Endothelin action in rat liver. Receptors, free Ca2+ oscillations, and activation of glycogenolysis.

High affinity binding sites for endothelin (ET) were identified on rat liver plasma membranes. Binding of 125I-ET-1 with its site was specific, saturable, and time dependent (kobs = 0.019 +/- 0.001 min-1), but dissociation of receptor-bound ligand was minimal. A single class of high affinity binding sites for 125I-ET-1 was identified with an apparent Kd of 32.4 +/- 9.8 pM and a Bmax of 1084 +/- 118 fmol/mg protein. ET-3 and big-ET-1 (1-38) (human) inhibited 125I-ET-1 binding with IC50 values of 1.85 +/- 1.03 nM and 43 +/- 6 nM, respectively. Aequorin measurements of cytosolic free Ca2+ in single, isolated rat hepatocytes showed that ET-1 at subnanomolar concentrations induced a series of repetitive, sustained Ca2+ transients. ET-1 had no effect on cAMP production. Finally, ET-1 caused a rapid and sustained stimulation of glycogenolysis in rat hepatocytes. A 1.8-fold maximal increase in glycogen phosphorylase alpha was observed at 1 pM ET-1, with an EC50 of 0.03 pM. Stimulation of the enzyme was specific for ET-1 since the order of potency of related peptides was similar to that in binding experiments (ET-1 greater than ET-3 greater than big ET-1). These data constitute the first demonstration of the presence of ET-1 binding sites in liver which is associated with a rise in cytosolic free Ca2+ and a potent glycogenolytic effect. We conclude that ET-1 behaves as a typical Ca2+ mobilizing hormone in liver.

Biochemical and pharmacological properties of SR 49059, a new, potent, nonpeptide antagonist of rat and human vasopressin V1a receptors.

SR 49059, a new potent and selective orally active, nonpeptide vasopressin (AVP) antagonist has been characterized in several in vitro and in vivo models. SR 49059 showed high affinity for V1a receptors from rat liver (Ki = 1.6 +/- 0.2) and human platelets, adrenals, and myometrium (Ki ranging from 1.1 to 6.3 nM). The previously described nonpeptide V1 antagonist, OPC-21268, was almost inactive in human tissues at concentrations up to 100 microM. SR 49059 exhibited much lower affinity (two orders of magnitude or more) for AVP V2 (bovine and human), V1b (human), and oxytocin (rat and human) receptors and had no measurable affinity for a great number of other receptors. In vitro, AVP-induced contraction of rat caudal artery was competitively antagonized by SR 49059 (pA2 = 9.42). Furthermore, SR 49059 inhibited AVP-induced human platelet aggregation with an IC50 value of 3.7 +/- 0.4 nM, while OPC-21268 was inactive up to 20 microM. In vivo, SR 49059 inhibited the pressor response to exogenous AVP in pithed rats (intravenous) and in conscious normotensive rats (intravenous and per os) with a long duration of action (> 8 h at 10 mg/kg p.o). In all the biological assays used, SR 49059 was devoid of any intrinsic agonistic activity. Thus, SR 49059 is the most potent and selective nonpeptide AVP V1a antagonist described so far, with marked affinity, selectivity, and efficacy toward both animal and human receptors. With this original profile, SR 49059 constitutes a powerful tool for exploring the therapeutical usefulness of a selective V1a antagonist.

Growth inhibitory properties of endothelin-1 in activated human hepatic stellate cells: a cyclic adenosine monophosphate-mediated pathway. Inhibition of both extracellular signal-regulated kinase and c-Jun kinase and upregulation of endothelin B receptors.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate, and synthetize fibrosis components. We have shown that endothelin-1 (ET-1) inhibits the proliferation of activated human HSC via endothelin B (ETB) receptors. We now investigate the transduction pathway involved in the growth inhibitory effect of ET-1 in activated HSC. Endothelin-1 and the ETB receptor agonist, sarafotoxin-S6C, increased synthesis of PGI2 and PGE2, leading to elevation of cAMP. The cyclooxygenase inhibitor ibuprofen and the adenylyl cyclase inhibitor SQ22536 both blunted the growth inhibitory effect of ET-1. Analysis of early steps associated with growth inhibition indicated that: (a) similar to ET-1, forskolin decreased c-jun mRNA induction without affecting c-fos and krox 24 mRNA expression; (b) ET-1, sarafotoxin-S6C, as well as forskolin, reduced activation of both c-Jun kinase and extracellular signal-regulated kinase. Finally, forskolin, PGI2, and PGE2 raised by fivefold the number of ET binding sites after 6 h, and increased the proportion of ETB receptors from 50% in control cells to 80% in treated cells. In conclusion, ET-1 inhibits proliferation of activated HSC via ETB receptors, through a prostaglandin/cAMP pathway that leads to inhibition of both extracellular signal-regulated kinase and c-Jun kinase activities. Upregulation of ETB receptors by prostaglandin/cAMP raises the possibility of a positive feedback loop that would amplify the growth inhibitory response. These results suggest that ET-1 and agents that increase cAMP might be of interest to limit proliferation of activated HSC during chronic liver diseases.

Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist.

SR 121463A, a potent and selective, orally active, nonpeptide vasopressin V2 receptor antagonist, has been characterized in several in vitro and in vivo models. This compound displayed highly competitive and selective affinity for V2 receptors in rat, bovine and human kidney (0.6 < or = Ki [nM] < or = 4.1). In this latter preparation, SR 121463A potently antagonized arginine vasopressin (AVP)-stimulated adenylyl cyclase activity (Ki = 0.26+/-0.04 nM) without any intrinsic agonistic effect. In autoradiographic experiments performed in rat kidney sections, SR 121463A displaced [3H]AVP labeling especially in the medullo-papillary region and confirmed that it is a suitable tool for mapping V2 receptors. In comparison, the nonpeptide V2 antagonist, OPC-31260, showed much lower affinity for animal and human renal V2 receptors and lower efficacy to inhibit vasopressin-stimulated adenylyl cyclase (Ki in the 10 nanomolar range). Moreover, OPC-31260 exhibited a poor V2 selectivity profile and can be considered as a V2/V1a ligand. In normally hydrated conscious rats, SR 121463A induced powerful aquaresis after intravenous (0.003-0.3 mg/kg) or oral (0.03-10 mg/kg) administration. The effect was dose-dependent and lasted about 6 hours at the dose of 3 mg/kg p.o. OPC-31260 had a similar aquaretic profile but with markedly lower oral efficacy. The action of SR 121463A was purely aquaretic with no changes in urine Na+ and K+ excretions unlike that of known diuretic agents such as furosemide or hydrochlorothiazide. In addition, no antidiuretic properties have been detected with SR 121463A in vasopressin-deficient Brattleboro rats. Thus, SR 121463A is the most potent and selective, orally active V2 antagonist yet described and could be a powerful tool for exploring V2 receptors and the therapeutical usefulness of V2 blocker aquaretic agents in water-retaining diseases.

Key amino acids located within the transmembrane domains 5 and 7 account for the pharmacological specificity of the human V1b vasopressin receptor.

In mammals, the vasopressin V(1b) receptor (V(1b)-R) is known to regulate ACTH secretion and, more recently, stress and anxiety. The characterization of the molecular determinant responsible for its pharmacological selectivity was made possible by the recent discovery of the first V(1b) antagonist, SSR149415. Based upon the structure of the crystallized bovine rhodopsin, we established a three-dimensional molecular model of interaction between the human V(1b)-R (hV(1b)-R) and SSR149415. Four amino acids located in distinct transmembrane helices (fourth, fifth, and seventh) were found potentially responsible for the hV(1b)-R selectivity. To validate these assumptions, we selectively replaced the leucine 181, methionine 220, alanine 334, and serine 338 residues of hV(1a)-R by their corresponding amino acids present in the hV(1b)-R (phenylalanine 164, threonine 203, methionine 324, and asparagine 328, respectively). Four mutants, which all exhibited nanomolar affinities for vasopressin and good coupling to phospholipase C pathway, were generated. hV(1a) receptors mutated at position 220 and 334 exhibited striking increase in affinity for SSR149415 both in binding and phospholipase C assays at variance with the hV(1a)-R modified at position 181 or 338. In conclusion, this study provides the first structural features concerning the hV(1b)-R and highlights the role of few specific residues in its pharmacological selectivity.

Vasopressin receptors in human adrenal medulla and pheochromocytoma.

The nature of vasopressin (VP) receptors present in normal and tumoral human adrenal was investigated using various experimental approaches. Specific VP-binding sites were detected by autoradiography using [3H]arginine VP as a radioligand in adrenal cortex and medulla. The V1a receptor subtype was expressed in the two parts of the gland, as shown by pharmacological studies and RT-PCR experiments. By contrast, the V1b receptor subtype was only expressed in medullary chromaffin cells. This was confirmed by the characterization of V1b transcripts detected in adrenal medulla tissues. In pheochromocytoma, we also detected functional V1b receptors. These receptors triggered intracellular calcium mobilization from intracellular pools and were involved in catecholamine secretion. Binding experiments performed on pheochromocytoma plasma membrane preparations also revealed V1a vasopressin-binding sites, whose roles and cellular localization have not yet been determined. RT-PCR experiments confirmed these data; 100% and 80% of the five tumors tested exhibited V1a and V1b transcripts, respectively. Perifusion experiments also demonstrated that some pheochromocytomas may secrete large amounts of VP. Our findings imply that VP locally secreted by human adrenal medulla may regulate adrenal function by acting on V1a or V1b receptors. More interestingly, we demonstrate that one pheochromocytoma oversecretes VP. In this particular case, this may contribute to the increase in blood pressure observed.

SR 120819A, an orally-active and selective neuropeptide Y Y1 receptor antagonist.

An orally-active antagonist of neuropeptide Y (NPY) Y1 receptors, SR 120819A, has been characterized. This compound displays highly selective and competitive affinity for rat, guinea-pig and human (Ki = 15 nM) NPY Y1 receptors. In vitro, SR 120819A blocks the inhibitory effect of NPY on adenylyl cyclase activity in human SK-N-MC cells and that of the selective Y1 agonist, [Leu31,Pro34]NPY, on rabbit vas deferens contraction (pA2 = 7.20 +/- 0.07). In vivo, by intravenous route, this compound acts as an antagonist in anesthetized guinea-pigs and, notably, after oral administration, SR 120819A counteracts the pressor response of [Leu31,Pro34]NPY (5 micrograms/kg i.v.) with a long duration of action (> 4 h at 5 mg/kg p.o.). Thus, SR 120819A is the first orally-effective NPY Y1 receptor antagonist yet described. It could be a useful tool for exploring the role of NPY and the therapeutic relevance of an antagonist at NPY Y1 receptors.

Characterization of NPY receptors controlling lipolysis and leptin secretion in human adipocytes.

In order to characterize neuropeptide Y (NPY) receptors present in human adipocytes, we used selective ligands together with specific molecular probes able to recognize the different NPY receptor subtypes. RT-PCR experiments revealed the presence of Y(1) receptor transcripts with Y(4) and Y(5) and absence of Y(2) signals. Binding studies, using selective radioiodinated ligands, detected a high number (B(max)=497+/-124 fmol/mg protein) of a high affinity binding site only with [(125)I]peptide YY (PYY) and [(125)I](Leu(31), Pro(34))PYY. These sites exhibited a typical Y(1) profile as indicated by the rank order of affinity of NPY analogs and the high affinity of two selective NPY receptor antagonists, SR120819A and BIBP3226. In [(35)S]GTPgammaS binding experiments, PYY activation was totally inhibited by SR120819A and BIBP3226. Both compounds antagonized, with similar efficiency, the antilipolytic effect exerted by NPY in isolated adipocytes. Finally, PYY and Y(1) ligands enhanced adipocyte leptin secretion, an effect totally prevented by SR120819A. Thus, highly expressed in human adipocytes, the Y(1) receptor sustains the strong antilipolytic effect of NPY and exerts a positive action on leptin secretion.

Characterization and localization of leptin receptors in the rat kidney.

Characterization and localization of leptin binding sites were investigated in rat kidneys using [125I]leptin as a ligand. [125I]Leptin specific binding was found in high amounts in rat renomedullary membranes. This binding was specific, saturable, time-dependent (K(obs) = 0.055 +/- 0.008 min(-1)) and the dissociation of receptor-bound ligand was slowly reversible (K(-1) = 0.048 +/- 0.013 min(-1)). From saturation experiments, a single class of high-affinity binding sites for leptin was identified with an apparent K(d) of 0.57 +/- 0.14 nM and a B(max) of 45 +/- 10 fmol/mg protein. [125I]Leptin binding was inhibited in a dose-dependent manner by cold leptin and was highly selective since not displaceable by a number of other hormones or peptides. Autoradiographic experiments performed on adult rat kidney sections showed the intense presence of [125I]leptin receptors only in specific areas of the renal inner medulla and also consistent labeling associated with vascular structures in the corticomedullary region. The study of the postnatal developmental expression of leptin receptors in the kidney showed very low expression during the early postnatal period (8-21 days). Full expression of leptin sites was achieved at about 30 days and remained stable throughout adulthood (60 days and upwards). Moreover, in vivo administration of leptin (0.5 mg/kg i.p.) induced a significant and rapid diuretic effect in normally hydrated conscious rats. Thus, these data constitute the first characterization and mapping of [125I]leptin specific binding sites in the rat kidney and raise the possibility of a renal control by leptin.

Binding properties of three neuropeptide Y receptor subtypes from zebrafish: comparison with mammalian Y1 receptors.

Neuropeptide Y (NPY) and peptide YY (PYY) are two related 36-amino-acid peptides found in all vertebrates and are involved in many physiological processes. Five receptor subtypes have been cloned in mammals (Y1, Y2, Y4, Y5, and y6). We have recently cloned three NPY/PYY receptor subtypes in zebrafish, called Ya, Yb, and Yc. Here we report on a direct comparison of the pharmacological properties of these three receptors in vitro using porcine NPY with alanine substitutions in positions 33-36 as ligands and three analogues with internal deletions: [Ahx(8-20)]NPY, [Ahx(8-20), Pro(34)]NPY, and [Ahx(5-24)]NPY. In all cases, the zYc receptor was the most sensitive to the modifications of the NPY molecule and zYa was the least sensitive (except for the Arg --> Ala replacement at position 33). Our data identified zYa as a receptor that can bind ligands specific for Y1, Y2, and Y4 receptors, while zYb and zYc were more Y1-like. All peptides with internal deletions bound to the zYa receptor with affinities similar to that of intact pNPY. Neither the Y1-selective antagonists BIBP3226 and SR120819A nor the Y2-selective BIIE0246 bound to any of the zebrafish receptors, although the amino acids identified as important for BIBP3226 binding were almost completely conserved. These results may prove helpful in molecular modeling of the three-dimensional receptor structure.

Effect of a new, potent, non-peptide V1a vasopressin antagonist, SR 49059, on the binding and the mitogenic activity of vasopressin on Swiss 3T3 cells.

The effects of SR 49059, a new non-peptide, selective arginine vasopressin (AVP), V1a antagonist, were investigated both on AVP's receptors and on the mitogenic effects of AVP on Swiss 3T3 fibroblasts. We characterized the AVP V1a receptors on Swiss 3T3 cell membranes using the new highly specific AVP V1a radioiodinated ligand, 125I-linear AVP antagonist. Specific binding of the 125I-linear AVP antagonist was saturable, time-dependent and reversible. A single class of high affinity binding sites was identified with an apparent Kd of 40 +/- 20 pM and a Bmax of 63 +/- 20 fmol/mg protein. 125I-Linear AVP antagonist binding to its receptors was potently inhibited in a concentration-dependent manner by AVP, by the peptide V1a antagonist d(CH2)5Tyr(Me)AVP and by the synthetic V1a antagonist, SR 49059 (IC50 in the nanomolar range) while OPC-21268, another non-peptide compound, was about 100-fold less potent. Both DDAVP, a selective V2 agonist, and oxytocin exhibited low affinity (IC50 > 1 microM) in agreement with the AVP V1a nature of the site identified on Swiss 3T3 cells. In addition, the broad-spectrum antiproliferative agent [Arg6, D-Trp7,9, MePhe8] substance P (6-11), was also able to interact at 3T3 AVP V1a receptors (IC50 = 395 +/- 170 nM). The mitogenic effects of AVP on quiescent Swiss 3T3 cells, assessed through [3H]thymidine incorporation, were selectively, stereospecifically and strongly inhibited by SR 40959 (IC50 = 14 +/- 2 nM) while OPC-21268 was inactive up to 220 nM. SR 49059 was even about six times more efficient than d(CH2)5Tyr(Me)AVP in inhibiting AVP-induced DNA synthesis. Moreover, SR 49059 fully inhibited Swiss 3T3 fibroblast proliferation since it completely blocked AVP-stimulated 3T3 cell growth from the G1/G0 into the S/G2M phase, as evidenced by cell cycle analysis using a cytofluorometer. In summary, SR 49059, through direct interaction at AVP V1a receptors, exerts the most potent antiproliferative effect yet described for any V1a antagonist on Swiss 3T3 cells.

Nonpeptide vasopressin receptor antagonists: development of selective and orally active V1a, V2 and V1b receptor ligands.

The involvement of vasopressin (AVP) in several pathological states has been reported recently and the selective blockade of the different AVP receptors could offer new clinical perspectives. During the past few years, various selective, orally active AVP V1a (OPC-21268, SR49059 (Relcovaptan)), V2 (OPC-31260, OPC-41061 (Tolvaptan), VPA-985 (Lixivaptan), SR121463, VP-343, FR-161282) and mixed V1a/V2 (YM-087 (Conivaptan), JTV-605, CL-385004) receptor antagonists have been intensively studied in various animal models and have reached, Phase IIb clinical trials for some of them. For many years now, our laboratory has focused on the identification of nonpeptide vasopressin antagonists with suitable oral bioavailability. Using random screening on small molecule libraries, followed by rational SAR and modelization, we identified a chemical series of 1-phenylsulfonylindolines which first yielded SR49059, a V1a receptor antagonist prototype. This compound displayed high affinity for animal and human V1a receptors and antagonized various V1a AVP-induced effects in vitro and in vivo (intracellular [Ca2+] increase, platelet aggregation, vascular smooth muscle cell proliferation, hypertension and coronary vasospasm). We and others have used this compound to study the role of AVP in various animal models. Recent findings from clinical trials show a potential interest for SR49059 in the treatment of dysmenorrhea and in Raynaud's disease. Structural modifications and simplifications performed in the SR49059 chemical series yielded highly specific V2 receptor antagonists (N-arylsulfonyl-oxindoles), amongst them SR121463 which possesses powerful oral aquaretic properties in various animal species and in man. SR121463 is well-tolerated and dose-dependently increases urine output and decreases urine osmolality. It induces free water-excretion without affecting electrolyte balance in contrast to classical diuretics (e.g. furosemide and hydrochlorothiazide). Notably, in cirrhotic rats with ascites and impaired renal function, a 10-day oral treatment with SR121463 (0.5 mg/kg) totally corrected hyponatremia and restored normal urine excretion. This compound also displayed interesting new properties in a rabbit model of ocular hypertension, decreasing intraocular pressure after single or repeated instillation. Thus, V2 receptor blockade could be of interest in several water-retaining diseases such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH), liver cirrhosis and congestive heart failure and deserves to be widely explored. Finally, further chemical developments in the oxindole family have led to the first specific and orally active V1b receptor antagonists (with SSR149415 as a representative), an awaited class of drugs with expected therapeutic interest mainly in ACTH-secreting tumors and various emotional diseases such as stress-related disorders, anxiety and depression. However, from the recently described tissue localization for this receptor, we could also speculate on other unexpected uses. In conclusion, the development of AVP receptor antagonists is a field of intensive pharmacological and clinical investigation. Selective and orally active compounds are now available to give new insight into the pathophysiological role of AVP and to provide promising drugs.

Importance of the phenotypic state of vascular smooth muscle cells on the binding and the mitogenic activity of endothelin.

Smooth muscle cells of the rabbit aorta, when grown in vitro, express distinguishable forms of phenotypes (contractile and synthetic). On contractile cells, ET-1 specifically bound to a single class of high affinity (KD = 128 pM) and high capacity (Bmax = 66,000 sites/cell) binding sites. But, whereas affinity of [125I]-ET-1 was not significantly affected by phenotypic modulation, synthetic cells displayed a 10-fold lower [125I]-ET-1 binding capacity than contractile smooth muscle cells. Similarly, the mitogenic effect of ET-1 on smooth muscle cells was considerably lower for synthetic than for contractile cells. The ET-1 receptor on primary cells was recognized by sarafotoxin S6b and the different ET-related peptides with an order of potency [ET-1 greater than S6b greater than ET-3 greater than Big ET-1 much greater than ET(16-21)] identical to that inducing smooth muscle cell growth. Therefore, these data indicate that the binding and the mitogenic effects of ET-1 on smooth muscle cells might be of different magnitudes depending on the phenotypic state of these cells.

The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors.

We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.

Aquaretic and hormonal effects of a vasopressin V(2) receptor antagonist after acute and long-term treatment in rats.

A single oral administration of 1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzene sulfonyl]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy)cyclohexane]indo l-2 -one SR121463 (0.3-3 mg/kg), a vasopressin non-peptide V(2) receptor antagonist, to rats induced dose-dependent aquaresis which was accompanied by Na(+), K(+), aldosterone and arginine vasopressin excretion over 6 h after dosing. However, no solute excretion was observed over 24 h. As a result of aquaresis, hemoconcentration and increases in plasma angiotensin II and adenocorticotrophin hormone were seen with 3 mg/kg at 2 h after dosing. Chronic treatment with SR121463 (3 mg/kg/dayx28 days) induced a marked aquaresis associated with aldosterone and vasopressin excretion. After a week of treatment, urine volume and aldosterone excretion were reduced ( approximately 40%) and then stabilised, while urine vasopressin excretion remained almost constant throughout the study. There were no changes in arterial pressure, plasma osmolality, plasma sodium concentration, or in number and affinity of liver vasopressin V(1A) and kidney V(2) receptors 24 h after the last treatment. These results indicate that SR121463 is a potent aquaretic agent and might be useful for the chronic management of water-retaining diseases in humans.

Acute renal response to the non-peptide vasopressin V2-receptor antagonist SR 121463B in anesthetized rats.

Vasopressin V2-receptor antagonists are promising agents for the use in water-retaining diseases. Potential renal mechanisms of action include effects on water permeability in the collecting duct as well as on electrolyte transport in the thick ascending limb of Henle's loop (TALH). To elucidate sites of action upstream of the distal tubule, e.g., in TALH, micropuncture experiments were performed in anesthetized rats during application of the V2-receptor antagonist SR 121463B. As compared to vehicle-treated rats, SR 121463B (0.3 mg/kg i.v.) did not affect mean arterial blood pressure (means +/- SEM, n=10 rats per group: 108+/-4 mmHg vs. 107+/-4 mmHg), whole kidney GFR (1.1+/-0.1 ml/min vs. 1.1+/-0.1 ml/min), or whole kidney fractional reabsorption (FR) of potassium (66+/-5% vs. 68+/-4%). The drug, however, reduced whole kidney FR of fluid (92+/-1% vs. 99+/-1%), increased urinary flow rate (84+/-7 microl/min vs. 8+/-1 microl/min) and electrolyte-free-water clearance (72+/-8 microl/min vs. 2+/-1 microl/min), and reduced urinary osmolality (148+/-11 mosmol/kg vs. 1,200+/-185 mosmol/kg). This pronounced diuretic response was associated with a minor reduction in whole kidney FR of sodium (99.6+/-0.1% vs. 99.9+/-0.1%) and chloride (98.3+/-0.2% vs. 98.9+/-0.1%). As compared to vehicle application, SR 121463B did not significantly alter single nephron GFR (39+/-2 nl/min vs. 39+/-1 nl/min, n=22 and 23 nephrons, respectively) or the FR up to the early distal tubule of fluid (76+/-2% vs. 76+/-1%), sodium (92+/-1% vs. 93+/-1%), potassium (91+/-1% vs. 90+/-1%) or chloride (90+/-1% vs. 91+/-1%). Together these data indicate a predominant aquaretic effect of SR 121463B which is located downstream of the early distal tubule. This response is compatible with blockade of vasopressin V2-receptors in the collecting duct and, as directly demonstrated by immunohistochemistry, subsequent retrieval of aquaporin-2 from apical plasma membrane, which inhibits water permeability and transport.

Effects of SR 49059, a non-peptide antagonist of vasopressin V1a receptors, on vasopressin-induced coronary vasoconstriction in conscious rabbits.

The effect of SR 49059, a new potent non-peptide vasopressin (AVP) V1a receptor antagonist, was investigated on AVP-induced electrocardiogram modifications. A high intravenous dose of AVP (0.5 IU or 1.23 micrograms/animal) produced an important transient t-wave elevation (from 4.7 +/- 0.2 to 8.9 +/- 0.7 mm) and heart rate decrease (from 199 +/- 5 to 99 +/- 6 bpm) in conscious rabbits. The t-wave increase was a significant index of coronary vasoconstriction-induced cardiac ischemia. SR 49059 had potent protective effects in this model both by intravenous (0.125 to 0.5 mg/kg) and oral (2.5 to 10 mg/kg) routes. After a 30-min pre-treatment, SR 49059 showed dose-dependent protection on t-wave elevation and heart rate decrease with ED50's of 95 (95% CL:168-22) and 30 (95% CL:54-6) micrograms/kg i.v., respectively. Complete blockade occurred with doses of 2 mg/kg i.v. and upwards. By the oral route, SR 49059 was rapidly absorbed and a dose of 10 mg/kg displayed a protective effect lasting more than 6 hours on both electrocardiogram parameters. Moreover, SR 49059 exerted a high stereospecific inhibitory effect since its enantiomer was totally inactive at 0.5 mg/kg i.v., suggesting that protection occurred by interaction with vascular AVP V1a receptors. Thus, SR 49059 is the first specific non-peptide V1a antagonist with long-lasting oral activity on AVP-induced coronary vasoconstriction and bradycardia. With this original profile, SR 49059 could be a promising therapeutical antivasospastic agent for preventing AVP-induced cardiac damage.

A nonpeptide vasopressin V(1a) receptor antagonist, SR 49059, does not prevent cisplatin-induced emesis in piglets.

We determined the pharmacological and the antiemetic properties of SR 49059, a selective nonpeptide V(1a) receptor antagonist, on cisplatin-induced emesis in the piglet. Firstly, we clearly demonstrate that SR 49059 is a potent V(1a) receptor antagonist in vitro and in vivo in the piglet. In binding studies, [3H]-SR 49059 exhibited high affinity for V(1a) receptors in piglet liver membranes (K(d) of 0.76 +/- 0.12 nM and B(max) of 138 +/- 22 fmol/mg prot.). In vivo, in decerebrate piglets, SR 49059 (1 mg/kg iv) antagonized AVP (500 ng/kg iv)-induced hypertension for at least 150 min and also blocked, for at least 270 min at 3 mg/kg iv, the pressor responses to exogenous LVP. After single and repeated iv or icv administration, we studied the antiemetic properties of SR 49059 on cisplatin-induced emesis in piglets. Animals receiving an emetic dose of cisplatin (5.5 mg/kg, iv) were observed continuously for 60 h. Piglets acting as controls were iv administered with vehicle 15 min prior to cisplatin infusion (T0(-15min)), while experimental animals received a single iv administration of SR 49059 at the dose of 1 or 3 mg/kg. In additional piglets, we administered SR 49059 (3 mg/kg) every 12 h from T0(-15min) to T48(-15min) (cumulative dose, 15 mg/kg). Another set of animals - observed only during the acute phase - was administered with SR 49059 (10 mg/kg) every 3 h from T0(-15min) to T15(-15min) (cumulative dose, 60 mg/kg). Lastly, 10 piglets were given a bilateral icv injection of SR 49059 (500 microg and 1500 microg/side) 1 h prior to cisplatin infusion. In all groups treated with SR 49059, the latency of the first emetic episode and the incidence of vomiting during the acute, the delayed and the cumulative phases remained statistically similar to that observed in controls, suggesting that V(1a) receptors are not involved in the onset and completion of nausea and vomiting.

Nonpeptide antagonists for vasopressin receptors. Pharmacology of SR 121463A, a new potent and highly selective V2 receptor antagonist.

Involvement of AVP in several pathological states is now established and specific modulation of the different AVP receptor subtypes (V1a, V1b and V2) offers new clinical perspectives for treating major diseases. Recent years have marked a turning point with the design and the use of the first nonpeptide vasopressin receptor antagonists expressing various selectively profile. In that field, we report here the characterization of SR 121463A a highly selective, orally-active antagonist of vasopressin V2 receptors in several models in vitro and in vivo. This compound displayed competitive nanomolar affinity for V2 receptors in various species including man and exhibited a highly selective AVP V2 profile. In vitro, SR 121463A potently antagonized AVP-stimulated adenylyl cyclase activity in human kidney preparations (Ki = 0.26 +/- 0.04 nM) without any intrinsic agonistic effect. In normally-hydrated rats, SR 121463A induced dose-dependent powerful and long-lasting aquaresis after intravenous (0.003 to 0.3 mg/kg) or oral (0.03 to 10 mg/kg) administration. The action of SR 121463A is purely aquaretic with no changes in urine Na+ and K+ excretions unlike that of known diuretic agents such as furosemide or hydrochlorothiazide. In vasopressin-deficient Brattleboro rats, SR 121463A is devoid of any V2 antidiuretic agonist properties. In addition, this compound potently antagonized DDAVP extrarenal V2 effects on hemostasis factor release (FVIII, vW and t-PA) in dogs (ID50 approximately 10 micrograms/kg i.v.). Thus, SR 121463A is the most potent and selective, orally-active V2 antagonist yet described. It is a useful ligand for exploring V2 receptors and the therapeutical usefulness of pure V2 aquaretic agents in several water-retaining diseases and congestive heart failure.

Vasopressin regulates adrenal functions by acting through different vasopressin receptor subtypes.

In mammals, vasopressin is known to be synthesized in the hypothalamus and released in the blood stream at the pituitary level. This neuropeptide is also synthesized and secreted by the adrenal medulla in many species including human. Moreover, agents like acetylcholine and corticotropin releasing factor stimulates its basal secretion. V1a vasopressin receptors are present in the adrenal cortex and are involved in steroids secretion (aldosterone in the zona glomerulosa and glucocorticoids in the zona fasciculata of some species). These receptors are coupled to phospholipase C beta and to dihydropyridine-sensitive calcium channels via heterotrimeric G proteins differing by their sensitivities to pertussis toxin. The adrenal medulla, from many species, exhibits V1a vasopressin receptors. In rat adrenal medulla, functional V1b vasopressin receptors could also be characterized. These receptors stimulate catecholamines secretion via activation of phospholipase C beta and subsequent mobilization of intracellular calcium. The adrenal medulla secretes AVP and exhibits functional vasopressin receptors. The adrenal cortex also possesses functional vasopressin receptors and is in contact with adrenal medulla via "medullary rays". We may thus reasonably conclude that AVP physiologically regulates adrenal gland functions via autocrine/paracrine mechanisms.