Effects of YM218, a nonpeptide vasopressin V1A receptor-selective antagonist, on human vasopressin and oxytocin receptors.

The binding and signal transduction characteristics of YM218 ((Z)-4'-{4,4-difluoro-5-[2-oxo-2-(4-piperidinopiperidino)ethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl}-2-methyl-3-furanilide hemifumarate), a newly synthesized, potent arginine vasopressin (AVP) V(1A) receptor-selective antagonist, were examined using cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells and human uterine smooth muscle cells (USMCs) expressing oxytocin receptors. YM218 potently inhibited specific binding of [(3)H] AVP to V(1A) receptors, exhibiting a K(i) value of 0.30 nM. In contrast, YM218 exhibited much lower affinity for V(1B), V(2) and oxytocin receptors, exhibiting K(i) values of 25,500 nM, 381 nM and 71.0 nM, respectively. In CHO cells expressing V(1A) receptors, YM218 potently inhibited the AVP-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), exhibiting an IC(50) value of 0.25 nM. However, in human USMCs expressing oxytocin receptors, YM218 exhibited a much lower potency in inhibiting the oxytocin-induced [Ca(2+)](i) increase, showing an IC(50) value of 607 nM, and had no effect on the AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM218 did not potently inhibit the production of cAMP stimulated by AVP, showing an IC(50) value of 62.2 nM. In all assays used, YM218 did not exhibit any agonistic activity. These results demonstrate that YM218 is a potent, nonpeptide human V(1A) receptor-selective antagonist, and that YM218 will be a valuable new tool to gain further insight into the physiologic and pharmacologic actions of AVP.     

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139     

Distinct receptor subtypes mediate arginine vasopressin-dependent ACTH release and intracellular calcium mobilization in rat pituitary cells

In the present study, adrenocorticotropic hormone (ACTH) release and intracellular calcium ([Ca(2+)](i)) increase induced by arginine vasopressin (AVP) were characterized in collagenase-dispersed and 3-day cultured rat anterior pituitary cells. AVP and the selective vasopressin V(1b) receptor agonist, [1-deamino-4-cyclohexylalanine]AVP (d[Cha(4)]AVP) induced ACTH release with nanomolar potencies in both cell preparations, and produced a maximal stimulation that was about 1.5 fold greater in the 3-day cultured cells, indicating that the vasopressin V(1b) receptor-ACTH release pathway is enhanced over time in culture. In dispersed cells, AVP, oxytocin and d[Cha(4)]AVP induced [Ca(2+)](i) increases with nanomolar potencies. The selective vasopressin V(1a) receptors antagonist, SR49059 (100 nM), together with the selective oxytocin receptors antagonist (d(CH(2))(5)(1)Tyr(Me)(2),Thr(4),Orn(8),Tyr-NH(2)(9)-vasotocin (100 nM), inhibited the maximal AVP response by ~70%, without affecting the response to d[Cha(4)]AVP, suggesting that the V(1b) receptor was only partially responsible for the AVP-induced [Ca(2+)](i) increase. In contrast, in 3-day cultures, AVP induced an increase in [Ca(2+)](i), while oxytocin and d[Cha(4)]AVP did not. The response to AVP was completely antagonized by SR49059, whereas the vasopressin V(1b) receptor antagonists, SSR149415 and (d(CH(2))(5)(1)Tyr(Me)(2),Thr(4),Orn(8),Tyr-NH(2)(9))-vasotocin had no effect, indicating that the [Ca(2+)](i) increase was mediated exclusively by vasopressin V(1a) receptors. In conclusion, the enhancement of vasopressin V(1b) receptor-mediated ACTH release and the lack of a detectable vasopressin V(1b) receptor coupling to [Ca(2+)](i) increase in cultured cells suggests the activation of a different/additional signaling pathway in the molecular mechanism of ACTH release.     

Binding and signal transduction characteristics of the nonpeptide vasopressin V1A receptor-selective antagonist YM218 in cultured rat mesangial cells

Vasopressin (AVP) causes mesangial cell contraction, proliferation and hypertrophy. The present study investigated the effects of YM218, a potent, nonpeptide AVP V(1A) receptor-selective antagonist, on rat mesangial cells using binding, signal transduction and cell growth assays. Specific binding of (3)H-AVP to rat mesangial cell plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with the expected V(1A) receptor profile. YM218 showed high affinity for V(1A) receptors, exhibiting a K(i) value of 0.19 nmol/l. AVP concentration-dependently increased intracellular Ca(2+) ([Ca(2+)](i)) levels, stimulated mitogen-activated protein (MAP) kinase and induced hyperplasia. Conversely, YM218 potently suppressed [Ca(2+)](i) elevation, activation of MAP kinase and hyperplasia induced by AVP. These results indicate that YM218 displays both high affinity for rat mesangial cell V(1A) receptors and high potency in inhibiting AVP-induced signal transduction and growth response. Therefore, YM218 is a useful pharmacologic tool for investigating the physiologic and pathophysiologic roles of AVP in kidney, and may have clinical application in the prevention or regression of mesangial cell growth.     

Selectivity of d[Cha4]AVP and SSR149415 at human vasopressin and oxytocin receptors: evidence that SSR149415 is a mixed vasopressin V1b/oxytocin receptor antagonist

1 A possible role of arginine vasopressin (AVP) V(1b) receptor subtype in stress-related disorders has been recently highlighted by the discovery of the agonist [1-deamino-4-cyclohexylalanine] AVP (d[Cha(4)]AVP) and the antagonist SSR149415. Both compounds have been proposed to target specifically V(1b) receptors, since the reported affinities for the related V(1a), V(2) and oxytocin receptors are in the micromolar or submicromolar range. In the present study, we further investigated the binding affinities of d[Cha(4)]AVP and SSR149415 at recombinant human vasopressin V(1b) (hV(1b)) and oxytocin (hOT) receptors expressed in Chinese hamster ovary (CHO) cells and functional properties of both compounds at hV(1b), hV(1a), hV(2) and hOT receptors. 2 d[Cha(4)]AVP bound to hV(1b) receptors and hOT receptors with pK(i) values of 9.68+/-0.06 and 7.68+/-0.09, respectively. SSR149415 showed pK(i) values of 9.34+/-0.06 at hV(1b) and 8.82+/-0.16 at hOT receptors. 3 d[Cha(4)]AVP stimulated [Ca(2+)](i) increase in hV(1b)-CHO cells with a pEC(50) value of 10.05+/-0.15. It showed pEC(50) values of 6.53+/-0.17 and 5.92+/-0.02 at hV(1a) and hV(2) receptors, respectively, and behaved as a weak antagonist at hOT receptors (pK(B)=6.31+/-0.12). SSR149415 inhibited the agonist-induced [Ca(2+)](i) increase with pK(B) values of 9.19+/-0.07 in hV(1b)-CHO and 8.72+/-0.15 in hOT-CHO cells. A functional pK(i) value of 7.23+/-0.10 was found for SSR1494151 at hV(1a) receptors, whereas it did not inhibit 20 nM AVP response at hV(2) receptors up to 3 microM. 4 Data obtained confirmed the high potency and selectivity of d[Cha(4)]AVP at hV(1b) receptors, but revealed that SSR149415, in addition to the high potency at hV(1b) receptors, displays a significant antagonism at hOT receptors.     

Pharmacological characterization of YM471, a novel potent vasopressin V(1A) and V(2) receptor antagonist

The pharmacologic profile of YM471 ((Z)-4'-[4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl]-2-phenylbenzanilide monohydrochloride), a novel potent vasopressin V(1A) and V(2) receptor antagonist, was investigated using several in vitro and in vivo techniques. YM471 showed high affinity for rat vasopressin V(1A) and V(2) receptors, exhibiting K(i) values of 0.16 and 0.77 nM, respectively. In contrast, YM471 exhibited much lower affinity for rat vasopressin V(1B) and oxytocin receptors, with K(i) values of 10.5 microM and 31.0 nM, respectively. In conscious rats, oral administration of YM471 (0.1-3.0 mg/kg) produced dose-dependent inhibition of the pressor response caused by exogenous vasopressin and increased urine excretion and decreased urine osmolality; this effect lasted more than 8 h. In all biological assays used, YM471 exhibited no agonistic activity. These results demonstrate that YM471 exerts potent and long-lasting antagonistic activity on both vasopressin V(1A) and V(2) receptors, and that this compound may be a useful tool for clarifying the physiologic and pathophysiologic roles of vasopressin and the therapeutic usefulness of the vasopressin receptor antagonist.     

Pharmacological characterization of YM087, a potent, nonpeptide human vasopressin V1A and V2 receptor antagonist

The effects of YM087 (4’-[(2-methyl-1,4,5,6-tetrahydroimidazo[4,5-d][1]benzazepin-6-yl)-carbonyl]-2-phenylbenzanilide monohydrochloride), a novel nonpeptide vasopressin (AVP) receptor antagonist, on [³H]AVP binding to human AVP receptors (V_1A, V_1B and V₂) cloned and transiently expressed in COS-1 cells generated from monkey renal tissue were studied. Scatchard analysis of saturation isotherms for the specific binding of [³H]AVP to membranes, prepared from COS-1 cells transfected with human V_1A, V_1B and V₂ receptors, yielded an apparent equilibrium dissociation constant (K _d) of 0.67nM, 0.28nM and 2.14nM and a maximum receptor density (B _max) of 2180fmol/mg protein, 369fmol/mg protein and 2660fmol/mg protein, respectively. YM087 showed high affinity for AVP V_1A and V₂ receptors with K _i values of 6.3 and 1.1nM, respectively, but had no effect on [³H]AVP binding to AVP V_1B receptors. In COS-1 cells expressing either AVP V_1A or V_1B receptors, AVP caused a concentration-dependent increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). YM087 inhibited the AVP-induced increase in [Ca²⁺]_i in COS-1 cells expressing AVP V_1A receptors in a concentration-dependent manner with an IC₅₀ value of 14.3nM, but did not influence this increase in AVP V_1B-receptor expressing cells. In contrast, stimulation of COS-1 cells expressing AVP V₂ receptors resulted in an accumulation of cAMP. YM087 inhibited AVP-induced cAMP production in COS-1 cells expressing AVP V₂ receptors in a concentration-dependent manner with an IC₅₀ value of 1.95nM. In all assays used, YM087 was devoid of any agonistic activity. These results suggest that YM087 is a potent nonpeptide dual human AVP V_1A and V₂ receptor antagonist, and that YM087 will be a powerful tool in investigation of the physiological and pathophysiological roles of AVP. Received: 15 July 1997 / Accepted: 23 September 1997     

Characterization of a novel nonpeptide vasopressin V(2)-agonist, OPC-51803, in cells transfected human vasopressin receptor subtypes

We discovered the first nonpeptide arginine-vasopressin (AVP) V(2)-receptor agonist, OPC-51803. Pharmacological properties of OPC-51803 were elucidated using HeLa cells expressing human AVP receptor subtypes (V(2), V(1a) and V(1b)) and compared with those of 1-desamino-8-D-arginine vasopressin (dDAVP), a peptide V(2)-receptor agonist. OPC-51803 and dDAVP displaced [(3)H]-AVP binding to human V(2)- and V(1a)-receptors with K(i) values of 91.9+/-10.8 nM (n = 6) and 3.12+/-0.38 nM (n = 6) for V(2)-receptors, and 819+/-39 nM (n = 6) and 41.5+/-9.9 nM (n = 6) for V(1a)-receptors, indicating that OPC-51803 was about nine times more selective for V(2)-receptors, similar to the selectivity of dDAVP. OPC-51803 scarcely displaced [(3)H]-AVP binding to human V(1b)-receptors even at 10(-4) M, while dDAVP showed potent affinity to human V(1b)-receptors with the K(i) value of 13.7+/-3.2 nM (n = 4). OPC-51803 concentration-dependently increased cyclic adenosine 3', 5'-monophosphate (cyclic AMP) production in HeLa cells expressing human V(2)-receptors with an EC(50) value of 189+/-14 nM (n = 6). The concentration-response curve for cyclic AMP production induced by OPC-51803 was shifted to the right in the presence of a V(2)-antagonist, OPC-31260. At 10(-5) M, OPC-51803 did not increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in HeLa cells expressing human V(1a)-receptors. On the other hand, dDAVP increased [Ca(2+)](i) in HeLa cells expressing human V(1a)- and V(1b)-receptors in a concentration-dependent fashion. From these results, OPC-51803 has been confirmed to be the first nonpeptide agonist for human AVP V(2)-receptors without agonistic activities for V(1a)- and V(1b)-receptors. OPC-51803 may be useful for the treatment of AVP-deficient pathophysiological states and as a tool for AVP researches.     

Effect of YM218, a nonpeptide vasopressin V(1A) receptor-selective antagonist, on rat mesangial cell hyperplasia and hypertrophy

Mesangial cell growth constitutes a key feature of progressive glomerular injury. Vasopressin (AVP), a potent peptide vasoconstrictor, acts on mesangial cells through the V(1A) receptors, inducing contraction and cell proliferation. This study examined the effects of YM218, a nonpeptide AVP V(1A) receptor-selective antagonist, on the mitogenic and hypertrophic effects of AVP in rat mesangial cells. When added to mesangial cells whose growth was arrested, AVP concentration-dependently induced hyperplasia and hypertrophy. YM218 potently prevented AVP-induced hyperplasia and hypertrophy of these cells. Furthermore, AVP stimulated endothelin (ET)-1 secretion from mesangial cells in a concentration-dependent manner and this effect was potently inhibited by YM218. ET-1 also induced hyperplasia and hypertrophy in mesangial cells and this effect was completely abolished by ET(A) receptor-selective antagonist YM598. In addition, AVP-induced hyperplasia and hypertrophy were partly inhibited by YM598. These results suggest that AVP may modulate mesangial cell growth not only by its direct action but also through the stimulation of ET-1 secretion. YM218 displays high potency in inhibiting the AVP-induced physiologic responses of mesangial cells via the V(1A) receptors and is a potent pharmacologic probe for investigating the physiologic and pathophysiologic roles of AVP in several renal diseases.     

Effect of conivaptan, a combined vasopressin V(1a) and V(2) receptor antagonist, on vasopressin-induced cardiac and haemodynamic changes in anaesthetised dogs.

The neurohormonal factor arginine vasopressin (AVP) produces potent systemic vasoconstriction as well as water retention in the kidneys via the V(1a) and V(2) receptors, respectively. Therefore, AVP may be considered as an aggravating factor of cardiac failure. In the present study, the effects of intravenous (i.v.) infusion of AVP on cardiovascular parameters and the effect of conivaptan (YM087, 4'-(2-methyl-1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine-6-carbonyl)-2-phenylbenzanilide monohydrochloride), a vasopressin V(1a)/V(2) receptor antagonist, on AVP-induced cardiac and haemodynamic changes were investigated in pentobarbitone-anaesthetised dogs. The i.v. infusion of AVP (0.12-4mUkg(-1)min(-1)) dose-dependently produced decreases in the cardiac contractility indicator LV dP/dt(max) and cardiac output (CO) and increases in left ventricular end-diastolic pressure (LVEDP) and total peripheral resistance (TPR). These changes accurately mimic the cardiovascular symptoms of congestive heart failure. The i.v. bolus injection of conivaptan (0.1mgkg(-1)) rapidly attenuated the AVP (4mUkg(-1)min(-1))-induced decrease in CO and reversed the AVP-induced elevation in both LVEDP and TPR. In conclusion, i.v. infusion of AVP produced cardiac dysfunction and vasoconstriction in pentobarbitone-anaesthetised dogs. Conivaptan demonstrated the ability to dramatically improve the impaired cardiovascular parameters induced by AVP. The results suggest the potential usefulness of conivaptan in treating congestive heart failure.     

Pharmacology of conivaptan hydrochloride (YM087), a novel vasopressin V1A/V2 receptor antagonist

Pharmacology of conivaptan hydrochloride (YM087) was investigated in in vitro and in vivo studies. In radioligand binding study, YM087 showed high affinity for both V1A and V2 receptors in animal and human species. Affinity of YM087 for V1A and V2 receptors was comparable to that of vasopressin (AVP). In functional antagonistic activity study, YM087 concentration-dependently inhibited AVP-induced intracellular Ca2+ elevation via human V1A receptors and AVP-stimulated cAMP accumulation via human V2 receptors. Intravenous administration of YM087 dose-dependently inhibited AVP-induced pressor responses and produced a dose-dependent aquaresis in rats and dogs. Oral administration of YM087 showed a potent and long-lasting antagonistic activity on V1A and V2 receptors. YM087 was effective in dogs with heart failure and in heart failure rats with hyponatremia and edema. These results reveal that YM087 is the first orally active V1A/V2 receptor antagonist and suggest that YM087 may be useful in the treatment of congestive heart failure and hyponatremia.     

Pharmacological characterization of F-180: a selective human V(1a) vasopressin receptor agonist of high affinity

1. The pharmacological properties of F-180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments revealed that F-180 exhibited a high affinity for the human V(1a) receptor subtype (K(i)=11 nM) and was selective for this receptor subtype. 2. Functional studies performed on CHO cells expressing human V(1a) receptors indicate that similarly to AVP, F-180 can stimulate the accumulation of inositol phosphate. The activation constant (K(act)) for both F-180 and AVP was 1.7 nM. F-180 was also an agonist for the human V(2) and V(1b) receptor subtypes and an antagonist for the human OT receptor. 3. Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F-180 on various mammalian species. F-180 showed high affinity and good selectivity for human and bovine V(1a) receptors, but weak affinity and non selective properties for rat V(1a) receptors. 4. To assess the functional properties of F-180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F-180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency. 5. In conclusion, we demonstrate that F-180 is the first selective V(1a) agonist described for human and bovine vasopressin receptors. Therefore F-180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V(1a) receptor subtypes.     

Pharmacologic properties of YM218, a novel, potent, nonpeptide vasopressin V1A receptor-selective antagonist

The pharmacologic profile of YM218, (Z)-4'-{4,4-difluoro-5-[2-oxo-2-(4-piperidinopiperidino)ethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl}-2-methyl-3-furanilide hemifumarate, a newly synthesized, nonpeptide vasopressin (AVP) receptor antagonist, was investigated using several in vitro and in vivo methods. YM218 exhibited high affinity for V1A receptors isolated from rat liver, with a Ki value of 0.50 nM. In contrast, YM218 exhibited much lower affinity for rat pituitary V1B, kidney V2, and uterus oxytocin receptors, with Ki values of 1510 nM, 72.2 nM, and 150 nM, respectively. In vivo studies revealed that YM218 dose-dependently inhibited pressor response to exogenous AVP in pithed rats (intravenous) and in conscious normotensive rats (intravenous or oral) with a long duration of action (>8 h at 3 mg/kg, p.o.). In contrast, oral administration of YM218 did not increase urine excretion in conscious rats. These results demonstrate that YM218 is a potent nonpeptide AVP V1A receptor-selective antagonist that will be useful in future studies to help clarify the physiologic and pathophysiologic roles of AVP.     

Effects of YM218, a nonpeptide vasopressin V(1A) receptor-selective antagonist, on vasopressin-induced growth responses in human mesangial cells

Mesangial cells are centrally-located glomerular pericytes with contractile, endocrine, and immunity-regulating functions. These cells are thought to maintain normal glomerular function, since mesangial cell proliferation and extracellular matrix formation are hallmarks of chronic glomerular disease. Vasopressin causes mesangial cell contraction, proliferation and hypertrophy. Consequently, the effects of YM218, a potent, nonpeptide vasopressin V(1A) receptor-selective antagonist, on the growth responses of human mesangial cells to vasopressin were investigated. YM218 showed high affinity for vasopressin V(1A) receptors, exhibiting a K(i) value of 0.18 nM. Vasopressin concentration-dependently increased intracellular Ca(2+) levels and induced hyperplasia and hypertrophy in cultured mesangial cells, YM218 potently inhibited these vasopressin-induced responses. These results clearly show that YM218 has both strong affinity for human mesangial cell vasopressin V(1A) receptors and great potency in inhibiting the vasopressin-induced growth responses of mesangial cells controlled by the vasopressin V(1A) receptors. The hyperplasia and hypertrophy of mesangial cells in vitro caused by vasopressin indicate its possible in vivo role in glomerular disease pathogenesis. Therefore, YM218 is a potent pharmacologic probe to investigate the physiologic and pathophysiologic roles of vasopressin in the development of renal disease.     

Pharmacological characterization of RWJ-676070, a dual vasopressin V(1A)/V(2) receptor antagonist

The dysregulation of arginine vasopressin (AVP) release and activation of vasopressin V(1A) and V(2) receptors may play a role in disease. The in vitro and in vivo pharmacology of RWJ-676070, a potent, balanced antagonist of both the V(1A) and V(2) receptors is described. RWJ-676070 binding and intracellular functional antagonist activity was characterized using cells expressing V(1A), V(1B) or V(2) receptors. Its inhibition of V(1A) receptor-mediated contraction of vascular rings and platelet aggregation was determined. V(2) receptor-medated aquaresis was determined in rats, dogs and monkeys. V(1A) receptor-mediated inhibitory activity was assessed in vivo in a vasopressin-induced hypertension model and in normotensive rats and in two hypertensive rat models. RWJ-676070 inhibited AVP binding to human V(1A) and V(2) receptors (Ki=1 and 14 nM, respectively). RWJ-676070 inhibited V(1A) receptor-induced intracellular calcium mobilization and V(2) receptor-induced cAMP accumulation with Ki values of 14 nM and 13 nM, respectively. The compound was slightly less potent against rat V(1A) receptors. RWJ-676070 inhibited V(1A) receptor-mediated vasoconstriction in rat and dog vascular rings and AVP-induced human platelet aggregation. Dose dependent aquaresis was demonstrated in rats, dogs and monkeys following oral administration. RWJ-676070 inhibited AVP-induced hypertension in rats but had no effect on arterial pressure in normotensive and spontaneously hypertensive rats but did decrease arterial pressure in Dahl, salt-sensitive hypertensive rats. RWJ-676070 is a new, potent antagonist of V(1A) and V(2) receptors that may be useful for treatment of diseases benefiting from balanced inhibition of both V(1A) and V(2) receptors.     

Impaired arginine-vasopressin-induced aldosterone release from adrenal gland cells in mice lacking the vasopressin V1A receptor

We examined aldosterone release in response to stimulation with arginine-vasopressin (AVP) using adrenal gland cells. AVP caused a significant increase in aldosterone release from the dispersed adrenal gland cells of wild-type mice (V1AR+/+) at concentrations from 0.1 microM to 1 microM. In contrast, AVP-induced aldosterone release was impaired in adrenal gland cells from mice lacking the vasopressin V1A receptor (V1AR-/-), while adrenocorticotropic hormone (ACTH)-induced aldosterone release in V1AR-/- mice was not significantly different from that in V1AR+/+ mice. In addition, a vasopressin V1A receptor-selective antagonist 1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone (OPC-21268) potently inhibited AVP-induced aldosterone release. Thus, our study clearly demonstrates that AVP-induced aldosterone release from adrenal gland cells is mediated via the vasopressin V1A receptor in mice.     

Characterisation of the effects of SR146131, a novel non-peptide CCK(1) receptor agonist, on IMR-32 human neuroblastoma cells

The effect of ?2-[4-(4-chloro-2, 5-dimethoxy-phenyl)-5-[2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoy l]-5, 7-dimethyl-indol-1-yl?-acetic acid (SR146131), a novel non-peptide agonist of cholecystokinin (CCK) CCK(1) receptors, was compared to the effect of sulphated cholecystokinin octapeptide (CCK-8-S) on CCK(1) receptors of the human neuroblastoma cell line IMR-32. SR146131 inhibited [125I]CCK-8-S binding to IMR-32 cells at nanomolar concentrations. SR146131 and CCK-8-S increased intracellular free Ca(2+) levels ([Ca(2+)](i)) in the same concentration range (EC(50)=6+/-2.3 and 1.3+/-0.14 nM, respectively). Although the shape of the [Ca(2+)](i) increase induced by CCK-8-S and SR146131 was slightly different, extracellular Ca(2+) removal affected the response of both compounds to a similar degree, and the response of both compounds was essentially due to Ca(2+) release from intracellular stores. This was also confirmed by measuring the [Ca(2+)](i) response of single cells: both compounds induced [Ca(2+)](i) oscillations at subnanomolar concentrations and elicited a large peak increase in [Ca(2+)](i) at higher concentrations (EC(50)=0.5+/-0.04 and 5.7+/-1.9 nM for CCK-8-S and SR146131, respectively). Both CCK-8-S and SR146131 induced a sustained increase of phosphoinositide turnover in these cells, and acted at similar concentrations (EC(50)=2.7+/-0.7 and 6+/-3.1 nM, respectively), although the maximal effect of SR146131 was somewhat lower than the effect of CCK-8-S. These data show that SR146131 activates human CCK(1) receptors on IMR-32 cells in a manner and with a potency similar to that of CCK-8-S.     

Evidence against adenosine analogues being agonists at the growth hormone secretagogue receptor

Adenosine and adenosine analogues have been reported to act as agonists or partial agonists at the growth hormone secretagogue receptor 1a (GHSR1a). We have re-examined this question. A concentration-dependent increase in intracellular calcium concentration ([Ca(2+)](i)) was observed in GHSR1a transfected HEK 293-EBNA cells stimulated with adenosine (EC50: 0.2 microM) or 2-chloroadenosine (EC50: 1.1 microM) but also in untransfected HEK 293-EBNA cells stimulated with 2-chloroadenosine (EC50: 0.67 microM) or 5'-N-ethylcarboxamidoadenosine (NECA) (EC50: 0.045 microM). These findings support endogenous expression of adenosine receptors, presumably A(2B) receptors in HEK 293-EBNA cells. In GHSR1a transfected CHO cells, lacking adenosine receptors, the GHSR1a agonist hGhrelin (EC50: 2.4 nM) increased [Ca(2+)](i), but no effects of adenosine, 2-chloroadenosine or NECA were detected. An inverse agonist of GHSR1a, [d-Arg-1, d-Phe-5, d-Trp-7, 9, Leu-11] substance P, reduced hGhrelin effects but adenosine, 2-chloroadenosine or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not. NECA increased the [Ca(2+)](i) in co-transfected (GHSR1a and A(2B) receptor) CHO cells (EC50: 0.053 microM), but no additive or synergistic effects on [Ca(2+)](i) or cAMP formation were observed after stimulation with NECA in the absence or in the presence of hGhrelin. In binding studies on GHSR1a transfected CHO cell membranes, [(125)I]-hGhrelin binding could be displaced by the GHSR1a agonist MK-0677 (IC50: 0.34 nM), hGhrelin (IC50: 1.5 nM), and the substance P analogue (IC50: 0.64 microM) but not by adenosine or 2-chloroadenosine. We conclude that adenosine and analogues do not act as agonists or partial agonists at the GHSR1a and that cross-talk between the GHSR1a and A(2B) receptors is limited.