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BackgroundFisherfolk are a most-at-risk population for HIV being prioritized for the scale up of HIV treatment in Uganda. Heavy alcohol use and potential drug use may be a major barrier to treatment adherence for men in this setting.ObjectiveThis study examines the prevalence of substance use, and its influence on antiretroviral treatment (ART) adherence, among male fisherfolk on ART in Wakiso District, Uganda.MethodsThis cross-sectional study included structured questionnaires (N = 300) with men attending HIV clinics near Lake Victoria. Using generalized logistic modeling analyses with a binomial distribution and logit link, we conducted multivariate models to test the association between each alcohol variable (quantity and frequency index, hazardous drinking) and missed pills, adjusting for covariates, and tested for interactions between number of pills prescribed and alcohol variables.ResultsThirty-one percent of men reported sub-optimal adherence. Half (46.7%) reported drinking, of which 64.8% met criteria for hazardous drinking. Illicit drug use was low (6%). In the multivariate model, men with greater scores on the alcohol frequency and quantity index were more likely to report missed pills compared to those reporting no drinking (AOR: 1.60, 95% CI: 1.29-1.97). Hazardous drinking had a greater effect on missed ARV doses among men taking twice daily regimens compared to once daily (AOR: 4.91, 95% CI: 1.68-14.37).ConclusionsOur findings highlight the need for targeted alcohol-reduction interventions for male fisherfolk on ART who drink at high quantities to improve ART adherence and to prevent the known negative health effects of alcohol for HIV-infected individuals.

The purpose of this study was to investigate the effects of endothelial denudation, inhibitors of nitric oxide (NO) and prostanoid synthesis on vasoconstrictor responses in the perfused rabbit ovarian vascular. The experiments were conducted using an in vitro perfusion system, where the ovarian vascular bed (en bloc) was perfused with Krebs' solution delivered at a constant flow rate using a peristaltic pump. Changes in perfusion pressure, which reflected peripheral resistance, were measured. Results showed that noradrenaline (NA) (10(-9) to 10(-6) mol) induced reproducible dose-dependent vasoconstrictor responses. Vasoconstrictor effects of low doses of noradrenaline were not affected by perfusion of the vascular bed with CHAPS (4.7 mg/ml for 30 s) to remove the endothelium. The same treatment however, significantly reduced responses induced by the higher doses of noradrenaline, thus depressing the maximum response. KCl-induced vasoconstriction was not affected by CHAPS. L-N(G)-nitroarginine (L-NOARG) (10(-5) mol/l) enhanced NA-induced vasoconstriction. D-NOARG, the inactive isomer of L-NOARG and aminoguanidine, an inhibitor of inducible nitric oxide synthase reduced rather than enhanced noradrenaline-induced responses. Methylene blue (3 x 10(-5) mol/l) and LY 83583 (10(-5) mol/l) produced a potentiation of NA-induced vasoconstrictor responses. Indomethacin (3 x 10(-6) mol/l) did not significantly enhance NA-induced vasoconstriction. The nonselective endothelin antagonist, SB 209670 (10(-7) and 10(-6) mol/l) did not inhibit the vasoconstriction to NA. In conclusion, results are interpreted to suggest that NA-induced vasoconstriction in the perfused rabbit ovarian vascular bed is accompanied by a release of NO and possibly endothelium-derived contracting factor. There was no evidence for a modulation of vasoconstrictor responses by products of arachidonic acid metabolism or endothelins released from the endothelium.

The effects of Ca2+ withdrawal and agents affecting Ca2+ translocation on alpha1-adrenoceptor-mediated vasoconstrictor responses in the perfused rabbit ovarian vascular bed were studied. Noradrenaline-induced vasoconstriction was lost in a Ca(2+)-free Krebs' solution, and the rate of loss of the response was accelerated by EGTA (2 mM). Noradrenaline-induced vasoconstriction and SDZ NVI 085-induced vasoconstriction were concentration-dependently inhibited by verapamil and nifedipine. These agents were, however, more effective against KCl-induced responses. Cyclopiazonic acid, an intracellular Ca(2+) depletor, concentration-dependently inhibited noradrenaline-induced responses and abolished the response in Ca(2+)-free Krebs' solution. GF 109203X and polymyxin B, inhibitors of protein kinase C (PKC), had no significant effect on noradrenaline-induced responses. Tyrosine kinase inhibitors, genistein and erbstatin, inhibited noradrenaline-induced vasoconstriction in the perfused rabbit ovarian vascular bed. The results would suggest that both extracellular Ca2+ and intracellular Ca2+ participate in noradrenaline-induced vasoconstrictor responses in the perfused rabbit ovarian vascular bed. The results would also suggest that tyrosine kinase and not protein kinase C activation has a role in such effects.

The effects of chronic hypoxia on the responses of rat large pulmonary arteries and veins to vasoactive agents have been examined. Endothelin-1-induced contractions of pulmonary arteries and pulmonary veins were reduced by chronic hypoxia. In contrast, chronic hypoxia augmented sarafotoxin 6c-induced contractile responses in pulmonary veins but not in pulmonary arteries. Chronic hypoxia augmented the constrictor effect of phenylephrine in pulmonary arteries, but not in pulmonary veins. The thromboxane receptor agonist, U46619 (9,11-dideoxy-9alpha,11alpha-epoxy-methanoprostaglandin++ + f2alpha) contracted pulmonary arteries and pulmonary veins, and although maximal responses were not altered in chronically hypoxic preparations, the EC50 value in pulmonary arteries was increased following chronic hypoxia. The relaxant effects of acetylcholine and isoprenaline on pulmonary arteries were potentiated by chronic hypoxia. In contrast, ionomycin-mediated relaxations of pulmonary arteries and pulmonary veins were reduced, while sodium nitroprusside-induced relaxation of pulmonary arteries and veins were not altered by chronic hypoxia. Previous studied have looked primarily at the effects of chronic hypoxia on pulmonary arteries. This data provides evidence that chronic hypoxia also causes selective changes in the reactivity of large pulmonary veins.

The mechanism of cold-induced bronchoconstriction is poorly understood. Our previous results show that cooling-induced contractions (CIC) do not involve activation of cell surface receptor systems nor innervation nor Ca2+ uptake. However, the results show that CIC is mainly dependent on intracellular Ca2+ [32]. Isolated tracheal strips and bronchiolar segments were suspended in organ baths containing Krebs' solution for isometric tension recording. Tissue responses during stepwise cooling from 37 to 5 degrees C were examined. Cooling ovine tracheal strips and bronchiolar ring segments to 20 degrees C caused a rapid contraction which decreased slowly until it reached the basal level in approximately 30 min. There is a significant inhibition of 45Ca2+ efflux at 20 degrees C to those incubated at 37 degrees C. This prompted this study whose aim was to determine the role of ion-pump and ion-carrier systems on cooling mechanisms. Inhibition of the Na+/K+ pump with ouabain (10 micrometer) evoked contraction in tracheal and bronchiolar preparations. When cooling was superimposed on this contraction the cooling-induced contractions were reduced in the bronchiolar segments. In the tracheal strips, at temperatures down to 15 degrees C ouabain converted the contractions to a relaxation, but further cooling to 10 and 5 degrees C resulted in contractions that were similar to control at 5 degrees C. Staurosporine, a protein kinase inhibitor (1 micrometer) enhanced CIC in trachea and bronchiole. Vanadate, a Ca2+-ATPase pump inhibitor (1 mm) potentiated CIC in the two preparations. Trifluoperazine and W-7, calmodulin antagonists (10 and 100 micrometer) enhanced CIC in tracheal preparations but not in the bronchiolar segments. Thapsigargin and cyclopiazonic acid (CPA), inhibitors of sarcoplasmic reticulum (SR) Ca2+-ATPase pump (1 and 10 micrometer) potentiated CIC in tracheal but not in bronchiolar preparations. Amiloride, Na+/H+ and Na+/Ca2+ exchange system inhibitor (1 mm) abolished CIC in both trachea and bronchiole. These results show a strong relationship between cooling and the activity of ion transport systems and indicate that CIC is due to inhibition of calcium removal mechanisms as a result of inhibition of these ion-pump and ion carrier systems., (Copyright 1999 The Italian Pharmacological Society.)

The mechanism of cold-induced bronchoconstriction is poorly understood. This prompted the present study whose aim was to determine the step-wise direct effect of cooling on smooth muscle of isolated ovine airways and analyse the role of calcium in the mechanisms involved. Isolated tracheal strips and bronchial segments were suspended in organ baths containing Krebs' solution for isometric tension recording. Tissue responses during stepwise cooling from 37 to 5 degrees C were examined. Cooling induced a rapid and reproducible contraction proportional to cooling temperature in ovine tracheal and bronchial preparations which was epithelium-independent. On readjustment to 37 degrees C the tone returned rapidly to basal level. Maximum contraction was achieved at a temperature of 5 degrees C for trachea and 15 degrees C for bronchiole. Cooling-induced contractions (CIC) was resistant to tetrodotoxin (1; 10 micrometer), and not affected by the muscarinic antagonist atropine (1 micrometer) or the alpha-adrenergic antagonist phentolamine (1 micrometer), or the histamine H1-antagonist mepyramine (1 micrometer) or indomethacin (1 micrometer). Ca2+ antagonists (nifedipine and verapamil) and Mn2+ raised tracheal but not bronchiolar tone and augmented CIC. Incubation in Ca2+-free, EGTA-containing Krebs' solution for 5 min had no effect on CIC, although it significantly reduced KCl-induced contraction by up to 75%. Cooling inhibited Ca2+ influx measured using 45Ca2+ uptake. Caffeine (100 micrometer) significantly inhibited CIC. The results show that cooling-induced contractions do not appear to involve activation of nerve endings, all surface reception systems or Ca2+ influx. However, CIC is mainly dependent on release of intracellular Ca2+., (Copyright 1999 The Italian Pharmacological Society.)

Electrical field stimulation of ovine trachealis muscle produced neurogenic atropine-sensitive contractions under resting conditions. However, when the tissues were precontracted with 5-hydroxytryptamine in the presence of atropine, electrical field stimulation induced a frequency-dependent tetrodotoxin-sensitive relaxation. The relaxation was biphasic, consisting of fast and slow phases. The fast component was attenuated by propranolol, indicating an action on beta-adrenoceptors. The slow phase was attenuated by capsaicin and, therefore, involved release of a peptide. These results showed that excitatory responses in ovine trachealis muscles are cholinergically mediated, while both adrenergic and peptidergic components mediate electrically induced relaxation in the trachea. We also examined the influence of lowering bath temperature to 20 degrees C on electrically evoked responses. These were significantly reduced by cooling. At 20 degrees C, under resting conditions, the time-to-peak tension was lengthened, and the amplitude of the contractile responses was significantly (p < 0.05) reduced. In the same preparation, carbachol-induced contractions were not reduced by cooling, indicating that the reduction in electrically induced contractions was probably due to a reduction in transmitter release. Cooling also abolished the fast inhibitory phase (adrenergic in nature) without significantly inhibiting the slow (non-adrenergic, non-cholinergic) component. Propranolol (1 micromol/l) and capsaicin (100 micromol/l) did not affect significantly the slow relaxation observed during cooling. It was concluded that cooling inhibited cholinergically mediated, electrically induced contractions and selectively abolished the adrenergic component of electrically induced relaxant responses.

In lungs from chronically hypoxic (CH, 3 weeks at 10% inspired O2) rats, oxygenation (20% O2, 5% CO2, 75% N2; PO2 121 mmHg) of the perfusate increases pulmonary perfusion pressure (PPP) and lung weight (LW). Hypoxic perfusate (95% N2, 5% CO2; PO2 5.5 mmHg) had no effect on PPP in lungs from CH rats. Indomethacin and nitro-L-arginine (L-NOARG) augmented the oxygen-induced increase in PPP. In contrast, the free radical scavengers superoxide dismutase (SOD) plus catalase delayed the onset of oxygen-induced vasoconstriction, while the endothelin (ET)B receptor antagonist BQ788 inhibited it. The ET(A) receptor antagonist BQ123 did not affect the PPP changes. This suggests a role for endogenous endothelins and ET(B) receptors in mediating the oxygenation-induced pulmonary vasoconstriction. Indomethacin had no effect on oxygen-induced lung weight (LW) changes while BQ788 and L-NOARG reduced the LW increase. This evidence shows that ET(B) receptor activation and NO generation are involved in the LW changes. In conclusion, oxygenation of the perfusate in isolated lungs from CH rats leads to pulmonary vasoconstriction which involves endothelins and activation of ET(B) receptors. In addition, increased NO production associated with ET(B) receptor activation is the prime stimulus for observed LW increase.

Sodium nitroprusside (SNP) (10(-9)-10(-6) mol) and carbachol (10(-9)-10(-6) mol) induced dose-dependent vasodilatation in the perfused rabbit isolated ovarian vascular bed. Carbachol, but not SNP-induced vasodilatation was abolished by treatment with CHAPS (4. 7 mg ml-1, 30 s) to remove the endothelium. Carbachol-induced responses were also significantly attenuated by LY 83583 (10(-5) M) and methylene blue (3x10(-5) M). L-NOARG (10(-5) M) reduced carbachol-induced vasodilatation. None of these compounds affected SNP-induced vasodilator responses. Both SNP- and carbachol-induced vasodilatation were attenuated by raising the [K+] in the Krebs' solution to 40 mM. The responses were also reduced by TEA (20 mM) but not by glibenclamide. It was therefore concluded that SNP induced cGMP-independent vasodilator responses in the perfused rabbit ovarian vascular bed. This vasodilator response involved membrane hyperpolarisation since it was lost in high [K+] Krebs' solution. (c) 1998 The Italian Pharmacological Society.

We investigated the effect of systemic hypoxia (Krebs-Henseleit solution gassed with 5% CO2/95% N2) on an isolated, perfused rat lung. Hypoxia resulted in a slowly developing sustained increase in pulmonary perfusion pressure (PPP) accompanied by an increase in lung weight (LW). The endothelin (ET) receptor antagonists BQ123 (3 and 10 microM), BQ788 (3 microM) and bosentan (1.5 and 5 microM) all attenuated the hypoxia-induced increases in LW and PPP. In addition, phosphoramidon (1 microM), an ET-converting enzyme inhibitor, also significantly attenuated the hypoxia-induced increases in PPP and LW. The use of two agents that alter peptide secretion, phalloidin (10 and 50 nM) and colchicine (100 nM), and the peptide synthesis inhibitor cycloheximide (5 microM) all significantly attenuated the hypoxia-induced increases in PPP and LW. The increase in PPP and LW after the onset of hypoxia was accompanied by an increase in perfusate levels of ET-1 compared with normoxic time-matched controls. The results show that in this model, systemic hypoxia is capable of causing a sustained vasoconstriction and increased LW. The fact that these increases can be attenuated by an ET-converting enzyme inhibitor, ET receptor antagonists and agents that block peptide synthesis and secretion, together with the increase in perfusate levels of ET-1, suggests that ET production and release contribute to the changes seen.

Objective and Design: The study examines the effects of nitric oxide synthase (NOS) inhibitors on the development of neurological EAE and the levels of nitrite in the central nervous system (CNS) during established disease., Materials: EAE was induced in male Lewis rats (200-250 g)., Treatment: Rats received NG-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg body weight) day 7 to 12 post-inoculation (P.I.), 7-nitroindazole (10 mg/kg) day 7 to 11 P.I. or aminoguanidine (200 or 400 mg/kg) day 1 to 12 P.I., Methods: Neurological symptoms were assessed and CNS cytosol nitrite and protein levels measured. Results were analysed using the Mann Whitney U-test and the Fischer exact probability test., Results: Symptoms of EAE were associated with a significant elevation in CNS nitrite (p < 0.001). Treatment with NOS inhibitors caused a marked reduction in nitrite levels (p < 0.01). However, in some experiments, vehicle administration also reduced CNS nitrite content (p < 0.05). Although neurological disease was suppressed in EAE-sensitised rats receiving L-NAME (2 +/- 0.3 vs 3 +/- 0.3 mean peak severity +/- SEM) and 7-nitroindazole (1 +/- 0.3 vs 3 +/- 0.3, p < 0.01) comparable inhibition was achieved by respective vehicle treatment (p < 0.01). In contrast, neither aminoguanidine nor corresponding vehicle altered disease development., Conclusions: Drug-induced reductions in CNS nitrite levels during EAE are not always associated with a suppression of neurological symptoms, which suggests NO is not of primary importance in disease pathogenesis. In addition, the study emphasises the strict requirement for appropriate controls when assessing the efficacy of drugs on the course of EAE.

1. The aims of the study were to assess the contribution of prostaglandins and nitric oxide (NO) to the effects of endothelin (ETs) and sarafotoxin 6c (SX6c) in perfused rat lungs. This was carried out by using indomethacin, a cyclo-oxygenase inhibitor and NG-nitro-L-arginine (L-NOARG), a NO synthase inhibitor. Responses were studied under basal perfusion conditions and in other experiments after the elevation of vascular tone with the thromboxane-mimetic, U46619. The sub-types of ET receptors involved were characterized by use of ET receptor antagonists and cross-tachyphylaxis. 2. Pulmonary perfusion pressure (PPP), lung weight and pulmonary inflation pressure (PIP), were continuously recorded. Although L-NOARG (100 microM) did not alter basal parameters it markedly augmented the vasoconstriction and lung weight increases induced by ET-1 (50-400 pmol) or SX6C (25-200 pmol) while vasoconstrictor responses to phenylephrine were not affected by L-NOARG. 3. L-NOARG markedly potentiated the bronchoconstriction induced by ET-1 or SX6C whereas it had no effect on responses to carbachol. 4. When vascular tone was elevated, low doses (1.25-40 pmol) of ET-1, ET-3 and SX6C produced falls in PPP. The vasodilator potencies were SX6C > ET-1 = ET-3. The ETA receptor antagonist, BQ123, did not affect these depressor responses whereas the mixed ETA/ETB antagonist, bosentan, blocked them. 5. Indomethacin (10 microM) partially inhibited vasodilator response to ET-1, whereas it had no effect on SX6C-induced vasodilation. 6. L-NOARG plus indomethacin completely blocked ET-1 induced vasodilation, whereas responses to SX6C were blocked by L-NOARG alone. 7. Repeated injections of submaximal doses of ET-1 or SX6C caused tachyphylaxis to vasodilator responses. Subsequent injections of SX6C or ET-1 did not elicit depressor responses showing cross tachyphylaxis had occurred. 8. These findings indicate that under basal conditions the pulmonary vasoconstrictor, lung weight and bronchoconstrictor responses to ET-1 and SX6C are attenuated by evoked release of nitric oxide (NO). When vascular tone was elevated, lower doses of ETs and SX6C produced vasodilatation. These vasodilator responses are indirect, those to SX6C being mediated via NO production, whereas those to ET-1 involve both NO and prostanoid(s). Tachyphylaxis and ET antagonist experiments indicate that the same receptor subtype is involved in mediating the vasodilatation and that this is of the ETB type located on the endothelium. However the post-receptor vasodilator events triggered by ET-1 or SX6C appear to be different.

1. alpha 1-Adrenoceptor agonists, noradrenaline, phenylephrine, methoxamine, oxymetazoline and SDZ NVI 085 but not alpha 2-adrenoceptor agonists, UK 14304, tizanidine or clonidine evoked dose-dependent vasoconstriction of the isolated perfused rabbit ovarian vascular bed. The rank order of agonist potency was noradenaline > oxymetazoline > phenylephrine > SDZ NVI 085 > methoxamine. 2. Prazosin (10(-8) M - 10(-5) M) displaced agonist dose-response curves to the right. The pA2/pKB values ranged between 7.27 and 7.66 against noradrenaline, phenylephrine, methoxamine and SDZ NVI 085 and were not significantly different from each other. Prazosin was however significantly less potent against oxymetazoline (pA2 6.38). Yohimbine (10(-6) M - 10(-5) M) was not very effective against any of the agonists. 3. WB 4101 (10(-8) M - 10(-5) M) displaced agonist dose-response curves to the right. The pA2/ pKB values ranged between 7.08 and 7.93 against noradrenaline, phenylephrine, methoxamine and SDZ NVI 085. WB 4101 was significantly less potent against oxymetazoline (pKB 6.85). 4. SZL-49 (5 x 10(-6) M) but not chloroethylclonidine (3 x 10(-5) M) significantly reduced vasoconstrictor responses to all the agonists. 5. Electrical field stimulation of the ovarian bed produced frequency-dependent vasoconstrictor effects which were abolished by 6-OHDA. The responses were also antagonized in a concentration-dependent by prazosin (10(-7) M - 10(-5) M) and WB 4101 (3 x 10(-8) M - 3 x 10(-7) M). Yohimbine reduced the response to electrical stimulation by 20% at 10(-5) M. The vasoconstrictor effect was also inhibited by SZL-49 but not by chloroethylclonidine. 6. These results would suggest that the vasoconstrictor responses of the ovarian vascular bed to adrenergic agonists and to electrical stimulation are mediated via the alpha 1A-adrenoceptor subtype.

1. Endothelin (ET) receptors within the vasculature and airways were studied in a rat perfused lung model in which pulmonary perfusion pressure (PPP), pulmonary inflation pressure (PIP) and lung weight were continuously monitored. 2. The vascular potencies of ETs (ET-1 > ET-2 > ET-3) suggest an action via ETA receptors. This was confirmed by use of the antagonist, BQ123 (2 microM). The vasoconstrictor effects of sarafotoxin 6c (SX6C) also indicated the presence of ETB receptors. 3. Lung weight increases induced by ETs appeared to be a consequence of their vasoconstrictor potencies. The mixed ET receptor antagonist, bosentan (5 microM), markedly attenuated the responses of ET-1 and SX6C on PPP and lung weight, further implicating activation of both ETA and ETB receptors in these responses. 4. Endothelin-1 (ET-1) induced an accumulation of albumin-bound Evans blue dye in orthogradely perfused lungs. Retrograde perfusion attenuated the extravasation and increase in lung weight due to ET-1 but significantly augmented those induced by SX6C. 5. The bronchoconstrictor actions of ETs (ET-1 = ET-2 = ET-3) and SX6C suggest this is an ETB-mediated response. However SX6C was more potent than ETs and the dose-response curve was significantly steeper and achieved a higher maximum. 6. Indomethacin did not affect the vascular or bronchial responses to ET-1 or SX6C. 7. These findings indicate that rat pulmonary vasculature contains both ETA and ETB receptors. Retrograde perfusion suggests that ETB receptors are located arterially whereas ETA receptors are predominantly venous in distribution. Differences in the bronchoconstrictor potency of SX6C (compared to ETs) and the antagonism by bosentan may indicate ETB receptor heterogeneity in the airways.

A ventilated perfused model of rat lung is described enabling continuous simultaneous measurement of pulmonary perfusion pressure (PPP), pulmonary inflation pressure (PIP) and lung weight. The preparation was stable for 2h before oedema formation commenced. Phenylephrine injected into the pulmonary artery produced selective increases in PPP (ED50 4.0 +/- 0.7 nmol, n = 5) without affecting airway tone or lung weight. Carbachol under basal perfusion conditions caused bronchoconstriction (ED50 4.25 +/- 0.4 nmol, n = 7) without affecting PPP and lung weight. Bradykinin (BK) caused increases in PPP (ED50 22 +/- 3 nmol, n = 8), lung weight and PIP (ED50 35.6 +/- 3.6 nmol, n = 8). Low doses of BK (25-50 nmol) caused increases in lung weight which were reversible. Higher doses (100-200 pmol) caused irreversible increases in lung weight which albumin-bound dye infusion showed were associated with albumin accumulation. Manipulation of venous outflow pressure revealed that the changes in lung weight induced by BK were potentially explicable as sequelae of vascular hydrostatic pressure changes possibly resulting from venoconstriction. It is concluded that this model is useful for studying drug actions on bronchial tone, pulmonary vasculature tone and vascular permeability.

1. When the isolated small intestine of the rat is perfused via the mesenteric artery, an oxytocic principle (Gaddum's substance R) is released which is detectable in the perfusate after 30 min and is present in samples collected 8 h later. 2. The oxytocic activity of substance R is lost after boiling but is unaffected by treatment with thioglycolate. Furthermore, atropine, methysergide and indomethacin failed to antagonize uterine contractions to substance R. 3. Neither substance R nor urinary kallikrein alone induce a contraction of the guinea-pig isolated ileum. However, in the presence of kininogen both substance R and urinary kallikrein produce a slow and prolonged contraction of the guinea-pig ileum. 4. The oxytocic and kininogenase properties of both substance R and urinary kallikrein are inhibited by Trasylol. 5. Soy bean trypsin inhibitor (SBTI) selectively inhibited both the oxytocic and the kininogenase activities of substance R but not those of urinary kallikrein. 6. Gel filtration of substance R resolved a single peak of oxytocic activity with an estimated molecular weight of 40 kDa. 7. We conclude that substance R is a kininogenase enzyme which may be distinguished from plasma kallikrein by its molecular weight and from urinary kallikrein by its susceptibility to SBTI. The exact nature of this enzyme remains to be elucidated.

We studied, in four normal men, the metabolism of 2-methoxyestrone (2-MeOE1) using pulse injections of either [3H]2MeOE1 (two men) or [14C]methoxy-2-MeOE1 plus [3H]2-MeOE1 (two men) by analysis of blood samples drawn at increasing time intervals after the pulse and of urine collected for 5 days. The disappearance from the blood of radioactivity as 2-MeOE1 could be characterized as a function that was the sum of three exponentials. The mean +/- SE value for the initial volume of distribution was 32 +/- 9 liters, and the mean MCR was 2470 +/- 770 liters/day. The disappearance of total 3H radioactivity from the blood was considerably slower, with a mean MCR of 290 +/- 30 liters/day, indicating the presence of a slowly turning over pool of 2-MeOE1 metabolites, probably including the 2-MeOE1 3-sulfate conjugate. The disappearance of total 14C radioactivity was slower than that of total 3H, indicating considerable demethylation of 2-MeOE1 with a very slow excretion of 14C from the released methyl group. In none of the subjects could we find in the blood radioactivity as unconjugated [3H]2-hydroxyestrone ( [3H]2-OHE1). However, examination of the urine indicated that considerable demethylation of [3H]2-MeOE1 had occurred. At least 64% of the urinary 3H-containing metabolites from the mixed dose had lost the 14C-bearing methoxylcarbon atom. The fractionated metabolites were qualitatively and quantitatively similar to those found earlier for [3H]2-OHE1. We conclude that 2-MeOE1, which of itself has little biological activity, can act as a pool of potentially active 2-OHE1 in the tissues.

Human myometrium taken between 15 and 42 weeks of pregnancy released a material in vitro which possessed anti-aggregatory activity. This material parallelled authentic prostacyclin in this action; it possessed a similar stability at alkaline pH and generation of activity was inhibited by indomethacin. Production of the prostacyclin-like material by the myometrium was similar at weeks 15 and 38 of pregnancy but a 4-fold increase in synthesis had occurred by 40 weeks. It is suggested this increase may be important for parturition.

1 Prostacyclin (PGI(2)) production by chopped segments of rat pregnant uterus was low compared with synthesis by separated myometrial tissue. Incubation of separated myometrium with decidua (2:1 by weight) led to an inhibition of myometrial PGI(2) output.2 Boiling decidual tissue abolished the inhibitory influence on myometrial PGI(2) output. Preincubation of decidua with 5,8,11,14-eicosatetraynoic acid (ETA) (30 mug/ml) also suppressed decidual inhibitory activity but indomethacin (30 mug/ml) was ineffective.3 Incubation of decidual and myometrial tissue with arachidonic acid (AA) 10 mug/ml did not increase the inhibition of myometrial PGI(2) synthesis, even if the decidua were pre-incubated with indomethacin.4 Myometrial PGI(2) production was reduced if the chopped tissue was pre-incubated with soya bean lipoxidase for 10 min at 4 degrees C. This reduction was reversed if the lipoxidase was incubated with ETA (30 mug/ml) for 30 min at 37 degrees C before addition to the myometrial tissue.5 Perfusion of the uterus to remove blood elements removed the inhibitory action that the decidua exerted upon myometrial PGI(2) production. PGI(2) synthesis by separated decidual and whole uterine tissue was markedly elevated.6 The addition of rat blood platelets (0.75 x 10(9)/ml) to incubations of perfused decidual tissue reduced PGI(2) output and restored the inhibitory action that the decidua exerted on myometrial PGI(2) synthesis.7 It is concluded that a lipoxygenase enzyme contained in blood platelets trapped within the decidual vasculature produces a hydroperoxy acid which inhibits decidual PGI(2) production or myometrial PGI(2) synthesis when the tissues are incubated together. It is suggested that perfusion is a pre-requisite before study of PGI(2) synthesis in highly vascularised tissues.8 The pathophysiological importance of such platelet lipoxygenase products is discussed.

1 The scraped myometrium of the pregnant uterus of the rat, when chopped and incubated, released an antiaggregatory material closely resembling prostacyclin (PGI2). The material was conclusively identified as PGI2 by identification of its hydrolysis product 6-oxo-prostaglandin F12 (6-oxo-PGF1 alpha) by gas chromatography-mass spectrometry (GC-MS). 2 Peak concentrations of PGI2 were detected after 15 min incubation of 20 degrees C. These concentrations were significantly higher than those detected at 37 degrees C, when largest amounts of prostacyclin were formed after 3 min incubation. The concentrations of 6-oxo-PGF1 alpha detected were similar at the different temperatures. 3 When samples were incubated at pH 8 and 20 degrees C, peak concentrations of prostacyclin were maintained between 15 and 35 min of incubation. When pH 7.4 was employed, prostacyclin concentration in the incubate fell to undetectable limits within this time. 4 Incubation of the chopped myometrium with arachidonic acid or phospholipase A2 stimulated prostacyclin production. 5 Preincubation of myometrial tissue for 10 min at 37 degrees C with inhibitory drugs before chopping reduced prostacyclin output. The doses needed to reduce PGI2 output by 50% (ID50) were: mepacrine (280 micrometer/ml), indomethacin (20 microgram/ml), 5,8,11,14 eicosatetraynoic acid (23 microgram/ml), 15-hydroperoxy arachidonic acid (23 microgram/ml) and tranylcypromine (225 microgram/ml). 6 It is suggested that due to the large amounts of material available, the rat pregnant myometrium is a useful model for the study of factors affecting prostacyclin synthesis.

The release of prostaglandin-like material and these spontaneous contractions of individual horns from the pregnant rat uterus in vitro have been studied on day 22 of pregnancy - the expected day of delivery. Removal of foetuses (retaining placentae in utero) from one or both uterine horns on day 16 or 17 significantly reduced prostaglandin F release and spontaneous activity. Rats which had been made unilaterally pregnant after ligation of one uterine horn, exhibited a decrease in prostaglandin F output from both horns. Uterine activity and prostaglandin release were increased in quiescent uteri by the addition of arachidonic acid (5 mug/ml) or phospholipase A (160 mu./ml); the effects were abolished by indomethacin (20 mug/ml). However, the stimulation of uterine activity by PGF2alpha (30-60 ng/ml) was not affected by indomethacin. It is concluded that the release of prostaglandins from the pregnant rat uterus in vitro at term is related to the presence of viable foetuses.

The preparation of a microsomal fraction from the decidual tissue of pregnant rat uteri is described. Incubation of such microsomes with a mixture of radiolabelled and cold arachidonic acid (51 micrometer) plus cofactors resulted in a 30% substrate conversion. Products were resolved into four peaks (A, B, C and D) by thin-layer chromatography. Combined gas-liquid chromatography-mass spectrometry and further thin-layer chromatography identified the products as PGF2 alpha (A); thromboxane B2 (B); a mixture of 6-OXO PGF1 alpha and PGE2 (C); PGD2 and PGE2 (D). PGE2 was the major product.

A mixture of 4-3-H and 4-14-C-mestranol was administered orally to four women. Reactions involving position 4 were no greater than 1.7-3% of the dose as measured by liberation of 3-H into body water. The extent of de-ethynylation in vivo was no greater than 1-2% of the dose as measured by urinary estrone metabolites. Mestranol (0.7 and 0.32% of the dose), 17alpha-ethynylestradiol (6.6 and 11.3%) and 2-hydroxy-17alpha-ethynylestradiol (0.64 and 0.7%) were identified as metabolite aglycons by reverse isotope dilution after Ketodase hydrolysis of the urine from two of the women.

Prostacyclin (PGI2) is a potent vasodilator and inhibitor of platelet aggregation, however, its half-life in aqueous solutions at neutral or acid pH is very short. In this study we have investigated the stability of PGI2 in plasma from different species and have demonstrated that the half-life of the anti-aggregatory and the hypotensive activity of PGI2 is considerably prolonged in human plasma. Interestingly the activity of PGI2 was not maintained on incubation in rat or rabbit plasma indicating that the stabilising factor may be confined to human plasma. Whether such stabilisation prolongs the half-life of PGI2 in the human circulation merits investigation.

A direct product isolation assay for quantifying the formation of 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from [6,7-3H]estradiol by rabbit hypothalami in vitro was developed, and the assay was used to characterize some properties of estrogen-2- and 4-hydroxylase activity in this tissue. The reaction was carried out under conditions that minimized further metabolism of enzymatically formed catechol estrogens. A simple two-step separation procedure, involving the use of a neutral alumina column, followed by thin layer chromatography, was developed to isolate the enzymatically formed catechol estrogens in a radiochemically homogeneous form. The detergent, Tween-80, was found to activate the enzyme and was used routinely at a concentration of 0.1% in the assay. The formation of 2-OHE2 was linear up to 10 min and with increasing protein concentrations up to 150 micrograms/incubation. Similar values were obtained for 4-OHE2. Maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 190 and 270 pmol/mg protein . 10 min, respectively. The apparent Km values with respect to estradiol for 2-OHE2 and 4-OHE2 were 125 and 150 microM, respectively. The highest specific activity for the enzyme was present in the 100,000 X g supernatant (S3), while the activity in the microsomal fraction (P3) was less than that in the original homogenate. Enzyme activity depended on the presence of NADPH and oxygen and was inhibited by CO as well as by high concentrations of SKF-525A. Estrogen-2- and 4-hydroxylase activity in rabbit hypothalamus differed from that in rat liver in two respects. In the liver, enzyme activity was localized in the microsomal fraction and was virtually abolished by Tween-80. In contrast, enzyme activity in rabbit hypothalamus was maximal in the soluble fraction (100,000 X g supernatant)and was stimulated by the detergent.

Using both pulse injections and constant infusions of 3-H-mestranol (3H-ME) (1) and 3-H-ethinyl estradiol (3H-EE) we have studied the metabolism of these compounds in non-users and users of oral contraceptives. Following pulse injection of 3-H-ME the disappearance of radioactivity could be described as a function which was the sum of two exponentials. Studied by both types of administration there was no difference in the metabolism of 3H-ME in the two groups; the overall mean plus or minus SE metabolic clearance rate (MCRM) was 690 plus or minus 45 1/day/m2, the mean ratio of the concentrations of radioactivity as EE following administration of ME (CRM-BB,E) was 0.23 plus or minus 0.02 and the mean (p)M,EBB (fraction of administered ME measured in blood as EE) was 0.19 (95% confidence limits equals 0.15 - 0.23). Following pulse injection of 3-H-EE the disappearance of radioactivity was best described as a function which is the sum of three exponentials. Results from both types of administration revealed no difference in the metabolism of 3H-EE between non-users. The overall mean plus or minus SE MCREE was 630 plus or minus 30 1/day/M2. The MCREE is significantly (0.02 greater than P greater than 0.01) less than the mean MCR for estradiol reported previously, in both non-users and users of oral contraceptives. The use of oral contraceptives containing estrogens and progestins does not appear to influence the metabolism of the estrogen used. Approximately 20% of mestranol is converted to and appears in the blood as ethinyl estradiol.

Prostacyclin, an unstable metabolite of arachidonic acid metabolism, has been shown to have potent platelet antiaggregatory and vasodilator activity. Available evidence suggests that arterial prostacyclin production is diminished in the diabetic state. In this study we have examined the sensitivity of platelets from diabetic subjects to the antiaggregatory effects of prostacyclin in vitro and report that platelets from some diabetic patients show diminished sensitivity to the antiaggregatory effects of prostacyclin. Thus diabetic subjects may not only have diminished prostacyclin production but also their platelets may be less sensitive to its effects. These findings may help to explain the increase in vascular disease observed in diabetic patients.

The release of prostacyclin from chopped myometrial fractions of 18-20 day pregnant rats was assayed by inhibition of ADP-induced aggregation of citrated rabbit platelet-rich plasma. Preincubation of myometrial tissue with oxytocin 10 mU/ml increased prostacyclin generation from 2.25 +/- 0.48 (control) to 3.75 +/- 0.73 ng/mg over 15 minutes. Bradykinin 20 microgram/ml also caused a significant increase in myometrial prostacyclin output from 2.26 +/- 0.19 to 4.26 +/- 0.64 ng/mg. PGF2 alpha 1 microgram/ml did not increase prostacyclin release significantly. Pretreatment of myometrial tissue with the phospholipase inhibitor mepacrine significantly reduced the peptide-stimulated release of prostacyclin. The results suggest that prostacyclin production may play an important role in modulating the actions of oxytocin and bradykinin in the pregnant rat myometrium.

1 The sensitivity of a wide range of isolated tissues to oxytocin was investigated. The longitudinal muscle strip of duck pulmonary vein proved the most suitable tissue for use with the superfusion technique, contracting to concentrations to oxytocin as low as 10 muu/ml.2 The duck pulmonary vein superfused with Krebs solution was contracted by oxytocin, the vasopressins, adrenaline, noradrenaline, 5-hydroxytryptamine, histamine and angiotensin II. Pre-treatment of the preparation with phenoxybenzamine (1-2 mug/ml) abolished the contractions to catecholamines and reduced the effects of histamine and 5-hydroxytryptamine without affecting the sensitivity to oxytocin.3 The pulmonary vein contracted when superfused with blood from an anaesthetized dog. This contraction was accompanied by a non-specific loss of responsiveness. When the pulmonary vein was superfused with Krebs solution that had been dialysed against blood the initial contraction was greatly reduced or abolished as was the loss in responsiveness.4 Oxytocin was stable in circulating dog's blood but approximately 50% was bound to plasma proteins. Oxytocin was not destroyed in the pulmonary circulation or the hind limbs. In the doses used oxytocin had a half-life of 60-90 s in the circulation of the dog under steady-state conditions. Disappearance occurred chiefly in the visceral vascular beds.

A mixture of 2-3H and 4-14C-17beta-estradiol 3-methyl ether was administered orally to a man and to a woman. 34 and 35 percent of the 3H was liberated into the body water of the man and of the woman, respectively, reflecting reactions involving position 2. The metabolism of estradiol methyl ether was qualitatively similar to that observed previously for radioactive estradiol administered intravenously to the same subjects, as judged by the measurement of various urinary metabolites by reverse isotope dilution. Evidence was obtained for hydroxylation at position 2 without demethylation by the isolation of urinary 2-hydroxyestrone 3-methyl ether which retained 33% of the original 3H. This 3H was presumably at position 1, resulted from an NIH shift which does not occur during hydroxylation of estrone or estradiol. This was confirmed by subsequent administration of a mixture of 4-14C and 3H-(methoxyl)-estradiol 3-methyl ether to the man. There was no evidence (by reverse isotope dilution) for 1-hydroxyestrone, 1-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether or 4-hydroxyestradiol 3-methyl ether as urinary metabolites of estradiol 3-methyl ether.

A mixture of 2-3H and 4-14C-mestranol was administered orally to five women and 2-3H-mestranol alone to one woman. Reactions involving position 2 were extensive as judged by liberation of 3H into body water (14-45% of the dose). 17alpha-Ethynylestradiol, 2-hydroxy-17alpha-ethynylestradiol, 2-methoxy-17alpha-ethynylestradiol, 2-hydroxy-17alpha-ethynylestradiol 3-methyl ether and 16geta-hydroxy-17alpha-ethynylestradiol were measured in the "glucuronide" and pH1 fractions and mestranol, D-homoestrone-17a and D-homoestradiol-17abeta were also measured in the "glucuronide" fraction frum the urine to two of the women by reverse isotope dilution. Radioactive 2-methoxyestradiol accounted for less than 0.011% of the 14C dose in the "glucuronide" fraction of one of the women, consistent with the extent of de-ethynylation previously reported (Steroids, 25, 343 (1975).

Using strips of rat pregnant uterus, treated with indomethacin to suppress spontaneous contractility, the oxytocic activity of prostacyclin was compared with other prostaglandins. A prostacyclin concentration of 32 ng/ml elicited uterine contractions in all experiments. In this respect prostacyclin was 80 times more active than 6-oxo-PGF1 alpha but less active than PGE2 or PGF2 alpha. Apart from a direct stimulant effect, prostacyclin also exhibited an indirect potentiating action. In threshold concentrations prostacyclin caused a 3-fold potentiation of threshold doses of oxytocin. A lesser 1.5-fold potentiation of PGE2 alpha was also observed. The implications of these findings in relation to prostacyclin playing a role in parturition are discussed.