Biomedical subjects

J R Vane

Publications and source records attributed to J R Vane.

At least 109 records · Page 6Linked to original sources

PubMed1993

The metabolism of endothelin-1 and big endothelin-1 by the isolated perfused kidney of the rabbit.

The activity of human big endothelin-1 (bET-1) or endothelin-1 (ET-1) was investigated in the isolated perfused kidney of the rabbit. In some experiments the effluent superfused a rabbit jugular vein (RbJV) or rat colon (RC), and bolus doses of bET-1 or ET-1 were administered either directly over the tissue (OT) or through the kidney (TK). When injected OT, the contractile responses of the RbJV to ET-1 were > or = 100-200 times more than those to bET-1. However, at least 10 times the ET-1 dose given OT was needed TK to produce an equivalent contraction of the RbJV, showing that ET-1 was being inactivated or removed by the kidney. Injections of bET-1 TK produced increases in perfusion pressure of a magnitude 1/25th of those of ET-1. In some experiments administration of bET-1 TK was associated with the release into the perfusate of an ET-1-like factor that contracted the RbJV, but not the RC (used to detect any angiotensin II release), at doses that had little effect when administered OT, suggesting that bET-1 was being activated by the kidney. Phosphoramidon (10 microM) infused TK blocked (92 +/- 1% inhibition) the renal responses to bET-1 and reduced the overflow of ET-1-like material onto the tissues without affecting ET-1-induced renal vasoconstriction. Incubation of bET-1 with rabbit renal cortical microsomes (100 micrograms protein) resulted in the generation of ET-1-like activity, as assessed by bioassay, which was inhibited by phosphoramidon (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effects of phosphoramidon in endothelial cell cultures on the endogenous synthesis of endothelin-1 and on conversion of exogenous big endothelin-1 to endothelin-1.

Several studies have shown that phosphoramidon (PHOS) reduces the release of endothelin-1 (ET-1) from cultured endothelial cells. Moreover, the main endothelin-converting enzyme (ECE) activity in these cells is a membrane-bound metallopeptidase that is also inhibited by PHOS. We have investigated further the role of the PHOS-sensitive ECE in the conversion of big ET-1 to ET-1. ET-1 was measured using a radioimmunoassay specific for the C-terminal ET[16-21] sequence. The effect of PHOS on the production of ET-1 from endogenous precursors was determined using cultured bovine aortic endothelial cells (BAECs) and the human endothelial cell line (EA.hy 926). The concentrations of ET-1 accumulating in the medium over 24 h from BAECs were lowered by PHOS (-27% 10 microM, -76% 100 microM). In contrast, with EA.hy 926 cells, the same concentrations of PHOS increased by five- to sixfold the amount of ET-1 present in the medium after 24-h incubation. In other experiments, incubation of big ET-1 (1 microM) with intact BAECs or EA.hy 926 cells resulted in the generation of ET-1, and with both cell types this was inhibited by PHOS (IC50: BAECs = 6.4 microM; EA.hy 926 = 0.26 microM). These results are consistent with both cell types having a PHOS-sensitive ECE that is readily accessible to exogenous big ET-1 and is therefore probably located on the plasma membrane. Furthermore, another intracellular ECE may play a part in the endogenous intracellular formation of ET-1 in EA.hy 926 cells.

Amino Acid Sequence↗

PubMed1993

Vasoconstriction in the rat kidney induced by endothelin-1 is blocked by PD 145065.

We have previously shown that the receptors mediating the renal and systemic vasoconstrictor effects of endothelin-1 (ET-1) are of two distinct endothelin receptor subtypes. Here, we evaluate the effect of PD 145065, a nonselective endothelin receptor antagonist, on the renal vasoconstrictor effects of ET-1 in the rat kidney. ET-1 induced concentration-dependent increases in perfusion pressure in the isolated perfused kidney of the rat. The ETA receptor-selective antagonists BQ-123 (10 microM) and FR 139317 (10 microM) lowered the ET-1-induced rise in perfusion pressure by 57% and 61%, respectively, at 3 x 10(-10) M ET-1. By comparison, the ET-1-induced vasoconstriction was fully antagonized (96% inhibition) by PD 145065 (10 microM). In the anesthetized rat, ET-1 produced a dose-dependent increase in mean arterial pressure, which was attenuated by PD 145065. The initial depressor response to ET-1 was completely blocked by PD 145065, as were the reduction in renal blood flow and increase in renal vascular resistance induced by ET-1. These results suggest that both the ETA and a non-ETA receptor subtype play an important role in mediating the vasoconstrictor effects of ET-1 in the rat kidney.

Amino Acid Sequence↗

PubMed1993

Comparative studies with the endothelin receptor antagonists BQ-123 and PD 142893 indicate at least three endothelin receptors.

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

Amino Acid Sequence↗

PubMed1993

Heterogeneous receptors mediate endothelin-1-induced changes in blood pressure, hematocrit, and platelet aggregation.

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

Animals↗

PubMed1993

Incomplete inhibition of the pressor effects of endothelin-1 and related peptides in the anaesthetized rat with BQ-123 provides evidence for more than one vasoconstrictor receptor.

1. The effects of the ETA receptor antagonist, BQ-123 on blood pressure changes induced by various members of the endothelin (ET)/sarafotoxin (SX) peptide superfamily were investigated in the anaesthetized rat. 2. ET-1 (1 nmol kg-1, i.v. bolus) induced a sustained increase in mean arterial pressure (MAP, maximum increase 44 +/- 3 mmHg). Intravenous injection of BQ-123 at 0.2, 1.0 or 5.0 mg kg-1 5 min before ET-1 inhibited the pressor response by 18, 50 and 61%, respectively. The ET-1 pressor response was inhibited by 75% when the peptide was given 60 min after the start of a 120 min i.v. infusion of BQ-123 (0.2 mg kg-1 min-1). 3. In addition to ET-1, BQ-123 (1 mg kg-1, i.v. bolus) attenuated the pressor responses to big ET-1 (1 nmol kg-1, i.v., bolus, maximum increase in MAP: 68 +/- 7 mmHg), ET-3 (3 nmol kg-1, i.v., bolus, maximum response: 30 +/- 3 mmHg), SX6b (1 nmol kg-1, i.v., bolus, maximum response: 41 +/- 5 mmHg) and SX6c (1 nmol kg-1, i.v., bolus, maximum response: 24 +/- 4 mmHg) by 65, 60, 88 and 50%, respectively. 4. With the exception of big ET-1, all the peptides used in this study induced an initial transient depressor response (-32 +/- 3 mmHg, n = 18). Although BQ-123 (1 mg kg-1, i.v., bolus) did not affect the absolute magnitude of the fall in MAP, the ETA receptor antagonist significantly prolonged the depressor responses induced by ET-3 and SX6b. 5. Thus, BQ-123 attenuates the pressor, but not the depressor effects of ET-1, big ET-1, ET-3, SX6b and SX6c. Complete inhibition of the pressor responses could not be achieved, suggesting that a component of the pressor response is not mediated via the ETA receptor.

Amino Acid Sequence↗

PubMed1993

Mediation via different receptors of the vasoconstrictor effects of endothelins and sarafotoxins in the systemic circulation and renal vasculature of the anaesthetized rat.

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

Anesthesia↗

PubMed1993

Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock.

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

Amino Acid Oxidoreductases↗

PubMed1993

Selective inhibition of agonist-induced but not shear stress-dependent release of endothelial autacoids by thapsigargin.

1. The effects of the Ca(2+)-ATPase inhibitor, thapsigargin, on the shear stress-dependent and on the agonist-stimulated release of endothelium-derived relaxing factor, i.e. nitric oxide (NO), and prostacyclin (PGI2) were studied in bovine and human cultured endothelial cells as well as in endothelium-intact arterial segments of the rabbit. 2. Preincubation with thapsigargin (1 microM for 10 min) had no effect on the shear stress-dependent release of NO from bovine aortic endothelial cells grown on beads, but abolished the release of NO induced by ADP, bradykinin, ionomycin or poly-L-lysine. Similarly, thapsigargin completely abrogated the agonist-stimulated PGI2 release from these cells, but had no effect on the shear stress-dependent release of PGI2. 3. The acetylcholine-induced release of NO from the luminally perfused thoracic aorta and femoral artery of the rabbit was suppressed by pretreatment with thapsigargin (1 microM). In contrast, thapsigargin did not affect the shear stress-dependent release of NO from the femoral artery. 4. Administration of thapsigargin to these vascular preparations or to cultured endothelial cells alone produced a substantial release of both NO and PGI2. This release declined towards previous values after washout of thapsigargin. 5. In human and bovine cultured endothelial cells, thapsigargin (1-1000 nM) caused a dose-dependent sustained rise in [Ca2+]i, an effect that was abolished in the absence of extracellular Ca2+. Stimulation of these cells with bradykinin, histamine, ADP or ionomycin after previous exposure to thapsigargin (30-1000 nM) no longer caused an increase in [Ca2+]i. of the release of these endothelial autacoids caused by shear stress or receptor-dependent and independent agonists.

Adenosine Diphosphate↗

PubMed1993

Support of renal blood flow after ischaemic-reperfusion injury by endogenous formation of nitric oxide and of cyclo-oxygenase vasodilator metabolites.

1. Ischaemia-reperfusion injury in the kidney is associated with a loss of autoregulation, an increase in renal vascular resistance (RVR), a decrease of renal blood flow (RBF) and ultimately acute renal failure. The aim of this study was to investigate the role of the release of endogenous nitric oxide (NO) in the recovery of RBF after ischaemic injury of the renal vascular bed. 2. Anaesthetized rats (thiopentone sodium; 120 mg kg-1, i.p.) were submitted to acute renal ischaemia followed by 2 or 6 h of reperfusion (I/R). Reperfusion was associated with a significant reduction in RBF, an increase in RVR, and an impairment of the vasodilator effect of acetylcholine (ACh). 3. NG-nitro-L-arginine methyl ester (L-NAME, 30 micrograms kg-1 min-1, i.v., n = 5) significantly prevented the recovery of RBF after I/R injury. Similarly, inhibition of prostanoid formation with indomethacin (5 mg kg-1, i.v., n = 4) significantly enhanced the rise in RVR associated with I/R injury. 4. Infusion of L-arginine (L-Arg; 1 or 3 mg kg-1 min-1, i.v., n = 5 and 4, respectively) or D-Arg (1 mg kg-1 min-1, i.v., n = 6), starting 30 min after occlusion, did not improve the recovery of RBF. Furthermore, infusion of L-Arg (20 mg kg-1 min-1 for 15 min; n = 4) had no effect on the I/R-induced impairment of the vasodilator responses to ACh. 5. To elucidate the relative importance of the constitutive and inducible NO synthase isoforms for the formation of NO after I/R, calcium-dependent (constitutive) and calcium-independent (inducible) NO synthase activities were measured in kidney homogenates obtained from ischaemic or non-ischaemic kidneys. A calcium-independent NO synthase activity was not detectable in kidney homogenates obtained from either sham-operated control rats or from animals subjected to I/R. Moreover, dexamethasone(3 mg kg-1, i.v., 60 min prior to I/R, n = 6), an inhibitor of the induction of NO synthase,had no effect on either RBF or RVR in rats subjected to I/R. In contrast to I/R, lipopolysaccaride(LPS, endotoxin; 5 mg kg-1, i.p., n = 3) caused a significant induction of a calcium-independent NO synthase activity in the kidney.6. These results confirm the importance of the release of vasodilator cyclo-oxygenase metabolites in the compromised renal circulation and indicate that the formation of NO derived from the constitutive, but not the inducible NO synthase, is also important for the maintenance of RBF after I/R injury of the renal vascular bed.

Amino Acid Oxidoreductases↗

PubMed1993

Induction by endotoxin of nitric oxide synthase in the rat mesentery: lack of effect on action of vasoconstrictors.

1. Male Sprague-Dawley or Wistar rats were injected with bacterial lipopolysaccharide (LPS; 5 mg kg-1, i.p.) and killed after 1, 3, 6, 15, and 24 h. The brains, mesenteries, spleens, lungs, livers, kidneys, hearts, aortae and diaphragms were removed and frozen immediately. Control rats were injected with sterile saline and killed after 6 h. 2. The organs were homogenized in a semi-frozen state and NO synthase (NOS) activity measured in tissues from both LPS-treated and saline-treated groups by the ability of homogenates to convert [3H]-L-arginine to [3H]-L-citrulline in a NADPH-dependent manner. 3. The NOS activity in all organs taken from control animals was found to be calcium-dependent, with the highest activity being in the brain. After LPS-treatment an induced calcium-independent NOS was detected in all tissues tested, with the exception of the brain. The spleen, lung, mesentery and liver had the highest amounts of LPS-induced NOS activity. No induction of calcium-dependent NOS was detected. 4. Induction of NOS was maximum 6 h after administration of LPS and had returned to control levels in 24 h. 5. The constitutive NOS in brain and mesentery and the LPS-induced activities in the spleen, lung, liver and mesentery were inhibited by NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME) according to concentration. The IC50 for L-NAME was 2.5 microM against the constitutive NOS from brain, and 20-25 microM against the inducible NOS. For L-NMMA the IC50 was 20-25 microM against either NOS isoform. 7. The vascular responses to endothelin-I (ET-1), the thromboxane A2-mimetic 11 alpha,9 alpha-epoxymethanoprostaglandin F2alpha (U46619), phenylephrine (PE) or 5-hydroxytryptamine (5-HT) were measured in the simultaneously perfused arterial and venous mesenteric vascular beds from both control and LPS-treated(6 h) rats. Vasoconstrictor responses to all agonists tested were unaffected by LPS treatment. In the presence of L-NAME (100 microM) vasoconstrictor responses were potentiated in both the arterial and venous portion of the mesenteric beds from both control and LPS-treated rats. The potentiation of responses to U46619 was significantly greater in beds from LPS-treated rats.8. Injection of LPS i.p. is associated with induction of NOS in all organs tested, except for the brain. In the mesentery this is not accompanied by a hyporesponsiveness to constrictor agents suggesting an increased sensitivity, particularly to U46619. This may explain the poor perfusion and tissue damage in the splanchnic circulation associated with sepsis.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1993

Mediation of endothelin-1-induced inhibition of platelet aggregation via the ETB receptor.

1. The effects of FR139317 (ETA antagonist) or PD145065 (non-selective ETA/ETB antagonist) on endothelin-1 (ET-1)-induced changes in blood pressure and inhibition of ex vivo platelet aggregation were investigated in the anaesthetized rabbit. 2. ET-1 (1 nmol kg-1, i.a. bolus) caused a sustained increase in mean arterial pressure (MAP) (peak increase 47 +/- 5 mmHg, n = 8). Intravenous infusion of FR139317 at 0.2 (n = 4) or 0.6 mg kg-1 min-1 (n = 4) inhibited the ET-1 pressor response by 83 or 89%, respectively. Infusion of PD145065 at 0.2 (n = 4) or 0.6 mg kg-1 min-1 (n = 4) inhibited the ET-1-induced increase in MAP by 79 or 75%, respectively. 3. The transient depressor response (-16 +/- 3 mmHg) which preceded the rise in blood pressure induced by ET-1 (1 nmol kg-1, i.a., n = 8) was enhanced by an intravenous infusion of FR139317 (0.6 mg kg-1 min-1) to -35 +/- 5 mmHg (P < 0.05, n = 4). This enhancement was abolished by indomethacin (5 mg kg-1, i.v.) pretreatment (-17 +/- 1 mmHg, n = 4). PD145065 (0.2 mg kg-1 min-1, i.v.) attenuated the ET-1-induced fall in blood pressure to -9 +/- 1 mmHg (n = 4), while a higher dose of this antagonist (0.6 mg kg-1 min-1, i.v.) completely abolished the ET-1-mediated depressor response. 4. ET-1 (1 nmol kg-1, n = 8) inhibited ex vivo platelet aggregation by 96% at 5 min after injection of the peptide. FR139317 (0.2 or 0.6 mg kg-1 min-1, i.v.) or PD145065 (0.2mg kg-1 min-1, i.v.) did not affect the inhibition of ex vivo platelet aggregation in response to ET-1. In contrast, intravenous infusion of PD145065 (0.6 mg kg-1 min-1) abolished the anti-aggregatory effects of ET-1.5. Thus, FR139317 inhibits the pressor, but not the depressor actions of ET-1 and has no effect on the ET-l-induced inhibition of ex vivo platelet aggregation. In contrast, PD145065 antagonizes the pressor and depressor responses to ET-1 and abolishes the anti-aggregatory effects of the peptide.6. These results strongly suggest that ET-1-induced vasoconstriction in the anaesthetized rabbit is primarily mediated via the ETA receptor while the depressor and antiaggregatory actions of ET-1 are due to activation of the ETB receptor.

Animals↗

PubMed1993

Role of tumour necrosis factor in the induction of nitric oxide synthase in a rat model of endotoxin shock.

1. This study investigates the role of tumour necrosis factor (TNF) in the induction of nitric oxide synthase (NOS) by bacterial endotoxin (lipopolysaccharide; LPS) in a rat model of endotoxin shock. 2. In anaesthetized rats, pretreatment with a monoclonal antibody for TNF (TNFab; 20 mg kg-1, s.c., at 16 h prior to LPS) ameliorated the fall in mean arterial blood pressure (MAP) in response to LPS (2 mg kg-1, i.v.). For instance, endotoxaemia for 180 min resulted in a fall in MAP from 114 +/- 6 (control) to 84 +/- 5 mmHg (P < 0.01; n = 7). In contrast, animals pretreated with TNFab prior to LPS injection maintained significantly higher MAP when compared to LPS-control (MAP at 180 min; 118 +/- 3 mmHg; P < 0.01, n = 5). 3. Three hours of endotoxaemia was also associated with a significant reduction of the contractile effects of noradrenaline (NA) (10(-8)-10(-6) M) on the thoracic aorta ex vivo. This hyporeactivity to NA was partially restored by in vitro treatment of the vessels with NG-nitro-L-arginine methyl ester (L-NAME, 20 min, 3 x 10(-4) M). Pretreatment of rats with TNFab (20 mg kg-1; at 16 h prior to LPS) significantly (P < 0.05) attenuated the LPS-induced hyporeactivity of rat aortic rings ex vivo. L-NAME did not enhance the contractions of aortic rings obtained from TNFab pretreated LPS-rats. 4. At 180 min after LPS there was a significant elevation of the induced NOS activity in the lung (5.14 +/- 0.57 pmol citrulline mg-1 min-1, n = 8). TNFab pretreatment significantly attenuated this induction of NOS in response to LPS by 37 +/- 6% (n = 5; P<0.05).5. We conclude that the formation of endogenous TNF contributes to the induction of the calcium in dependent isoform of NOS in response to LPS in vivo. Thus, the beneficial effects of agents which inhibit either the release or the action of TNF in circulatory shock may be, in part, due to inhibition of NOS induction.

Amino Acid Oxidoreductases↗

PubMed1993

Dissociation of the anti-ischaemic effects of cloricromene from its anti-platelet activity.

1. Cloricromene is a non-anticoagulant coumarin derivative with anti-platelet and anti-leukocyte properties, which has beneficial effects in various models of ischaemia and shock. 2. We have assessed the effects of cloricromene on (a) ex vivo platelet aggregation, and (b) infarct size using a model of myocardial ischaemia in the anaesthetized rabbit. 3. Cloricromene (1-1000 micrograms kg-1 min-1 for 15 min) induced a dose-dependent inhibition of ex vivo platelet aggregation, causing only a minimal increase in heart rate and no change in mean arterial blood pressure. The inhibitory activity was considerably stronger when platelet aggregation was induced by collagen than by ADP. 4. Cloricromene inhibited ex vivo platelet aggregation in rabbits pretreated with indomethacin (5 mg kg-1) and this inhibition persisted for 30-60 min. 5. The model of myocardial ischaemia involved 1 h occlusion of the first antero-lateral branch of the left coronary artery followed by 2 h of reperfusion. Infusion of cloricromene (30 or 300 micrograms kg-1 min-1), ibuprofen (80 micrograms kg-1 min-1) or vehicle began 15 min prior to occlusion, and continued throughout the experiment. 6. While area at risk was similar for all groups studied, cloricromene (30 or 300 micrograms kg-1 min-1) or ibuprofen caused a reduction in infarct size, and decreased myeloperoxidase activity in the tissue of the infarcted myocardium. 7. Cloricromene at 300 micrograms kg-1 min-1 also reduced the occlusion-induced elevation of the ST-segment of the rabbit electrocardiogram, and inhibited platelet aggregation ex vivo. Ibuprofen or cloricromene at 30 fg kg-1 min-1 had no effect on either the ST-elevation or platelet reactivity.8. Thus, cloricromene exhibits a cardioprotective activity via an inhibition of leukocyte infiltration, in the presence (300 microg kg-l min-1) or absence (30 microg kg-1 min-1) of inhibition of platelet activity ex vivo.The anti-aggregatory activity of cloricromene acts via a mechanism that is either different from, or in addition to, inhibition of cyclo-oxygenase, and is of long duration.

Animals↗

PubMed1993

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF.

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

Amino Acid Sequence↗

PubMed1993

Characterization of endothelin receptors mediating the effects of the endothelin/sarafotoxin peptides on autonomic neurotransmission in the rat vas deferens and guinea-pig ileum.

1. To characterize the receptors mediating the effects of the endothelin/sarafotoxin family of peptides on the responses to electrical stimulation of the rat vas deferens (RVD) and guinea-pig ileum (GPI) we have used endothelin-1 (ET-1), ET-3, sarafotoxin 6b (SX6b) and SX6c as agonists and the endothelin-receptor antagonists BQ-123 (ETA receptor selective) and PD 142893 (non-selective). 2. In the RVD, ET-1 and SX6b increased the twitches induced by field stimulation starting at a threshold concentration of 10(-10) M while the threshold concentration for ET-3 was 3 x 10(-9) M. SX6c (up to 3 x 10(-8) M) did not potentiate the twitches. SX6b produced significantly (P < 0.05) greater potentiations than ET-1 at concentrations of 3 x 10(-9) M and higher, and 10(-7) M ET-3 also produced a significantly greater effect than ET-1 at the same concentration. Thus, at threshold the rank order of peptides was ET-1 = SX6b > ET-3 >>> SX6c, and at concentrations of 3 x 10(-8) M and higher, SX6b > ET-3 > ET-1 >>> SX6c. 3. In the presence of BQ-123 or PD 142893 (10(-5) M) the threshold concentrations for ET-1 to augment the twitches were increased 30 fold. In the same conditions neither SX6b nor ET-3 potentiated the responses. The relative activities of the endothelin/sarafotoxin peptides and the effectiveness of the endothelin receptor antagonists are consistent with postjunctional ETA receptors mediating these effects. 4. In the transmurally stimulated GPI the endothelin/sarafotoxin peptides produced two effects; an increase in the basal tension of the tissues and an inhibition of the twitch responses. To increase the basal tension the peptides had the order of potency ET-1 > SX6b>> ET-3 = SX6c. These direct effects of ET-1 or SX6b were strongly antagonized (100 fold) by either BQ-123 (10-5M) or PD 142893(10-5 M). Thus, ETA receptors mediate contractions of the GPI induced by these peptides.5. The endothelin/sarafotoxin peptides were approximately equipotent at depressing twitches of the GPI in response to transmural stimulation (EC50s, 4 x 10-11 to 1.5 x 10-10 M). The depressions induced byET-1 were unaffected by either BQ-123 (10-5 M) or PD 142893 (10-5 M). BQ-123 produced a small(three fold) antagonism of the inhibitory effects of ET-3 or SX6c. These results indicate that a receptor of the ETB type mediates the inhibitory effects of the endothelin/sarafotoxin peptides on neurotransmission in the GPI.6. Thus, both ETA receptors and ETB receptors mediate the effects of the endothelin/sarafotoxinpeptides on neurotransmission.

Amino Acid Sequence↗

PubMed1993

Platelet-activating factor contributes to the induction of nitric oxide synthase by bacterial lipopolysaccharide.

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

Amino Acid Oxidoreductases↗

PubMed1993

Nifedipine inhibits the induction of nitric oxide synthase by bacterial lipopolysaccharide.

We investigated the effect of the calcium channel antagonist nifedipine on the induction of nitric oxide synthase (NOS) by bacterial endotoxin (lipopolysaccharide; LPS) in J774.2 macrophages, in cultured rat aortic smooth muscle cells and in a rat model of endotoxin shock. Stimulation by LPS for 24 hr increased nitrite accumulation in the supernatant of both cell types. NOS induction accounts for this nitrite accumulation, as both NG-methyl-L-arginine and cycloheximide reduced nitrite production in both cell types. Dexamethasone inhibited LPS-stimulated nitrite production in macrophages, but not in rat aortic smooth muscle cells. Nifedipine inhibited the production of nitrite in these LPS-treated cell types, with a more pronounced effect on macrophages. However, nifedipine did not inhibit the production of nitrite in J774.2 cells in which NOS had already been induced by prior exposure to LPS, and any possible further induction was inhibited by cycloheximide. In anesthetized rats subjected to LPS, pretreatment with nifedipine or dexamethasone ameliorated the fall in mean arterial blood pressure and the vascular hyporeactivity to norepinephrine at 180 min after LPS injection. At 180 min after LPS, an increase in a calcium-independent (induced) NOS activity was measured in lung homogenates. This induced NOS activity was reduced in lungs from rats treated with nifedipine or dexamethasone before LPS. Thus, nifedipine inhibits the induction of NOS in response to LPS in cultured cells in vitro and in the anesthetized rat.

Amino Acid Oxidoreductases↗