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T D Warner

Publications and source records attributed to T D Warner.

At least 73 records · Page 4Linked to original sources
PubMed1997

Characterization of the induction of nitric oxide synthase and cyclo-oxygenase in rat aorta in organ culture.

1. Within vessels, the formation of nitric oxide (NO) or prostaglandins is normally catalysed in the endothelium by constitutive isoforms of NO synthase (eNOS) and cyclo-oxygenase (COX-1), respectively. However, during inflammatory conditions, the underlying smooth muscle acquires the ability to release NO and prostaglandins after the expression of inducible isoforms of NOS (iNOS) and COX (COX-2). The co-induction of iNOS and COX-2 has been studied over 24 h in isolated vascular smooth muscle cells in vitro. However, due to the limitation of using cultured cells, the relationship between the activities of iNOS and COX over longer periods has not been addressed. Moreover, the relative contribution of the endothelium to the production of NO and prostaglandins under inflammatory conditions is not completely understood. 2. Here using an organ culture system, we have determined the profile of COX (6-keto prostaglandin F1 alpha (6-keto PGF1 alpha), PGE2, thromboxane B2 (TXB2) and NOS (nitrite and nitrate) metabolites released over a period of 10 days from segments of rat aorta. In each case, segments from the same animal were left untreated or treated with bacterial lipopolysaccharide (LPS; 10 micrograms ml-1) in order to induce iNOS and COX-2. Prostaglandins were measured by radioimmunoassay whilst nitrite and nitrate were measured, respectively, by Greiss reaction alone, or following a nitrate reductase step. The isoforms of NOS and COX responsible for metabolite release were characterized pharmacologically by use of inhibitors and at the molecular level by reverse transcription polymerase chain reaction with specific primers for iNOS, eNOS, COX-1 and COX-2. In separate experiments the role of the endothelium in the release of nitrite, nitrate and prostaglandins and in the expression of iNOS, eNOS, COX-1 and COX-2 was determined by comparing responses in endothelium denuded and endothelium-intact segments of rat aorta. 3. Under control culture conditions vessels released prostaglandins in the following rank order 6-keto PGF1 alpha = PGE2 > > TXB2. LPS increased the release of 6-keto PGF1 alpha and PGE2 but not of TXB2, an effect that was inhibited by the protein synthesis inhibitor cycloheximide (1 microM), the anti-inflammatory steroid dexamethason (1 microM), the nonsteroidal anti-inflammatory drug indomethacin (30 microM) and, where tested, the selective COX-2 inhibitor NS-398 (30 microM). Similarly, segments of rat aorta released detectable levels of nitrite and nitrate, which were reduced by NG-nitro-L-arginine methyl ester (L-NAME, 1 mM), which inhibits all isoforms of NOS, and by dexamethasone (1 microM), which inhibits the induction of iNOS. The proportion of nitrate to nitrite released over the 10 day period varied greatly from approximately 1:1 on days 5 to 8 to 5:1 on day 9. However, the sum of nitrite and nitrate (NOx) as well as PGE2 remained elevated over the whole 10 day period. The formation of 6-keto PGF1 alpha peaked on days 1 and 2. 4. In freshly prepared tissue, mRNAs for eNOS, COX-1, iNOS and COX-2 were detected. After 24 h in culture, there was an apparent increase in the level of mRNAs for iNOS and COX-2 but not for eNOS or COX-1, an effect that was further enhanced when LPS was included in the culture medium. The expressions of mRNA for eNOS, COX-1, iNOS or COX-2 were not greatly different in vessels with intact or disrupted endothelium. Similarly the release of NOx or PGE2 by vessels after the 1st or 9th day in culture were not significantly different from vessels prepared with or without endothelium. 5. Thus, COX-2 and iNOS are co-induced in intact vessels in culture, with the vascular smooth muscle being the main site of mediator generation. In contrast to data from isolated cells in culture (observed usually over 1 day), both COX and NOS activities in cultured blood vessels were elevated for at least 10 days. Also, unlike isolated cells in culture, the COX and NOS pathways were active independently; L-NAME had little effect on the activity of COX and indomethacin had little effect on the activity of NOS.

6-Ketoprostaglandin F1 alpha↗
PubMed1997

Growing placebo response rate: the problem in recent therapeutic trials?

Recently the development of several promising new compounds for anxiety and depression was discontinued because of difficulty demonstrating therapeutic effects. This article explores alternatives to "increasing placebo response rate" as explanations. We reanalyzed a study of 81 panic patients treated with placebo, alprazolam 2 mg or 6 mg, or imipramine 225 mg daily to investigate the effect of baseline pathology and selective effects of treatment on biological and cognitive components of panic disorder. The regression of endpoint on baseline number of spontaneous panic attacks differed among treatment groups, with lower slopes for the more active compounds. Only patients with many panic attacks at baseline benefited from the active compounds. Also, treatment effects declined progressively on the more cognitive aspects of the disorder (situational panic attacks and phobia ratings) for alprazolam and were entirely absent for imipramine. Implications for the etiology of panic disorder, its treatment, and therapeutic research are discussed.

Alprazolam↗
PubMed1996

Cardiovascular responses induced by injection of endothelin-1 into the superior colliculus of rats.

Microinjection of endothelin-1 (1-10-100 pmol) into the superficial layer of the superior colliculus of anaesthetised rats caused dose-dependent decreases in blood pressure. This is unlikely to be a non-specific effect, for angiotensin II (1 nmol) caused increases in blood pressure. Injection of endothelin-1 (10 pmol) into the superior colliculus also caused falls in renal blood flow as measured by an ultrasonic flow probe. Decreases in blood pressure and falls in renal blood flow induced by injection of endothelin-1 into the superior colliculus were unaffected by bilateral vagotomy suggesting that attenuations in the activity of the sympathetic nervous system were involved in these events.

Animals↗
PubMed1996

What turns on the endothelins?

Production of the potent vasoconstrictor peptide endothelin-1 (ET-1) within the circulation is increased markedly in a number of pathologies, such as the damage following from ischaemia and reperfusion, vasculitis, congestive heart failure, and systemic inflammatory response (septic shock syndrome) and related pathological states. All these conditions are associated with marked increases in the production of cytokines such as tumour necrosis factor-alpha and interleukin-2. Our experiments indicate that in rats administration of either of these cytokines results in a rapid increase in the circulating levels of ET-1 and a very pronounced ET-1-dependent coronary vasoconstriction. Furthermore, in rats suffering from adjuvant polyarthritis, in which there is marked joint inflammation and associated cytokine production, there are dramatic increases in coronary perfusion pressure which are absent when rats are treated with an endothelin receptor antagonist. This does not imply that inflammation must be associated with coronary vasoconstriction and myocardial dysfunction, but rather that in the rat, where ET-1 is a strong and almost irreversible constrictor of coronary vessels, coronary perfusion pressure serves as a very good bioassay for ET-1 activity. What our results do imply is that the ET-1 system is latent, but that in numerous disease states it can become rapidly upregulated following endothelial activation by cytokines.

Animals↗
PubMed1996

California dreamin' 'bout endothelin: emerging new therapeutics.

Progress in our understanding of endothelins has been extremely rapid since their discovery in 1988. A number of pharmaceutical companies have developed potent, orally active, nonpeptide endothelin receptor antagonists with efficacies in a wide variety of animal disease models and potential for treating human disease. However, only time will tell whether or not these compounds are developed into drugs that are perceived as worthy of a place in the therapeutic marketplace. If this is the case, endothelin will have been promoted from the status of striking scientific discovery to therapeutic target in a remarkably short period.

Animals↗
PubMed1996

Endothelin-1 and the periaqueductal gray area of the rat: an autoradiographic and functional pharmacological study.

Endothelin-1 (ET-1) injected centrally induces pressor effects and associated haemodynamic changes. Here we have evaluated the effects on systemic and regional cardiovascular parameters of injection of ET-1 into the periaqueductal gray (PAG) area of anaesthetized rats. In addition, we have used the ETA receptor-selective antagonist, FR 139317, the ETB receptor-selective antagonist, BQ-788, and the ETA/ ETB receptor non-selective antagonist, SB 209670, to identify the receptor(s) mediating these effects. We have also used in vitro autoradiography to identify binding sites for ET-1 in the PAG. 2. In vitro autoradiography showed dense binding of [125I]-PD 151242 (for ETA receptors) in the PAG area, with the binding sites being homogeneously distributed within the dorsal, lateral and ventral subregions. Tissues incubated with [125I]-BQ 3020 (for ETB receptors) had little binding. 3. Injection of ET-1 (0.1, 1 and 10 pmol per rat) in the dorsolateral PAG area significantly increased, in a dose-dependent manner the mean arterial blood pressure (MAP). The highest dose of ET-1 (10 pmol) also decreased the heart rate by 18 +/- 1%, n = 6 (P < 0.05). Increases in blood pressure induced by ET-1 (1 pmol; 31 +/- 6.6 mmHg, n = 6) were greatly reduced by pre-administration to the PAG area of FR 139317 (5 nmol per rat) or SB 209670 (3 nmol per rat) (97 and 94%, respectively), but were unaffected by BQ-788 (5 nmol per rat). Similarly, FR 139317 and SB 209670 prevented the decrease in heart rate induced by ET-1 while BQ-788 did not affect it. 4. Injection of ET-1 to the PAG area caused falls in renal blood flow (RBF) as measured by an ultrasonic flow probe, and increased renal vascular resistance (RVR). Pre-treatment of the PAG with FR 139317 or SB 209670, but not with BQ-788, prevented this ET-1-induced effect. 5. Injection of ET-1 (10 pmol) also increased total peripheral resistance (TPR; control, 2.39 +/- 0.2 mmHg ml-1 min 100 g body weight) by 100 +/- 9% (n = 5) and reduced the cardiac output (CO; control, 94.7 +/- 3.1 ml min-1) by 30 +/- 3% (n = 5), as determined by radioactive microspheres. Vascular resistances were increased in other organs, such as skeletal muscle (88 +/- 5%, n = 4), the colon (55 +/- 7%, n = 4) and the stomach (47 +/- 3%, n = 4). Pretreatment of the PAG area with FR 139317 or SB 209670 reduced the increases in TPR and vascular resistance, and the reduction in CO caused by ET-1. BQ-788 did not effect the responses to ET-1. 6. Thus, there are predominantly ETA binding sites within the PAG area and injection of ET-1 into the PAG area causes complex haemodynamic changes which are sensitive to ETA receptor antagonism. ETA receptors are, therefore, the predominant mediators of the actions of ET-1 in the PAG of the rat.

Animals↗
PubMed1996

Influence of endothelial mediators on the vascular smooth muscle and circulating platelets and blood cells.

The vascular endothelium regulates the tone of the underlying smooth muscle and the reactivity of blood elements such as platelets and neutrophils by the release of mediators, in particular nitric oxide, prostacyclin and endothelin-1. The first two of these are potent vasodilators which also inhibit platelet and neutrophil aggregation and adhesion, while endothelin-1 is the most potent mammalian vasoconstrictor peptite yet found. There are also interactions between these mediators. For instance, endothelin-1 acts on specific receptors on the endothelium to increase the release of nitric oxide and prostacyclin, while nitric oxide depresses the production and/or release of endothelin-1 from endothelial cells. Endothelin-1 and prostacyclin do not appear to be involved in the basal regulation of blood pressure whereas nitric oxide does for inhibition of its production in normotensive animals produces a marked elevation in blood pressure. Conversely, numerous vascular disease states have been associated with elevations in the production and/or release of endothelin-1 and it has been implicated in the deleterious changes associated with ischaemia/reperfusion injury, subarachnoid haemorrhage and hypertension. Endothelin-1 has also been proposed to be pro-atherogenic, as have reductions in the production of the vasodilator mediators nitric oxide and prostacyclin.

Blood Platelets↗
PubMed1995

Endothelin 1 mediates ex vivo coronary vasoconstriction caused by exogenous and endogenous cytokines.

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

Animals↗
PubMed1995

Rapid degradation of endothelin-1 by an enzyme released by the rat isolated perfused mesentery.

1. In vivo the effects of endothelin-1 (ET-1) are limited by its rapid removal from the circulation and possibly by its metabolism by enzymes such as neutral endopeptidase 24.11, deamidase or carboxypeptidase A. Here, using as a model the isolated perfused mesenteric arterial bed of the rat, we have examined the involvements of these enzymatic activities in the vascular responses to ET-1. 2. Samples of Krebs buffer which had been recirculated through the mesenteric arterial bed for 30 min rapidly destroyed the activity of ET-1 as assessed either by bioassay on rings of rat thoracic aorta or by high performance liquid chromatography (h.p.l.c.). For instance, after 15 min incubation with the recirculated-Krebs solution (recirc-K) the contraction induced by 3 x 10(-9) M ET-1 was reduced by more than 90%. Contractions induced by sarafotoxin 6b (3 x 10(-9) M) were similarly suppressed by preincubation with recirc-K whereas those to Arg-vasopressin (3 x 10(-9) M) were unaffected. 3. The degradation of ET-1 by recirc-K was prevented by 1,10-phenanthroline (10(-3) M), abolished by heating the recirc-K solution to 90 degrees C for 15 min, and reduced by EGTA (5 x 10(-3) M) or ET-1(16-21) (10(-5) M). For instance, in the presence of ET-1(16-21) (n = 6) the contraction induced by ET-1 was reduced by only 40% after 15 min incubation with recirc-K buffer. Leupeptin (3 x 10-4 M), dichloroisocoumarin(5 x 10-5 M), phenylmethyl-sulphonyl fluoride (10-3 M), a combination of bacitracin (300 mg ml-1),bestatin (10-5 M), captopril (10-5 M), phosphoramidon (10-4 M) and thiorphan (10-4 M) or Polypep (aproprietary protein digest) did not inhibit the degradation of ET-1 by recirc-K.4. In experiments examining directly the vascular responses of the isolated perfused mesentery of the rat, the addition of cumulative concentrations of ET-1 to the recirculating Krebs solution caused small concentration-dependent increases in perfusion pressure. The inclusion of ET-1(16-2l), ET-1(17-21), or ET-1(18-21) (10-5M) greatly potentiated these responses, but not those to Arg-vasopressin or methoxamine.The effects of 1,10-phenanthroline or EGTA could not be examined in this system because these agents both depressed non-specifically the vasoconstrictor responses of the mesenteric vascular bed.5. Thus, the rat mesentery releases an enzyme that very rapidly destroys ET-1 or the very closely related peptide, sarafotoxin 6b but not Arg-vasopressin. This enzyme is most probably a metallopeptidase because of its sensitivity to inhibition by 1,10-phenanthroline or EGTA. It is particularly interesting that a simple vascular bed such as the mesentery produces such a powerful endothelin metabolising enzyme. It is tempting, therefore, to speculate that the endothelin degrading enzyme active at neutral pH that- we have found is important in the metabolism of ET-1 throughout the vasculature.

Animals↗
PubMed1995

Coronary vasoconstriction in vitro in the hearts of polyarthritic rats: effectiveness of in vivo treatment with the endothelin receptor antagonist SB 209670.

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

Animals↗
PubMed1995

Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids.

1. Lipopolysaccharide (LPS) co-induces nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) in J774.2 macrophages. Here we have used LPS-activated J774.2 macrophages to investigate the effects of exogenous or endogenous nitric oxide (NO) on COX-2 in both intact and broken cell preparations. NOS activity was assessed by measuring the accumulation of nitrite using the Griess reaction. COX-2 activity was assessed by measuring the formation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Western blot analysis was used to determine the expression of COX-2 protein. We have also investigated whether endogenous NO regulates the activity and/or expression of COX in vivo by measuring NOS and COX activity in the lung and kidney, as well as release of prostanoids from the perfused lung of normal and LPS-treated rats. 2. Incubation of cultured murine macrophages (J774.2 cells) with LPS (1 microgram ml-1) for 24 h caused a time-dependent accumulation of nitrite and 6-keto-PGF1 alpha in the cell culture medium which was first significant after 6 h. The formation of both 6-keto-PGF1 alpha and nitrite elicited by LPS was inhibited by cycloheximide (1 microM) or dexamethasone (1 microM). Western blot analysis showed that J774.2 macrophages contained COX-2 protein after LPS administration, whereas untreated cells contained no COX-2. 3. The accumulation of 6-keto-PGF1 alpha in the medium of LPS-activated J774.2 macrophages was concentration-dependently inhibited by chronic (24 h) exposure to sodium nitroprusside (SNP; 1-1000 microM). Sodium nitroprusside (1-1000 microM) also acutely (30 min) inhibited COX-2 activity in broken cell preparations of LPS-activated (12 h) J774.2 macrophages, in a similar concentration dependent manner. Addition of adrenaline (5 mM) and glutathione (0.1 mM) increased the activity of COX-2 in broken cell preparations. In the presence of these co-factors, SNP inhibited prostanoid production only at the highest concentration used (1 mM). When J774.2 cells were incubated in the presence of LPS (1 microg ml-1) and NG-monomethyl-L-arginine (L-NMMA: 1 mM) for 12 h, SNP at the highest concentration used (1 mM) acutely (30 min) inhibited the activity of COX-2 in cell homogenates with co-factors. However, when J774.2 macrophages were incubated for 24 or 12 h with LPS (1 microg ml-1)and L-NMMA (1 mM), the addition of SNP (0.001-1I000 microM) increased in a concentration-dependent manner the accumulation of 6-keto-PGF1a in intact cells (measured at 24 h) and COX-2 activity in cell homogenates in the presence of co-factors (determined at 12 h). SNP (1 mM; together with LPS for 12 h)decreased the amount of COX-2 protein induced by LPS in J774.2 macrophages.4. Indomethacin (30 1AM) abolished the formation of 6-keto-PGFa by LPS-activated macrophages, but had no effect on the release of nitrite. Conversely, L-NMMA, at the highest concentrations used (1 and 10 mM), increased the release of 6-keto-PGFIa an effect which was reversed by excess L-arginine (3 mM)but not by D-arginine. Similarly, the decrease in nitrite formation caused by L-NMMA was partially reversed by L-arginine (3 mM), but not by D-arginine. L-NMMA (10 mM; together with LPS for 12 h)increased the amount of COX-2 protein induced by LPS in J774.2 macrophages.5. In separate experiments, J774.2 macrophages were activated with LPS (1 microg ml-1), and L-NMMA(10 mM) was added for various times (0.5-24 h) before the collection of mediun at 24 h. L-NMMAenhanced the release of 6-keto-PGFI,, in a time-dependent manner, with the maximal enhancement seen when the NOS inhibitor was incubated with the cells for 24 h. 6. In experiments on male Wistar rats, we investigated the effect of L-NMMA on the release of prostanoids (6-keto-PGF1a prostaglandin E2, thromboxane B2) elicited by arachidonic acid (AA,30nmol) from ex vivo perfused kidneys and lungs. The release from the organs from normal and LPS-treated rats was unaffected by L-NMMA intraperitoneally (30 mg kg-1) for 6 h together with LPS(5 mg kg-1) or LPS vehicle. Similarly, acute (5 min) in vitro exposure to L-NMMA (1 mM) of the perfused organs from control and LPS-treated animals did not change the release of prostanoids elicited by AA (30 nmol).7. These results show that LPS causes the induction of iNOS and COX-2 in J774.2 macrophages. The co-release of NO and PGI2 induced by LPS is dependent on protein synthesis and occurs after a lag-time of 6-12 h. The formation of COX metabolites has no effect on NOS activity whereas NO inhibits both COX-2 activity and induction. These results demonstrate that NOS and COX can be co-induced in vitro and that under these conditions large amounts of NO inhibit the degree of COX expression and activity.In the absence of endogenous NO, lesser amounts of exogenous NO increase the activity of COX-2. In those situations in vivo when the level of NO induction is relatively low, NO does not regulate the increased activity of COX.

Amino Acid Oxidoreductases↗
PubMed1995

Contractile activity of endothelin precursors in the isolated gallbladder of the guinea-pig: presence of an endothelin-converting enzyme.

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

Animals↗
PubMed1995

Comparison of effects of chronic and acute administration of NG-nitro-L-arginine methyl ester to the rat on inhibition of nitric oxide-mediated responses.

1. Vascular responses to acetylcholine and sodium nitroprusside in vivo and in vitro, in the isolated perfused kidney and in rings of rat thoracic aorta, were measured in rats treated chronically with NG-nitro-L-arginine methyl ester (L-NAME; approx, 70 mg kg-1) and compared to responses in age-matched control animals, and age-matched animals after the acute administration of L-NAME (3-100 mumol kg-1). Parallel experiments examined alterations in responsiveness in rings of trachea and anococcygeus muscles taken from the same animals. 2. Chronic oral administration of L-NAME elevated the blood pressure in anaesthetized animals from 114 +/- 5 mmHg to 153 +/- 11 mmHg (n = 5). The hypotensive responses to both acetylcholine (1 nmol kg-1) and sodium nitroprusside (10 nmol kg-1) were enhanced by chronic L-NAME treatment (n = 5-7) whereas acute L-NAME administration enhanced only the response to sodium nitroprusside (n = 5). 3. After chronic treatment with L-NAME, the basal perfusion pressure in the isolated perfused kidney was elevated. However, vasodilator responses to either acetylcholine (1 nmol) or sodium nitroprusside (3 nmol) were unaltered (n = 5-7). The vasodilatation induced by acetylcholine was inhibited in a concentration-dependent manner by the administration of acute L-NAME (0.1 - 100 microM; n = 5), such that significant inhibition was seen at 10 microM L-NAME. The response to sodium nitroprusside was unaffected by L-NAME. 4. The relaxations of isolated rings of rat thoracic aorta induced by acetylcholine were inhibited in tissues prepared from rats treated chronically with L-NAME (n = 5-7). Acute administration of L-NAME (0.1-100 microM) concentration-dependently inhibited the relaxations induced by acetylcholine in this preparation, with significant inhibition occurring at 1 microM L-NAME (n = 5). Responses to sodium nitroprusside were unaffected by either chronic or acute exposure to L-NAME (n = 5-7).5. Relaxations of precontracted anococcygeus muscles induced by electrical field stimulation, or contractions of rings of trachea induced by carbachol or endothelin-1, were unaffected by chronic oral administration of L-NAME (n = 4-6). Acute addition of L-NAME (0.1-100 microM) to the organ baths inhibited in a concentration-dependent manner the relaxations of anococcygeus muscles taken from control animals, with a significant effect being seen at a concentration of 10 micro.M (n = 4-6).6. Our cardiovascular data are consistent with chronic oral administration of L-NAME inhibiting the production of nitric oxide (NO) within the vasculature, although the pattern of inhibition is not uniform between different tissues. Despite the inhibition of endothelial NO production, chronic L-NAME does not alter the vasodepressor activity of acetylcholine in vivo or in the isolated perfused kidney. This maybe explained by an enhanced responsiveness of guanylyl cyclase pathways, the increased release of vasodilators other than nitric oxide or a decreased importance of nitric oxide in resistance vessels compared with conductance vessels. The resistance of peripheral neuronal NO responses to chronic treatment with L-NAME indicates that selective inhibition of different isoforms of NOS may be achieved in vivo.

Acetylcholine↗
PubMed1995

Characterization of receptors for kinins and neurokinins in the arterial and venous mesenteric vasculatures of the guinea-pig.

1. In the present work, we have studied the microvascular reactivity of the arterial and venous mesenteric beds of the guinea-pig to bradykinin, neurokinins and other agents. 2. The vasoactive properties of three selective agonists for neurokinin receptors, namely [Sar9, Met (O2)11]SP (NK1), [beta-Ala8]NKA(4-10) (NK2) and [MePhe7]NKB (NK3), were evaluated on precontracted arterial and venous mesenteric vasculatures of the guinea-pig. The NK1-selective agonist, [Sar9,Met(O2)11]SP (1 to 1000 pmol), induced an endothelium-dependent and N omega-nitro-L-arginine methyl ester (L-NAME)-sensitive relaxation of the arterial vasculature precontracted with methoxamine, whereas the NK2 and NK3-selective agonists were virtually inactive at high doses (1000 pmol). 3. The three selective neurokinin receptor agonists were inactive in the non-precontracted arterial and venous mesenteric vasculatures as well as in the precontracted venous mesenteric vasculature. 4. Bradykinin (0.1 to 100 pmol) induced a marked dose- and endothelium-dependent vasodilatation of the precontracted arterial and venous vasculatures. ED50 values were 5.5 pmol on the arterial side and 1.9 pmol on the venous side. In contrast, desArg9-bradykinin was inactive at doses up to 1000 pmol. Furthermore, on the arterial and venous sides, a higher dose of bradykinin (1000 pmol), induced a biphasic effect, a transient constriction followed by a marked and sustained vasodilatation. The vasodilator effects of bradykinin were abolished by Hoe 140 (0.1 microM) and CHAPS, markedly reduced by L-NAME and were unaffected by [Leu8]desArg9-bradykinin (0.1 microM) on both sides of the mesenteric vasculature. Hoe 140 also abolished the arterial vasoconstrictions induced by high doses of bradykinin. 5. Noradrenaline, angiotensin II and endothelin-1 produced contractions on both sides of the mesenteric circulation, while acetylcholine (arterial side) and sodium nitroprusside (arterial and venous sides) caused vasodilatation.6. Our study supports the view that NK1 receptors responsible for vasodilatation are present solely in the endothelium of the arterial mesenteric vasculature of the guinea-pig. On the other hand, bradykinin(0.1 to 100 pmol) exerts predominantly vasodilator effects on both sides of the mesenteric vasculature via selective activation of B2 receptors located on the endothelium. The same receptor type located on the smooth muscle appears to be responsible for the arterial and venous constriction with high doses of bradykinin.

Animals↗
PubMed1995

Roles of endothelin receptors in the regional and systemic vascular responses to ET-1 in the anaesthetized ganglion-blocked rat: use of selective antagonists.

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

Anesthesia↗
PubMed1995

The contribution of tumour necrosis factor-alpha and endothelin-1 to the increase of coronary resistance in hearts from rats treated with endotoxin.

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

Animals↗