In the present study, we demonstrate that the intravenous infusion of endothelin-1 (3 and 10 ng/kg/min) causes a decrease in the mean micturition volume of rats in addition to an increase in mean arterial pressure. These effects are blocked by both the ETA/ETB-non-selective and the ETA-selective endothelin antagonists SB 217242 and SB 247083 respectively (both 30 mg/kg). However, it was also observed that the ETB-selective agonist sarafotoxin 6c (3 and 10 ng/kg/min) had similar effects on both mean arterial pressure and micturition volume. Initial experiments indicated that spontaneously hypertensive rats have a much lower mean micturition volume than normal rats. Binding studies comparing the total number and ratio of ETA/ETB receptors in spontaneously hypertensive, Wister-Kyoto and Sprague-Dawley rats revealed no significant differences in receptor expression. However, the magnitude of the response to endothelin-1 was greater in spontaneously hypertensive versus normal rats.

The role of angiotensin II (AII) and angiotensin IV (AIV) as inducers of PAI-1 expression during hypertension was studied in vivo. A 2-week infusion of AII (300 ng/kg/min) via an osmotic pump increased systolic blood pressure (171 ± 2 vs. 138 ± 6 mm Hg), urinary protein excretion (32 ± 6 vs. 14 ± 2 mg/day), and renal (2.2 ± 0.5 vs. 1.0 ± 0.1) and cardiac (1.8 ± 0.3 vs. 1.0 ± 0.1) gene expression of plasminogen activator inhibitor 1 (PAI-1). AIV infusion did not affect any of the above with the exception of PAI-1 gene expression which was increased in the left ventricles (1.7 ± 0.3 vs. 1.0 ± 0.1). AII-infused rats displayed a decreased creatinine clearance (538 ± 75 vs. 898 ± 96 ml/min) and hypertrophic left ventricles (0.275 ± 0.006 vs. 0.220 ± 0.011 g/100 g). Our results demonstrate that AII but not AIV infusion is associated with increased renal PAI-1 gene expression.

In the present study, the role of transforming growth factor-beta (TGFbeta) on the production of the extracellular matrix component, fibronectin, in the prostate has been studied. The mRNA levels of fibronectin, TGFbeta and the two TGFbeta receptors, ALK5 (activin like kinase) and type II, were measured using reverse-transcription polymerase chain reaction (RT-PCR). TGFbeta increased fibronectin mRNA and protein (7-fold) in a concentration-dependent fashion. An interesting relationship between the two TGFbeta receptors was found in that TGFbeta caused an upregulation of its type I receptor mRNA (5-6-fold) and a downregulation of the type II receptor mRNA (5-fold). Time-course experiments revealed that the change in expression of the TGFbeta receptors reached maximum at 24 h with an early increase at 4-5 h, whereas the fibronectin gene expression was not significantly stimulated until about 24 h. These data provide evidence that TGFbeta stimulates extracellular matrix production in prostate cells.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.

Endothelin-1 (ET-1) has been suggested to have a potential function as an inflammatory mediator. The study reported here assessed the putative inflammatory/nociceptive actions of the ET isopeptides using endothelin-B (ET(B))-receptor knockout (KO) mice and ET(A)- (SB 234551) and ET(B)- (A192621) selective antagonists. Phenylbenzoquinone (PBQ)-induced algesia was evident in the wild-type (WT) ET(B) (+/+) mice, attenuated by 80% in the heterozygous ET(B) (+/-) mice, and absent in the ET(B) (-/-) homozygotes. This was reproduced pharmacologically in WT ET(B) (+/+) mice where the algesic effect of PBQ was inhibited 74% by A192621, but unaffected by SB 234551 (both at 25 mg/kg p.o.). Similar observations were made in a model of cutaneous inflammation: ET(B) (+/+) mice had a marked inflammatory response to topical arachidonic acid, ET(B) (+/-) and ET(B) (-/-) mice had significantly reduced edema responses (37% and 65% inhibition). Neutrophil infiltration was reduced in the ET(B) (+/-) and ET(B) (-/-) mice (51% and 65% reduction, respectively). Topical administration of A192621 (500 microg/ear) inhibited arachidonic acid-induced swelling (39%) in WT ET(B) (+/+) mice. Collectively, these results support a role for the ET(B)-receptor in the mediation of inflammatory pain and cutaneous inflammatory responses. As such, the development of ET(B)-receptor-selective antagonists may be of therapeutic utility in the treatment of inflammatory disorders.

Adrenomedullin is a potent vasodilatory peptide that increases cAMP in a number of different systems including rat mesangial cells. Since mesangial cells play a significant role in glomerular matrix production, we evaluated the effects and molecular mechanisms of adrenomedullin action on hyaluronic acid release, an important extracellular matrix component. Adrenomedullin increased hyaluronic acid release in mesangial cells in a concentration-dependent manner. Forskolin, an adenylate cyclase activator, and dibutyryl-cAMP, a cell permeable cAMP analog, also increased hyaluronic acid release significantly. Adrenomedullin-stimulated hyaluronic acid release was inhibited by the adrenomedullin receptor antagonist, adrenomedullin-(22-52). Inhibition of protein kinase A with H89 [[N-[2-(( p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride)]], a potent protein kinase A inhibitor did not affect adrenomedullin-stimulated hyaluronic acid release; however, H89 [[N-[2-(( p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride]] inhibited forskolin- and dibutyryl-cAMP-induced hyaluronic acid production. In addition, SB203580 [[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-im idazole), a P38 mitogen-activated protein kinase (P38 MAPK) inhibitor attenuated adrenomedullin-, forskolin-, and dibutyryl-cAMP-stimulated hyaluronic acid release. Hyaluronic acid release induced by adrenomedullin, forskolin and dbcAMP was also inhibited by wortmannin [[1S-(1alpha, 6balpha, 9abeta, 11alpha, 11bbeta)]-11-(Acetyloxy)-1, 6b, 7, 8, 9a, 10, 11, 11b-octahydro-1-(methoxymethyl)-9a, 11b-dimethyl-3H-furo[4, 3, 2-de]indeno[4, 5-h]-2-benzopyran-3, 6, 9-trione]. We conclude that adrenomedullin, forskolin and dbcAMP cause an increase in hyaluronic acid release in rat mesangial cells through a pathway that involves activation of wortmannin-sensitive kinase and P38 MAPK. Although cAMP stimulation and protein kinase A activation can induce hyaluronic acid release. adrenomedullin-stimulated hyaluronic acid release appears to be independent of protein kinase A activation. These data provide the first demonstration of the involvement of P38 MAPK- and wortmannin-sensitive kinase pathways in the stimulation of hyaluronic acid production by rat mesangial cells.

The role of endothelin B (ET(B)) receptors in inflammation and nociception was examined using ET(B) receptor knockout mice. Genotyping studies were used with tissues from ET(B)((+/+)), ET(B)((+/-)), and ET(B)((-/-)) mice to confirm the loss of ET(B) receptors. Algesia induced by phenylbenzoquinone was evident in the (+/+) mice, reduced by approximately 80% in the (+/-) mice, and absent in the (-/-) mice. Phenylbenzoquinone-induced algesia in (+/+) mice was inhibited 74% by the ET(B) receptor-selective antagonist A192621 (25 mg/kg p.o.), but unaffected by the ET(A) receptor-selective antagonist SB 234551 (25 mg/kg p.o.). Noninflammatory pain, induced by hotplate, was equivalent between (+/+) and (-/-) mice. The cutaneous inflammatory response to topical arachidonic acid (AA) also was evaluated. Whereas (+/+) mice had a marked inflammatory response to AA, the (+/-), and (-/-) mice had significantly reduced fluid phase responses (37 and 65% inhibition, respectively). Neutrophil infiltration also was reduced in the (+/-) and (-/-) mice (51 and 65% reduction, respectively). Topical administration of A192621 (500 microg/ear) in (+/+) mice inhibited AA-induced swelling (39%), whereas SB 234551 (500 microg/ear) was without effect. Collectively, these results implicate the ET(B) receptor in mediation of inflammatory pain and cutaneous inflammatory responses in mice.

The effects of a nonpeptide, orally active mixed endothelin (ET) ETA/ETB receptor antagonist, SB 217242, and an angiotensin-converting enzyme (ACE) inhibitor, ramipril, were evaluated after inter-renal aortic banding in the rat. Separate sham, vehicle, and treatment groups were compared in each study. In vehicle-treated animals in the ramipril group, aortic banding for 4 weeks produced significant cardiac hypertrophy (247 +/- 5 mg/100 g bw vs. 305 +/- 11 mg/100 g bw; p < 0.001), right (upstream) renal hypertrophy (380 6 mg/100 g bw vs. 559 28 mg/100 g bw; p < 0.001), and significant left (downstream) renal atrophy (405 4 mg/100 g bw vs. 192 25 mg/100 g bw; p < 0.001). Continuous ramipril treatment (1 mg/kg p.o. once daily), begun 3 days before aortic banding, inhibited cardiac hypertrophy (305 11 mg/100 g bw vs. 266 7 mg/100 g bw; p < 0.05) but did not alter renal hypertrophy or atrophy. In a similarly designed study, SB 217242 (30 mg/kg p.o. b.i.d.) had no effect on the development of cardiac hypertrophy (298 7 mg/100 g bw vs. 310 12 mg/100 g bw) or renal hypertrophy (561 15 mg/100 g bw vs. 575 19 mg/100 g bw), but abolished the development of renal atrophy (158 16 mg/100 g bw vs. 395 19 mg/100 g bw; p < 0.001). [I-125]ET-1 radioligand binding experiments indicated that the density of both ETA and ETB receptors was increased dramatically (three- to fourfold) in the atrophic kidney cortex compared to sham or hypertrophic kidneys, In situ hybridization studies indicate an upregulation of ETB receptor mRNA in the glomeruli of atrophic kidneys within 5 days of aortic banding. In conclusion, an angiotensin-dependent mechanism may mediate cardiac hypertrophy associated with aortic banding, whereas ET-dependent mechanisms may mediate an atrophic response in the hypoperfused kidney, perhaps through an interaction with upregulated ETA and/or ETB receptors.

Infusion of endothelin-1 (ET-1) into conscious, chronically instrumented dogs (10 ng/kg.min i.v.) resulted in a significant increase in mean arterial pressure and significant reductions in renal plasma flow, glomerular filtration rate, and sodium excretion. Intravenous infusion of SB 209670 (30 micrograms/kg.min, i.v.) abolished these responses, whereas infusion of SB 234551 (30 micrograms/kg.min, i.v.) resulted in significant increases in renal plasma flow and urinary sodium excretion. These data indicate that SB 234551 can unmask ETB receptor-induced renal vasodilatation and inhibition of sodium reabsorption.

Structurally and pharmacologically diverse vasodilators are known to lower blood pressure, increase heart rate, and produce acute injury to right coronary arteries in the dog. Administration of low concentrations of endothelin-1 (ET-1) to anesthetized dogs causes coronary vasoconstriction and reductions in coronary blood flow. Therefore, pharmacologic blockade of endothelin receptors (ETA and ETB) with the mixed ET receptor antagonist SB 209670 could lead to coronary vasodilatation. In toxicology studies, continuous administration of SB 209670 to dogs for 5 days at 50 micrograms/kg/min was associated with minor but sustained increases in heart rate (10-30 beats/min), slight decreases in mean arterial pressure (10-15 mm Hg), and medial hemorrhage and necrosis of extramural coronary arteries in the right atria. Doses of 10 micrograms/kg/min had no effect. The lesions in the right atrium were associated with the highest density of ET receptors, approximately 470 fmol/mg compared to 170-200 fmol/mg in the ventricles and septum. Because changes in systemic cardiovascular parameters are minimal, the coronary arterial lesion is most likely due to a local vasodilatory effect in the coronary bed.

The data presented in this manuscript describe the binding characteristics of the ETA-selective antagonist, [125I]TTA 386 (hexamethyleneimino carbonyl-Leu-Tri-Ala-β-Ala-Tyr-Phe). This radioligand bound with high affinity and specificity to cloned human ETA receptors and rat mesenteric artery ETA receptors. The apparent dissociation constants (Kds) and maximum binding capacities were 1.0 nM and 8.5 pmol/mg for cloned human ETA receptors and 0.8 nM and 170 fmol/mg for rat mesenteric artery membranes respectively. Binding of [125I]TTA 386 was fast reaching equilibrium by 45 min and 15 min for human ETA and rat mesenteric artery membrane, respectively. Addition of excess unlabeled ligand resulted in the dissociation of bound radioligand from both preparations. Competition of [125I]TTA 386 binding by unlabeled ET-1, ET-3, TTA 386 and BQ123 revealed appropriate ETA pharmacology. This radioligand did not display any binding to cloned human ETB receptors.

This study demonstrates the presence of a novel endothelin (ET) receptor subtype that displays high affinity for both ETA- and ETB-selective ligands. This subtype has been identified in canine spleen membranes using ETB-selective agonists ET-3, IRL-1620, sarafotoxin 6c (S6c) as well as ETA-selective antagonists BQ123 and related cyclic pentapeptides. Binding of 125I-ET-3 to canine spleen membranes was specific and saturable with an apparent dissociation constant of 130 pM and maximum binding (Bmax) of 240.0 fmol/mg protein. Although the apparent affinities obtained with 125I-ET-1 and 125I-ET-3 were comparable (90 and 130 pM, respectively), the maximum binding obtained with 125I-ET-3 was approximately 35% of that obtained with 125I-ET-1, which indicates that canine spleen possesses both ETA and ETB receptors in the ratio 65:35. Competition binding experiments using 125I-ET-3 and unlabeled ET-1, ET-3, S6c, and IRL-1620 suggested that although ET-1 and ET-3 displayed similar high affinity, S6c and IRL-1620 were 20-300-fold weaker than ET-1 and ET-3 in competing for 125I-ET-3 binding to canine spleen membranes. In addition, BQ123, an ETA-selective antagonist, displaced 125I-ET-3 binding from canine spleen with an IC50 value of 30 nM. Similar profiles were obtained with related cyclic pentapeptides. Electrophysiological studies performed on Xenopus laevis oocytes injected with canine spleen poly(A)+ RNA indicated that the ETB receptor present in these tissues is functional and displays the same pharmacology as that observed in binding studies using these membranes. As a comparison, both binding and functional studies were performed in canine lung and the data indicate that the ETB receptor present in this tissue is similar to that of the cloned human ETB receptor but different from that present in canine spleen. These observations were further confirmed by performing cross-linking experiments on these membranes. Although canine lung and cloned human ETB receptors displayed the same molecular weight bands with similar pharmacology, canine spleen ETB receptors displayed different molecular weight bands and different pharmacology. In addition, the ETB receptors present in canine spleen were also identified in canine bladder, monkey spleen and human spleen. Thus, the data presented in this manuscript provide evidence for the presence of a novel ETB receptor in different tissues as well as different species including human.

Endothelin receptors are widely distributed throughout a number of tissues. A novel ETB receptor splice variant (ETB-SVR) was identified from a human placental cDNA library. Sequence analysis indicated that the ETB-SVR is 436 amino acids long and shares 91% identity to the human ETB-R. Northern blot analysis indicated an mRNA species of 2.7 kilobases, which is expressed in the lung, placenta, kidney, and skeletal muscle. Ligand binding studies of the cloned ETB-SVR and ETB-R receptors expressed in COS cells showed that ET peptides exhibited similar potency in displacing 125I-ET-1 binding. Functional studies showed that ET-1, ET-3, and sarafotoxin 6c displayed similar potencies for inositol phosphates accumulation in ETB-R-transfected COS cells, whereas no increase in inositol phosphate accumulation was observed in ETB-SVR-transfected cells. In addition, exposure of ETB-R-transfected cells to ET-1 caused an increase in the intracellular acidification rate whereas ETB-SVR-transfected cells did not respond to ET-1. These data suggest that the ETB-SVR and ETB-R are functionally distinct and the difference in the amino acid sequences between the two receptors may determine functional coupling. Availability of cDNA clones for endothelin receptors can facilitate our understanding of the role of ET in the pathophysiology of various diseases.

The data presented in this manuscript describes the binding characteristics of [3H]SB 209670, a potent nonpeptide tritium-labeled endothelin (ET) receptor antagonist. The binding of this antagonist to cloned human ETA and ETB receptors was specific, saturable and of high affinity. The apparent dissociation constants were 0.20 and 1.0 nM for ETA and ETB receptors, respectively. The maximum binding was 4.7 and 22.5 pmol/mg protein for ETA and ETB receptors, respectively. Unlike [125]ET-1, the binding of [3H]SB 209670 was reversible. The half-times (T1/2) for dissociation of this ligand from ETA and ETB receptors were approximately 60 and 10 min, respectively. Competition binding studies using [3H]SB 209670 and unlabeled agonists ET-1, ET-3 and S6c indicated that these agonists displayed similar affinities for human ETB receptors, whereas with ETA receptors, ET-1 was approximately 50-fold and 1500-fold more potent than ET-3 and S6c, respectively. Of the peptide antagonists tested, BQ123 (ETA-selective peptide antagonist), displayed Ki values of 40 and > 2300 nM for ETA and ETB, whereas RES701 (ETB-selective antagonist) displayed Ki values of > 1600 and 81 nM for ETA and ETB receptors, respectively. The nonselective peptide antagonist, PD 142893, was approximately 2-fold more potent for ETA compared with ETB receptors. Similar observations were made with nonselective nonpeptide antagonists, Bosentan, (+/-) SB 209670, SB 209670, and (-) SB 209670. All these compounds were 2 to 10 times more potent for ETA than ETB receptors.