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PubMed1985

Functional involvement of vasopressin in the maintenance of systemic arterial blood pressures after phenoxybenzamine or phentolamine administration: studies in Long-Evans and Brattleboro rats.

The alpha adrenoceptor antagonists phenoxybenzamine and phentolamine are reported to have opposite effects on vasopressin release, the former inhibiting and the latter enhancing it. In this study we have assessed the functional involvement of vasopressin in the maintenance of blood pressure in conscious rats after administration of either phenoxybenzamine or phentolamine. In normal (Long-Evans) rats, phenoxybenzamine caused a small fall in arterial blood pressure, whereas phentolamine initially caused a profound hypotension which was followed by a fluctuating recovery back to normotensive levels. Similar effects were seen in rats deficient in hypothalamic vasopressin (Brattleboro strain). Administration of an antagonist of the cardiovascular actions of vasopressin [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid)-8-D-arginine vasopressin] in the presence of either alpha adrenoceptor antagonist alone was without effect in Long-Evans or Brattleboro rats, but, under these conditions, subsequent administration of captopril caused a profound and sustained hypotension in both strains. Administration of captopril in the presence of either alpha adrenoceptor antagonist alone caused a prompt fall in blood pressure which was sustained for the duration of the experiment in the Brattleboro rats. However, under these conditions, the blood pressure of the Long-Evans rats showed some recovery over the subsequent 45 min; this recovery was antagonized by [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid)-8-D-argine vasopressin]. It is concluded that after alpha adrenoceptor antagonism with either phenoxybenzamine or phentolamine, the renin-angiotensin system exerts a major pressor influence. However, after captopril administration in the presence of phenoxybenzamine or phentolamine, vasopressin contributes to the maintenance of arterial blood pressure in Long-Evans rats; the magnitude of this contribution is similar irrespective of the alpha adrenoceptor antagonist used.

Adrenergic alpha-Agonists↗
PubMed1996

Central vasopressin blockade enhances its peripheral release in response to peripheral osmotic stimulation in conscious rats.

Increased plasma osmolality results in increased central as well as peripheral release of vasopressin. Experiments were carried out to determine whether, in this circumstance, vasopressin can act centrally to modulate its peripheral release. Prior to the start of a thirty-min i.v. infusion of 2.5 M or 0.15 M NaCl, the rats were given an intracerebroventricular (i.c.v.) injection of a peptide V1/V2 vasopressin antagonist (2 micrograms), OPC-31260 (60 micrograms), a non-peptide V2 antagonist, or 1-desamino-8-D-arginine vasopressin (dDAVP, 5 ng), a V2 agonist. Experiments with the peptide antagonist were carried out in male and non-estrous female rats. Since there were no differences between males and females in the measured responses, experiments with the other two drugs were carried out only in males. Pretreatment with either the V1/V2 antagonist or the V2 antagonist enhanced the increase in plasma vasopressin levels in response to the hypertonic saline infusion by about 50% at the end of 30 min. dDAVP, on the other hand, had no effect. None of the i.c.v. drugs had an affect on either the pressor or bradycardic responses to hypertonic saline infusion. These observations suggest that vasopressin can act centrally in a negative feedback fashion to attenuate its own release into the peripheral circulation in response to increased plasma osmolality.

Animals↗
PubMed1996

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.

Acute Disease↗
PubMed2005

Vasopressin for cardiac arrest: a systematic review and meta-analysis.

BACKGROUND: The current guidelines for cardiopulmonary resuscitation recommend vasopressin as an alternative to epinephrine for the treatment of adult shock-refractory ventricular fibrillation. The objective of this study was to determine the effectiveness of vasopressin in the treatment of cardiac arrest. METHODS: We performed a systematic review and meta-analysis of 1519 patients with cardiac arrest from 5 randomized controlled trials that compared vasopressin and epinephrine. Two reviewers conducted a systematic search of electronic databases, complemented by hand searches, to identify randomized trials. Reviewers evaluated the quality of the trials, extracted data, and derived pooled estimates using a random-effects model. RESULTS: There were no statistically significant differences between the vasopressin and epinephrine groups in failure of return of spontaneous circulation (risk ratio [RR], 0.81; 95% confidence interval [CI], 0.58-1.12), death before hospital admission (RR, 0.72; 95% CI, 0.38-1.39), death within 24 hours (RR, 0.74; 95% CI, 0.38-1.43), death before hospital discharge (RR, 0.96; 95% CI, 0.87-1.05), or combination of number of deaths and neurologically impaired survivors (RR, 1.00; 95% CI, 0.94-1.07). Subgroup analysis based on initial cardiac rhythm showed no statistically significant difference in the rate of death before hospital discharge between the vasopressin and epinephrine groups in any of the 3 subgroups: ventricular fibrillation or ventricular tachycardia (RR, 0.97; 95% CI, 0.79-1.19), pulseless electrical activity (RR, 1.02; 95% CI, 0.95-1.10), or asystole (RR, 0.97; 95% CI, 0.94-1.00). CONCLUSIONS: There is no clear advantage of vasopressin over epinephrine in the treatment of cardiac arrest. Guidelines for Advanced Cardiac Life Support should not recommend vasopressin in resuscitation protocols until more solid human data on its superiority are available.

Bias↗
PubMed1978

Radioimmunoassayable plasma vasopressin associated with surgery.

Plasma vasopressin concentration was measured by radioimmunoassay before, during, and after anesthesia and surgery in ten subjects. During the short period between the onset of anesthesia and the start of operation, small elevations of vasopressin level were noted. Surgery itself was associated with significant elevations of up to 82 pg/ml. Highest levels of vasopressin were noted with major intra-abdominal surgery and lowest levels with limb surgery. The immediate postoperative period was marked by plasma vasopressin levels that were often higher than during surgery itself. Levels gradually fell to their preoperative state after three to four days. The elevated levels of vasopressin can be associated with oliguria and excessive water retention. Among the possible mechanisms for the stimulus to vasopressin secretion are pain, stress, positive pressure respiration and anoxia. This study confirms by radioimmunoassay the changes in plasma vasopressin level with surgery that have been previously described by bioassay.

Anesthesia, General↗
PubMed1984

Cerebrospinal fluid vasopressin and increased intracranial pressure.

Cerebrospinal fluid and plasma vasopressin were measured in patients with cerebral disorders associated with varying levels of elevated intracranial pressure. The mean cerebrospinal fluid vasopressin concentration was significantly increased in patients with pseudotumor cerebri (2.0 +/- 0.2 [SEM] pg/ml), intracranial tumor (2.3 +/- 0.4 pg/ml), and intracranial hemorrhage (1.9 +/- 0.3 pg/ml) compared with control patients (1.2 +/- 0.1 pg/ml). A significant relationship was found between intracranial pressure and the cerebrospinal fluid vasopressin concentration within all groups of patients and in the whole sample as well (r = 0.79; p less than 0.001). In the groups of patients with intracranial tumor, hydrocephalus, and intracranial hemorrhage, some individuals showed plasma vasopressin concentrations inappropriate to the corresponding plasma osmolality, but no relationship was found between intracranial pressure and plasma vasopressin concentration. It is suggested that increased intracranial pressure is a stimulus to centrally released vasopressin. The clinical importance of increased cerebrospinal fluid vasopressin concentrations is still not known.

Adolescent↗
PubMed1985

Stimulus for vasopressin release during elective intra-abdominal operations.

In 12 patients undergoing an upper abdominal operation, blood pressure and peripheral venous blood samples were taken at intervals throughout the procedure. There was no significant increase in plasma vasopressin concentration after induction of anaesthesia or skin incision; within 3 min of opening the peritoneum and commencing intraperitoneal manipulation there was a highly significant rise (P less than 0.01), maintained with fluctuations until closure of the abdomen. There was no correlation between the changes of blood pressure and those in plasma vasopressin level. In 16 patients undergoing elective cholecystectomy similar observations were made to coincide with events believed, on the basis of the first study, to be related to changes in the concentration of vasopressin. There was a significant rise in vasopressin concentration (P less than 0.01) after incision of the peritoneum, 1 min after the start of intraperitoneal manipulation (P less than 0.01) and after deliberate traction on the stomach (P less than 0.01). During operative cholangiography, when there was no intraperitoneal manipulation, there was a significant fall in the vasopressin level. There was no correlation between changes in vasopressin concentration and blood pressure. These findings indicate that during an abdominal operation nervous stimuli, arising from within the peritoneal cavity and probably mediated via the autonomic system, are an important factor responsible for the increased secretion of vasopressin, and, at least in the absence of major changes in blood pressure and osmolality, the determinant factor.

Abdomen↗
PubMed1992

Intracellular calcium-mediated activation of hepatic Na+/H+ exchange by arginine vasopressin and phenylephrine.

The effect of Ca++ mobilizing agonists arginine vasopressin and phenylephrine on Na+/H+ exchange was studied in freshly isolated hepatocytes and isolated perfused rat livers. The activity of Na+/H+ exchange was determined from the rate of H+ efflux, 22Na uptake and pHi recovery. Arginine vasopressin and phenylephrine stimulated H+ efflux and 22Na uptake in isolated rat hepatocytes and increased the rate of pHi recovery from acid-loaded hepatocytes. These effects were inhibited by amiloride. Arginine vasopressin- and phenylephrine-induced increases in H+ efflux were also dependent on extracellular Na+. Arginine vasopressin- and phenylephrine-induced increases in intracellular Ca++ concentration, H+ efflux, 22Na uptake and intracellular pH recovery were decreased in hepatocytes preloaded with the Ca(++)-buffering agent [bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid] (MAPTA). Na+/H+ exchange-dependent intracellular pH recovery from cytosolic acidification was stimulated by thapsigargin, which increases intracellular calcium concentration by inhibiting endoplasmic reticulum Ca++ ATPase. Arginine vasopressin- and phenylephrine-induced increases in intracellular pH recovery were not dependent on extracellular Ca++ and were inhibited by calmidazolium, a calmodulin inhibitor. Arginine vasopressin and phenylephrine also increased H+ efflux in the absence but not in the presence of amiloride in perfused rat livers without affecting biliary HCO3- excretion. These results indicate that arginine vasopressin and phenylephrine activate Na+/H+ exchange in rat hepatocytes, an effect mediated in part by intracellular Ca++ and calmodulin kinase. Furthermore, sinusoidal Na+/H+ exchange does not appear to be involved in biliary HCO3- excretion.

Animals↗
PubMed1994

Reduction of gastric hyperemia by glypressin and vasopressin administration in cirrhotic patients with portal hypertensive gastropathy.

Gastric mucosal perfusion is increased in portal-hypertensive gastropathy, and this may contribute to gastric bleeding from these lesions. Therefore drugs reducing gastric mucosal perfusion may be beneficial in the treatment of overt bleeding from portal-hypertensive gastropathy. In this study gastric mucosal perfusion was assessed in 28 cirrhotic patients with portal-hypertensive gastropathy under basal conditions and after double-blind intravenous administration of vasopressin (0.4 U/min), glypressin (2-mg injection) or placebo, with laser-Doppler flowmetry and reflectance spectrophotometry. Vasopressin and glypressin induced a significant increase in blood pressure and a decrease in heart rate. These effects were more pronounced in the vasopressin group. Both vasopressin and glypressin induced a sustained and similar reduction in gastric mucosal perfusion as assessed by laser-Doppler flowmetry (-36% +/- 8% and -34% +/- 6%, respectively; p < 0.05 with respect to basal values and with respect to the control group), whereas placebo had no effect. Both drugs significantly reduced the oxygen content of the gastric mucosa; however, the impairment in mucosal oxygenation was greater (p < 0.05) in the vasopressin group (-17% +/- 3%) than in the glypressin group (-6% +/- 0.1%). We conclude that the increased gastric perfusion in cirrhotic patients with portal-hypertensive gastropathy may be reduced by either vasopressin or glypressin. These findings support the use of these drugs in clinical trials treating bleeding portal-hypertensive gastropathy. The lower reduction in gastric mucosal oxygen content observed with glypressin could decrease the incidence of ischemic adverse events associated with the use of vasopressin.

Double-Blind Method↗
PubMedDate not supplied

Regulated expression of GRP78 during vasopressin-induced hypertrophy of heart-derived myocytes.

Although the development of cellular hypertrophy is widely believed to involve Ca(2+) signaling, potential supporting roles for sequestered Ca(2+) in this process have not been explored. H9c2 cardiomyocytes respond to arginine vasopressin with an initial mobilization of Ca(2+) stores and reduced rates of mRNA translation followed by repletion of Ca(2+) stores, up-regulation of translation beyond initial rates, and the development of hypertrophy. Rates of synthesis of the endoplasmic reticulum (ER) chaperones, GRP78 and GRP94, were found to increase preferentially at early times of vasopressin treatment. Total GRP78 content increased 2- to 3-fold within 8 h after which the chaperone was subject to post-translational modification. Preferential synthesis of GRP78 and the increase in chaperone content both occurred at pM vasopressin concentrations and were abolished at supraphysiologic Ca(2+) concentrations. Co-treatment with phorbol myristate acetate decreased vasopressin-dependent Ca(2+) mobilization and slowed appearance of new GRP78 molecules in response to the hormone, whereas 24 h pretreatment with phorbol ester prolonged vasopressin-dependent Ca(2+) mobilization and further increased rates of GRP78 synthesis in response to the hormone. Findings did not support a role for newly synthesized GRP78 in translational up-regulation by vasopressin. However up-regulation, which does not depend on Ca(2+) sequestration, appeared to expedite chaperone expression. This report provides the first evidence that a Ca(2+)-mobilizing hormone at physiologic concentrations signals increased expression of GRP78. Translational tolerance to depletion of ER Ca(2+) stores, typifying a robust ER stress response, did not accompany vasopressin-induced hypertrophy.

Animals↗
PubMed1995

Glucocorticoid amplifies vasopressin-induced phosphoinositide hydrolysis in aortic smooth muscle cells.

It has been reported that glucocorticoid modifies phosphoinositide (PI) hydrolysis stimulated by vasoactive agents in vascular smooth muscle cells. In the present study, we investigated the point at which glucocorticoid affects vasopressin-induced PI hydrolysis in primary cultured rat aortic smooth muscle cells. The pretreatment with dexamethasone significantly amplified the formation of inositol trisphosphate (IP3) induced by vasopressin in a dose-dependent manner in a range of 1 pM to 10 nM. The effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone had little effect on the number of vasopressin receptor and its affinity to vasopressin. The pretreatment with dexamethasone also amplified the formation of IP3 induced by NaF, a GTP-binding protein activator, or angiotensin II. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase C (PKC)-activating phorbol ester, significantly reduced the dexamethasone-induced enhancement of IP3 formation stimulated by vasopressin, angiotensin II or NaF 4 alpha-Phorbol-12, 13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the enhancement by dexamethasone. These results strongly suggest that glucocorticoid amplifies vasopressin-induced PI hydrolysis at a point downstream from GTP-binding protein in primary cultured rat aortic smooth muscle cells, and that the activation of PKC has a negative feedback effect on the amplification by glucocorticoid of vasopressin-induced PI hydrolysis.

Animals↗
PubMed1994

Synergistic stimulation of DNA synthesis by bradykinin and vasopressin in Swiss 3T3 cells.

Vasopressin and bradykinin bind to receptors coupled to GTP-binding proteins and rapidly induce polyphosphoinositide breakdown leading to Ca2+ mobilization and activation of protein kinase C. Both peptides are known to induce mitogenesis in the presence of growth factors that act through receptors with intrinsic tyrosine kinase activity. Surprisingly, addition of a combination of vasopressin and bradykinin to Swiss 3T3 cells synergistically stimulates DNA synthesis in the absence of any other growth factors. This effect is induced at nanomolar concentrations of the peptides and could be inhibited by addition of specific receptor antagonists or broad spectrum neuropeptide antagonists. Bradykinin, which stimulates transient activation of protein kinase C, induces DNA synthesis in synergy with substances that cause long-term activation of protein kinase C, like vasopressin or phorbol 12,13-dibutyrate. Down-regulation of protein kinase C inhibited the induction of mitogenesis by the combination of vasopressin and bradykinin, thus demonstrating the importance of long-term activation of this enzyme for DNA synthesis. Analysis of tyrosine phosphorylated proteins of M(r) = 110,000-130,000 and M(r) = 70,000-80,000 revealed a biphasic response after stimulation with bradykinin, whereas the response induced by vasopressin declined after the initial maximum. The combination of bradykinin with vasopressin caused an enhanced and prolonged increase in tyrosine phosphorylation of these proteins as compared with the individual peptides. Inhibition of tyrosine phosphorylation by tyrphostin was paralleled by inhibition of DNA synthesis. Together, these results demonstrate synergistic stimulation of DNA synthesis by bradykinin and vasopressin via prolonged stimulation of multiple signaling pathways and imply that the interactive effects of Ca(2+)-mobilizing peptides on mitogenesis may be more general than previously thought.

3T3 Cells↗
PubMed1988

Demonstration of glucocorticoid receptor-like immunoreactivity in glucocorticoid-sensitive vasopressin and corticotropin-releasing factor neurons in the hypothalamic paraventricular nucleus.

Many parvocellular neurons in the paraventricular nucleus of the hypothalamus express high levels of corticotropin releasing factor (CRF) or vasopressin following adrenalectomy. To determine whether glucocorticoids feed back directly on these neurons, a mouse monoclonal antibody directed against the rat liver glucocorticoid receptor was used in combination with polyclonal antisera directed against either vasopressin or CRF to permit simultaneous visualization of either peptide with glucocorticoid receptor-like immunoreactivity (IR). Rats were adrenalectomized (ADX) for 2 weeks to optimize numbers of vasopressin - and CRF-IR neurons. Six hours prior to sacrifice, a separate group of adrenalectomized rats was treated with corticosterone (40 mg/kg). This short-term replacement resulted in nuclear localization of glucocorticoid receptor-like-IR but did not attenuate the increased numbers of CRF- and vasopressin-IR neurons observed after adrenalectomy. It was therefore possible to visualize vasopressin- or CFR-IR and nuclear glucocorticoid receptor-like-IR simultaneously. Cell counts of double-labeled neurons in the paraventricular nucleus of the hypothalamus (PVH) demonstrated that glucocorticoid receptor-like-IR is colocalized in virtually all the CRF and vasopressin immunoreactive parvocellular neurons studied, which respond to adrenalectomy by increased peptide expression. These data suggest that a major feedback effect of glucocorticoids on the hypothalamic-pituitary-adrenal axis is exerted directly within nuclei of CRF and vasopressin neurons.

Adrenalectomy↗
PubMed1979

Effect of metabolic inhibitors on vasopressin-stimulated transport systems in the toad bladder.

Vasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin-stimulated water flow or vasopressin-stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally. Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP-stimulated water flow but showed no effect on urea transport. We would conclude that water and urea transport, as examples of hormone-stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non-nucleotide pathway may be involved in the action of vasopressin.

Adenylyl Cyclases↗
PubMed1998

Protein kinase C: modulation of vasopressin-induced calcium influx and release in A7r5 vascular smooth muscle cells.

This study was guided by the hypothesis that specific isoforms of protein kinase C may participate in modulating increases in intracellular Ca2+ that are induced by stimulation of vascular smooth muscle cells with vasopressin. Immunoblot analysis revealed that A7r5 vascular smooth muscle cells expressed conventional (alpha), novel (delta and epsilon), and atypical (iota/lambda and mu) isoforms of protein kinase C. Stimulation of fura-2-loaded cells with 20 nM vasopressin induced a rapid transient increase in the intracellular concentration of calcium that was followed by a slowly declining component which was above baseline throughout the period of observation. Cell fractionation studies showed that the calcium response was associated with (a) transient translocation of the alpha and delta isoforms of protein kinase C from the cytosolic fraction to the particulate-membrane fraction, (b) sustained translocation of the epsilon isoform, and (c) no translocation of iota/lambda or mu isoforms. Ratiometric and isobestic fluorescence analysis showed that vasopressin-induced Ca2+ influx and release were markedly inhibited in cells that were preincubated with either 1 microM phorbol 12-myristate 13-acetate, or 10 microM 1, 2 dioctanoyl-sn-glycerol, two structurally different activators of protein kinase C. In contrast, vasopressin-induced increases in intracellular Ca2+ were not significantly altered following preincubation with either 1 microM 4alpha-phorbol or 4alpha-phorbol 12,13-didecanoate, analogs of phorbol 12-myristate 13-acetate that do not activate protein kinase C. Moreover, the inhibitory effects of phorbol 12-myristate 13-acetate were prevented by treatment with 1 microM GF109203X, a potent inhibitor of protein kinase C. Taken together, these results show that direct activation of protein kinase C can negatively modulate vasopressin-induced Ca2+ influx and release in cultured vascular smooth muscle cells. They also show that stimulation with vasopressin induces translocation of specific isoforms of protein kinase C, an observation suggesting that one or more of these isoforms may participate in modulation of vasopressin-induced increases in intracellular Ca2+.

Animals↗
PubMed1996

Endothelin-1 and vasopressin activate Ca(2+)-permeable non-selective cation channels in aortic smooth muscle cells: mechanism of receptor-mediated Ca2+ influx.

The effects of vasopressin and endothelin-1 on cultured aortic smooth muscle cell lines (A7r5) were investigated by measurements of intracellular calcium [Ca2+]i and the patch-clamp techniques. Vasopressin and endothelin-1 (100 nM) evoked an initial peak followed by a smaller sustained rise of [Ca2+]i in the presence of extracellular calcium [Ca2+]o. In the absence of [Ca2+]o, only the initial peak of [Ca2+]i was observed. Therefore, the initial peak of [Ca2+]i was mainly due to calcium release from the storage sites, whereas the later sustained rise of [Ca2+]i was due to the calcium entry from outside. The sustained rise of [Ca2+]i was unaffected by nifedipine (10 microM) significantly, but was completely abolished by La3+ (1 mM). Under current clamp conditions with K(+)-internal solution, vasopressin and endothelin-1 (100 nM) produced hyperpolarization, then followed by depolarization. Under voltage clamp conditions at a holding potential of -40 mV, both vasopressin and endothelin-1 first activated the outward current, then followed by a long-lasting inward current with a high noise level. The first outward current was abolished by charybdotoxin (100 nM), Cs+ in the patch pipette and high EGTA (10 mM) in the pipette, suggesting that it was a Ca(2+)-sensitive K+ current (IK.Ca). The inward current was still elicited with the patch pipette containing Cs(+)-internal solution, and reversed at about 0 mV. The reversal potential was not significantly altered by the replacement of [Cl-]i or [Cl-]o, proposing that the inward current is a cation selective channel (IN.S.). The inward current was also observed even when extracellular cations are Ca2+. La3+ (1 mM), Cd2+ (1 mM) completely abolished the vasopressin-induced (IN.S.), however, nifedipine (10 microM) failed to inhibit it significantly. Single channel activities were recorded in the cell-attached configurations when vasopressin or endothelin-1 was applied to the bathing solution. The unitary conductance of the channels was approximately 20 pS with 140 mM Na+, Cs+, or K+ in the pipette, but was 15 pS with 110 mM Ca2+ in the pipette. Permeabilities sequence calculated from the reversal potentials was Na+ not equal to Cs+ not equal to K+ > Ca+. These results provide evidence that calcium entry and membrane depolarization elicited by vasopressin or endothelin-1 are mediated by a receptor-mediated Ca(2+)-permeable non-selective cation channel in aortic smooth muscle cells.

Aorta, Thoracic↗
PubMed1998

Molecular aspects of vasopressin receptor function.

The molecular mechanisms governing the G protein coupling selectivity of different members of the vasopressin receptor family were studied by using a combined molecular genetic/biochemical approach. While the V1a and V1b vasopressin receptors are selectively linked to G proteins of the Gq/11 class, the V2 vasopressin receptor is preferentially coupled to Gs. Systematic functional analysis of V1a/V2 hybrid receptors showed that the second intracellular loop of the V1a receptor is required and sufficient for efficient coupling to Gq/11, whereas the third intracellular loop of the V2 receptor is required and sufficient for coupling to Gs. By using a strategy involving the coexpression of the wild type V1a receptor with chimeric G protein alpha s/alpha q subunits, two C-terminal alpha q/11 residues were identified that are critical for proper receptor recognition. We previously demonstrated -in transiently transfected COS-7 cells- that selected mutant V2 vasopressin receptors (all of which have been identified in X-linked nephrogenic diabetes insipidus patients) containing inactivating mutations in the C-terminal third of the receptor protein (including missense, frameshift, or nonsense mutations) can be functionally rescued by coexpression with a C-terminal V2 receptor fragment (V2-tail) spanning the region where the various mutations occur. Co-immunoprecipitation experiments and a newly developed sandwich ELISA revealed that the V2-tail polypeptide directly interacts with the mutant V2 receptors thus creating a functional receptor protein. To study the potential therapeutic usefulness of these findings, CHO cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors (E242stop, Y280C, and W284stop) were created and infected with a recombinant adenovirus coding for the V2-tail polypeptide. Following adenovirus infection, arginine vasopressin (AVP) gained the ability to stimulate cAMP formation in all CHO cell clones studied. Adenovirus-mediated gene transfer also proved to be a highly efficient method to achieve expression of the V2-tail fragment (as well as of the wild type V2 vasopressin receptor) in MDCK renal tubular cells. We therefore speculate that the targeted expression of receptor fragments in vivo may represent a novel strategy in the treatment of human diseases caused by inactivating mutations in distinct G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1989

Frequency-dependent effects of activation and inhibition of protein kinase C on neurohypophysial release of oxytocin and vasopressin.

Isolated rat neurohypophyses were superfused in vitro and the release of vasopressin and oxytocin into the medium was determined by specific radioimmunoassays. Hormone secretion was increased by electrical stimulation of the pituitary stalk at different frequencies. The effects of several phorbol esters, known to activate phorbol 12,13-dibutyrate, PDB) or not to affect (4 alpha-phorbol 12,13-dideconate and phorbol 12-monoacetate) protein kinase C, and of the direct protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) were tested. Electrical stimulation with 450 pulses caused the release of about 45 microU vasopressin and 55 microU oxytocin, when a frequency of 3 Hz was applied, and of about 500 microU vasopressin and oxytocin, when a frequency of 15 Hz was used. PDB (1 mumol/l) increased the release of vasopressin evoked by 15 Hz stimulation maximally by about 40-50% and that evoked by 3 Hz stimulation by about 150%. The release of oxytocin evoked by 15 Hz stimulation was increased by about 150% and that evoked by 3 Hz stimulation by about 400-500% in the presence of PDB. Both inactive phorbol esters had no effects on the evoked release of vasopressin or oxytocin. The effect of PDB on the release of vasopressin and oxytocin was blocked by H7 (10-30 mumol/l). H7 (30 mumol/l) alone reduced the release of vasopressin evoked by stimulation at 15 Hz by 50%. The release of oxytocin was not significantly affected by H7. In the presence of naloxone (1 mumol/l) the release of oxytocin evoked by 3 and 15 Hz stimulation was increased by about 175 and 105%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗