Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01185 (vasopressin)
 23,126 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adenylate cyclase and the [8-lysine]vasopressin receptor were solubilized from pig kidney medulla membranes using the nonionic detergent Triton X-100. Optimal conditions for solubilization were under continuous stirring in a medium containing 0.5% (/v) Triton X-100, 100 mM Tris-HCl, pH 8, and 10 mM MgCl2. Both adenylate cyclase activity and [3H][8-lysine]vasopressin binding activity were recovered in a -26,000 X g supernatant of detergent-treated membranes. The yield of solubilized adenylate cyclase was nearly 100%. The soluble enzyme was no longer sensitive to antidiuretic hormone but was slightly activated by sodium fluoride. The affinity of the soluble receptor for [8-lysine]vasopresin was les than that of the membrane-bound receptor (mean apparent Km values, respectively 10(-7) M and 2 X 10(-8) M), however binding cooperativity was preserved. Hill coefficients were 1.42 for the soluble receptor and 1.50 for the membrane receptor. The soluble receptor discriminated as efficiently as did the membrane receptor between [8-lysine-a1vasopressin and oxytocin. The yield of spolubilized receptor was only 30% despite the fact that all binding activity had disappeared from the residual pellet of detergent-treated membranes. When the membranous receptors were occupied before solubilization and the latter was performed under conditions in which dissociation of the hormone-receptor comples is slow, i.e. at low temperature, 65% to 100% of the hormone-receptor complex was recovered in the soluble fraction. The soluble hormone-receptor complex partially dissociated on rewarming whereas the free hormone concentration was kept unchanged in the medium. The residual binding capacity, which was 30% of the initial value, was identical with that determined when the receptor was solubilized in free form before incubation with labeled hormone. It was concluded that (a) solubilization of the receptor molecules was complete, (b) during solubilization two forms of the receptor appear, of which only one is accessible to the hormone, (c) occupancy of the receptor by the hormone prevented the formation of the nonaccessible form, and (d) some component or components of the soluble fraction might be responsible for the loss in apparent affinity.
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PMID:Solubilization of the [8-lysine]vasopressin receptor and adenylate cyclase from pig kidney plasma membranes. 17 Feb 74

During the period of life that precedes weaning, the facial nucleus of the newborn rat is rich in 3H-vasopressin binding sites, and exogenous arginine vasopressin (AVP) can excite facial motoneurons by interacting with V1 (vasopressor-type) receptors. We have investigated the mode of action of this peptide by carrying out single-electrode voltage-clamp recordings in coronal brainstem slices from the neonate. Facial motoneurons were identified by antidromic invasion following electrical stimulation of the genu of the facial nerve. When the membrane potential was held at or near its resting level, vasopressin generated an inward current whose magnitude was concentration related; the lowest peptide concentration still effective in eliciting this effect was 10 nM. The vasopressin-induced current, IAVP, was resistant to tetrodotoxin (TTX) and was insensitive to a reduction in extracellular calcium concentration. It was sustained, was inward at all potentials tested (-120 to -25 mV), and increased in magnitude during depolarization. IAVP was not generated by the blockade of a potassium current, because it did not reverse at hyperpolarized potentials, was not affected by a two-fold increase in the transmembrane potassium gradient, and was not modified by the potassium channel blockers tetraethylammonium bromide (TEA), 4-aminopyridin (4-AP), barium, cesium, quinine, glibenclamide, and apamin. Also, IAVP was not affected by changes in the transmembrane chloride gradient. In contrast, it could be reduced by partially substituting extracellular sodium with equimolar N-methyl-D-glucamine or Tris. Our results suggest that vasopressin increases the excitability of facial motoneurons by generating a persistent sodium-dependent membrane current that is voltage gated and TTX resistant.
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PMID:Vasopressin generates a persistent voltage-dependent sodium current in a mammalian motoneuron. 164 97

1. Intracellular current and voltage clamp recordings were obtained from rat supraoptic nucleus neurones in superfused hypothalamic explants in order to evaluate their response to dopamine and to D1 and D2 agonists. 2. With one exception, exposure to dopamine (10-200 microM) depolarized supraoptic neurones. When tested for an effect on twenty-one spontaneously active supraoptic neurones, dopamine enhanced the firing of all eleven continuous-firing (possibly oxytocin-secreting) neurones and prolonged the burst in all ten phasic-firing (vasopressin-secreting) neurones. 3. In sixty-seven of sixty-eight neurones where current injection was used to maintain membrane potential below threshold for action potential generation, current clamp data revealed that exposure to dopamine (10-200 microM) was followed in 10-17 s by a gradual 3-7 mV membrane depolarization that lasted for 4-15 min and was accompanied by a 12-23% reduction in input resistance. Exposure to quinpirole, a D2 agonist (10-200 microM), induced a similar response with comparable onset, duration and change in input resistance. In contrast, tests on sixteen cells indicated little or no response to a D1 agonist SKF38393. 4. Under voltage clamp, dopamine was noted to induce an inward current, accompanied by a 7.5-40% increase in membrane conductance over the corresponding time course. 5. Voltage-current plots for dopamine-induced depolarizations were linear in the range -50 to -110 mV. Dopamine and quinpirole depolarizations had extrapolated mean reversal potentials of -25 +/- 10 mV (mean +/- S.D.) and -20 +/- 15 mV respectively. This approximated the mean reversal potential of -20 +/- 8 mV measured from the dopamine-induced inward current using single-electrode voltage clamp. 6. The actions of dopamine were selectively antagonized by two D2 receptor antagonists, sulpiride and spiperone, but neither influenced membrane depolarizations induced by equimolar concentrations of noradrenaline. Dopamine-induced depolarizations also persisted following selective blockade of alpha 1-adrenergic receptors by prazosin; under these conditions, noradrenaline induced membrane hyperpolarization. 7. Following complete substitution of external Na+ with Tris, the reversal potential for the dopamine-induced response was shifted to -70 +/- 9.8 mV. This value was consistently less negative than the estimated potassium equilibrium potential. 8. The depolarization action of dopamine persisted in media containing tetrodotoxin and with an external calcium concentration ([Ca2+]o) of 0 mM-Ca2+ with 6 mM-Mg2+ or Mn2+, but was abolished following intracellular injection of [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a Ca2+ chelator.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Dopamine D2 receptor activation depolarizes rat supraoptic neurones in hypothalamic explants. 168 25

The potent vasoconstrictor endothelin leads to smooth muscle cell depolarization and increases in intracellular Ca2+. Although effects of endothelin on calcium channels have been described, it also has been speculated that endothelim may activate additional ion channels. The purpose of the present study was to identify an alternative ion current that could play a role in depolarizing cells in response to vasoconstrictors like endothelin and vasopressin. The effects of endothelin, vasopressin, sarafotoxin S6b, and phenylephrine were assessed using whole-cell patch-clamp recordings from primary dissociated rat aortic or mesenteric arterial smooth muscle cells cultured for 24-72 hours. From the usual resting potentials of these cells of -50 to -60 mV, endothelin (1-100 nM) induced a depolarization via an increase in membrane conductance. This depolarization was phasic, oscillating repeatedly from the resting potential to a relatively depolarized level and back to the resting potential. From a holding potential of -60 mV, endothelin-1, endothelin-3, vasopressin, or sarafotoxin S6b (but not phenylephrine) induced transient inward currents that also could be phasic. In external sodium, lithium, or cesium (but not Tris) and in internal potassium or cesium, these currents reversed near 0 mV. Although nifedipine-insensitive, the inward currents were absent in zero calcium, barium, or strontium, or in the presence of cobalt or nickel. These results represent the first report of a nonselective cation current in primary vascular smooth muscle cells that is calcium dependent and that could be responsible for the depolarizations induced from the resting potential by vasoconstrictors such as endothelin.
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PMID:Endothelin induces a nonselective cation current in vascular smooth muscle cells. 171 35

Stimulation of an amiloride-sensitive Na+ influx pathway, which mediates Na+/H+ exchange, has been postulated to be an important step in the initiation of DNA synthesis in quiescent human fibroblasts. If the elevation of intracellular Na+ or the alkalinization of intracellular pH resulting from the activation of this system is a trigger for subsequent mitogenic events, then its inactivation may also be important to cellular functions. We investigated the duration of the activation of Na+ influx by serum in human foreskin fibroblasts (HSWP). It was found that activation of Na+ influx by 10% serum was transient, declining with a t 1/2 = 15 min. Similarly, the Na+ content of the cells rose rapidly following serum addition and decreased with a t 1/2 = 15 min. In addition, both the lys-bradykinin- and the vasopressin-stimulated Na+ influx and Na+ content declined with a t 1/2 of approximately 15 min. Similar results were obtained using both Tris-buffered and Hepes-buffered, amino-acid-free EMEM. Finally, the above experiments were repeated under conditions normally used to assess the mitogenic response of cells. It was found that in cells arrested in G0 by serum deprivation in CO2-buffered EMEM, the serum activated Na+ flux was also transient with a t 1/2 of approximately 20 min. The desensitization of cells to serum could be readily (t 1/2 = 20') reversed by a subsequent incubation of cells in serum-free medium. Stimulation of Na+ influx by both the divalent cation ionophore A23187 and the phospholipase activator melittin in also desensitized rapidly, suggesting the process is independent of receptor downregulation. The desensitization during serum preincubation occurred in both low Na+ and low pH medium suggesting that the process is not due to negative feedback on the transport system via a rise in cellular Na+ concentration or a rise in intracellular pH. Although the mechanism of desensitization is at present not known, it is likely to be a physiologically important event.
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PMID:Desensitization of the serum effect on Na+ influx in cultured human fibroblasts. 609 Apr 76

Kidney function is regulated by several hormones which act through adenylate cyclase-cyclic AMP system. The present study was undertaken to investigate cyclic AMP- and cyclic GMP-phosphodiesterase (cAMP-PDE and cGMP-PDE respectively) activities in the rat kidney, and also the effect of several hormones affecting the kidney function on these enzyme activities in vitro. Rat kidneys were separated into cortex and medulla. These were homogenized in 50 mM Tris-HCl buffer, pH 7.5, containing 0.32 M sucrose and fractionated by centrifugation. PDE activity was measured in all fractions, using the two-step assay system. A low substrate concentration (0.5 microM) was used, unless otherwise stated. Substantial activity was present in all of the fractions and most of the activity existed in the soluble fraction (105000 X g supernatant). Cyclic GMP-PDE activity was dominant in both cortex and medulla. The rat kidney contained two forms of cAMP-PDE, one of which had a Km of 2.0 X 10(-4) M and another which had a low Km of 2.5 X 10(-5) M, and one form of cGMP-PDE with a Km of 2.5 X 10(-5) M. These cAMP-PDE and cGMP-PDE were purified by Sepharose-6B column chromatography. Cyclic AMP-PDE activity was found in a broad area associated with two peaks and cGMP-PDE activity had one peak corresponding to the same peak as the high molecular weight cAMP-PDE. Calmodulin was eluted after the peak of cGMP-PDE activity. Both cAMP-PDE and cGMP-PDE activities were inhibited by calcium ion at a concentration of more than 5.0 X 10(-4) M. Cyclic GMP-PDE activity was not activated by calmodulin in the presence of enough calcium ion. The effect of 1 alpha, 25(OH)2 Vit D3, parathyroid hormone (PTH), antidiuretic hormone (ADH), calcitonin (CT), angiotensin II, and trichlormethiazide on the partially purified cAMP-PDE and cGMP-PDE activities were examined. 1 alpha, 25(OH)2 Vit D3 activated cAMP-PDE activity and did not affect cGMP-PDE activity. The concentrations of 1 alpha, 25(OH)2 Vit D3 producing 50% activation of cAMP-PDE activity were 5.0 X 10(-11) M (cortex) and 6.7 X 10(-10) M (medulla). CT and ADH inhibited both cAMP-PDE activities. The concentrations of CT producing 50% inhibition of cAMP-PDE activity were 4.0 X 10(-5) M (cortex) and 3.3 X 10(-7) M (medulla), and those of cGMP-PDE activity were 1.0 X 10(-5) M (cortex) and 1.0 X 10(-4) M (medulla). Concerning ADH, the concentrations required for 50% inhibition of cAMP-PDE activity were 5.3 X 10(-6) M (cortex) and about 1.0 X 10(-3) M (medulla), and those of cGMP-PDE activity were 5.3 X 10(-3) M (cortex) and 5.3 X 10(-8) M (medulla).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Effect of several hormones on cyclic 3',5'-nucleotide phosphodiesterase in rat kidneys]. 631 6

Specific binding sites for vasopressin (AVP) were located in subcellular particulate fractions of rat brain with tritiated vasopressin of high specific activity, 22.5 Ci/mmol. Rat brain tissue was dissected, placed in cold 0.32 M sucrose containing proteolytic inhibitors, homogenized and fractionated into a crude nuclear fraction (1K pellet), crude mitochondrial fractions (12K pellet), and plasma membranes and microsomes (100K pellet). Specific binding of vasopressin was found in the 12K and 100K pellets in the presence of a divalent metal ion with Ni greater than Co greater than Mg greater than Mn greater than no metal ion at pH 7.4 in 50 mM Tris-Maleate buffer. Maximum specific binding of 16 nM AVP was located in the 100K anterior cortex fraction which bound 350 fmoles/mg protein; striatum, midbrain/thalamus, cerebellum, and medulla oblongata and pons bound specifically about 200 fmoles/mg protein and frontal poles and parietal cortex about 100 fmoles/mg protein in the 100K pellet. In all of the brain regions studied, except hippocampus and septum, the 100K pellet bound specifically 2 to 4 times more 3H-AVP than the 12K pellet. In the hippocampus with 16 nM AVP, the 12K pellet bound specifically 150 fmoles/mg protein; the septum, 75 fmoles/mg protein. Little or no binding to the 100K pellet was present in these regions. Bound AVP could be dissociated rapidly from the membranes by the addition of EDTA. The 12K hippocampal pellet was further fractionated into myelin, mitochondria, and synaptosomes; purification was confirmed by marker enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of 3H-AVP binding sites in particulate preparations of rat brain. 641 23

To elucidate the precise localization of vasopressin (VP) V1 and V2 receptors in the kidney, we utilized in vitro macroautoradiography (macro-ARG) and microautoradiography (micro-ARG) of these receptors in Wistar rat kidneys. This was done by using OPC-21268 and OPC-31260, two newly developed selective V1 (OPC-21268) and V2 (OPC-31260) receptor antagonists. For macro-ARG, 10-microm kidney sections were incubated with Tris-HCl buffer containing [3H]-VP with or without unlabeled ligand (VP, OPC-21268, or OPC-31260) at 20 degrees C for 40 min. These sections were then loaded into X-ray cassettes with Hyperfilm-[3H] and exposed in the dark for 2 months. The autoradiograms were quantitatively analyzed by using the research analysis system RAS 1,000; the V1 and V2 receptors were quantitated by subtracting the nonspecific binding (incubated with OPC-21268 and OPC-31260, respectively) from the total binding. To assess a more precise localization of the V1 and V2 receptors, we also investigated the micro-ARG of the renal V1 and V2 receptors by dipping the kidney section slides used for macro-ARG into a photographic emulsion and observing the receptors under light microscopy. [3H]-VP binding to the rat kidney was completely displaced by unlabeled excess VP, but not by unlabeled angiotensin II, indicating that [3H]-VP binding was specific for VP receptors. Computerized quantification showed that V2 receptors, visualized by OPC-31260, were the predominant type of VP receptor in the kidney. Conversely, V1 receptors, visualized by OPC-21268, were fewer in number. V1 receptors were partly localized to the glomerulus, cortical vessels, interstitial cells, and the medullary vessels. The V2 receptors localized to the collecting ducts and medullary tubules. Our findings indicated that renal V1 and V2 receptors can be detected by in vitro macro- and micro-ARG by using OPC-21268 and OPC-31260.
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PMID:In vitro macro- and microautoradiographic localization of V1 and V2 receptors in the rat kidney using OPC-21268 and OPC-31260. 922 35

More efficient and faster separation conditions for qualitative as well as quantitative analysis of neuropeptides in human plasma using capillary zone electrophoresis (CZE) have been developed. The analysis method for neuropeptides has been improved specifically to study thyroid hormone related neuropetides for the regulation of thyroid disease. In this study, we investigated the pretreatment methods, composition of the running buffer and rinsing procedures between runs in order to obtain more sensitive and faster separation of trace neuropeptides in plasma by CZE. The tested neuropeptides were somatostatin (SOMA), vasopressin (VP), neurotensin (NT), and thyrotropin-releasing hormone (TRH). Plasma samples were pretreated by deproteinization and solid-phase extraction method. The fraction of neuropeptides was reconstituted in 40% acetonitrile followed by ultrafiltration, and then analyzed by CZE. Resolution and sensitivity was improved using the separation buffer composition with 100 mM Tris-phosphate buffer (pH 2.0) while the sensitivity was further improved via a stacking method using the sample buffer of 40% acetonitrile. These sample pretreatment methods and buffer condition permit quantitative analysis on tested neuropeptides at the 20 ng/mL level. The rinsing procedures between runs using 90% ethanol dramatically shortened the rinsing time to 30 min.
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PMID:Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis. 1150 49

Vasopressin, oxytocin as well as other active nonapeptides (vasotocin, etc) are difficult to isolate from tissues. Traditionally they were identified using cumbersome biological assays or immunoassays, commercially unavailable, and with some cross reactivity. Based on the fact that all these peptides have two Cysteines in their molecules we developed a simple, sensitive and specific method to detect them by HPLC after pre-column fluorescent derivatization with monobromobimane (mBBr). The peptides were separated on a Vydac C18 column after reduction with Tris (2-carboxyethyl) phosphine (TCEP) and derivatization with mBBr for 5 minutes in dark. Using this method we were able to detect specific peaks for arginine-, lysine-vasopressin, and vasotocin at levels as low as 10 pmol. The method can be used to detect other active peptides with cyst(e)ins in their molecule, as well.
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PMID:A chemical method to isolate hypothalamic nonapeptides by coupling cyst(e)in with bimane. 1206 2


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