Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies were performed to identify the receptor that mediates AVP-stimulated phosphoinositide (PI) hydrolysis in cultured rat inner medullary collecting tubule (RIMCT) cells. While the selective V1 receptor agonist [Ho1, Phe2, Orn8] VT has no effect on inositol trisphosphate (IP3) production over the range of 10(-13)-10(-7) M, the selective V2 receptor agonist VDAVP stimulates IP3 production in dose-dependent fashion. Oxytocin stimulates IP3 production in dose-dependent fashion as well. AVP-stimulated phospholipase C activity is not inhibited by the V1 receptor antagonist d(CH2)5Tyr(Me)AVP(10(-7) M) but is eliminated by the V2 receptor antagonist d(CH2)5DTyr(Et)VAVP (10(-7) M). Similarly, the response to oxytocin is eliminated by the V2 receptor antagonist. The selective oxytocin receptor agonist [Thr4, Gly7] oxytocin does not stimulate cAMP production in RIMCT cells but does promote PI hydrolysis. The selective oxytocin receptor antagonist desGlyNH2d(CH2)5[Tyr(Me)-Thr4]OVT (10(-7) M) does not inhibit AVP-stimulated cAMP production but eliminates IP3 production in response to AVP or the V2 receptor agonist VDAVP. These studies demonstrate that AVP or a V2 receptor agonist stimulate PI hydrolysis in cultured RIMCT cells via occupancy of the oxytocin receptor.
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PMID:Vasopressin-stimulated phosphoinositide hydrolysis in cultured rat inner medullary collecting duct cells is mediated by the oxytocin receptor. 164 53

Arginine-vasopressin (AVP) plays a determinant role in the normal ACTH response to stress in mammals. We cloned a human cDNA coding a 424 amino acid G-protein coupled receptor structurally related to the vasopressin/oxytocin receptor family. When expressed in COS cells, this receptor binds AVP with a high affinity (Kd = 0.55 +/- 0.13 nM) and is functionally coupled to phospholipase C. Competition studies with peptidic or non peptidic AVP analogues reveal that it is pharmacologically distinct from V1a and V2 AVP receptors and therefore it is designated V3. RT-PCR analysis shows that the human V3 receptor is expressed in normal pituitary and also in kidney, but is undetectable in liver, myometrium and adrenal gland. Northern blot analysis reveals a approximately 4.8 kb messenger in human corticotropic pituitary adenomas.
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PMID:Cloning and characterization of the human V3 pituitary vasopressin receptor. 780 41

Vasopressin (AVP), the antidiuretic hormone, is a cyclic nonapeptide that acts through binding to G protein-coupled specific membrane receptors pharmacologically divided into three subtypes (V1a, V1b, and V2) linked to distinct second messengers. Within the family of human AVP receptors, the V2 AVP receptor has been cloned, but the structure of the human V1a and V1b AVP receptors remains unknown. We report here the structure and functional expression of a human V1a AVP receptor complementary DNA isolated from human liver cDNA libraries. Cloning and sequencing of a full-length clone isolated a 1472-nucleotide sequence encoding a 418-amino acid polypeptide with seven putative transmembrane domains typical of G protein-coupled receptors. Amino acid sequence identity with the rat liver V1a AVP receptor, the human and rat V2 AVP receptors, and the human oxytocin receptor was 72, 36, 37, and 45%, respectively. Functional characterization of the cloned receptor was done by transient expression in COS-7 cells and stable expression in Chinese hamster ovary cells. Localization of the expressed receptor at the cellular surface was illustrated by using the fluorescent linear analog phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 coupled to fluorescein-avidin by dodecabiotin. Competition binding experiments with phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-[125I]Tyr-NH2 and AVP analogs revealed high affinity specific binding sites of the V1a subtype. Saturation binding experiments with [3H]AVP confirmed the presence of a single class of high affinity binding sites. Measurement of AVP-induced inositol phosphate production and calcium mobilization confirmed that the expressed V1a AVP receptor is coupled to phospholipase C via a pertussis toxin-insensitive pathway. Thus, the human V1a AVP receptor belongs to the superfamily of seven-transmembrane segment receptors with a significant sequence identity with the other members of the AVP-oxytocin family of receptors.
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PMID:Molecular cloning, sequencing, and functional expression of a cDNA encoding the human V1a vasopressin receptor. 810 69

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

Parturition results from the establishment of phasic regular uterine contractions. Contractility in myometrial smooth muscle is stimulated by an increase in intracellular calcium ([Ca2+i]) which activates myosin light chain phosphorylation leading to increased myosin ATPase activity and enhanced rate of acto-myosin cross bridge formation. G proteins play a pivotal role in smooth muscle activation and relaxation by coupling cell membrane receptors to effector enzymes and ion channels. G alpha(s) and G alpha(i) stimulate and inhibit adenylyl cyclase, respectively and control cAMP formation. G alpha(q) stimulates phospholipase C resulting in the formation of two second messengers: inositol 1,4,5-trisphosphate (InsP3) which releases Ca2+ from the sarcoplasmic reticulum, and 1,2-diacylglycerol which activates protein kinase C. The oxytocin receptor stimulates myometrial contractility by increasing [Ca2+i] through both pertussis toxin resistant (G alpha(q)) and pertussis toxin sensitive (?G alpha (i)) pathways. beta-Adrenoceptors and prostaglandin EP2 receptors promote relaxation via G alpha(s)-adenylyl cyclase. The concentration of myometrial oxytocin receptors is five-times higher in pregnant compared to non-pregnant myometrium but decreases in samples obtained during labour. When myometrial slices are challenged with oxytocin there is a rapid increase in InsP3 levels with a time course which is similar to the rise in [Ca2+i] provoked by oxytocin in cultured myometrial cells. The formation of InsP3 in response to oxytocin in myometrial tissue at term is similar in samples obtained before and after the onset of labour. G alpha(q) and G alpha(i) are expressed at similar levels in non-pregnant and in pregnant myometrium obtained before or during labour. By contract, G alpha(s) levels are higher in pregnant compared to non-pregnant myometrium and decrease in samples obtained during labor. These changes in G alpha(s) are paralleled by prostaglandin E2-induced adenylyl cyclase activity in the same tissues. Parturition may be the consequence of downregulation of pathways that favour uterine quiescence by increasing cAMP formation, resulting in a relative dominance of stimulatory receptors that increase InsP3/Ca2+ availability.
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PMID:Parturition: activation of stimulatory pathways or loss of uterine quiescence? 871 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 carboxyl terminus of the G protein alpha subunit is a key determinant of the fidelity of receptor activation. We have previously shown that the Gq alpha subunit (alpha q) can be made to respond to alpha i-coupled receptors by replacing its carboxyl terminus with the corresponding alpha i2, alpha o, alpha z residues. We now extend these findings in three ways: 1) carboxyl-terminal mutations of alpha q/alpha i chimeras show that the critical amino acids are in the -3 and -4 positions, 2) exchange of carboxyl termini between alpha q and alpha z allows activation by receptors appropriate to the carboxyl-terminal residues, and 3) we identify receptors that either do or do not activate the expected carboxyl-terminal chimeras (alpha q/alpha i, alpha q/alpha s, alpha s/alpha q). Replacement of the five carboxyl-terminal amino acids of alpha q with the alpha s sequence permitted an alpha s-coupled receptor (the V2 vasopressin receptor but not the beta 2-adrenergic receptor) to stimulate phospholipase C. Replacement of the five carboxyl-terminal amino acids of alpha z with residues of alpha q permitted certain alpha q-coupled receptors (bombesin and V1a vasopressin receptors but not the oxytocin receptor) to stimulate adenylyl cyclase. Thus, the relative importance of the G alpha carboxyl terminus in permitting coupling to a new receptor depends on the receptor with which it is paired. These studies refine our understanding and provide new tools with which to study the fidelity of receptor/G alpha activation.
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PMID:Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation. 886 34

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

Oxytocin (OT) induces PG synthesis by both uterine endometrial and amnion cells. We showed previously that CHO cells stably transfected with the rat oxytocin receptor (CHO-OTR cells) also synthesize PGE2 in response to OT. In the present work we have demonstrated that OTRs are coupled to both Gi and Gq/11, using immunoprecipitation of solubilized OTR complexes and ADP ribosylation. OT treatment caused the rapid phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2 or p42MAPK), which was partially inhibited by pertussis toxin (PTX), consistent with OTR-Gi coupling. The PTX-insensitive portion of ERK2 phosphorylation was linked to Gq, as inhibitors of both phospholipase C (U-73122) and protein kinase C (GF-109203X) blocked OT-induced ERK2 phosphorylation. OT-stimulated c-fos expression was also mediated by ERK2 phosphorylation. The ERK-c-fos pathway has been shown to be associated with cell proliferation, but OT had no effect on [3H]thymidine uptake by CHO-OTR cells. However, inhibition of OT-induced ERK2 phosphorylation with an ERK kinase inhibitor (PD-98059) markedly reduced OT-stimulated PGE2 synthesis, pointing to the importance of ERK2 activation in OT action.
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PMID:ERK2 mediates oxytocin-stimulated PGE2 synthesis. 957 24


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