Vasopressin V2 Receptor Agonist Research Articles

Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V₂ receptor (V₂R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V₂R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus.

Hyponatremia in Patients Treated With Terlipressin for Severe Gastrointestinal Bleeding Due to Portal Hypertension

Terlipressin is frequently used in acute variceal bleeding due to its powerful effect on vasopressin V1 receptors. Although terlipressin is also a partial agonist of renal vasopressin V2 receptors, its effects on serum sodium concentration have not been specifically investigated. To examine the effects of terlipressin on serum sodium concentration in patients with acute portal-hypertensive bleeding, 58 consecutive patients with severe portal-hypertensive bleeding treated with terlipressin were investigated. In the whole population, serum sodium decreased from 134.9 ± 6.6 mEq/L to 130.5 ± 7.7 mEq/L (P = 0.002). Thirty-nine patients (67%) had a decrease in serum sodium ≥ 5 mEq/L during treatment: in 18 patients (31%), between 5 and 10 mEq/L and in 21 patients (36%), greater than 10 mEq/L. In this latter group, serum sodium decreased from 137.2 ± 5 to 120.5 ± 5 mEq/L (P < 0.001). In multivariate analysis, the reduction in serum sodium was related to baseline serum sodium and Model for End-Stage Liver Disease (MELD) score; patients with low MELD and normal or near-normal baseline serum sodium had the highest risk of hyponatremia. Serum sodium returned to baseline values in most patients shortly after cessation of therapy. Three of the 21 patients with marked reduction in serum sodium developed neurological manifestations, including osmotic demyelination syndrome in one patient due to a rapid recovery of serum sodium (serum sodium in these three patients decreased from 135, 130, and 136 to 117, 114, and 109 mEq/L, respectively). An acute reduction in serum sodium concentration is common during treatment with terlipressin for severe portal-hypertensive bleeding. It develops rapidly after start of therapy, may be severe in some patients and is associated with neurological complications, and is usually reversible after terlipressin withdrawal.

The role of protein kinase C in the establishment of the mechanism of vasopressin antidiuretic action in the rat kidney during mammalian postnatal development

The kidney of immaturely born mammals in early postnatal development is insensitive to the effect of the antidiuretic hormone, vasopressin. It has been demonstrated that water permeability of the epithelial cells in the collecting ducts of a rat kidney increases during development; in this process, the response to desmopressin, an agonist of vasopressin V2 receptors, appears at the age of 20 days. The observed increase in water permeability is connected with an increased content of the water channel's proteins aquaporins AQP2 and AQP3 in the plasma membrane. The calcium-dependent protein kinase C isoforms are the likely components of the vasopressin signal's transduction and are possibly involved in the mechanisms underlying the maturation of sensitivity to this hormone. The contents of three protein kinase C isoforms (alpha, delta, and zeta) in rats at different periods of their postnatal development were estimated using Western blot hybridization. It has been shown that the contents of protein kinase C isoforms alpha and delta increase with development, whereas the content of isoform zeta remains constant. The most likely participant of the mechanism providing for maturation of the cell's hormonal competence for vasopressin is the calcium-dependent protein kinase Ca, because it's content in the plasma membrane is maximal on days 20-24, which coincides with the time when the vasopressin action appears.

Administration of vasopressin V2 receptor agonist compensated the peripheral but not the central behavioral effects of the vasopressin deficiency in Brattleboro rats

Administration of vasopressin V2 receptor agonist compensated the peripheral but not the central behavioral effects of the vasopressin deficiency in Brattleboro rats

New Benzylureas as a Novel Series of Potent, Nonpeptidic Vasopressin V2 Receptor Agonists

Vasopressin (AVP) is a hormone that stimulates an increase in water permeability through activation of V2 receptors in the kidney. The analogue of AVP, desmopressin, has proven an effective drug for diseases where a reduction of urine output is desired. However, its peptidic nature limits its bioavailability. We report herein the discovery of potent, nonpeptidic, benzylurea derived agonists of the vasopressin V2 receptor. We describe substitutions on the benzyl group to give improvements in potency and subsequent modifications to the urea end group to provide improvements in solubility and increased oral efficacy in a rat model of diuresis. The lead compound 20e (VA106483) is reported for the first time and has been selected for clinical development.

Lack of elevation of urinary albumin excretion among patients with chronic syndromes of inappropriate antidiuresis

A recent study has revealed that acute and chronic administration of the vasopressin V2 receptor (V2R) agonist dDAVP induced a marked increase of urinary albumin excretion (UAE) in healthy rats and humans (Bardoux P et al. Nephrol Dial Transplant 2003; 18: 497-506). The occurrence of an elevation of UAE among patients with chronic syndromes of inappropriate antidiuresis has not been reported. We looked for the elevation of UAE in 24-h urine samples of the following patients: nine chronic SIADH patients, two patients with acute post-operative SIADH, three patients of the same family with nephrogenic syndrome of inappropriate antidiuresis (NSAID) and two patients with hyponatraemia due to surdosage of dDAVP in the setting of central diabetes insipidus. There was no elevation of UAE in our patients (whether they presented with hyponatraemia or not), apart from a patient treated with supra-physiological doses of dDAVP. When she received 80 microg/day of dDAVP, her UAE was 42 mg/day. In this patient, UAE returned to the normal range (21 mg/day) when doses of dDAVP were tapered (20 microg/day). The present study shows that chronic V2R stimulation generally does not result in a rise in UAE. The discrepancy between our results and those of the above-mentioned study could be explained by a dose-dependent effect of V2R stimulation on UAE.

Vasopressin V2 receptor (V2R)‐mediated potentiation of platelet activating factor (PAF)‐induced increase in rat mesentery venular microvessel permeability

We tested the hypothesis that V2R activation potentiates the increase in microvessel permeability induced by PAF, an inflammatory mediator involved in sepsis. Individual venular microvessels in rat mesentery were perfused with 10mg/ml albumin in Ringer to measure hydraulic conductivity (Lp) by the modified Landis technique. The vessels were exposed to PAF (10nM), which induced an Lp increase peaking in 5–10min followed by return toward baseline values. The same vessels were then exposed to the selective V2R agonist desmopressin (dDAVP) for 40min followed by a second exposure to PAF in the presence of dDAVP. Peak PAF‐induced Lp significantly increased at dDAVP concentrations of 30pM (2.1 fold ±0.4 SEM, n=8) and 100pM (2.8 fold ±0.9 SEM, n=5) relative to peak Lp with PAF alone. In contrast, 10pM dDAVP did not increase PAF‐induced peak Lp. In control experiments, a second exposure to PAF alone did not cause an increase in peak Lp relative to the first PAF exposure. Also, dDAVP alone had no effect on baseline Lp. In further experiments, the V2R antagonist AdAc‐D‐Tyr(Et)‐Phe‐Val‐Asn‐Abu‐Pro‐Arg‐Arg‐NH2 (86nM) abolished potentiation by dDAVP (30pM) of PAF‐induced peak Lp (n=6). These results show that V2R activation potentiates PAF‐induced increase in vascular permeability, suggesting that V2R activation may exacerbate vascular leak syndrome in conditions such as sepsis. Supported by Ferring Research Institute Inc.

Vasopressin regulation of inner medullary collecting ducts and compensatory changes in mice lacking adenosine A1 receptors

Activation of adenosine A(1) receptors (A(1)R) can inhibit arginine vasopressin (AVP)-induced cAMP formation in isolated cortical and medullary collecting ducts. To assess the in vivo consequences of the absence of A(1)R, we performed experiments in mice lacking A(1)R (A(1)R(-/-)). We assessed the effects of the vasopressin V(2) receptor (V(2)R) agonist 1-desamino-8-d-arginine vasopressin (dDAVP) on cAMP formation in isolated inner medullary collecting ducts (IMCD) and on water excretion in conscious water-loaded mice. dDAVP-induced cAMP formation in isolated IMCD was significantly greater ( approximately 2-fold) in A(1)R(-/-) compared with wild-type mice (WT) and, in contrast to WT, was not inhibited by the A(1)R agonist N6-cyclohexyladenosine. A(1)R(-/-) and WT had similar basal urinary excretion of vasopressin, expression of aquaporin-2 protein in renal cortex and medulla, and acute increases in urinary flow rate and electrolyte-free water clearance in response to the V(2)R antagonist SR121463 or acute water loading; the latter increased inner medullary A(1)R expression in WT. Dose dependence of dDAVP-induced antidiuresis after acute water loading was not different between the genotypes. However, A(1)R(-/-) had greater inner medullary expression of cyclooxygenase-1 under basal conditions and of the P2Y(2) and EP(3) receptor in response to water loading compared with WT mice. Thus vasopressin-induced cAMP formation is enhanced in isolated IMCD of mice lacking A(1)R, but the adenosine-A(1)R/V(2)R interaction demonstrated in vitro is likely compensated in vivo by multiple mechanisms, a number of which can be "uncovered" by water loading.

Investigation of mechanism of desmopressin binding in vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors: Molecular dynamics simulation of the agonist‐bound state in the membrane–aqueous system

The vasopressin V2 receptor (V2R) belongs to the Class A G protein-coupled receptors (GPCRs). V2R is expressed in the renal collecting duct (CD), where it mediates the antidiuretic action of the neurohypophyseal hormone arginine vasopressin (CYFQNCPRG-NH2, AVP). Desmopressin ([1-deamino, 8-D]AVP, dDAVP) is strong selective V2R agonist with negligible pressor and uterotonic activity. In this paper, the interactions responsible for binding of dDAVP to vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors has been examined. Three-dimensional activated models of the receptors were constructed using the multiple sequence alignment and the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(alpha) (338-350) prototype (Slusarz, R.; Ciarkowski, J. Acta Biochim Pol 2004 51, 129-136) as a template. The 1-ns unconstrained molecular dynamics (MD) of receptor-dDAVP complexes immersed in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane model was conducted in an Amber 7.0 force field. Highly conserved transmembrane residues have been proposed as being responsible for V2R activation and G protein coupling. Molecular mechanism of the dDAVP binding has been suggested. The internal water molecules involved in an intricate network of the hydrogen bonds inside the receptor cavity have been identified and their role in the stabilization of the agonist-bound state proposed.

Quantitative Cell-Based High-Content Screening for Vasopressin Receptor Agonists Using Transfluor®Technology

The authors demonstrate the use of a simple, universal G-protein-coupled receptor (GPCR) assay to screen for agonists for a specific GPCR. Cells stably expressing a green fluorescent protein (GFP)-labeled beta-arrestin fusion protein and the vasopressin V2 receptor (V2R) were used in a high-content screening (HCS) assay to screen a small peptide library for V2R agonists. Cells were treated with the peptides at a final concentration of 500 nM for 30 min. Agonist stimulation causes V2R internalization into endosomes. GFP-beta-arrestin remains associated with the V2R in endosomes, resulting in a fluorescent pattern of intracellular spots. Assay plates were automatically imaged and quantitatively analyzed using an HCS imaging platform and a fast turnkey image analysis application optimized for detection of receptor activation and intracellular spots. Hits were further evaluated to determine their potency. The combination of unique biology, automated high-content analysis, and a powerful means of validating hits results in better leads.

Antidiuretic Effects of a Novel Nonpeptide Vasopressin V2-Receptor Agonist, OPC-51803, Administered Orally to Dogs

We elucidated the pharmacological properties of a novel nonpeptide vasopressin V2-receptor agonist, OPC-51803 ((5R)-2-[1-(2-chloro-4-(1-pyrrolidinyl)benzoyl-2,3,4,5-tetra-hydro-1H-1-benzazepine-5-yl]-N-isopropylacetamide), via both in vitro binding experiments incorporating canine kidney and platelet membrane fractions and in vivo experiments that would determine the compound’s antidiuretic effects after oral administration to water-loaded dogs. OPC-51803 displaced [3H]arginine vasopressin (AVP) binding to canine V2 and V1a receptors, as determined by resulting Ki values of 15.2 ± 0.6 nM (n = 4) and 653 ± 146 nM (n = 4), respectively. These data indicate that OPC-51803 was about 43 times more selective for V2 receptors than for V1a receptors. Antidiuretic studies showed that orally administered doses of OPC-51803 (0.03 to 0.3 mg · kg−1) decreased urine volume and increased urinary osmolality in a dose-dependent manner in water-loaded dogs. Intravenous OPC-51803 infusions (0.3 and 3 μg · kg−1 · min−1) did not affect renal or systemic hemodynamics in anesthetized dogs. Since these results confirm that OPC-51803 shows antidiuretic action in dogs, the compound may be useful for treating AVP-deficient pathophysiological states.

Increased contraction to noradrenaline by vasopressin in human renal arteries

Arginine vasopressin (AVP) not only acts directly on blood vessels through vasopressin V1 receptor stimulation but also may modulate adrenergic-mediated responses in animal experiments. The aim of the present study was to assess whether subpressor concentrations of AVP could contribute to an abnormal adrenergic contractile response of human renal arteries. Renal artery rings were obtained from 27 patients undergoing nephrectomy. The rings were suspended in organ bath chambers for isometric recording of tension. AVP (10(-10) mol/l) and the vasopressin V1 receptor agonist [Phe2, Orn8]-vasotocin (10(-10) mol/l) produced a leftward shift of the concentration-response curve to noradrenaline (half-maximal effective concentration decreased from 1.1 x 10(-6) mol/l to 3.1 x 10(-7) mol/l). The enhancement of noradrenaline-induced contractions was inhibited by the vasopressin V1 receptor antagonist d(CH2)5Tyr(Me)AVP (10-8 mol/l) and unaffected by endothelium removal or pretreatment with the inhibitor of nitric oxide (NO) synthase NG-monomethyl-l-arginine (l-NMMA). The vasopressin V2 receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP) (10(-10)-10(-8) mol/l) did not modify contractile responses to noradrenaline. In the presence of the dihydropyridine calcium antagonist nifedipine (10(-6) mol/l), vasopressin failed to enhance the contractile response to noradrenaline. The results demonstrate that subpressor concentrations of vasopressin potentiate the contractile effects of noradrenaline without intervention of the NO system. The effects appear to be mediated by vasopressin V1 receptor stimulation, which brings about an increase in calcium entry through dihydropyridine-sensitive calcium channels.

An Efficient Synthesis of a Key Inter- mediate for Vasopressin V2 Receptor Agonist OPC-51803 by Lipase-catalyzed Enantioselective Transesterification

An efficient and enantioselective synthesis of (R)-[1-(p-toluene-sulfonyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid (2), a key intermediate in the synthesis of (R)-2-{1-[2-chloro-4-(1-pyrrolidinyl)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}-N-isopropylacetamide (OPC-51803, 1), was accomplished. The chiral alcohol [(S)-3], the precursor of (R)-2, was separated from the racemic alcohol [(′)-3] using the lipase mediated transesterification in vinyl acetate. The obtained (S)-3 was fractionated without using column chromatography and it was converted to 2 without a decrease in the enantiomeric excess.

Responses of regional kidney perfusion to vasoconstrictors in anaesthetized rabbits: dependence on agent and renal artery pressure.

1. We tested the effects of intravenous infusions of angiotensin II (AngII; 300 ng/kg per min) and the vasopressin V1 receptor agonist [Phe2,Ile3,Orn8]-vasopressin (30 ng/kg per min) on regional kidney perfusion in an extracorporeal circuit model in anaesthetized rabbits in which renal artery pressure (RAP) can be set independently of systemic mean arterial pressure. To test whether the level of RAP can influence the renal vascular response to [Phe2,Ile3,Orn8]-vasopressin, we compared its effects when RAP was initially set at approximately 65 mmHg with those when RAP was set at approximately 130 mmHg. 2. When RAP was initially set at approximately 65 mmHg, a 20min infusion of AngII increased RAP (13%) and reduced renal blood flow (RBF; 50%) and cortical perfusion (CBF; 43%). Medullary perfusion (MBF) transiently increased during the first 10 min of infusion, but was not significantly different from control levels during the final 5 min of infusion. 3. When RAP was initially set at approximately 65 mmHg, a 20 min infusion of [Phe2,Ile3,Orn8]-vasopressin increased RAP (9%) and reduced RBF (21%); MBF was reduced by 57%, but CBF was reduced by only 15%. In contrast, when RAP was initially set at approximately 130 mmHg, infusion of [Phe2,Ile3,Orn8]-vasopressin reduced RAP (7%) and increased RBF (13%). In these experiments, MBF was reduced by 38%, but CBF increased by 6%. 4. Our experiments show that AngII preferentially reduces CBF, while [Phe2,Ile3,Orn8]-vasopressin preferentially reduces MBF. The renal vascular responses to [Phe2,Ile3,Orn8]-vasopressin appear to be profoundly affected by the level of RAP, because increasing RAP from approximately 65 to approximately 130 mmHg transforms its cortical vasoconstrictor effect into cortical vasodilatation while leaving the response of the medullary microvasculature relatively unchanged. Whether renal vascular responses to other vasoactive agents (e.g. AngII) are similarly affected by the level of RAP remains to be determined.

Vasopressin V2 (SR121463A) and V1a (SR49059) receptor antagonists both inhibit desmopressin vasorelaxing activity

Although [Arg 8]vasopressin is a potent vasoconstrictor, it possesses vasorelaxant properties manifested either after vasopressin V1 receptor blockade or directly in some vascular beds. The nature of the receptor involved in the vasorelaxant effect of [deamino-Cys 1 d-Arg 8]vasopressin (desmopressin), a vasopressin V2 receptor agonist, was studied on rat precontracted aortic rings by the use of highly selective new non-peptide vasopressin receptor antagonists. The present study demonstrates for the first time that desmopressin relaxant effect is antagonized by the vasopressin V2 receptor antagonist SR121463A, but also by the vasopressin V1A receptor antagonist SR49059, suggesting that desmopressin-induced relaxation is mediated by a receptor subtype sharing both V1A and V2 pharmacological profiles.

Capacitative calcium entry in smooth muscle cells from preglomerular vessels.

Calcium entry via voltage-gated L-type channels is responsible for at least half of the increase in cytosolic calcium ([Ca(2+)](i)) in afferent arterioles following agonist stimulation. We sought the presence of capacitative calcium entry in fresh vascular smooth muscle cells (VSMC) derived from rat preglomerular vessels. [Ca(2+)](i) was measured using fura-2 ratiometric fluorescence. Vasopressin V1 receptor agonist (V1R) (10(-7) M) increased [Ca(2+)](i) by approximately 100 nM. A calcium channel blocker (CCB), nifedipine or verapamil (10(-7) M), inhibited the response by approximately 50%. V1R in the presence of CCB increased [Ca(2+)](i) from 106 to 176 nM, confirming that calcium mobilization and/or entry may occur independent of voltage-gated channels. In nominally Ca(2+)-free buffer, V1R increased [Ca(2+)](i) from 94 to 129 nM, denoting mobilization; addition of CaCl(2) (1 mM) further elevated [Ca(2+)](i) to 176 nM, indicating a secondary phase of Ca(2+) entry. Similar responses were obtained when CCB was present in calcium-free buffer or when EGTA was present. In nominally Ca(2+)-free medium, the sarcoplasmic reticulum Ca(2+)-ATPase inhibitors (SRCAI), thapsigargin and cyclopiazonic acid (CPA), increased [Ca(2+)](i) from 97 to 128 and 143 nM, respectively, and to 214 and 220 nM, respectively, when 1 mM extracellular Ca(2+) was added. In the presence of verapamil, the results with CPA acid were nearly identical. In Ca(2+)-free buffer, the stimulatory effect of V1R or SRCAI on the Ca(2+)/fura signal was quenched by the addition of Mn(2+) (1 mM), demonstrating divalent cation entry. These studies provide evidence for capacitative (store- operated) calcium entry in VSMC freshly isolated from rat preglomerular arterioles.

Arginine vasopressin-mediated stimulation of nitric oxide within the rat renal medulla.

The present study was designed to determine whether arginine vasopressin (AVP) can stimulate nitric oxide (NO) production within the renal medulla and thereby modulate renal medullary blood flow. An in vivo microdialysis/NO trapping technique was used to determine changes in medullary interstitial [NO]. AVP (2 ng/kg per minute) was delivered into the renal medullary interstitium and resulted in a significant increase in renal medullary [NO] of 35%, which was blocked by pretreatment with nitro-L-arginine methyl ester (L-NAME) (1.3 microg/kg per minute) administered into the renal medullary interstitium. The vasopressin V2 receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP) resulted in a significant increase of 32% in renal medullary interstitial [NO]. No change in renal medullary interstitial [NO] was observed after selective vasopressin V1 receptor stimulation. Laser-Doppler flowmetry with implanted optical fibers was performed to measure cortical and medullary blood flow changes within the kidney. Renal interstitial infusion of dDAVP in rats pretreated with a vasopressin V1 receptor antagonist resulted in a 15% increase (P<0.05) in medullary blood flow, which was completely blocked by pretreatment with L-NAME (1.3 microg/kg per minute). This study demonstrates that AVP increases renal medullary interstitial [NO] through vasopressin V2 receptor stimulation, which in turn elevates blood flow to the renal medulla.

Effects of long-term vasopressin receptor stimulation on medullary blood flow and arterial pressure.

Studies were carried out using instrumented unanesthetized rats to determine the long-term effects of arginine vasopressin (AVP) and a specific vasopressin V1 receptor agonist (V1AG; [Phe2, Ile3, Orn8]- vasopressin) on the renal medullary blood flow and arterial blood pressure. It was hypothesized that the hypertension observed with chronic medullary infusion of a V1 receptor agonist may be associated with a sustained reduction of blood flow, whereas infusion of AVP may fail to produce a sustained reduction of blood flow and thereby be unable to produce hypertension. Uninephrectomized Sprague-Dawley rats were prepared with implanted renal cortical and medullary optical fibers for daily measurements of cortical and medullary blood flow using laser-Doppler flowmetry techniques. An implanted renal medullary interstitial infusion catheter delivered either AVP or a specific V1AG at a dose of 2 ng . kg-1 . min-1 over a period of 5 days. The V1AG produced no change of cortical blood flow but a chronic 35% reduction of medullary blood flow (P < 0.05) and mild hypertension (11 +/- 4 mmHg, P < 0.05). AVP produced only an initial, nonsignificant 1- to 2-day reduction of medullary blood flow (-13%) and failed to raise arterial pressure significantly. We conclude that a sustained V1AG response is necessary to achieve a chronic reduction of medullary blood flow and hypertension. The present data are consistent with the idea that chronic stimulation of V2 receptors by AVP offsets the vasoconstrictor and hypertension actions of AVP-induced stimulation of medullary V1 receptors.

Response of isolated rat descending vasa recta to bradykinin.

Outer medullary descending vasa recta (OMDVR) were dissected from the outer medullary vascular bundles of young rats, perfused in vitro, and loaded with fura 2 for measurement of intracellular calcium concentration ([Ca2+]i) by fluorescent ratio imaging. Fluorescent video images revealed that fura 2 selectively loads into endothelial cells but not pericytes. Bradykinin (BK), at concentrations > 10(-11) M, elicited an increase in [Ca2+]i from baseline values in the range from 50 to 100 nM to peak values of 600-800 nM followed by a sustained plateau of 150-250 nM. The vasopressin V1-receptor agonist [Phe2,Ile3,Orn8]vasopressin constricted OMDVR but yielded no observable [Ca2+]i response, a finding that is consistent with an endothelial cell origin for the fura 2 fluorescent signal. The BK [Ca2+]i response was blocked by the selective BK B2-receptor antagonists D-Arg-[Hyp3,Thi5.8,D-Phe7]BK and D-Arg-[Hyp3,D-Phe7,Leu8]BK but not the B1 antagonist des-Arg9-[Leu8]BK. BK vasodilated microperfused OMDVR that had been preconstricted with 10(-8) M angiotensin II. We conclude that the [Ca2+]i response of OMDVR endothelia can be selectively studied with fura 2, that BK increases endothelial [Ca2+]i via the B2 receptor, and that BK can vasodilate descending vasa recta.

Role of Vasopressin in Neurocardiogenic Responses to Hemorrhage in Conscious Rats

Vasovagal reflexes, such as hypotension and bradycardia, are induced by rapid hemorrhage and mimic neurocardiogenic reflexes in mammals. We examined the role of vasopressin in the neurocardiogenic responses to mild, rapid hemorrhage (1 mL/100 g for 30 seconds) and severe hemorrhage (1 mL/100 g body wt for 30 seconds repeated three times at 11-minute intervals) in homozygous Brattleboro and Long-Evans rats. Mild, rapid hemorrhage induced severe bradycardia and hypotension only in Long-Evans rats. Exogenous vasopressin (1.85 pmol/kg per minute for 1 hour) restored both the bradycardic and hypotensive responses in Brattleboro rats. DDAVP, a vasopressin V2-receptor agonist (0.19 pmol/kg per minute for 24 hours), did not affect the cardiovascular responses to hemorrhage in Brattleboro rats, although it maintained urine production within normal limits. However, OPC-31260 (21.6 mumol/kg IV), a vasopressin V2-receptor antagonist, attenuated both the hypotensive and bradycardic responses to hemorrhage in Long-Evans rats. A vasopressin V1-receptor antagonist attenuated bradycardia and delayed the recovery of arterial pressure after hemorrhage but did not affect the hypotension that occurred immediately after hemorrhage in Long-Evans rats. Methylatropine also attenuated both the bradycardic and hypotensive responses induced by hemorrhage, but propranolol had no effect on the cardiovascular responses to hemorrhage in Long-Evans rats. The recovery of arterial pressure after repeated hemorrhage was less adequate in Brattleboro rats than in Long-Evans rats. Our results suggest that the neurocardiogenic responses to hemorrhage, especially hypotension, may be related to vasodilation induced by a V2-receptor-mediated mechanism and by the vagal reflex, both of which are substantiated by the existence of vasopressin. The coexistence of V1- and V2-receptor mechanisms may be necessary for the hypotensive response to hemorrhage. We found that a V2-receptor antagonist attenuated the hypotension mediated by the so-called neurocardiogenic reflex.