'-oligoadenylate synthetase by poly(A).poly(U) in a dose-dependent manner. Moreover, it was established that poly(A).poly(U) elicited IL-6 production in J774A1 cells in a time-and dose-dependent manner. Consequently, the effect of IL-6 on 2',5'-oligoadenylate synthetase activity was studied. IL-6 either alone or in combination with IL-1 and TNF did not induce 2',5'-oligoadenylate synthetase activity. IL-6 did not potentiate IFN-gamma-induced 2'-5'-oligoadenylate synthetase activity. In contrast, addition of IL-6 to the incubation medium potentiated the stimulation of 2'-5'-oligoadenylate synthetase activity by IFN-alpha. These results suggest that IL-6 is a necessary but not sufficient factor in the induction of 2'-5'-oligoadenylate synthetase activity in the J774A1 cell line by poly(A).poly(U).

Increased vascular production of nitric oxide (NO) may contribute to the peripheral vasodilation and hyperdynamic state complicating advanced liver cirrhosis. In this study, we examined the effect on forearm blood flow of local brachial artery infusion of noradrenaline (NA) and NG-monomethyl-l-arginine (l-NMMA), an inhibitor of NO-synthase, in 10 alcoholic ascitic cirrhotic patients (patients with decompensated alcohol-induced liver disease: DALD group) and 10 patients with well-compensated alcohol-induced liver disease (CALD group). Forearm blood flow was measured by venous occlusion plethysmography. As compared with the CALD group, the DALD group had higher cardiac index and forearm blood flow as well as lower systemic blood pressure and vascular resistance. Infusions of NA and l-NMMA produced similar reduction in resting blood flow in the CALD group. However, in the DALD group, NA was significantly less effective than l-NMMA. The forearm vasoconstrictor response to NA was also significantly reduced in the DALD group when compared with the CALD group. In the DALD group, NA decreased forearm blood flow by 21.0 ± 6.2% and increased vascular resistance by 37.2 ± 12.3%, whereas respective changes in the CALD group were 41.8 ± 6.2% (P < .01) and 77.8 ± 9.9% (P < .02). In contrast, l-NMMA induced greater forearm vasoconstriction in the DALD group than in the CALD group. In decompensated patients, l-NMMA decreased forearm blood flow by 50.4 ± 2.7% and increased vascular resistance by 115.9 ± 14.4%, whereas changes in compensated patients were 38.2 ± 4.9% (P < .05) and 77.4 ± 16.2% (NS), respectively. These results are consistent with the hypothesis that increased vascular synthesis of NO contributes to the high dynamic state of patients with advanced cirrhosis.

Objectives. This study examined the possible association between endothelin and coronary atherosclerosis and evaluated the synthesis and release of endothelin in the presence of various stimuli that occur during cardiac catheterization. Background. Circulating endothelin has been reported to be increased in diffuse atherosclerosis and acute myocardial infarction. However, the relation between coronary artery disease and endothelin release remains unclear. Methods. We measured the plasma and urinary concentrations of endothelin immunoreactivity in 45 patients and 10 healthy control subjects. Results. In group IA (n = 9), simultaneous blood sampling in the coronary sinus and femoral artery during coronary angioplasty of the left anterior descending coronary artery demonstrated no immediate changes in plasma immunoreactive endothelin-1 (ir-ET-1) levels. In 11 patients in group IB undergoing coronary angioplasty of a major artery, we did not detect changes in peripheral plasma concentrations of ir-ET-1 within 24 h, but urinary ir-ET-1 levels increased from 9.2 ± 2.3 to 18.6 ± 4.9 pg/mg of creatinine a few hours after coronary angioplasty (mean ± SEM, p < 0.05). This increase in urinary endothelin excretion persisted 24 h later. Group II patients (n = 12) had coronary angiography without coronary angioplasty. Levels of both plasma and urinary ir-ET-1 did not change during the 24-h follow-up period. There was no relation between the severity of coronary atherosclerosis and the plasma or urinary concentrations of ir-ET-1. Systolic aortic pressure correlated with basal urinary excretion of endothelin (r = 0.54, p = 0.03, n = 15). In group III (n = 13), levels of ir-ET-1 in patients undergoing right heart catheterization without angiography did not differ from those in the control group. Conclusions. The presence or the severity, or both, of coronary atherosclerosis is not associated with a detectable increase in endothelin release. The diagnostic procedures of Catheterization do not modify endothelin concentrations in plasma and urine. Vascular stretch or injury, or both, during coronary angioplasty increases urinary ir-ET-1 levels a few hours after the procedure. This increase persists for at least 24 h but is not detectable by brief sampling of peripheral or coronary sinus blood.

The potent pulmonary vasodilating property of atrial natriuretic factor (ANF) may alter gas exchange in patients with COPD. We examined the hemodynamic and gas exchange responses to intravenous infusion of ANF (0.01 and 0.03 ng/min/kg body weight) in eight stable patients with COPD studied during spontaneous breathing, using the inert gas elimination technique. When compared with baseline, ANF infusion was associated with a dose-dependent decrease in pulmonary artery pressure (from 27.3 ± 2.5 to 23.9 ± 1.8 and 20.2 ± 1.7 mm Hg, respectively) and a dose-dependent increase in blood flow perfusing poorly ventilated and unventilated units (Va/Q 2 remained unchanged (70.2 ±3.6, 68.1 ± 3.8 65.4 ± 3.5 mm Hg, respectively) because of a significant increase in minute ventilation ( V . E ) from 8.6 ± 0.8 to 9.6 ±0.8 and 10.3 ±0.7 L/min (p 2 (from 117 ± 17 to 110 ± 15 and 96.4 ± 8.8 mm Hg, respectively; p

The interactions between L-arginine, the source of nitric oxide formation, and N G-nitro-L-arginine (L-NOARG), an inhibitor of nitric oxide synthase were examined in isolated rat aorta. Carbachol induced endothelium-dependent relaxation was used to study the constitutive form of the enzyme, whereas the loss of contraction tonicity to phenylephrine following incubation of endothelium free rings with interleukin 1 was used to investigate the smooth muscle inducible form. L-NOARG is a potent inhibitor of the two types of the enzyme in the rat aorta. However, the effects of L-NOARG on the constitutive (endothelial) vascular NO synthase were partially prevented, but not reversed, by L-arginine. In contrast, L-arginine fully reversed the action of L-NOARG on the smooth muscle inducible form of NO synthase. The selective reversibility of L-NOARG on the inducible, by comparison with the constitutive form of nitric oxide synthase present in the vasculature, may represent a limitation in the use of this type of com...

One of the major biological effects of the endothelium-derived peptide endothelin-1 (ET-1) is its receptor-mediated constrictive action on vascular smooth muscle. In this study, we have examined the effects on the ET-1 pathway of 18 h exposure at 37 degrees C of cultured rat aortic smooth muscle cells to dexamethasone (DEX) and phosphoramidon. ET-1 synthesis was evaluated by radioimmunoassay, ET-1 binding characteristics were determined with [125I]iodo-ET-1, and ET-1-induced intracellular calcium mobilization was measured using fura-2-loaded cells. DEX (100 nM) led to a 2- to 3-fold-increase of ET-1 production, it down-regulated ET-1 receptors and reduced ET-1-stimulated calcium mobilization by 70%. In contrast, phosphoramidon (100 microM) inhibited ET-1 production by 60%, up-regulated ET-1 receptors and potentiated ET-1-induced calcium mobilization by 75%. These results indicate that the regulatory effects of DEX and phosphoramidon on ET-1 receptors are mediated via ET-1 production by the cells. This suggests an autocrine control of ET-1 receptors by endogenous ET-1 synthesis in vascular smooth muscle cells.

A number of experimental models of L-DOPA-induced dyskinesia have been proposed, but these models result in a low to medium rate of dyskinetic animals with mild to severe symptoms. The objective of this study was to combine a model of 6-OHDA-induced parkinsonism and of L-DOPA-induced dyskinesia in rats to establish a reliable preclinical model. Two stereotaxic injections of 6-OHDA were administered in the left striatum. This model led to 90-100% of rats with a marked contralateral circling behaviour, significant limb use asymmetry (20%), a decrease in ipsilateral striatal dopamine content (70%) and degeneration of dopamine neurons in the substantia nigra (70%). Chronic treatment with L-DOPA was administered for 35 days and consisted of three phases with incremental daily doses. The third phase resulted in 83-90% of rats developing severe abnormal involuntary movements (AIMs) which included limb and locomotive dyskinesia, axial dystonia and orolingual dyskinesia. Reproducibility of the model, criteria of strict blinding, placebo-controlled design, randomization of study subjects and pretrial determination of sample size were used to measure efficacy of amantadine and istradefylline and to validate the protocol design. Acute or subchronic post-treatment with amantadine reduced the severity of dyskinesia while istradefylline punctually attenuated AIMs. Our experimental conditions using gradual development of dyskinesia induced by increasing doses of L-DOPA resulted in a reliable model of L-DOPA-induced dyskinesia with a high rate of dyskinetic rats.

Hypercholesterolemia and atherosclerosis are conditions associated with impaired endothelium-dependent relaxation. In hypercholesterolemic animals, intravenous administration of L-arginine, the precursor of nitric oxide, normalizes endothelium-dependent vasodilator activity. In the present study, we questioned whether intracoronary administration of L-arginine in patients with coronary artery disease could improve coronary vascular reactivity to acetylcholine. Thirteen hypercholesterolemic patients with diffuse coronary atherosclerosis but nonstenotic lesions of the left anterior descending (LAD) coronary artery were investigated. Quantitative coronary angiography and subselective intracoronary Doppler flow velocity measurements were performed to determine LAD diameters and coronary blood flow. Intracoronary infusion of acetylcholine was performed during 3 consecutive 3-minute periods at incremental rates adjusted to achieve estimated final concentrations of 5 x 10(-7), 10(-6) and 5 x 10(-6) M. After evaluation of the response to acetylcholine, L-arginine was infused into the LAD at the rate of 25 mg/min (10(-3) M) and the same stepwise 3-minute infusions of acetylcholine were repeated during infusion of L-arginine. Infusion of acetylcholine induced a dose-dependent reduction of distal epicardial LAD diameter reaching -48.5 +/- 17% at 5 x 10(-6) M (p0.01 vs control values). L-arginine alone had no effect on the distal LAD diameter but attenuated acetylcholine-induced vasoconstriction to -21 +/- 9% at 5 x 10(-6) M acetylcholine (p0.01). Coronary blood flow showed a biphasic response to acetylcholine, increasing by 41 +/- 12% at 5 x 10(-7) M (p0.01) and decreasing by 21 +/- 13% at 5 x 10(-6) M (p0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Initiation and development of proliferative responses to growth factors are often associated to an activation of the Na+/H+ exchange. The present work examined the effect of endothelin (ET-1) on cell proliferation and Na+/H+ exchange in cultured vascular smooth muscle cells. In rat aortic vascular smooth muscle, ET-1 (0.1 to 10 nmol/L) increased the [3H] thymidine uptake in a dose-dependent manner. This effect was enhanced in presence of insulin (0.1 micrograms/mL to 10 micrograms/mL) as a function of concentration. The Na+/H+ exchange, which is a necessary response for mitogenesis, was dose-dependently stimulated by increasing concentrations of ET-1 (1 to 1000 nmol/L) and presented a biphasic response: a transient acidification followed by a sustained alkalinization. Alkalinization induced by ET-1 was similar to that obtained by the phorbol 12-myristate 13-acetate (PMA). An inhibitor of protein kinase C, H7, or a long-term pretreatment of cells with PMA for 24 h inhibited the effect of ET-1 and PMA on Na+/H+ exchange. These results confirm that ET-1 could act as a growth factor for vascular smooth muscle cells and suggest that its mode of action depends for a large part to protein kinase C activation.

The effects of moderate cooling and of phenylarsine oxide on the contraction induced by two vasoactive peptides, angiotensin II (AII) and endothelin (ET-1), were investigated on endothelium-free rings of rat aortas. At 37 degrees C, the contraction induced by AII (0.1 microM) was transient. This decline in tension is unlikely to be due to rapid degradation of AII. In contrast, ET-1 (10 nM) induced a slowly developing and sustained contraction similar to the one observed with phorbol 12-13 dibutyrate (PDB, 22 nM). Moderate cooling (25 degrees C) significantly potentiated and prolonged the effect of AII but reduced the velocity of the ET-1 and PDB contraction, although the rate of the phenylephrine (1 microM) response remained unchanged. Phenylarsine oxide (100 microM) reduced the decline in tension in response to AII but inhibited the contraction elicited by ET-1 and PDB. In rings incubated in calcium-free medium (37 degrees C), AII induced a phasic contraction. This was followed by a second phasic contraction after calcium (2.5 mM) had been restored to the bath. The intensity of this second contraction decreased as the time between AII and calcium injection increased. This method, using regression analysis, permitted us to determine the time taken to reduce the contraction by half (4.8 min; r: 0.96), which may reflect the half-time of receptor sequestration. In calcium-free medium, the contractions induced by ET-1 and PDB were slow and sustained. Thus, rapid AII-receptor internalization leads to a short-term regulation of vascular tone whereas activation of protein kinase C by ET-1 may induce a long-term regulation.

BN82451 belongs to a new family of small molecules designated as multitargeting or hybrid molecules. BN82451 is orally active, has good central nervous system penetration, and elicits potent neuronal protection and antiinflammatory properties. Neuronal protection is due to Na(+) channel blockade, antioxidant properties, and mitochondria‐protecting activity, whereas inhibition of cyclooxygenases is mostly responsible for its antiinflammatory activity. BN82451 has been shown to exert a potent neuroprotective effect in various in vitro and in vivo animal models. BN82451 was found to exert a significant protection in experimental animal models mimicking aspects of cerebral ischemia, Parkinson disease, Huntington disease, and more particularly amyotrophic lateral sclerosis. Collectively, its pharmacological properties designate BN82451 as a promising neuroprotective agent.

There is substantial evidence that excitotoxicity and oxidative damage may contribute to Huntington's disease (HD) pathogenesis. We examined whether the novel anti-oxidant compound BN82451 exerts neuroprotective effects in the R6/2 transgenic mouse model of HD. Oral administration of BN82451 significantly improved motor performance and improved survival by 15%. Oral administration of BN82451 significantly reduced gross brain atrophy, neuronal atrophy and the number of neuronal intranuclear inclusions at 90 days of age. These findings provide evidence that novel anti-oxidants such as BN82451 may be useful for treating HD.

Nitric oxide (NO) and reactive oxygen species are both implicated in neuronal death due to cerebral ischemia. BN 80933, an original compound associating an inhibitor of neuronal NO synthase with an antioxidant, has been shown to reduce functional and histological damage in rat submitted to cerebral ischemia. The aim of the present study was to confirm these results in mice and to further examine the effects of BN 80933 on inflammatory response, including blood-brain barrier (BBB) disruption, brain edema, and neutrophil infiltration after transient middle cerebral artery occlusion (MCAO). Intravenous administration of BN 80933 at 3 and 10 mg/kg 3 h after MCAO significantly reduced by 26 to 36% the infarct volume evaluated 24 and 48 h after ischemia, and improved the neurological score. Furthermore, BN 80933 at both dosages decreased by 42 to 75% the extravasation of Evans blue in brain parenchyma observed 24 h after ischemia. This reduction in BBB disruption was associated with decreased brain edema as demonstrated by the 37% reduction in brain water content induced by BN 80933 at 3 mg/kg 24 h after MCAO. Neutrophil infiltration in brain parenchyma, evaluated by the myeloperoxidase activity, was also reduced by 45 to 56% in animals treated with BN 80933 at 3 and 10 mg/kg. Together, these results extend the protective capacity of BN 80933 against brain ischemic injury and confirm that BN 80933 represents a promising treatment for stroke.

A series of hybrid compounds possessing an nNOS pharmacophore linked to an antioxidant fragment has been synthesized. Among them, compound 8d, a propofol derivative, displayed the greatest dual potencies against nNOS (IC(50)=0.12 microM) and lipid peroxidation (IC(50)=0.4 microM) accompanied with e/nNOS selectivity (67.5). This shows that nNOS was able to accommodate very bulky groups such as di-tert-butyl or di-iso-propyl phenol in its active site.

Formation of the lipid peroxidation product 8-epi-prostaglandin2alpha (8-epi-PGF2alpha) a bioactive marker of oxidative stress, was quantified in in vitro and in vivo models of neuronal death. In culture media of primary rat cortical neurones exposed to hypoxia followed by reoxygenation, a 3.7-fold increase of 8-epi-PGF2alpha concentration was observed in comparison to control cells. In rats submitted to 2h middle cerebral artery occlusion followed by a 22h reperfusion period, a 27-fold increase of 8-epi-PGF2alpha was observed in the ischaemic hemisphere compared with the corresponding hemisphere of sham-operated rats. Treatment with the neuroprotective agent BN 80933 significantly reduced both 8-epi-PGF2alpha elevations in vitro and in vivo. These data suggest that 8-epi-PGF2alpha elevations might reflect the damaging free radical overproduction and subsequent lipid peroxidation during neuronal injury induced by hypoxia and ischaemia. Inhibition of 8-epi-PGF2alpha elevations participates to the neuroprotective effects of BN 80933.

The effects of endothelin-1 (ET-1) on blood pressure and platelet aggregation were studied in anesthetized beagle dogs. Platelet aggregation was monitored in vivo by a filter-loop technique and in vitro by using platelet-rich plasma and whole blood. ET-1 (0.03-0.3 nmol/kg) induced a transient dose-dependent hypotension and inhibited platelet aggregation in vivo. These changes were accompanied by dose-dependent elevation of plasma 6-keto-PGF1 alpha levels. Pretreatment of animals with indomethacin (2 mg/kg plus 3 mg/kg/h) completely abolished the antiaggregatory action of ET-1 and significantly attenuated the hypotensive response to ET-1. Intra-arterial infusion of prostacyclin at concentrations similar to those observed following ET-1 injections mimicked the antiaggregatory and hypotensive actions of ET-1. ET-1 (0.1-100 nM) did not modify platelet aggregation induced by ADP, collagen, and platelet-activating factor in vitro, suggesting that dog platelets do not possess ET-1 receptors. These findings indicate that the antiaggregatory and hypotensive actions of ET-1 in beagle dogs are mediated through release of prostacyclin.

The purpose of this study was to investigate the mechanisms involved in the angiotensin II-induced increase in the contractile response of the hypertensive wall after prolonged incubation in the organ-bath buffer. In 5-h incubated rings, the contractile response to angiotensin II in aortic rings with endothelium from spontaneously hypertensive rats (SHRs) was markedly exaggerated in comparison to 2-h incubated rings. No such potentiation was observed in SHR rings after removal of the endothelium or in intact and denuded Wistar-Kyoto (WKY) rat rings. Aspirin and SQ29548 inhibited and cycloheximide and actinomycin D reduced the time-dependent enhanced response to angiotensin II in rings with endothelium from SHRs. In SHR rings with endothelium incubated for 2 h, the contractions caused by angiotensin II were potently inhibited by piroxicam but were unaffected by NS-398. Conversely, in rings incubated for 5 h, the hyperresponsiveness to angiotensin II was inhibited to a greater extent by NS-398 than by piroxicam. Piroxicam but not NS-398 had a further inhibitory effect on the residual angiotensin II-induced contraction in actinomycin D-treated rings incubated for 5 h. In conclusion, our study shows that long-term incubation leads to hyperresponsiveness to angiotensin II in SHR aorta with endothelium. The enhanced response is associated with the induced release of vasoconstrictor prostanoids sensitive to the inhibitory effect of NS-398, a preferential inhibitor of COX-2.

Modifications of rat prostatic alpha1-adrenoceptors were investigated in testosterone-induced prostatic hypertrophy. [3H]prazosin bound to a single class of binding sites with a dissociation constant of 57.9+/-5.02 pM. The greater part of the binding capacity (24.6+/-1.02 fmol/mg protein) was made up of chloroethylclonidine-resistant binding sites that showed high-affinity for oxymetazoline and 5-methyl-urapidil, and was identified as alpha1A-adrenoceptors. The remaining chloroethylclonidine-sensitive binding sites that showed low-affinity for oxymetazoline and 5-methyl-urapidil were preferentially identified as alpha1B-adrenoceptors. mRNA for the three alpha1-adrenoceptors (alpha1a, alpha1b and alpha1d) was detected. Testosterone administration produced a 23% decrease of alpha1-adrenoceptor density, likely by an increase of prostatic glandular epithelium and a decrease in the relative proportion of smooth muscle, thus of alpha1-adrenoceptor density. The steady state level of mRNAs for alpha1-adrenoceptors was not modified by testosterone treatment. These results indicate that prostate alpha1-adrenoceptors are not affected in the prostatic hypertrophy induced by testosterone.

C-type natriuretic peptide (CNP) may affect renal and vascular functions differently from atrial natriuretic peptide (ANP). The objective of this study was to compare the renal and vascular actions of CNP to those of ANP in normal men. CNP or ANP (0.005, 0.01, and 0.05 μg ⋅ kg−1⋅ min−1) were given by infusion to eight healthy volunteers. CNP caused dose-dependent increases in natriuresis (UNa) and in the fractional excretion of sodium (FENa) with no effect on diuresis (UV), renal plasma flow, and glomerular filtration rate (GFR). Fraction of filtration (FF) increased only with the 0.05 μg ⋅ kg−1⋅ min−1CNP dose. ANP caused larger increases in UNa, FENa, and FF than CNP and also increased UV at 0.01 and 0.05 μg ⋅ kg−1⋅ min−1and GFR at 0.05 μg ⋅ kg−1⋅ min−1. Although the ANP and CNP infusions produced similar elevation in the respective peptides plasma levels, urinary and nephrogenous guanosine 3′,5′-cyclic monophosphate increased less in response to CNP than to ANP. Blood pressure, forearm blood flow, plasma renin activity, and aldosterone remained unaffected during the peptides infusion. Plasma ANP increased slightly during CNP infusion. Our data indicate a higher threshold of renal response to CNP than to ANP. In contrast to ANP, CNP probably may not act as an endocrine factor in humans.

The α 1 -adrenoceptor subtypes mediating contraction of rabbit aorta and urethra were pharmacologically characterized using an isolated organ bath technique. Although aorta was as sensitive as urethra to the contractile action of methoxamine, phenylephrine was about 10 times more potent as a contractile agonist on aorta than on urethra. In aorta, the rank order of agonist sensitivity was norepinephrine > phenylephrine > clonidine > methoxamine whereas the rank order in urethra was clonidine > methoxamine ≥ phenylephrinc > norepinephrine. A lack of significant correlation between the potency of different α 1 -adrenoceptor antagonists tested against the phenylephrine-induced contraction in aorta and in urethra indicated that different α 1 -adrenoceptor subtypes mediated the contractile response in the two preparations. The potency of different α 1 -adrenoceptor antagonists tested in rabbit urethra was significantly correlated with their affinity for the cloned human α 1c -, but not α 1a - or α 1b -, adrenoceptor subtype. Such a clear correlation with the potency of different α 1 -adrenoceptor antagonists tested in rabbit aorta and their affinity for one subtype of cloned human α 1 -adrenoceptor was not found. Chlorethylclonidine, which produced a 10 000-fold rightward shift in the phenylephrine concentration-response curve for rat aorta, had a weak inhibitory effect in rabbit aorta and urethra as well as in other rabbit tissues (spleen, fundus, renal artery, saphenous artery). The results indicate that significant heterogeneity exists among α 1 -adrenoceptor in rabbit aorta and urethra ( α 1c -adrenoceptor). However, chlorethylclonidine does not seem to be a suitable tool for the differentiation of α 1 -adrenoceptor subtypes in the rabbit.

Agmatine was about as potent as aminoguanidine to inhibit the activity of the inducible form of nitric oxide synthase (iNOS) in isolated rat aorta. Like aminoguanidine, agmatine was devoid of significant activity on the constitutive form of NOS. Agmatine inhibited the conversion of [3H] L -arginine in [3H] L -citrulline in partially purified iNOS from macrophages (IC50=262±39.9 μM). Thus, our data suggest that agmatine may act as endogenous inhibitor of iNOS.

Increased vascular production of nitric oxide (NO) may contribute to the peripheral vasodilation and hyperdynamic state complicating advanced liver cirrhosis. In this study, we examined the effect on forearm blood flow of local brachial artery infusion of noradrenaline (NA) and NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO-synthase, in 10 alcoholic ascitic cirrhotic patients (patients with decompensated alcohol-induced liver disease: DALD group) and 10 patients with well-compensated alcohol-induced liver disease (CALD group). Forearm blood flow was measured by venous occlusion plethysmography. As compared with the CALD group, the DALD group had higher cardiac index and forearm blood flow as well as lower systemic blood pressure and vascular resistance. Infusions of NA and L-NMMA produced similar reduction in resting blood flow in the CALD group. However, in the DALD group, NA was significantly less effective than L-NMMA. The forearm vasoconstrictor response to NA was also significantly reduced in the DALD group when compared with the CALD group. In the DALD group, NA decreased forearm blood flow by 21.0 +/- 6.2% and increased vascular resistance by 37.2 +/- 12.3%, whereas respective changes in the CALD group were 41.8 +/- 6.2% (P < .01) and 77.8 +/- 9.9% (P < .02). In contrast, L-NMMA induced greater forearm vasoconstriction in the DALD group than in the CALD group. In decompensated patients, L-NMMA decreased forearm blood flow by 50.4 +/- 2.7% and increased vascular resistance by 115.9 +/- 14.4%, whereas changes in compensated patients were 38.2 +/- 4.9% (P < .05) and 77.4 +/- 16.2% (NS), respectively. These results are consistent with the hypothesis that increased vascular synthesis of NO contributes to the high dynamic state of patients with advanced cirrhosis.

To investigate the mechanisms of sodium retention in patients with chronic obstructive lung disease (COLD), we examined the renal and hormonal responses to volume expansion with isotonic saline and to infusion of atrial natriuretic factor (ANF) in 10 hypercapnic (PaCO2 52 +/- 2 mm Hg) and 12 normocapnic patients (PaCO2 39 +/- 1 mm Hg). Sodium excreted within 4 h of loading (expressed as % sodium load) was 23.5 +/- 2.5% (p < 0.05) in normocapnic and 8.5 +/- 1.5% (p < 0.001) in hypercapnic patients, compared with 32.5 +/- 3.0% in 11 age-matched control subjects. Sodium excretion and renal blood flow correlated negatively with arterial PCO2 and positively with FEV1. Basal plasma ANF concentrations were 72 +/- 5 pg/ml in controls, 100 +/- 14 pg/ml in normocapnic patients, and 230 +/- 52 pg/ml in hypercapnic patients (p < 0.001). Plasma renin activity and aldosterone did not differ between groups. In response to volume expansion, plasma ANF increased in both normocapnic and controls (with a greater increase in normocapnic patients) but remained unchanged in hypercapnic patients. Exogenous ANF increased glomerular filtration rate, renal plasma flow, natriuresis, and diuresis in both groups of patients. Patients with COLD have depressed renal function that appears unrelated to activation of the renin-angiotensin-aldosterone system. An increased secretory response of ANF to volume expansion may help to maintain volume homeostasis in normocapnic patients, while a blunted secretory response of ANF may contribute to sodium retention in hypercapnic patients.

The endothelin receptors in rabbit isolated rings of saphenous artery and saphenous vein have been characterized using endothelin-1, endothelin-2, endothelin-3, sarafotoxin S6c, and BQ123. Although artery rings were more sensitive than those from vein to the contractile action of phenylephrine, endothelin-1 was about three times more potent as a contractile agonist on vein than on artery. In rings precontracted with phenylephrine, carbachol was 10 times more potent in vein than in artery rings to induce endothelium-dependent relaxation. However, in rings precontracted to a similar tone by endothelin-1, the relaxation elicited by carbachol was reduced in the vein but remained unchanged in the artery. In endothelium-denuded saphenous artery, endothelin-1 and endothelin-2 elicited contraction with equal potency, whereas endothelin-3 and sarafotoxin S6c were weak agonists. In saphenous vein, the rank order of sensitivity was sarafotoxin S6c > endothelin-2 > endothelin-1 = endothelin-3, whereas sarafotoxin S6c and, to a lesser extent, endothelin-3 act as partial agonists. The ETA receptor antagonist BQ123 shifted, to the right, the concentration–response curves of endothelin-1 on endothelium-denuded saphenous artery (pA2 = 7.25). In the endothelium-denuded saphenous vein, 10 μM BQ123 shifted to the right only the response to high concentrations of endothelin-1. In vein but not in artery, endothelin-1 and sarafotoxin S6c induced an endothelium-dependent relaxation, which was increased, in the case of endothelin-1, in the presence of BQ123. These results indicate that the rabbit saphenous vein contains a mixed population of ETA and ETB vasoconstrictor receptors located in the smooth muscle cells and vasorelaxant ETB receptors situated on endothelial cells. In contrast, the saphenous artery only possesses smooth muscle cell ETA receptors responsible for constriction.Key words: endothelium, endothelin, vein, artery, BQ123.

In primary rat cortical glial cell cultures lipopolysaccharide (LPS) induced a dose- and time-dependent increase of intracellular cyclic GMP concentration associated with a release of nitrite. The LPS-induced cyclic GMP and nitrite increase was enhanced by interferon-γ and was prevented by L-N G - nitroarginine, dexamethasone and cycloheximide. Thus indicates that LPS effect occured via the production of nitric oxide (NO) and involved new protein synthesis suggesting the induction of NO syntahse in these cells. Furthermore this induction was Ca 2+ -independent and was blocked by an inhibitor of the synthesis of tetrahydrobiopterin. The inducible NO synthase was also expressed by C6 glioma cells. In primary mixed cultures containing both neuronal and glial cells, the effects of LPS were less important than in primary glial cell cultures suggesting that glial cells rather than neurons expressed the inducible form of NO synthase. On the other hand no change on neuronal viability was observed after NO synthase induction by LPS in this culture type. This study indicates that glial cells are able to induce NO synthase without affecting neuronal survival.

To assess the ability of L-arginine to improve endothelium-dependent vasodilation in atheromatous coronary arteries, we determined whether intracoronary infusion of L-arginine could improve coronary vascular reactivity to acetylcholine (ACh) examining both conductance and resistance vessels. Coronary blood flow velocity was assessed by positioning a 3 Fr Doppler catheter into the proximal coronary artery segment (six left anterior descending and one circumflex coronary artery). Computed quantitative angiography allowed the measurement of distal diameters. After baseline measurements, ACh was infused at incremental infusion rates through the Doppler lumen catheter (3 min period each) to obtain the estimated concentrations of 5 x 10(-7), 10(-6), and 5 x 10(-6) M. After returning to baseline, L-arginine was infused at the rate of 25 mg/min (10(-3) M) through the Doppler lumen catheter. Infusion of ACh was then repeated according to the same protocol than in the absence of L-arginine. The heart rate and mean arterial blood pressure did not change at any step of the protocol. Infusion of ACh induced dose-dependent vasoconstriction of coronary distal segments with a reduction in coronary distal segments by 39 +/- 15% at 5 x 10(-6) M (p0.01). During infusion of L-arginine, the coronary diameter was reduced by only 16 +/- 10% (p0.05) at the highest ACh dose. The coronary blood flow velocity increased by 100 +/- 15% at 5 x 10(-7) M ACh (p0.05) but only to 16 +/- 15% at 5 x 10(-6) M ACh (NS).(ABSTRACT TRUNCATED AT 250 WORDS)

In anesthetized and ventilated guinea pigs intravenous injection of ET-1, ET-2, or ET-3 (1 or 2 nmol/kg) induced similar dose-dependent increases in pulmonary inflation pressure (PIP) associated with increases in mean arterial blood pressure (MBP). Pretreatment of the guinea pigs with 1 mg/kg intravenous indomethacin significantly inhibited the increase in PIP evoked by 2 nmol/kg of ET-1, ET-2, or ET-3. In contrast, the increase in MBP following injection of the various ET isotypes was not significantly affected by indomethacin. Injection of ET-1, ET-2, or ET-3 (40, 120, and 400 pmol) via the pulmonary artery of isolated and perfused guinea pig lungs induced dose-dependent increases in PIP and pulmonary perfusion pressure (PPP), thromboxane B2 (TXB2) release, and formation of lung edema. In keeping with the in vivo results, no marked differences were observed between the activities of ET-1, ET-2, and ET-3 on isolated and perfused guinea pig lungs. Indomethacin (5 microM) added to the perfusion medium significantly inhibited the alterations of PIP and PPP, TXB2 release, and edema formation evoked by 400 pmol ET-1, ET-2, or ET-3. High-affinity binding sites for ET-1, ET-2, and ET-3 exhibiting similar characteristics were identified on guinea pig lung membrane. Therefore ET-1, ET-2, and ET-3 exert comparable bronchopulmonary and pressor activities in the guinea pig and probably act via interaction with the same binding site. In addition, the ET-induced increase in PIP and pulmonary vasoconstriction are primarily mediated via the production of cyclooxygenase metabolites.

The effects of NG-monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide (NO) synthesis was tested on the endotoxin-induced alterations of alpha-adrenoceptor function. In isolated aorta, there was no significant difference in the tension induced by phenylephrine (PE, 10 microM) on rings removed from control and endotoxin injected rats (10 mg/kg, ip). However, a lack of tonicity of the contraction was observed in rings of shocked rats (8 +/- 2.9 and 86 +/- 4.6% relaxation at 105 min for sham and shocked rings respectively). The gradual tension decrease to PE was more potent in rings possessing endothelial cells. However, in both preparations, the loss of tonicity was significantly inhibited by NMMA (30 microM). In endothelium-free rings, L-arginine (100 microM) potentiated the loss of tonicity to PE and reversed the inhibitory effect of NMMA. NMMA, like methylene blue, was also able to restore the PE-contraction. The results indicate that the endotoxin-induced alterations of vascular reactivity may be due, in part, to NO formation from L-arginine independent of the endothelium.

Endothelin-1 was able to induce an immediate and transient increase in cytosolic free Ca2+ concentrations in the A10 cell line of vascular smooth muscle. This was associated with a strong stimulation of the Na+:H+ exchange, the Na+, K+ pump and the [Na+,K+,Cl-]-cotransport system. Pump stimulation appeared to be secondary to sodium entry through Na+:H+ exchange because it was absent in Na+ loaded cells and in the presence of ethyl-isopropyl-amiloride. Cotransport stimulation was blocked by indomethacin, suggesting the involvement of a cyclooxygenase product. In conclusion, the monovalent ionic perturbations associated to the vasoconstrictor and mitogenic actions of endothelin-1 are counterbalanced by activation of the Na+,K+ pump and the [Na+,K+,Cl-]-cotransport system.

1. To investigate the mechanisms leading to enhanced synthesis and release of atrial natriuretic factor during chronic hypoxia, we measured immunoreactive plasma atrial natriuretic factor, blood gases, packed cell volume, pulmonary artery pressure and systemic artery pressure in male Sprague–Dawley rats exposed to 1, 2 or 3 weeks of normobaric hypoxia. Rats were implanted with pulmonary and carotid artery catheters and studied conscious, 23 h after return to hypoxia. 2. The concentration of atrial natriuretic factor messenger RNA was measured in the right and left ventricular free walls of rats exposed to 3 weeks of hypoxia and in normoxic control rats. 3. There was a trend for plasma atrial natriuretic factor to increase with the duration of exposure to hypoxia but only the 3-week hypoxic rats had a significantly higher level (1080 ± 193 pg/ml) than the normoxic control rats (318 ± 46 pg/ml, P 4. Three weeks exposure to hypoxia produced a 3.4-and a 2.9-fold increase in atrial natriuretic factor messenger RNA concentration in the right and left ventricles, respectively, suggesting that the increase in pulmonary artery pressure and the resulting right ventricular hypertrophy may not be the only trigger for the increase in atrial natriuretic factor synthesis and that both ventricles may contribute to the increase in plasma atrial natriuretic factor.

To examine the interrelations between humoral systems involved in the circulatory and body fluid volume homeostasis of patients with chronic obstructive pulmonary disease (COPD), we measured plasma levels of renin activity (PRA), aldosterone (Aldo), and atrial natriuretic factor (ANF) in 14 patients with stable COPD who used continuous O2 therapy. Hemodynamics, blood gases, and plasma hormone levels were measured (1) while patients received supplemental O2; (2) after 30 min O2 discontinuation; and (3) after a 30-min period of 96% O2 breathing. Plasma immunoreactive ANF concentrations were 196 +/- 50 pg/ml during O2 breathing and were positively related to transmural pulmonary arterial wedge pressure (tPpaw, r = 0.90, p less than 0.001) and to PaCO2 (r = 0.57, p less than 0.02). Compared to normal subjects matched for age and sex, patients had higher plasma ANF levels (196 +/- 52 versus 72 +/- 6 pg/ml, p less than 0.01), similar PRA (2.1 +/- 0.5 versus 1.3 +/- 0.3 ng/ml/h, NS), and slightly lower plasma Aldo (98 +/- 17 versus 156 +/- 19 pg/ml, p less than 0.05). Discontinuation of O2 while decreasing PaO2 from 70 +/- 3 to 50 +/- 3 mm Hg resulted in a significant increase in pulmonary arterial pressure (Ppa) from 29 +/- 2 to 32.5 +/- 3 mm Hg (p less than 0.01) and cardiac index (Cl) from 3.6 +/- 0.1 to 3.9 +/- 0.1 L/min/m2 (p less than 0.01) and a decrease in systemic arterial pressure (Psa) from 96 +/- 3 to 91 +/- 2 mm Hg (p less than 0.05); transmural cardiac filling pressures and pulmonary vascular resistance (PVR) were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

In cultured rat aortic smooth muscle cells, ({sup 125}I)endothelin (ET-1) bound to an apparent single class of high affinity recognition sites with a dissociation constant of 1.84 +/- 0.29 nmol/L and a maximum binding of 62 +/- 10.5 fmol/10(6) cells. The binding was not affected by calcium antagonists or vasoactive substances, including angiotensin II, arginine vasopressin, atrial natriuretic factor and bradykinin. Exposure of the cells to ET-1 (0.01 nmol/L to 10 nmol/L) resulted in an apparent dose-dependent reduction of the number of endothelin binding sites with no significant modification of its binding affinity. The time course of the down-regulation of ET-1 binding sites showed that this effect was present after 30 min incubation and persisted after 18 h. This indicates that down-regulation of ET-1 binding sites can modulate the activity of ET-1 and suggests a rapid internalization of ET-1 in vascular cells.

Intravenous (i.v.) injection of endothelin in the anesthetized and ventilated guinea-pig induced a dose-dependent increase in pulmonary inflation pressure. The 1 nmol/kg dose rapidly enhanced pulmonary inflation pressure, reaching a peak at 30 s, as well as produced a marked and sustained increase in mean arterial blood pressure. A maximal increase in arterial blood pressure was observed 4 min after the injection of 1 nmol/kg endothelin and remained at a plateau for 20 min. The increase in pulmonary inflation pressure induced by endothelin was significantly enhanced by treatment of the guinea-pigs with propranolol (1 mg/kg i.v.). In contrast to what was observed in untreated guinea pigs, the injection of 1 nmol/kg endothelin in propranolol-treated animals produced within 2 min an irreversible and dramatic decrease in mean arterial blood pressure with a further decline for up to 90 min. The increase in pulmonary inflation pressure induced by 0.5 nmol/kg endothelin was also potentiated significantly in propranolol-treated guinea-pigs as compared to untreated animals. Endothelin 0.5 nmol/kg caused only a transient and non-significant increase in mean arterial blood pressure in both propranolol-treated and untreated animals. Injection of endothelin (40, 120 and 400 pmol) via the pulmonary artery into isolated guinea-pig lungs evoked significant increases in pulmonary inflation pressure and perfusion pressure accompanied by the dose-dependent release of TXB 2 but, in contrast, no release of histamine. The in vitro effect of endothelin on pulmonary inflation pressure and perfusion pressure was potentiated when propranolol (1 μM) was added to the perfusion medium. These results demonstrate that the bronchopulmonary effects of endothelin are, at least in part, dissociated from the vascular action of the peptide.

Two atrial natriuretic factor (ANF) receptor subtypes are present in vascular nooth muscle cells: the B receptors (or biologically active) coupled to a guanylate cyclase ad the C receptors (clearance) representing 95% of the total number of ANF binding sites but noncoupled to a guanylate cyclase. Using binding experiments with 125I-ANF and measurement of cGMP production stimulated by ANF, we were able to demonstrate that ANF receptors are sensitive to homologous (induced by ANF) and heterologous regulation (induced by angiotensin II, All) in rat cultured vascular smooth muscle cells. The effect of the two hormones showed marked differences, in their time course, their reversibility and their consequence on guanylate cyclase activity. Although both ANF and All reduced the total umber of ANF binding sites after 18 h, ANF induced a desensitization of the guanylate cyclase whereas All elicited a potentialization of this system. From these results, we have included that in vascular cells B receptors are sensitive to homologous regulation and C receptors are sensitive to heterologous regulation by All. This also highlights a specific interaction between ANF and All at the receptor level.