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Query: UNIPROT:P01178 (oxytocin)
 15,767 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

1. Membrane potentials have been recorded from cells of seminiferous tubules of rats in vitro using micro-electrodes. The value in 808 impalements was -28-2 +/- 0-3 mV (mean +/- S.E.) at 33 degrees C. 2. Increasing the potassium concentration depolarized the cells, a tenfold increase in concentration causing a depolarization of 16 mV. Removal of sodium from the bathing solution caused a hyperpolarization of 3 mV at a potassium concentration of 5-9 m-equiv/l. Removal of chloride and replacement with impermeant anions had no effect on potential. Removal of calcium from the bathing solution caused a minor but significant depolarization. 3. Ouabain (10-3 M), dinitrophenol (2-5 times 10-4 M) or removal of glucose from the bathing fluid all caused depolarization. The membrane potentials of the cells were sensitive to temperature over the range 10-33 degrees C, the apparent activation energy for the reactions maintaining the potential being approximately 6 kcal/mole. 4. Membrane potentials in seminiferous tubules were independent of age of the animal, were insensitive to previous hypophysectomy and were insensitive to a number of hormones (FSH, LH, HCG, oxytocin). In high concentration prostaglandin E1 caused depolarization. 5. Acetazoleamide (4 times 10-5 M) caused a rapid, but reversible, depolarization of the tubular cells. This was also true in conditions when the HCO'3/CO2 buffer system was replaced with Tris-buffer. Another carbonic anhydrase inhibitor (p-sulphonamido-benzoic acid) had similar effects on cell potentials as acetazoleamide. These results are discussed in relation to the nature of the ionic secretion produced in the tubules. 6. Occasional cells showed phasic variations in membrane potential. A possible connexion between these variations and the contractile activity of the tubules is discussed.
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PMID:Intracellular potentials in cells of the seminiferous tubules of rats. 115 7

Midtrimester abortion was successfully induced in 13 of 22 patients by serial intravaginal administration of 15(S)-15-methyl-prostaglandin F2alpha (THAM) suppositories. Nine patients, 4 nulliparas and 5 multiparas, failed to abort after 24 hours of prostaglandin administration and a concomitant infusion of oxytocin was initiated. Seven of the nine patients aborted within 7 hours of the combined therapy and one patient on methadone maintainence aborted after 17.5 hours of combined therapy, 41.5 hours after the first dose of prostaglandin. A single patient failed to abort, despite the concomitant prostaglandin-oxytocin administration and underwent surgical evacuation. The mean abortion time for the 21 successful abortions was 22.56 hours. Nulliparous patients aborted somewhat faster, mean 21.79 hours, than multiparous patients, mean 23.80 hours, but this difference was not statistically significant. In this study, one patient aborted in less than 12 hours, and 62% of the successful cases aborted within 24 hours. The plasma levels of 15-ME-PGF2alpha were analyzed by radioimmunoassay in 10 patients. Plasma prostaglandin levels rose significantly 30 minutes after the insertion of the first suppository, but there was a wide variation in levels from patient to patient. It was observed that the 2 patients with the highest levels had the fastest abortion times and episodes of gastro-intestinal side effects appeared related to a rise in prostaglandin levels. Sixty-four percent of the patients in this study had no gastro-intestinal side effect related to prostaglandin administration.
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PMID:Induction of midtrimester abortion by serial intravaginal administration of 15(S)-15-methyl-prostaglandin F2alpha (THAM) suppositories. 120 82

We describe a rapid method for the preparation and binding site labeling of cryostat sections for use in light microscopy. Instead of using antibodies to bind to specific sites, substance P, delta-sleep-inducing peptide, oxytocin, and dopamine were covalently attached to BSA and then the BSA-ligand complex was adsorbed on 5-nm colloidal gold particles. Bioassays carried out on isolated organs indicated that the physiological activity of the ligand GPL complex was maintained. Most of the technical steps included use of an ordinary microwave oven (MWO), with tissues exposed for less than 1 min in any given step. Cryostat sections of unfixed rat brain were pre-incubated for 50 sec in the MWO in a Tris-buffered solution (pH 7.4) containing 1.5% BSA, then further incubated for 50 sec in the MWO in Tris-buffered solution containing 1% gelatin and the diluted colloidal gold suspension. After washing, the preparations were postfixed for 30 sec in the MWO in 5% formaldehyde solution, pH 7.4. Finally, the cell-bound gold particles were enlarged by a silver-enhancing process and counterstained. Preparations observed at high magnification provided excellent resolution of the cell binding sites. Positive and negative controls performed by addition of BSA-conjugated ligands to the pre-incubation and incubation medium, and displacement of the markers by an excess of unbound ligand in the pre-incubation or the incubation medium, showed the specificity of the tissue labeling.
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PMID:Microwave-aided binding of gold-protein-ligand (GPL) complexes. Light microscopic observations in the rat brain. 137 31

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

We have analysed, with the aid of a novel radioiodinated oxytocin (OT)-receptor antagonist, the role of Mg2+ in uterine OT-receptor function. The antagonist-receptor interaction was characterized by high affinity, reversibility and stereospecificity in Tris/HCl buffer containing 3 mmol of Mg2+/litre as well as buffer free of Mg2+. By contrast, omission of Mg2+ decreased the affinity of the receptor for OT by about 1500-fold; moreover, the stereospecificity of agonist, but not antagonist, binding was lost. Since guanine nucleotides had relatively minor effects in this system (less than or equal to 2-fold decrease in OT affinity), we suggest that the agonist-binding site of OT receptors is directly modulated by Mg2+, unlike other receptors, where the effects of bivalent cations are exerted via guanine-nucleotide-binding (G-) proteins. Thus the ligand recognition mechanism of OT receptors may be novel in this respect.
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PMID:Essential role of magnesium in oxytocin-receptor affinity and ligand specificity. 253 90

Urinary bladders of frogs were exposed to a transepithelial proton and osmotic gradient (serosal pH 8.1, Tris or bicarbonate buffer; mucosal pH 5.8, unbuffered) while the alkalinization rate of the mucosal bath and the net water movement were simultaneously monitored. It was observed that 1) the mucosal alkalinization rate was dependent on serosal pH and buffer; 2) oxytocin increased the mucosal alkalinization rate only when serosal bicarbonate was employed, whereas the net water movement augmented both when serosal bicarbonate or Tris buffers were used; 3) amiloride did not modify the mucosal alkalinization rate either before or after oxytocin; 4) the increases in the mucosal alkalinization rate and in the net water movement induced by oxytocin (serosal bicarbonate) were negatively correlated. In other experiments intracellular pH (pHi) was estimated with the DMO distribution technique with the following results. 1) Oxytocin increased the pHi when either serosal bicarbonate or Tris buffers was used and even in the presence of a low mucosal pH (Tris buffer, pH 5.8). 2) Important cellular acidification was observed when CO2 was bubbled (to pH 5.8), whereas the hydrosmotic response to 8-bromo-cAMP was clearly inhibited. These results indicate that cellular alkalinization could play a pivotal role in action of ADH, show that ADH can modify the transepithelial pH equilibrium mechanism, and suggest that intracellular pH regulation and water permeability control can be linked regulatory processes.
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PMID:Intracellular pH, transepithelial pH gradients, and ADH-induced water channels. 630 8

Human leptin expressed by E. coli had been used to treat human obesity in American and scientists had achieved good effects, the researchers here wanted to know whether human leptin can be expressed in the mammary glands of transgenic animas. In this study, human leptin gene about 1.0 kb, the terminator of rabbit whey acid protein gene (rWAP) about 0.2 kb and the promoter including the distal upstream region and part of the first exon of rWAP gene about 6.3 kb were used to construct a expression vector. Before we did the subclonings, the sequences of the human leptin gene were sequenced by ABI377 DNA Sequencer, the results showed that the fragment of human leptin gene included the last nine base pairs of the first exon, the complete sequences of the second exon(172 bp) and parts of the third exon(including part of the encoding sequences and part of the 3' untranslated region). The final expression vector was digested with NotI and a fragment of 7.5 kb was collected and dissolved in TE(10 mmol/L Tris.Cl, pH7.4; 0.1 mmol/L EDTA) for later microinjection. The concentration of DNA was about 2 micrograms/mL, the copy number in 1 mL was about 2.4 x 10(11), every 1 to 2 pL of the prepared DNA solution was microinjected into the mouse embryos at pronucleus stage. After standard microinjection procedures, 48 live mice were obtained. The tails of the mice were cut(about 0.1 g) at four weeks of age, genomic DNA was extracted and digested completely with EcoRI, two were confirmed to be transgenic mice(both were female) by Southern hybridization using DIG labeled human leptin gene as probe, transgenic rate among the mice born was about 4% (2/48). The two female transgenic mice(2# and C3) were mated with nontransgenic male mice. The two founder transgenic mice were segregated with their baby mice for at least three hours at the fifth day after parturition and were milked by intraperitoneal injection of 0.3 IU of oxytocin and udder massage. SDS-PAGE was used to analyze whether there were expression of human leptin in the milk of the two founder transgenic mice with the milk of non-transgenic mouse at fifth day after parturition as control. SDS-PAGE results showed that compared with the control there was a new band in both of the founder transgenic mice milk, and its molecular weight was about 16 kD, which was quite similar with that of the human leptin. The researchers estimated that the expression level of this protein in the milk of the transgenic mice was about 1-2 mg/mL.
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PMID:[A study on the expression of human leptin in the mammary glands of transgenic mice]. 1133 Jan 96

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

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).
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PMID:Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection. 1523 89


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