Biomedical subjects

T J Opgenorth

Publications and source records attributed to T J Opgenorth.

At least 19 recordsLinked to original sources

PubMed1995

Characterisation of functional endothelin receptors in the canine isolated perfused spleen.

The endothelin receptor subtypes involved in the vasoconstriction, capsular smooth muscle contraction, prostaglandin E2 and prostacyclin release induced by endothelin-1 have been investigated in the canine isolated perfused spleen using both the endothelin ETA receptor antagonist FR 139317 and the endothelin ETB receptor agonist IRL 1620. THe isolated canine spleen was perfused with warmed (37 degrees C) and oxygenated (95% O2/5% CO2) Krebs solution at constant flow with continuous recording of splenic arterial perfusion pressure and spleen weight. Samples of splenic venous effluent were collected to determine the amounts of prostaglandin E2 and prostacyclin, measured by radioimmunoassay. Endothelin-1 (4-200 pmol) and IRL 1620 (20-1000 pmol) dose-dependently increased splenic arterial perfusion pressure but the former was more potent on a molar basis (the molar dose ratio IRL/endothelin-1 required to increase splenic arterial vascular resistance by 25% was approximately 33). The infusion of the nitric oxide inhibitor N omega-nitro-L-arginine methyl ester (10 microM), but not of the enantiomer N omega-nitro-D-arginine methyl ester, significantly potentiated the increase in splenic arterial vascular resistance induced by endothelin-1. The infusion of FR 139317 (1 microM) markedly attenuated the increased splenic arterial perfusion pressure induced by endothelin-1 without affecting that evoked by IRL 1620. At the highest dose (200 pmol), endothelin-1 induced a small but significant capsule contraction as reflected by the reduction in the spleen weight. The infusion of FR 139317 (1 microM) abolished this contractile effect. IRL 1620 (in doses up to 1000 pmol) did not significantly affect the capsule tone.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗

PubMed1995

Endothelin receptor antagonism.

Following the original report by Yanagisawa et al. (1988) more than 7 years ago, compelling evidence that ET plays an important role in the local regulation of smooth muscle tone and cell growth has been reported. In addition, many studies point to a significant role for endothelin in nonvascular function. The investigation of the endothelin system has been greatly advanced in the last 2 to 3 years through significant advances in the development of potent and selective ET receptor antagonists. These agents have proven to be essential tools for elucidating the biological significance of the ET system, leading to the realization that antagonism of the ET system may have significant therapeutic potential. As emphasized in this review, the importance of chronic blockade of the ET system may be a critical aspect of future research in this exciting area. Confounding issues remain the lack of information about the role of the ETB receptor, the apparent pharmacological evidence for additional ET receptor subtypes, and species variation in the tissue distribution of ET isoforms and receptor subtypes. Along with the greater ability to understand the endothelin system provided by potent and selective pharmacological agents, is the important contribution of modern molecular biology techniques, highlighted by the insights gained from recent reports of results from ET gene disruption studies. Kurihara et al. (1994) found that ET-1-deficient homozygous mice die at birth of apparent respiratory failure secondary to severe craniofacial abnormalities. Subsequently, Yanagisawa's laboratory has presented and published a series of complementary gene disruption studies. First, Hosoda et al. (1994) demonstrated remarkably, that ETA receptor knockout mice bear morphological abnormalities nearly identical to ET-1 knockout mice. Second, they found that disruption of the ET-3 peptide and ETB receptor genes result in homozygous mice that share identical phenotypic traits (i.e., coloration changes and aganglionic megacolon) which are similar to a previously known natural mutation, the Piebald-Lethal mouse (Hosoda et al., 1994; Baynash et al., 1994). This phenotype has a human corollary known as Hirschsprung's Disease and it is now known that the disease, though multigenic, results from a missense mutation of the ETB receptor gene in some individuals (Puffenberger et al., 1994). Taken together these data indicate that the endothelin system is essential to correct embryonic neural crest development, a completely novel finding within the superfamily of guanine-protein-linked receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Systemic hemodynamics and renal function during long-term pathophysiological increases in circulating endothelin.

Although recent studies have reported endogenous plasma endothelin levels to be elevated two- to fivefold in chronic pathophysiological states, whether such an increase in circulating endothelin levels alone can lead to significant long-term alterations in cardiovascular and renal function is not known. The purpose of this study was to examine the long-term systemic hemodynamic and renal effects of a pathophysiological increase in plasma endothelin concentration in chronically instrumented, conscious dogs (n = 7). Infusion of endothelin-1 (2.5 ng.kg-1.min-1) for 8 days increased plasma concentration of immunoreactive endothelin approximately two- to threefold from 6.7 +/- 0.4 to 16.0 +/- 2.2 pg/ml. Mean arterial pressure increased 21% from a control value of 86.7 +/- 2.1 to 105.0 +/- 2.5 mmHg during the endothelin infusion period. Cardiac output averaged 2,200 +/- 205 ml/min during control and fell by 33% on day 4 of endothelin infusion (1,484 +/- 146 ml/min) and was still 14% below control after day 8 of endothelin infusion (1,885 +/- 154 ml/min). Endothelin increased total peripheral resistance from 42.0 +/- 3.1 to 80.3 +/- 9.1 mmHg.l-1.min. Increasing plasma endothelin two- to threefold was associated with an increase in renal vascular resistance and decreases in glomerular filtration rate and renal plasma flow. Endothelin-1 had no long-term effect on plasma renin activity or aldosterone concentration. These data indicate the importance of pathophysiological levels of endothelin in controlling renal and cardiovascular function in chronic conditions. Furthermore, the results indicate that endothelin may play a role as a mediator of chronic hypertension in pathophysiological states associated with endothelial dysfunction.

Animals↗

PubMed1995

Chronic endothelin-induced pressor and renal actions in conscious dogs do not require altered ANG II formation.

Plasma endothelin levels are elevated approximately two- to threefold in a number of chronic pathophysiological conditions associated with hypertension. Results from recent studies indicate an important interaction between endothelin and the renin-angiotensin system (RAS). The role of the RAS in mediating the increases in arterial pressure produced by long-term pathophysiological elevations in circulating levels of endothelin is unknown. Therefore, the purpose of this study was to chronically increase circulating levels of endothelin within the pathophysiological range and determine the long-term cardiovascular and renal actions of endothelin in control dogs (n = 6) and in dogs pretreated with a converting-enzyme inhibitor (CEI) (n = 6) or CEI + angiotensin II (ANG II) replacement (n = 6). Infusion of endothelin-1 for 8 days at a rate of 2.5 ng.kg-1.min-1 increased plasma endothelin from 7.1 +/- 0.9 to 19.8 +/- 3.3 pg/ml. In control dogs, endothelin increased mean arterial pressure (MAP) by 19% (90 +/- 2 to 107 +/- 3 mmHg) while decreasing renal blood flow (RBF) by 30% and glomerular filtration rate (GFR) by 15-20%. Long-term elevation of circulating endothelin produced similar elevations in MAP in dogs pretreated with CEI (+16%) or CEI + ANG II (+17%). Similar decreases in RBF and GFR also occurred in response to endothelin in all three groups. These results indicate that although long-term increases in circulating endothelin within the pathophysiological range produce significant increases in arterial pressure, this effect does not appear to be mediated by the RAS.

Angiotensin II↗

PubMed1995

Endothelin receptor in human astrocytoma U373MG cells: binding, dissociation, receptor internalization.

Endothelin (ET) receptor in human astrocytoma U373MG cells was characterized. ET-1, ET-3, sarafotoxin S6C, IRL1620, BQ788, Ro46-2005 and PD142893 inhibited specific [125I]ET-1 binding with Ki values of 0.03 0.06, 0.74, 5.01, 4.45, 2275 and 157 nM, respectively. ETA selective antagonists BQ123 and FR139317 at 1 microM did not block [125I]ET-1 binding. Reverse transcription-polymerase chain reaction confirmed the results from competition studies that U373 cells expressed predominantly ETB receptor. The Bmax and KD values of [125I]ET-1 binding were 0.15 pmol/1 x 10(6) cells and 0.23 nM. The molecular mass for the receptor was 45 kDa. ET-1 binding did not stimulate Ca+2 mobilization, phosphatidylinositol hydrolysis or arachidonic acid release, nor did it affect the intracellular cAMP or cGMP level. Interestingly, a majority of ET (> 80%) bound to the receptor was rapidly internalized, consistent with emerging evidence that a major function of ETB receptor is to clear ET. [125I]ET-1 binding was time-dependent and bound [125I]ET-1 was difficult to dissociate. In contrast, bound antagonists were much easier to dissociate. The results suggest that agonists and antagonists of the ET receptor exhibited different dissociation characteristics, with antagonist binding more reversible than agonist binding.

Astrocytoma↗

PubMed1994

Endothelin receptor agonists and antagonists exhibit different dissociation characteristics.

Endothelins (ETs) are vasoconstricting peptides that bind to membrane receptors to initiate their physiological effects. This report compares the dissociation characteristics of selected ET agonists and antagonists, and studies the effects of any difference in dissociation characteristics on the potency of antagonists. Competition studies using various ET receptor ligands against [125I]ET-1 or [125I]ET-3 binding demonstrated that porcine cerebellum membranes contain predominantly ETB receptor. [125I]IRL1620 associated with the receptors in a time-dependent manner. Although bound [125I]IRL1620 was easier to dissociate than bound [125I]ET-3, both agonists exhibited a dissociation half life > 20 h. For non-radiolabeled ligands, bind-and-wash studies were employed in which membranes were pre-incubated with unlabeled ligand followed by extensive washing before assaying for [125I]ET-1 binding. Results from bind-and-wash studies confirmed that bound non-radiolabeled IRL1620 and ET were as difficult to dissociate as [125I]ligands. In contrast, bound PD142893 and Ro46-2005 were easily dissociated from ETB receptors. Consequently, the inhibitory effects of PD142893 and Ro46-2005 on [125I]agonist binding diminished following incubation time. In cloned human ETA and ETB receptors, bound ET-1 was also more difficult to dissociate than bound antagonists. These results suggest that the differences in the dissociation characteristics of ET receptor agonists vs. antagonists may account for the diminished potency of Ro46-2005 and PD142893 as a function of incubation time.

Amino Acid Sequence↗

PubMed1994

[3H]A-81988, a potent, selective, competitive antagonist radioligand for angiotensin AT1 receptors.

Abbott-81988 (A-81988), 2-(N-n-Propyl-N-[(2'-[1H-tetrazol-5-yl]biphenyl-4- yl)methyl]amino)pyridine-3-carboxylic acid is a potent, competitive, non-peptidic antagonist of angiotensin AT1 receptors. A-81988 was labeled with tritium to high specific activity (16 Ci/mmol) and radioligand binding assays performed in rat liver membranes. [3H]A-81988 bound with high affinity (KD = 0.57 nM) and the KD determined from kinetics assays was similar. Non-specific binding (defined with 10(-6) M angiotensin-II) was very low (< 6% at the KD). The binding of [3H]A-81988 was competitive and exhibited appropriate pharmacological specificity for compounds acting at angiotensin AT1 receptors. These properties demonstrate that [3H]A-81988 will be a useful radioligand for studies of angiotensin AT1 receptors in various tissues.

Angiotensin II↗

PubMed1994

Endothelin receptor antagonists exhibit diminishing potency following incubation with agonist.

Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. ET binding to receptors has been shown to be almost irreversible because bound ET is difficult to dissociate. This report studies the dissociation characteristics of receptor antagonists and further examines the effects of ET's difficult-to-dissociate binding on the potency of antagonists. In membranes prepared from porcine cerebellum, [125I]ET-1 binding was effectively blocked by ET-1 and ET-3 with similar IC50 values (0.08 nM vs. 0.17 nM), suggesting that porcine cerebellum contains predominantly the ETB receptor subtype. [125I]ET-3 binding was inhibited by Ro46-2005 and PD142893, two non-selective antagonists, with IC50 values of 570 +/- 50 nM and 410 +/- 100 nM, respectively. Consistent with previous observations, bound [125I]ET-1 in porcine cerebellum membranes was also difficult to dissociate. In contrast, bound Ro46-2005 or PD142893, but not bound ET-1, could be readily washed away from membranes, suggesting that antagonist binding was more reversible than ET-1 binding. Although Ro46-2005 or PD142893 at 0.5 microM inhibited 0.1 nM [125I]ET-1 binding by > 80% after 15 min of incubation, the inhibitory effect decreased to approximately 50% after 3 h of incubation, and further decreased to < 10% at 24 h. This decrease in antagonizing potency was further confirmed by the results that the IC50 values of the two antagonists against [125I]ET-3 binding increased with increasing incubation time. Control experiments indicate that the observed decrease in the potency of Ro46-2005 and PD142893 was not the result of ligand degradation. These results suggest that the potency of antagonists is critically dependent on the incubation time because antagonist binding is more reversible than ET binding.

Amino Acid Sequence↗

PubMed1994

Cardiovascular effects of orally administered ABBOTT-81988, an angiotensin II antagonist, in conscious spontaneously hypertensive rats.

ABBOTT-81988 (A-81988), 2-(N-propyl-N[(2'-[1H-tetrazol-5-yl]biphenyl- 4yl)methyl] amino) pyridine-3-carboxylic acid, a nonpeptide angiotensin II (AII) antagonist was studied in the conscious spontaneously hypertensive rate (SHR) (male, 18 to 21 weeks) for cardiovascular effects of oral administration. Oral A-81988 at 0.3 to 3 mg/kg produced a dose-related 10 to 29% decrease in mean arterial pressure (MAP) in SHR (control, 161 to 177 mm Hg; n = 19) for 12 to 24 h without changing heart rate. Oral A-81988 at 3 mg/kg daily maintained MAP in SHR at normotensive levels (97 to 120 mm Hg) during a 5-day protocol with no rebound hypertension at termination of treatment. There was an increase in plasma renin activity in nanograms AI/milliliter/hour in SHR treated with A-81988 (32 +/- 3, n = 6 v 5 +/- 2, n = 6 for vehicle) during its antihypertensive action. The oral potency of A-81988 was enhanced about 10-fold in furosemide-treated SHR. The pressor response to AII was inhibited selectively in SHR even after an 8-day treatment with A-81988 (approximately 3 mg/kg/day orally). Total peripheral resistance was lowered and cardiac output unchanged in SHR administered A-81988 (3 mg/kg/day orally for 2 days). A-81988 (3 mg/kg orally) did not cause orthostatic hypotension in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

PubMed1994

ETA receptor-mediated responses to endothelin-1 and big endothelin-1 in the rat kidney.

1. Renal clearance experiments were conducted in anaesthetized Sprague-Dawley rats to determine the effect of the ETA receptor antagonist, BQ-123, on the renal haemodynamic response to endothelin-1 (ET-1) and its precursor, big endothelin-1 (big ET-1) at doses that produce an equivalent degree of renal vasoconstriction. 2. Infusion of either big ET-1 at 100 pmol kg-1 min-1 or ET-1 at 12 pmol kg-1 min-1 for 60 min produced almost identical decreases in renal blood flow (RBF) and glomerular filtration rate (GFR). Big ET-1 produced an increase in mean arterial pressure (MAP) that was significantly larger than the increase produced by ET-1. 3. Co-infusion with BQ-123 (0.1 mg kg-1 min-1) prevented the rise in MAP produced by big ET-1 and completely blocked the renal response. Similarly, BQ-123 inhibited both the increase in MAP and the decrease in RPF and GFR produced by ET-1. 4. Big ET-1 but not ET-1, produced a significant increase in water and sodium excretion. BQ-123 had no effect on the diuretic and natriuretic response to big ET-1 consistent with possible ETB-mediated inhibition of tubular reabsorption. 5. We have previously shown that at higher doses of ET-1, BQ-123 was unable to inhibit the renal vasoconstrictor response despite blockade of the pressor response. Taken together, these results indicate that ET-1 activates primarily ETA receptors at moderately low doses to produce renal vasoconstriction while higher doses also involve non-ETA receptors. 6. Since ET-l has a similar binding affinity for both ETA and ETB receptors, we suggest that there maybe a third type of ET-1 receptor in the rat kidney with lower affinity for ET-1 that is coupled to a vasoconstrictor mechanism.

Amino Acid Sequence↗

PubMed1994

Endothelin receptors in human smooth muscle cells: antagonist potency differs on agonist-evoked responses.

The present study characterized endothelin (ET) receptors in human pericardium smooth muscle cells (HPSMC) and examined the potency of antagonists on ET-evoked signal transduction and DNA synthesis. HPSMC contain both ETA and ETB receptors. ET-1 binding was completely inhibited by a nonselective antagonist (Ro-46-2005) or a combination of ETA-selective and ETB-selective ligands (BQ-123 and ET-3). The molecular masses for ETA and ETB receptors were 69 and 42 kDa, respectively. ET-1, but not ET-3, stimulated phosphatidylinositol hydrolysis and arachidonic acid release in a dose- and time-dependent manner, reaching a plateau within 20-40 min. These immediate effects of ET-1 on signal transduction were completely inhibited by 1 microM, BQ-123, ET-1, but not ET-3, stimulated DNA synthesis in a dose-dependent manner, and the effect became prominent after 24 h. BQ-123 (1 microM) or Ro-46-2005 (10 microM) did not completely inhibit this mitogenic effect of ET-1. The reduced potency of BQ-123 and Ro-46-2005 on the delayed effect of ET-1 was not the result of ligand degradation or a difference in receptor internalization; rather, the decrease in potency was due to the fact that antagonist binding was more reversible than ET-1 binding.

Binding Sites↗

PubMed1994

Atrial natriuretic peptide and glomerular hyperfiltration during onset of spontaneous diabetes mellitus.

The mechanisms responsible for the elevation of glomerular filtration rate (GFR) in early stages of insulin-dependent diabetes mellitus (IDDM) are undefined. The objectives of this study were to define the temporal pattern of onset of glomerular hyperfiltration in the spontaneously diabetes-prone (BB/DP) rat and to evaluate the possible role of atrial natriuretic peptide (ANP) as the primary mediator of the observed alterations in renal hemodynamics. GFR was significantly higher (1.38 +/- 0.07 ml.min-1 x g-1; n = 5) in moderately hyperglycemic BB/DP rats (blood glucose > 270 mg/dl) 14 days after the onset of IDDM compared with age-matched diabetes-resistant rats (BB/DR), which averaged 1.03 +/- 0.07 ml.min-1 x g-1 (n = 7). Circulating ANP levels in moderately hyperglycemic BB/DP rats 1, 7, and 14 days after onset of IDDM were within the normal range, averaging 100 +/- 21, 57 +/- 12, and 65 +/- 6 pg/ml, respectively, and were not significantly different (P > 0.05) from ANP levels in age-matched normoglycemic BB/DR rats. To further test the role of ANP in glomerular hyperfiltration, an ANP receptor antagonist was infused into anesthetized BB/DP rats (n = 10) 14 days after onset of IDDM, after baseline measurements of mean arterial pressure, renal hemodynamics, and renal fluid and electrolyte excretions. ANP receptor antagonism caused a significant reduction in mean arterial pressure from 120 +/- 3 to 103 +/- 2 mmHg; however, there were no significant effects of ANP receptor blockade on GFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Endothelin ETA receptor antagonist reduces myocardial infarction induced by coronary artery occlusion and reperfusion in the rat.

The effect of the endothelin ETA receptor antagonist FR 139317 on myocardial infarction was studied in the rat. Under anesthesia, rats were subjected to 30 min of left main coronary artery occlusion and 3 h of reperfusion. FR 139317 (15, 35 and 70 mg/kg total dose) was continuously infused i.v. starting approximately 30 min before coronary artery occlusion and continuing throughout occlusion and reperfusion. The area at risk (AAR), determined using phthalocyanine dye, was in the range of 48-63% of the left ventricle (LV). The infarct zone (IZ) was evaluated by tetrazolium staining defect and its size was calculated as a percent of AAR. The IZ/AAR (%) was significantly reduced in rats treated with FR 139137 (15 mg/kg: 20 +/- 4%, n = 6; 35 mg/kg: 24 +/- 2%, n = 6, and 70 mg/kg: 26 +/- 4%, n = 8) compared to the vehicle group (36 +/- 2%, n = 22) (p < 0.05). When rats were treated beginning just prior to reperfusion, FR 139317 (35 mg/kg) also produced a significant reduction in infarct size (IZ/AAR: 22 +/- 1% for FR 139317, n = 6 vs. 39 +/- 6% for vehicle, n = 6, p < 0.05). These data suggest an important role for the ETA receptor-mediated effects of ET in the pathophysiology of myocardial infarction. ETA receptor antagonism may provide a novel therapeutic approach for cardioprotection in myocardial infarction.

Animals↗

PubMed1994

Characterization of antihypertensive activity of ABBOTT-81988, a nonpeptide angiotensin II antagonist in the renal hypertensive rat.

2-(N-Propyl-N[(2'-[1H-tetrazol-5-yl]biphenyl-4yl)methyl]amin o) pyridine-3-carboxylic acid (ABBOTT-81988), a novel nonpeptide angiotensin II (AII) antagonist, was evaluated to characterize its antihypertensive activity in the conscious renal hypertensive rat. Oral or i.v. administration of ABBOTT-81988 at 0.03 to 0.3 mg/kg produced a dose-dependent, sustained decrease in mean arterial pressure (MAP; control 162-173 mm Hg, n = 27) of approximately 20 to 70 mm Hg. At a dose of 0.3 mg/kg p.o., ABBOTT-81988 lowered MAP to a normotensive level for more than 24 hr and did not change heart rate. During its antihypertensive effect (delta MAP, -28% approximately -35%), ABBOTT-81988 (0.1-03 mg/kg i.v.) decreased total peripheral resistance (delta resistance, -31% approximately -43%), and cardiac output remained either unchanged or slightly elevated. ABBOTT-81988 (0.3 mg/kg i.v.) produced an additional antihypertensive effect (delta MAP, -12 +/- 2%, n = 5) in captopril-pretreated (10 mg/kg i.v.) hypertensive rats, but captopril (10 mg/kg i.v.) had no effect in ABBOTT-81988-pretreated (0.3 mg/kg i.v.) rats. In the normotensive rat, ABBOTT-81988 (0.3 mg/kg p.o.) had no effect on basal MAP, but it inhibited the AII-induced (0.1 microgram/kg i.v.) pressor response by 51% to 91% for 24 hr, whereas the responses to norepinephrine (0.3 microgram/kg i.v.), vasopressin (0.03 IU/kg i.v.) and bradykinin (3 micrograms/kg i.v.) were not affected. It is concluded that ABBOTT-81988 is a safe and efficacious AII antagonist that may have use in the treatment of human hypertension.

Angiotensin II↗

PubMed1993

Potent and selective inhibitors of an aspartyl protease-like endothelin converting enzyme identified in rat lung.

Two structurally distinct series of potent and selective inhibitors of an aspartyl protease-like endothelin converting enzyme (ECE) activity identified in the rat lung have been developed. Pepstatin A, which potently inhibits the rat lung ECE, served as the basis for the first series. Alternatively, selected renin inhibitors containing the dihydroxyethylene moiety were shown to be inhibitors of rat lung activity. Subsequent modifications improved inhibition of the rat lung ECE while eliminating renin activity. Both series of ECE inhibitors demonstrated a range of selectivity over Cathepsin D. Water-solubilizing moieties were appended onto selected compounds to facilitate in vivo testing. Partial reduction of the pressor response to exogenously administered Big ET-1 was observed with selected rat lung ECE inhibitors.

Amino Acid Sequence↗

PubMed1993

In vivo characterization of a phosphoramidon-sensitive endothelin-converting enzyme in the rat.

Experiments were conducted to characterize the nature of a phosphoramidon-sensitive endothelin-converting enzyme in vivo by evaluating the pressor response to a bolus intravenous (i.v.) injection of endothelin family peptides following administration of phosphoramidon in anesthetized Sprague-Dawley rats. Phosphoramidon given i.v. at 10 mg/kg completely prevented the pressor response to human big endothelin-1-(1-38) (big ET-1). The EC50 for phosphoramidon was determined to be in the range of 1 to 3 mg/kg. The pressor response to big ET-1 60 min after phosphoramidon injection was attenuated by roughly 60% indicating a long inhibitory half-life. Very high doses of big ET-1 (> 20 mg/kg) were capable of over-riding the effect of phosphoramidon and produced characteristic pressor responses suggesting that the inhibition by phosphoramidon can be considered competitive in nature. Human big endothelin-3-(1-41) (big ET-3) produced significant increases in arterial pressure although with less potency and efficacy compared to big ET-1. The pressor response to big ET-3 was also inhibited by phosphoramidon. Phosphoramidon does not act indirectly by interfering with ET-1 receptor-mediated actions since the inhibitor has no effect on the in vivo pressor response to ET-1 and does not antagonize [125I]ET-1 receptor binding or constrictor responses in vitro. These results are consistent with the idea that a phosphoramidon-sensitive endothelin-converting enzyme is capable of cleaving both big ET-1 and big ET-3 to the active peptides in the rat.

Animals↗

PubMed1993

Prolonged treatment by phosphoramidon modulates the number of endothelin receptors in cultured Swiss 3T3 fibroblasts.

Endothelin (ET) is generated from prepro-ET initially by dibasic pair proteolysis, followed by a specific proteolytic cleavage between Trp21 and Val22. Currently, intense research is focused on the investigation of a metalloprotease that is inhibited by phosphoramidon (PHOS) only, but not by other protease inhibitors. In this report, we show that ET binding was increased significantly in cultured Swiss 3T3 fibroblasts with PHOS pretreatment. The effect of PHOS was dose- and time-dependent. Other protease inhibitors, such as thiorphan, pepstatin-A, E-64, phenylmethyl sulfonyl fluoride, and aprotinin, failed to exert a similar effect. Control experiments indicated that the effect of PHOS was not due to inhibition of 125I-ET-1 degradation. Binding studies using whole cells or membranes prepared from cell show that ET binding sites increased from 23,000 to 133,000 sites/cell in control versus PHOS-treated cells. The effect of PHOS treatment on the ET receptor may be due to the inhibition of a protease responsible for ET-receptor processing. This effect is likely to complicate interpretation of results from studies using PHOS to block the putative ET-converting enzyme.

3T3 Cells↗

PubMed1993

Stimulation of endogenous endothelin release in the anesthetized rat.

Although many agents have been shown to stimulate release of endothelin (ET) in vitro, the factors regulating and affecting production and release of ET in intact animals remain obscure. Experiments were conducted to determine the effect of surgical preparation and infusion of plasma and/or saline on circulating immunoreactive (ir)-ET concentrations and to characterize the mechanism of endotoxin-induced increases in plasma ir-ET. Male Sprague-Dawley rats (210-325 g) anesthetized with Inactin were placed on a surgical heating table for a period of about 2 h before blood was collected via puncture of the abdominal aorta. The plasma concentration of ir-ET was 8.1 +/- 0.9 pg/ml in rats that did not undergo surgical preparation or receive any further treatment. Standard surgical preparation, as for renal clearance experiments (catheters in the trachea, femoral artery, femoral vein, and bladder), resulted in a dramatic rise in ir-ET to 17.7 +/- 3.0 pg/ml (p < 0.05). Infusion of plasma from donor rats (5 ml/kg over 20 min) resulted in an additional significant increase to 32.6 +/- 3.5 pg/ml (p < 0.05). In contrast, infusion of saline (0.9% NaCl) in a similar manner produced no further increase in ir-ET levels (23.2 +/- 3.2 pg/ml). Infusion of endotoxin in anesthetized, surgically prepared rats significantly increased ir-ET. This increase was not blocked by a platelet-activating factor antagonist (Esai 6123), despite blockade of endotoxin-induced hypotension. Phosphoramidon, which has been shown to block in vivo conversion of exogenous big ET to ET, was unable to prevent endotoxin-induced increases in ir-ET.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia↗