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1. Vitamin C may influence NO-dependent relaxation independently of effects on oxidant stress. 2. We investigated effects of vitamin C (0.1 -- 10 mmol l(-1)) on relaxation of pre-constricted rabbit aortic rings to acetylcholine (ACh), authentic NO and the NO-donors glyceryl trinitrate (GTN), nitroprusside (NP) and S-nitroso-N-acetyl-penicillamine (SNAP). DETCA (2 -- 6 mmol l(-1)), a cell permeable inhibitor of endogenous Cu-Zn superoxide dismutase (SOD) was used to increase intracellular superoxide anion (O(2)(-)). 3. Vitamin C reduced the response to ACh (71 +/- 7% inhibition of maximum relaxation at 10 mmol l(-1)) and inhibited relaxation to authentic NO. Vitamin C inhibited relaxation to GTN but potentiated relaxations to NP and SNAP, causing a parallel shift to a lower concentration range of the log dose-response curve by approximately one log unit at the highest dose. 4. Vitamin C increased the concentration of NO in bath solution (plus EDTA, 1.0 mmol l(-1)) following the addition of SNAP from 53 +/- 14 to 771 +/- 101 nmol l(-1) over the range 0.1-3.0 mmol l(-1). 5. DETCA inhibited relaxation to ACh (71 +/- 9% inhibition of maximum relaxation). This inhibition was abolished by a cell permeable SOD mimetic, but not by vitamin C. DETCA inhibited relaxation to SNAP but not that to NP nor to GTN. 6. Vitamin C inhibits endothelium-dependent relaxations of rabbit aortic rings to ACh and authentic NO and does not reverse impaired relaxation resulting from increased intracellular oxidant stress. Vitamin C potentiates relaxation to the NO-donors NP and SNAP by a mechanism that could involve release of NO from nitrosothiols.

1. We have investigated the effect of cytoplasmic pH (pHi) on the relationship between platelet cytoplasmic Ca2+ concentration ([Ca2+]i) and eicosanoid biosynthesis. Stirred gel-filtered human platelets loaded with fluorescent indicators of Ca2+ and H+ were suspended in balanced salt solutions at 37 degrees C. [Ca2+]i was controlled by calcium ionophore (ionomycin). Increased [Ca2+]i was associated with increased production of thromboxane A2 (TXA2) as determined by radioimmunoassay of its stable hydrolysis product TXB2, and of 12-hydroxy eicosatetraenoic acid (12-HETE) measured by high performance liquid chromatography. 2. Varying pHi with a K+/H+ ionophore (nigericin) in platelets suspended in K+ rich solutions of pH 6.8, 7.4 or 7.8 with subsequent resuspension in solution of pH 7.4 containing albumin (1 g l-1) and Ca2+ (1 mM) resulted in pHi of 6.72 +/- 0.05, 7.31 +/- 0.02 and 7.71 +/- 0.04 (mean +/- s.e. mean, n = 5). Ionomycin (1.2 microM) increased [Ca2+]i by 97.1 +/- 17.6, 191.9 +/- 48.7 and 322.8 +/- 55.7 nM at the different values of pHi respectively; TXB2 production was 0.7 +/- 0.2, 2.1 +/- 0.4 and 10.7 +/- 3.3 ng micrograms-1 protein, and 12-HETE production was 150.9 +/- 68.2, 184.4 +/- 77.9 and 302.3 +/- 62.8 ng micrograms-1 protein. 3. Ammonium chloride (50 mM) caused a small reduction in pHo while increasing pHi from 7.32 +/- 0.04 to 7.89 +/- 0.05 and increasing ionomycin (1.2 microM)-induced [Ca2+]i responses from 94.1 +/- 67.3 to 721.6 +/- 288.3 nM. TXB2 production increased from 3.1 +/- 2.1 to 17.3 +/- 8.2 and 12-HETE production increased from 100.5 +/- 26.7 to 203.2 +/- 36.4 ng microg-1 protein. Responses of [Ca2+]i and TXB2 production to epoxymethano prostaglandin H2 (U46619, an endoperoxide-thromboxane receptor agonist) increased significantly in the presence of NH4C1.4. Alterations of pHi (such as may occur under pathological conditions) influence [Ca2+]i responses and eicosanoid synthesis in human platelets.

1. We compared the effects of NG-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor, on vasodilatation produced by acetylcholine and methacholine in human forearm vasculature. 2. Acetylcholine (83 nmol min-1) infused into the brachial artery of 8 healthy volunteers caused a submaximal increase in forearm blood flow, measured by venous occlusion plethysmography, from 3.3 +/- 0.5 (mean +/- s.e. mean) to 13.3 +/- 1.7 ml min-1 100 ml-1. 3. Co-infusion of L-NMMA (4 mumol min-1) with acetylcholine (83 nmol min-1) over 6 min resulted in a 58% +/- 12% fall in the response to acetylcholine whereas during co-infusion of saline over the same time period in the same subjects (n = 8) on a different day the response to acetylcholine fell by only 9% +/- 17% (P < 0.01). 4. Methacholine (1.5 and 15 nmol min-1) increased forearm blood flow from 2.5 +/- 0.4 to 5.9 +/- 0.9 and from 3.2 +/- 0.4 to 17.0 +/- 1.9 ml min-1 100 ml-1 respectively. 5. Co-infusion of L-NMMA (4 mumol min-1) had no significant effect on the response to methacholine (1.5 or 15 nmol min-1) when compared with saline control (n = 8). Co-infusion of a higher dose of L-NMMA (8 mumol min-1) with methacholine (1.5 nmol min-1) did not significantly inhibit the vasodilator response (n = 7). 6. These results suggest that, in human forearm vasculature, methacholine acts predominantly through mechanisms other than the L-arginine/nitric oxide pathway.

1. To obtain direct evidence of abnormal eicosanoid biosynthesis in rats injected with anti-glomerular-basement-membrane antibodies (a-GBM), products derived from thromboxane A2 (TXA2) and prostacyclin (PGI2) were measured in 24 h urine collections before and after a-GBM. 2. Administration of a-GBM (9.5 mg) caused albuminuria, decreased creatinine clearance, increased numbers of intra-glomerular neutrophils and increased excretion of TXB2, 2,3-dinor-TXB2 (products of TXA2) and 6-oxo-PGF 1 alpha and 2,3-dinor-6-oxo-PGF 1 alpha (products of PGI2) at 24 h. 3. Interleukin-1 (IL-1 beta; 5 micrograms) alone caused an increase in PGI2 metabolite excretion but had no effect on TXA2 metabolites. It had no effect on creatinine clearance but increased numbers of glomerular neutrophils by approximately 4-5 fold compared to a-GBM. 4. Pretreatment of rats with IL-1 beta before a-GBM synergistically increased albumin excretion but only additively increased eicosanoid excretion. Numbers of intra-glomerular neutrophils and creatinine clearance were unchanged compared to IL-1 beta alone. 5. The cyclo-oxygenase inhibitor, ibuprofen (10 mgkg-1 i.p., twice daily for 4 days) inhibited both serum TXB2 production and urinary prostaglandin excretion. It also caused an almost complete attenuation of albumin excretion. Creatinine clearance and glomerular neutrophils remained unchanged after a-GBM/IL-1 beta. 6. We conclude that the 50% inhibition of thromboxane production induced by ibuprofen does not modify the fall in creatinine clearance of accumulation of neutrophils in the glomerulus caused by the a-GBM. This degree of inhibition of eicosanoid production was associated with a striking decrease in proteinuria, but this may reflect a haemodynamic rather than a disease modifying action.

1. The effect on platelet aggregation of glyceryl trinitrate in the presence of cultured vascular smooth muscle cells was determined turbidometrically. U46619 (a thromboxane mimetic) was used as agonist and experiments were performed in the presence of aspirin. Inorganic nitrite production from glyceryl trinitrate by vascular smooth muscle cells was also measured, to provide an indirect index of nitric oxide synthesis. 2. The combination of vascular smooth muscle cells together with glyceryl trinitrate, at concentrations that had little effect individually, profoundly inhibited platelet aggregation. 3. The inhibitory effect on platelet aggregation of vascular smooth muscle cells together with glyceryl trinitrate was markedly attenuated by haemoglobin, an inhibitor of nitric oxide. 4. These results show that vascular smooth muscle cells inhibit platelet aggregation when exposed to glycerol trinitrate and suggest that this is due to generation of nitric oxide from glyceryl trinitrate by vascular smooth muscle.

1 Fresh rat aortic rings were incubated in HEPES-buffered salt solutions (pH 8.0) in the presence or absence of amiloride analogues. The effect of these drugs on prostacyclin (PGI2) synthesis was determined by radioimmunoassay of the stable hydrolysis product 6-oxo-prostaglandin (PG)F1 alpha. 2 Amiloride and phenamil (potent inhibitors of epithelial Na+ transport) had no significant effect on basal or Ca2+-stimulated PGI2 synthesis. 3 Several analogues previously reported to inhibit Na+/Ca2+ exchange caused a dose-related increase in 6-oxo-PGF1 alpha production in media containing NaCl 120 mM and CaCl2 2.5 mM. 2',3'-Benzobenzamil was the most potent analogue with a maximum stimulation of 4.51 +/- 0.89 fold, and an EC50 of 3 x 10(-5) M. 4 Amiloride analogues bearing substituents on the 5-amino group of the pyrazine ring have been reported to inhibit Na+/H+ exchange more potently than Na+/Ca2+ exchange. Three of these compounds inhibited Ca2+-stimulated 6-oxo-PGF1 alpha production at concentrations that did not significantly influence basal 6-oxo-PGF1 alpha production.

Bradykinin, angiotensin II, arginine vasopressin (AVP) or des-amino-D-arginine vasopressin (DDAVP) were administered by intravenous infusion to 10 healthy men. The concentration of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha), the stable hydrolysis product of prostacyclin (PGI2), was measured in plasma using gas chromatography/negative ion chemical ionisation mass spectrometry. Dose-related increases in plasma concentrations of 6-oxo-PGF1 alpha occurred during administration of bradykinin (100-3200 ng kg-1 min-1). The concentrations of 6-oxo-PGF1 alpha rose from baseline values in the range less than 1.0-4.9 pg ml-1 to 24.9-47.6 pg ml-1 at maximum tolerated infusion rates. There were no changes in the concentrations of 6-oxo-PGF1 alpha during administration of angiotensin II, AVP or DDAVP at infusion rates which caused haemodynamic changes.

Synthesis of vascular epoprostenol (PGI2) and platelet thromboxane (TX) A2 is influenced by the coagulation cascade in incompletely understood ways. To elucidate this, prostanoids were determined in human blood anticoagulated by different drugs and incubated with and without rat aortic rings. Control incubations were performed in Hanks balanced salt solution. PGI2 and TXA2 synthesis were assessed by radioimmunoassay of their stable hydrolysis products 6-oxo-prostaglandin (PG) F1 alpha and TXB2. Fresh aortic rings incubated in Hanks solution with a thrombin inhibitor (TCK) synthesized similar quantities of 6-oxo-PGF1 alpha in the presence or absence of sodium citrate. In contrast, the intracellular calcium antagonist TMB-8 inhibited 6-oxo-PGF1 alpha synthesis. In contrast to the finding in Hanks solution, sodium citrate inhibited 6-oxo-PGF1 alpha synthesis by fresh aortic rings incubated in blood anticoagulated with TCK. However, TXB2 synthesis was not affected by citrate. Blood incubated alone at 37 degrees C in plain glass tubes generated a small amount of immunoreactive 6-oxo-PGF1 alpha. A thromboxane synthase inhibitor, OKY1581, increased immunoreactive 6-oxo-PGF1 alpha. However, blood anticoagulated with TCK and incubated similarly, generated no detectable 6-oxo-PGF1 alpha either in the presence or absence of OKY1581, showing that 6-oxo-PGF1 alpha synthesis in the previous experiments was dependent on the vascular rings. OKY1581 had little or no effect on 6-oxo-PGF1 alpha synthesis in incubations of fresh aortic rings with blood anticoagulated with TCK, despite inhibition of TXB2 synthesis. However, OKY1581 increased 6-oxo-PGF1 alpha synthesis by rings pretreated with acetylsalicylic acid (ASA) when incubated in blood, presumably by diversion of platelet endoperoxide to vascular PGI2 synthase. Sodium citrate did not influence the increase in 6-oxo-PGF1 alpha synthesis by ASA pretreated aortic rings caused by OKY1581 in whole blood. This implies that the PGI2 stimulating activity of whole blood in the absence of citrate exerts its effect proximal to PGI2 synthase. It is concluded that a low molecular weight serum factor formed during activation of the intrinsic coagulation pathway in blood, modulates PGI2/TXA2 balance by an action on vascular cyclo-oxygenase, possibly by an effect on intracellular calcium.

Fresh rat aortic rings were incubated in HEPES-buffered salt solutions. Extracellular Ca2+ stimulated the production of prostacyclin (PGI2), as determined by radioimmunoassay of its stable hydrolysis product 6-oxo-prostaglandin F1 alpha. This action of Ca2+ was modified by H+ over the pH range 8.0-6.5. Stimulation by calcium ionophore A23187 was not pH-dependent. In parallel incubations of aortic rings with 45Ca2+, followed by washing in the presence of La3+, tissue uptake of 45Ca2+ increased progressively as extracellular pH was increased from 6.5-8.0. Over this range intracellular pH, estimated by the distribution of [14C]-dimethadione, varied from 5.9-7.4. Stimulation by Ca2+ of PGI2 synthesis was concentration-dependent over the range 0.7-20 mM. The maximum effect was an increase of approx. 4 fold. Nifedipine, but not verapamil or diltiazem, inhibited Ca2+-stimulated PGI2 synthesis. A dihydropyridine compound that activates voltage-dependent Ca2+ channels, Bay K 8644, did not increase PGI2 synthesis. 8-(N, N-diethylamino)-octyl-3,4,5 timethoxybenzoate (TMB-8), an antagonist of intracellular Ca2+ mobilisation, inhibited basal and Ca2+-stimulated PGI2 synthesis to a similar extent. A solution containing 40 mM K+ reduced Ca2+-stimulated PGI2 production. Mg2+ stimulated PGI2 synthesis in a pH-dependent manner but was less potent than Ca2+. Other divalent cations (Mn2+, Ba2+ and Sr2+), and La3+ had little or no effect on basal or Ca2+-stimulated PGI2 synthesis.

Bradykinin, prostaglandin E2 (PGE2), PGD2 and vehicle (saline) were each administered intravenously on separate occasions to 6 healthy men for a period of 60 min. 13,14-Dihydro-15-oxo-PGF2 alpha was identified in plasma samples obtained during intravenous infusions of bradykinin and PGE2 but not during infusions of PGD2 or saline. The structure of this metabolite was verified by comparison of three different derivatives with authentic standards, using gas chromatography/electron capture mass spectrometry. Bradykinin increased plasma concentrations of 13,14-dihydro-15-oxo-PGF2 alpha from baseline values in the range less than 5-10 pg ml-1 to 28-403 pg ml-1. PGE2 increased plasma concentrations of 13,14-dihydro-15-oxo-PGF2 alpha from baseline values in the range less than 5-17 pg ml-1 to 160-603 pg ml-1. Neither PGD2 nor the vehicle affected 13,14-dihydro-15-oxo-PGF2 alpha concentrations. We conclude that bradykinin-stimulated 13,14-dihydro-15-oxo-PGF2 alpha may be derived from PGE2 or PGF2 alpha. The possibility that these prostaglandins are synthesized by stimulation of microvascular endothelium during bradykinin infusion is discussed.

1 Bradykinin was infused intravenously into conscious rabbits to determine its effect on the concentration of prostaglandins in plasma. 6-Oxo-prostaglandin (PG) F1 alpha, the stable hydrolysis product of prostacyclin, and 13,14-dihydro-15-oxo-PGF2 alpha, a metabolite derived from PGE2 and PGF2 alpha, were measured by gas chromatography-electron capture mass spectrometry. 2 Incremental infusions of bradykinin (0.4-3.2 micrograms kg-1 min-1) increased plasma concentrations of both 6-oxo-PGF1 alpha and 13,14-dihydro-15-oxo-PGF2 alpha. 3 Aspirin (10 mg kg-1, i.v.) inhibited bradykinin-stimulated 6-oxo-PGF1 alpha and 13,14-dihydro-15-oxo-PGF2 alpha synthesis at 30 min and 6 h. At 24 h, the mean bradykinin-stimulated 13,14-dihydro-15-oxo-PGF2 alpha concentration was 66% of its original value, whilst 6-oxo-PGF1 alpha remained substantially inhibited. 4 The different rates of recovery of bradykinin-stimulated production of the two prostaglandins after inhibition by aspirin suggests that intravenous bradykinin stimulates prostacyclin and PGE2/PGF2 alpha production in distinct cell populations which synthesize cyclo-oxygenase at different rates.

1. Rabbit coeliac artery rings were mounted in tissue baths containing Krebs solution at 37 degrees C in order to determine whether their response to bradykinin is initiated by B1- or B2-receptors. Tension was recorded isometrically. 2. Phenylephrine contracted the tissue. Subsequent addition of bradykinin or des Arg10-kallidin caused relaxation which was not dependent on an intact endothelium. Des Arg10-kallidin, a B1-receptor selective agonist, was more potent than bradykinin. 3. [beta-Thienyl alanyl6,9, D-Phe8]-kallidin and [Leu9]-des Arg10-kallidin antagonized bradykinin and des Arg10-kallidin. [Leu9]-des Arg10-kallidin, a B1-receptor selective antagonist, was more potent than [Thi6,9, D-Phe8]-kallidin, a less selective drug that acts on both B1- and B2-receptors. 4. Kinin-induced relaxation was reversibly antagonized by ibuprofen (a cyclo-oxygenase inhibitor) and by 5-(N,N-hexamethylene)amiloride (an inhibitor of Na+/H+ exchange). Ibuprofen caused a parallel shift to the right of a semi-logarithmic plot of the agonist concentration-effect relationship, whereas the amiloride analogue depressed the maximum response and reduced the slope. 5. We conclude that bradykinin and des Arg10-kallidin relax rabbit coeliac artery by combining with B1-receptors. The response is mediated by a cyclo-oxygenase product and may be influenced by cellular Na+/H+ exchange.

The effect of aspirin on prostacyclin (PGI2) and thromboxane B2 (TXB2) synthesis was studied in rabbits. Tissues were removed from animals killed at intervals after injection of aspirin, and incubated with Hanks' solution. PGI2 synthesis was monitored by radioimmunoassay of its hydrolysis product, 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha). TXB2 production in clotted blood, also measured by radioimmunoassay, was determined as an index of platelet cyclo-oxygenase activity. 6-oxo-PGF1 alpha and TXB2 production 0.5 h after aspirin were similarly inhibited to less than 5% of control in all incubations. Subsequent recovery of PGI2 synthesis occurred more rapidly in aortic endothelium than in other tissues, including aorta denuded of endothelium. Recovery of TXB2 production was slower than that of PGI2. Intravenous cycloheximide prevented the partial recovery of PGI2 synthesis that otherwise occurred 6 h after aspirin, while intravenous epidermal growth factor increased recovery. It is concluded that in the rabbit, cyclo-oxygenase is synthesized more rapidly in aortic endothelium than in deep layers of aorta, or in the other tissues studied.