UNIPROT:P01178 - FACTA Search

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Query: UNIPROT:P01178 (oxytocin)
15,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The entire coding region of an ovine endometrial oxytocin receptor (OTR) cDNA was generated by PCR, subcloned into the SV40 major late promoter expression vector pSVLJ and transiently expressed in Cos-7 cells. A specific OTR antagonist, 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin (OTA), was used to describe the binding kinetics of the expressed receptor which had a Kd of 4.5 nM and Bmax of 2.4 nM/mg protein (6.8 x 10(5) receptor molecules/transfected cell). The functional properties of the expressed OTR were determined by measuring oxytocin-induced phosphoinositide (PI) hydrolysis. Oxytocin increased PI turnover in OTR transfected cells fourfold in excess of residual endogenous activity, and stimulated phospholipase C (PLC) activity in a dose- and time-dependent manner, confirming that the expressed OTR cDNA was functional. Arginine vasopressin also stimulated PI turnover in a dose-dependent manner; thresholds of responses to oxytocin and arginine vasopressin were 10(-9) M and 10(-7) M respectively. OTA did not increase PI turnover and competitively inhibited the oxytocin-induced response. Direct activation of the pathway by aluminium fluoride and guanosine (3'-O-thio)-triphosphate (GTP gamma S) confirmed that the OTR was G-protein linked. Co-incubation of GTP gamma S with oxytocin shifted the PI-response threshold from 10(-7) M to 10(-9) M and significantly increased the level of response, suggesting that maximum PI turnover was agonist-dependent. The G-protein involved in mediating the signal transduction pathway was pertussis toxin-insensitive and, therefore, probably a member of the Gq subfamily. The PLC inhibitor, U73122, had no effect on oxytocin-induced PI turnover, consistent with the response in endometrial tissue. These data suggest that the signalling pathway mediated by expressed OTR is similar to that attributed to OTR occupancy in ovine endometrium.
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PMID:Functional characterisation of an ovine endometrial oxytocin receptor cDNA transiently expressed in Cos-7 cells. 869 Oct 97

Oxytocin increases myometrial intracellular free calcium by promotion of calcium entry and release of calcium from intracellular stores. Calcium release from intracellular stores is secondary to an increase in phosphoinositide (PI) turnover and generation of IP3. We have explored the biochemical basis for the coupling of oxytocin (OT) to phospholipase C (PLC). Rat myometrial membranes contain PLC beta, gamma, and delta isoforms as well as the GTP-binding proteins G alpha(q) and G alpha(11). Oxytocin stimulates both GTPase and PLC activity in rat and human myometrial membranes. These data and available structural information suggest that the oxytocin receptor couples to PLC through a GTP-binding protein. In support of this hypothesis, an antibody generated against the specific C-terminal region of G alpha(q) and G alpha(11) inhibits both the oxytocin-stimulated GTPase and PLC activities. This inhibition is reversed by neutralization of the antibody with the antigenic peptide. The data indicate that the oxytocin receptor couples to PLC, presumably of the beta subclass, via interaction with proteins of the G alpha(q/11) subclass. In the nonpregnant, estrogen-primed rat, the stimulation of PI turnover by oxytocin is inhibited by the hormone relaxin and by pertussis toxin. The effects of both of these agents are mediated by the action of cAMP-dependent protein kinase. In plasma membranes, GTP-stimulated PLC activity can also be inhibited by treatment with protein kinase A. These data suggest that cAMP-dependent phosphorylation at a step involving GTP-binding protein/PLC coupling can exert a negative effect on the stimulation of IP3 formation by oxytocin and thereby affect contraction/relaxation in the myometrium.
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PMID:Mechanisms regulating oxytocin receptor coupling to phospholipase C in rat and human myometrium. 871 99

The present studies were undertaken to better characterize the pharmacological properties of oxytocin receptors (OTRs) of the porcine kidney cell line, LLC-PK1, in their natural membranous environment and after solubilization. In intact membranes, binding of a selective radioligand was rapid, reversible, saturable, and of high affinity. High-affinity agonist binding was reduced by a GTP analogue, suggesting that these OTRs are associated with G-protein(s). After solubilization with the zwitterionic detergent CHAPSO, OTRs retained their high affinity for the radioligand and rank order potency for oxytocin analogues, and agonist binding remained biphasic and GTP sensitive.
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PMID:Solubilization of oxytocin receptors in porcine renal LLC-PK1 cell membranes. 880 30

Ionic mechanisms responsible for histamine-induced prolonged depolarization in supraoptic nucleus neurons were investigated using whole-cell patch recordings in horizontally prepared brain slices from adult male rats. Bath application of histamine (1-10 microM) in control medium induced membrane depolarization in nine of 12 phasically firing, putative vasopressin cells, but not in continuous firing, putative oxytocin cells (none of five cells). Depolarization, usually accompanied by increased firing rate, started within 20 s after histamine reached the slices, lasting for 3-13 min, after which they repolarized, and this was repeatable upon washout. Chelation of intracellular Ca2+ with 11 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate and perfusion of slices with Ca(2+)-free medium blocked neither histamine-induced membrane depolarizations nor increased firing rates in 24 of 30 cells recorded. Depolarizations were always associated with decreases in membrane conductance. Following treatment with promethazine (H1 receptor antagonist) in six cells excited previously by histamine, subsequent application induced neither membrane depolarization nor increased firing. H1 receptor agonists mimicked histamine-induced depolarization (four of six cells) but the H2 receptor agonist, dimaprit (10 microM), had no effect (all of nine cells). In medium containing 0 mM Ca2+, 2 mM Co2+ and 1-2 microM tetrodotoxin, with internal Ca2+ chelation, bath application of histamine induced an apparent inward current in 15 of 20 supraoptic neurons tested. The peak of inward current evoked by 1-10 microM histamine at holding potentials around -50 mV varied from 10 to 50 pA (27.3 +/- 0.3 pA, mean +/- S.E.M.). Ramp voltage tests revealed that this inward current decreased as membrane potential was hyperpolarized and had a reversal potential of -90.1 +/- 3.8 mV (n = 10). Subtraction of current obtained before from that during histamine application revealed a current that was linear against membrane potential. Increasing external K+ concentration or introduction of K+ channel blockers in the medium attenuated or abolished histamine-induced inward current at membrane potentials close to -50 mV. When external Cl- concentration was reduced, histamine-induced inward current was still seen in five of seven supraoptic cells tested. Neither inward current nor change in conductance was observed following bath application of histamine in 11 of 12 neurons recorded using patch pipettes containing guanosine 5'-O-(2-thiodiphosphate), and in seven of eight neurons using pipettes containing guanosine 5'-O-(3-thiotriphosphate). These results suggest that histamine depolarizes supraoptic neurons, at least in part, by inhibiting a K+ leakage current mediated by H1 receptors linked to GTP-binding proteins and Ca(2+)-independent pathways. This study provides initial evidence for the second messengers regulating K+ leakage current.
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PMID:Histamine-induced prolonged depolarization in rat supraoptic neurons: G-protein-mediated, Ca(2+)-independent suppression of K+ leakage conductance. 884 19

The neuropeptide oxytocin (OT) is synthesized in the hypothalamus and can be released either as a hormone from the neurohypophysis or as a neurotransmitter in various brain regions. The present studies were undertaken to better characterize the pharmacological properties of brain oxytocin receptors (OTRs) using a radioligand selective for OTRs. Based on kinetic analysis, brain membranes obtained from 10-day-old rats display rapid and reversible binding to this ligand. In addition, saturation isotherm studies demonstrated that binding was saturable and of high affinity. Indicative of the selectivity of these receptors, compounds known to be ligands for OTRs in other tissues were able to displace the radioligand with high affinity. Consistent with the divalent cation requirement of OTRs in other tissues, OT binding was greatly reduced in rat brain membranes by the removal of magnesium from the incubation. To examine the possible GTP regulation of these receptors, binding was examined in the presence of a GTP analog. High affinity agonist, but not antagonist, binding was reduced by the GTP analog, indicating that these OTRs are likely to be associated with G proteins.
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PMID:Guanine nucleotide regulation and cation sensitivity of agonist binding to rat brain oxytocin receptors. 892 1

1. Oxytocin is known to act on autoreceptors of oxytocin neurones in the supraoptic nucleus (SON). We investigated whether oxytocin modulates putative oxytocin neurones by suppressing the GABAA receptor-mediated synaptic inputs on these cells. 2. GABAergic inhibitory postsynaptic currents (IPSCs) were recorded from SON neurones in hypothalamic slices from young rats. Oxytocin specifically reduced the amplitude of both spontaneous and evoked IPSCs, without altering their current kinetics. 3. The effect of oxytocin was observed in 70% of the magnocellular neurones recorded from the dorsomedial part of the SON. d(CH2)5OVT, a specific antagonist of oxytocin receptors, blocked the effect of oxytocin on the IPSCs. Vasopressin had no effect on oxytocin-sensitive SON neurones. 4. The intervals between spontaneous IPSCs were not affected by oxytocin. This suggested that oxytocin had a postsynaptic effect on SON neurones. 5. This postsynaptic origin was further substantiated by application of TTX, which blocked all evoked release but did not prevent the suppressive effect of oxytocin on the amplitude of the spontaneous IPSCs still present in the recording. The selective effect of oxytocin on IPSC amplitude was also maintained in nominally zero extracellular calcium. 6. Intracellular perfusion of SON neurones with GTP gamma S mimicked the effect of oxytocin on IPSCs, while GDP beta S, similarly applied, abolished the effect of oxytocin. 7. Application of calcium mobilizers such as thapsigargin and caffeine also reduced the amplitude of spontaneous IPSCs without significantly altering the frequency at which IPSCs occurred. 8. Thus, oxytocin depresses GABAergic synapses in the SON via modulation of the postsynaptic GABAA receptors. This would lead to disinhibition of SON neurones sensitive to oxytocin and could, therefore, be a powerful means of controlling the firing of oxytocin neurones.
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PMID:Postsynaptic mechanism of depression of GABAergic synapses by oxytocin in the supraoptic nucleus of immature rat. 896 Nov 90

In order to probe the nature of oxytocin receptor (OTR)/G alpha(q/11) protein coupling, we examined the effect of co-expression of OTR intracellular domains on oxytocin-stimulated phosphoinositide turnover in COSM6 cells overexpressing OTR and G alpha(q). Co-expression of G alpha(q) enhanced the oxytocin response maximally at a pOTR/pG alpha(q) plasmid transfection ratio of 1:0.16. In cells co-expressing OTR and G alpha(q/11), oxytocin stimulated phosphoinositide turnover with an EC50 of 48 nM. Co-transfection with plasmids expressing OTR intracellular domains inhibited oxytocin-stimulated phosphoinositide turnover by 23 +/- 6% (1i), 37 +/- 4% (2i), 55 +/- 6% (3i), and 40 +/- 6% (4i), respectively (P < 0.01). Expression of the 3i loop of the alpha(1B)-adrenergic receptor, which also couples to G alpha(q/11), inhibited phosphoinositide turnover by 35 +/- 2% (P < 0.01), while expression of the 3i loop of the dopamine 1A receptor, which couples to G alpha(s), had no effect. While these data indicate a functional role for the OTR 3i loop, they also suggest that interactions with more than one intracellular domain probably mediate the coupling of OTR to the G alpha(q/11) class of GTP-binding proteins.
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PMID:Evidence for the involvement of several intracellular domains in the coupling of oxytocin receptor to G alpha(q/11). 948 84

Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified. Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghalpha is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem. J. 315, 739-744]. To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor-Ghalpha-PLC-delta1 complex. PLC-delta1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghalpha (GTP-bound Ghalpha). Furthermore the stimulated PLC-delta1 activity resulting from activation of Ghalpha via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP. Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-delta1 in the three-component preparation by anti-Gh7alpha antibody resulted in the PLC-delta1 being tightly coupled to activated Ghalpha on stimulation of the oxytocin receptor. These results indicate that PLC-delta1 is the effector for Ghalpha-mediated oxytocin receptor signalling.
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PMID:Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as an effector. 951 91

The neuropeptide oxytocin can depolarize parasympathetic preganglionic neurons in the dorsal motor nucleus of the vagus nerve of the rat by generating a sustained inward current, which is sodium-dependent and tetrodotoxin-insensitive. The second messenger activated by oxytocin receptor binding is, however, not yet known. In the present study, we attempted to characterize it by using the whole-cell recording technique and brainstem slices. When loaded with GTP-gamma-S, a non-hydrolysable analogue of GTP, vagal neurons generated a persistent inward current in the absence of agonist and the oxytocin effect was suppressed, suggesting that the peptide-evoked current was mediated by G-protein activation. Loading vagal neurons with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA) suppressed a calcium-dependent, slowly decaying potassium aftercurrent but did not affect the oxytocin response, suggesting that the latter was not mediated by an agonist-induced increase in the intracellular calcium concentration. Protein kinase C (PKC) activation was probably not involved, since the peptide-evoked current was not modified by loading neurons with the PKC inhibitor H7. Thus, the oxytocin-evoked current in vagal neurons was probably not mediated by phospholipase C-beta (PLC-beta) activation. Loading neurons with 8-Br-cAMP or with an adenylyl cyclase activator (forskolin) reduced the oxytocin-evoked current by about half. SQ 22536, an adenylyl cyclase inhibitor, reduced this current by a similar amount. However, the peptide-evoked current was unaffected by Rp-cAMPS and Sp-cAMPS, an inhibitor and an activator, respectively, of cAMP-dependent protein kinase (PKA). We suggest that oxytocin activates two distinct signalling pathways in vagal neurons: one which is cAMP-dependent, but PKA-independent, and one, unidentified, which is PLC-beta-and cAMP-independent. Each pathway accounts for about half of the peptide effect and both appear to involve G-protein activation.
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PMID:The oxytocin-induced inward current in vagal neurons of the rat is mediated by G protein activation but not by an increase in the intracellular calcium concentration. 951 66

A series of studies was conducted to characterize changes in components of the cell signalling cascade that mediates oxytocin-induced prostaglandin F2 alpha (PGF2 alpha) synthesis at the onset of luteolysis in sheep. In the first experiment, caruncular tissue was dissected from 20 ewes on days 12-15 of the oestrous cycle, and incubated for the measurement of phospholipase C (PLC) activity or secretion of PGF2 alpha. Activation of GTP-binding proteins with aluminium fluoride stimulated both inositol phosphate accumulation and PGF2 alpha secretion on all days examined. However, oxytocin did not stimulate PLC activity or PGF2 alpha accumulation until day 13. While the ability of oxytocin to stimulate PLC activity increased after day 13, oxytocin-induced PGF2 alpha secretion declined slightly from day 13 to 15, suggesting that cell signalling components downstream from PLC modulate the response to oxytocin after day 13. Oxytocin failed to stimulate PGF2 alpha synthesis on day 14 after oestrus. Secretion of endogenous luteal oxytocin may have rendered uterine tissues collected on day 14 refractory to oxytocin in vitro. Therefore, a second study was conducted in ovariectomized, steroid replaced ewes. Ovarian steroids were administered to mimic endogenous changes in progesterone and oestradiol. The temporal patterns of PGF2 alpha synthesis in response to oxytocin and pharmacological agents were similar to uterine tissues from cyclic ewes in the first experiment; however, the magnitude of the response was less. These data suggest that oxytocin receptors are absent or are not coupled to PLC until day 13 after oestrus.
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PMID:Oxytocin- and aluminium fluoride-induced phospholipase C activity and prostaglandin F2 alpha secretion during the ovine luteolytic period. 964 Feb 61

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