Increase In Phosphoinositide Hydrolysis Research Articles

Regulation of Gβγi-dependent PLC-β3 activity in smooth muscle: inhibitory phosphorylation of PLC-β3 by PKA and PKG and stimulatory phosphorylation of Gαi-GTPase-activating protein RGS2 by PKG.

In gastrointestinal smooth muscle, agonists that bind to Gi-coupled receptors activate preferentially PLC-β3 via Gβγ to stimulate phosphoinositide (PI) hydrolysis and generate inositol 1,4,5-trisphosphate (IP3) leading to IP3-dependent Ca(2+) release and muscle contraction. In the present study, we identified the mechanism of inhibition of PLC-β3-dependent PI hydrolysis by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Cyclopentyl adenosine (CPA), an adenosine A1 receptor agonist, caused an increase in PI hydrolysis in a concentration-dependent fashion; stimulation was blocked by expression of the carboxyl-terminal sequence of GRK2(495-689), a Gβγ-scavenging peptide, or Gαi minigene but not Gαq minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-β3 and inhibited CPA-induced PI hydrolysis, Ca(2+) release, and muscle contraction. The effect of isoproterenol on all three responses was inhibited by PKA inhibitor, myristoylated PKI, or AKAP inhibitor, Ht-31, whereas the effect of GSNO was selectively inhibited by PKG inhibitor, Rp-cGMPS. GSNO, but not isoproterenol, also phosphorylated Gαi-GTPase-activating protein, RGS2, and enhanced association of Gαi3-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant Gαi mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit Gβγi-dependent PLC-β3 activity by direct phosphorylation of PLC-β3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with Gαi-GTP. This allows RGS2 to accelerate Gαi-GTPase activity, enhance Gαβγi trimer formation, and inhibit Gβγi-dependent PLC-β3 activity.

Activation of long‐chain free fatty acid receptor FFAR1 (GPR40) and FFAR4 (GPR120) causes release of brain‐derived neurotropic factor from enteric glial cells (905.2)

Recent deorphanization of several G protein‐coupled receptors (GPRs) as endogenous receptors for free fatty acids (FFARs) has increased our understanding of these nutrients as signaling molecules and the integral role of the gut microbiota in the regulation of host defense mechanisms, energy metabolism, gastrointestinal motility, and secretion. Enteric glial cells and neurotrophins such as BDNF, glial cell line‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) are essential for the development and integrity of enteric nervous system in the gut. The role of free fatty acid receptors in the regulation of BDNF release from enteric glial cells, however, is not known. Aim. To determine the expression of FFARs and the signaling pathways coupled to FFAR1 and FFAR4, receptors for long chain fatty acids, that mediate BDNF release from enteric glial cells. Results. Quantitative RT‐PCR and western blot studies showed expression of FFAR1, FFAR2, FFAR3, FFAR4 and GPR84 in enteric glial cells; expression of FFAR1, however, was more abundant than other receptors. Linolenic acid which activates FFAR1/FFAR4 caused an increase in phosphoinositide hydrolysis (measured in [3H]myo‐inositol labeled cells), intracellular Ca2+ (measured in Fura‐2 loaded cells) and BDNF release (measured by ELISA). Conclusion. Enteric glial cells express receptors for free fatty acids and activation of FFAR1 and FFAR4 causes BDNF release via stimulation of PLC‐β/IP3/Ca2+ pathway.Grant Funding Source: Supported by DK34153

Glutamate receptors: emerging players in melanomagenesis

Glutamate receptors: emerging players in melanomagenesis

Endothelins participate in the central and peripheral regulation of submandibular gland secretion in the rat

We previously reported that endothelins (ETs) are involved in the rat central and peripheral regulation of bile secretion. In this study we sought to establish whether ET-1 and ET-3 modulated submandibular gland secretion when locally or centrally applied. Animals were prepared with gland duct cannulation to collect saliva samples and jugular cannulation to administer sialogogues. ETs were given either into the submandibular gland or brain lateral ventricle. Intraglandularly administered ETs failed to elicit salivation per se. However, ET-1, but not ET-3, potentiated both cholinergic- and adrenergic-evoked salivation through ET(A) receptors. ET-1 decreased cAMP content but increased phosphoinositide hydrolysis, whereas ET-3 attenuated both intracellular pathways. The expression of ET(A) and ET(B) receptor mRNAs as well as that of ETs was revealed in the submandibular gland by RT-PCR. Immunohistochemical studies showed that ET(A) receptor staining was localized around the interlobular ducts and acini, compatible with the myoepithelial cells' location, whereas ET(B) receptor staining was restricted to small blood vessels. When applied to the brain, both ETs induced no salivation but enhanced cholinergic- and adrenergic-evoked salivary secretion through parasympathetic pathways. ET-1 response was mediated by brain ET(A) receptors, whereas that of ET-3 was presumably through nonconventional ET receptors. Present findings show that ETs are involved in the brain regulation of cholinergic- and adrenergic-stimulated submandibular gland secretion through the activation of distinct brain ET receptors and parasympathetic pathways. However, when ETs were administered into the gland, only ET-1 enhanced cholinergic and adrenergic salivation likely through myopithelial cell contraction by activating ET(A) receptors coupled to phospholipase C. The presence of ETs and ET receptors suggests the existence of an endothelinergic system in the submandibular gland.

Activation of Human Phospholipase C-η2 by Gβγ

Phospholipase C-eta2 (PLC-eta2) was recently identified as a novel broadly expressed phosphoinositide-hydrolyzing isozyme [Zhou, Y., et al. (2005) Biochem. J. 391, 667-676; Nakahara, M., et al. (2005) J. Biol. Chem. 280, 29128-29134]. In this study, we investigated the direct regulation of PLC-eta2 by Gbetagamma subunits of heterotrimeric G proteins. Coexpression of PLC-eta2 with Gbeta 1gamma 2, as well as with certain other Gbetagamma dimers, in COS-7 cells resulted in increases in inositol phosphate accumulation. Gbeta 1gamma 2-dependent increases in phosphoinositide hydrolysis also were observed with a truncation mutant of PLC-eta2 that lacks the long alternatively spliced carboxy-terminal domain of the isozyme. To begin to define the enzymatic properties of PLC-eta2 and its potential direct activation by Gbetagamma, a construct of PLC-eta2 encompassing the canonical domains conserved in all PLCs (PH domain through C2 domain) was purified to homogeneity after expression from a baculovirus in insect cells. Enzyme activity of purified PLC-eta2 was quantified after reconstitution with PtdIns(4,5)P 2-containing phospholipid vesicles, and values for K m (14.4 microM) and V max [12.6 micromol min (-1) (mg of protein) (-1)] were similar to activities previously observed with purified PLC-beta or PLC-epsilon isozymes. Moreover, purified Gbeta 1gamma 2 stimulated the activity of purified PLC-eta2 in a concentration-dependent manner similar to that observed with purified PLC-beta2. Activation was dependent on the presence of free Gbeta 1gamma 2 since its sequestration in the presence of Galpha i1 or GRK2-ct reversed Gbeta 1gamma 2-promoted activation. The PH domain of PLC-eta2 is not required for Gbeta 1gamma 2-mediated regulation since a purified fragment encompassing the EF-hand through C2 domains but lacking the PH domain nonetheless was activated by Gbeta 1gamma 2. Taken together, these studies illustrate that PLC-eta2 is a direct downstream effector of Gbetagamma and, therefore, of receptor-activated heterotrimeric G proteins.

Different Mechanisms Regulate Lysophosphatidic Acid (LPA)-dependent Versus Phorbol Ester-dependent Internalization of the LPA1 Receptor

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA 1-5. The LPA 1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA(1) receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not beta-arrestin. Agonist-dependent internalization of LPA 1, however, requires a cluster of serine residues (amino acids 341-347) located proximal to the dileucine motif, beta-arrestin, and to a lesser extent clathrin AP-2. The serine cluster of LPA 1 is required for beta-arrestin2-GFP translocation to the plasma membrane and signal desensitization. In contrast, the dileucine motif (IL) is required for both basal and PMA-induced internalization. Evidence for the beta-arrestin independence of PMA-induced internalization of LPA 1 comes from the observations that beta-arrestin2-GFP is not recruited to the plasma membrane upon PMA treatment and that LPA 1 is readily internalized in beta-arrestin1/2 knock-out mouse embryonic fibroblasts. These results indicate that distinct molecular mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA 1 receptor.

PKC‐dependent regulation of the receptor locus dominates functional consequences of cysteinyl leukotriene type 1 receptor activation

Leukotrienes are important lipid mediators of asthma that contribute to airway inflammation and bronchoconstriction. Critical mechanisms for physiological regulation of the main G protein-coupled receptor (GPCR) mediating the leukotriene responses in asthma, cysteinyl leukotriene type 1 receptor (CysLT1R), have not been delineated. Although desensitization of GPCRs is a well-established phenomenon, studies demonstrating its physiological relevance are lacking. Here, we demonstrate that relief of PKC-mediated desensitization of endogenous CysLT1Rs augments multiple LTD4-stimulated cellular functions, with associated increases in intracellular signaling events. In analyses of airway smooth muscle contraction ex vivo, PKC inhibition augmented LTD4-stimulated contraction, and increased phosphoinositide hydrolysis and calcium flux in both murine and human airway smooth muscle cells. Similarly, for human monocytes, PKC inhibition augmented LTD4-stimulated calcium flux and cell migration assessed in transwell chamber experiments and also augmented LTD4-induced production of monocyte chemotactic protein assessed by ELISA. In contrast, PKC inhibition had no effect or slightly attenuated these cell functions and signaling events promoted by other receptor agonists, suggesting that despite antithetical effects on downstream events, desensitization of the CysLT1R is the principal mechanism by which PKC regulates the functional consequences of CysLT1R activation.

Pharmacological Evidence for a Functional Serotonin-2B Receptor in a Human Uterine Smooth Muscle Cell Line

The present study investigated the serotonin-induced increase in phosphoinositide hydrolysis and mobilization of intracellular Ca2+ ([Ca2+]i) in human uterine smooth muscle cells (HUSMCs) to identify the serotonergic receptor positively coupled to phospholipase C in these cells. In phosphoinositide (PI) assays, serotonin (5-HT) and alpha-methyl-5-HT were potent, full agonists (EC50 = 20 and 4.1 nM, respectively), whereas the phenylethylamine, R-(-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride, was less active (EC50 = 63 nM). Proposed 5-HT2B-selective agonists, BW-723C86 [alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride] and (+)-norfenfluramine, exhibited strong agonist potency and efficacy comparable with 5-HT (EC50 = 18 and 33 nM, respectively) and approximately 15-fold more potency than (-)-norfenfluramine (EC50 = 500 nM). 5-HT2C receptor agonists m-chlorophenylpiperazine and MK-212 [6-chloro-2-(1-piperaxinyl)pyrazine] were weak agonists in these cells, with potencies of 110 and 880 nM, respectively. A similar rank order of potency was observed in [Ca2+]i mobilization assays (r = 0.9, p < 0.005) in the HUSMC and with contraction of rat stomach fundus strips that contain a 5-HT2B receptor (r = 0.9, p < 0.001). Antagonist studies revealed that a 5-HT2B-selective antagonist, RS-127445 [2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine] (Ki = 0.13 nM), was significantly more effective at inhibiting 5-HT-induced activity than a 5-HT2A antagonist, M-100907 (R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol]) (Ki= 914 nM) and the 5-HT2C antagonists RS-102221 (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylsulfo-amido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione hydrochloride) (Ki = 2.5 microM) and SB-242084 (6-chloro-5-methyl-1-[6-92-methylpyridin-3-yloxy) pyridine-3-ylcarbamoyl] indoline) (Ki = 42.4 nM) in the HUSMC PI turnover assays. Taken together, these studies strongly suggest the presence of a functionally active 5-HT2B receptor subtype in HUSMCs. The physiological role of this receptor in these cells remains to be defined.

PAR1 Cleavage and Signaling in Response to Activated Protein C and Thrombin

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.

Activation of PLC-δ1 by Gi/o-coupled receptor agonists

The mechanism of phospholipase (PLC)-delta activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca(2+) stimulated an eightfold increase in PLC-delta1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three G(i/o)-coupled receptor agonists (somatostatin, delta-opioid agonist [D-Pen(2),D-Pen(5)]enkephalin, and A(1) agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-delta1(E341R/D343R; 65-76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca(2+) influx and was not observed in the absence of extracellular Ca(2+), but was partly inhibited by nifedipine (16-30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca(2+) channels. Treatment of the cells with a G(q/13)-coupled receptor agonist, CCK-8, caused only transient, PLC-beta1-mediated PI hydrolysis. Unlike G(i/o)-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-delta1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or Galpha(13) minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-delta1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca(2+) influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine ( approximately 25%) and strongly inhibited by SKF-96365 ( approximately 75%) and in cells expressing PLC-delta1(E341R/D343R). Agonist-independent Ca(2+) release or Ca(2+) influx via voltage-gated Ca(2+) channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-delta1 antibody or nifedipine. We conclude that PLC-delta1 is activated by G(i/o)-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca(2+) influx via store-operated Ca(2+) channels.

Bradykinin specificity and signaling at GPR100 and B2 kinin receptors

British Journal of Pharmacology (2004) 143, 931–932. doi:10.1038/sj.bjp.0706031

An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis

A novel approach to measuring receptor-stimulated phosphoinositide hydrolysis was developed based on the principles of immobilized metal ion affinity chromatography (IMAC) and scintillation proximity assay (SPA). Hard Lewis metal ions, such as Zr 4+, Ga 3+, Al 3+, Fe 3+, Lu 3+, and Sc 3+, were immobilized on SPA beads via metal chelate and utilized as affinity ligands to entrap inositol phosphates. [ 3H]Inositol phosphates bound to IMAC-SPA beads through the strong interaction of their phosphate group with the immobilized metal ions. The binding brought [ 3H]inositol phosphates in close proximity to the scintillant embedded in the SPA beads, thereby allowing the radioactivity to be quantified. Quantification of [ 3H]inositol phosphate production in cells preincubated with [ 3H]inositol provided a highly sensitive measurement of phosphoinositide hydrolysis. The utility of this approach was demonstrated in measuring the response mediated by the G-protein-coupled neurokinin NK1 receptor and the tyrosine kinase-linked platelet-derived growth factor (PDGF) receptor. Substance P stimulated phosphoinositide hydrolysis concentration-dependently in CHO cells expressing NK1 receptors with a maximal 12-fold increase in inositol phosphate production. Similarly, PDGF-BB stimulated a 5-fold increase in phosphoinositide hydrolysis in quiescent Swiss 3T3 cells. This new approach is highly sensitive, fast, simple, easily performed on 96-well plates, and amenable for high-throughput screening.

Cyclosporin A: Effects on the Secretory Process and Noradrenergic Activity in the Submandibular Gland of the Rat

Objectives: The aim of the present work was to study the effect of long-term cyclosporine (CSA) administration on norepinephrine (NE) metabolism and adrenergic-evoked secretion in the rat submandibular gland (SMG). Methods: Dose-response curves to adrenergic agonists (methoxamine, isoproterenol, NE) were performed in control and CSA (10 and 30 mg/kg every 2 days for 1 month)-treated rats after SMG duct cannulation. In SMG tissue neuronal NE uptake, release, synthesis and endogenous content were determined. In addition phosphoinositide intracellular signaling was also investigated. Results: CSA administration caused an increase in salivary secretion evoked by methoxamine (α-adrenergic agonist) and NE but failed to modify salivation evoked by β-adrenergic stimulation (isoproterenol). Long-term CSA administration decreased NE release and synthesis whereas it enhanced the amine uptake and phosphoinositide hydrolysis in the SMG. Conclusions: The administration of CSA for 30 days induced salivary gland sensitization likely mediated by diminished adrenergic input. Present results suggest that the decreased sympathetic activity evoked by long-term CSA administration in the rat SMG may lead to sensitization of the gland supported by increased phosphoinositide hydrolysis and enhanced adrenergic-evoked salivation.

Human urotensin II mediates vasoconstriction via an increase in inositol phosphates

The cyclic peptide urotensin II has recently been cloned from human and reported to potently constrict primate blood vessels. To elucidate the cellular signalling mechanisms of this peptide, we investigated a possible relationship of vasomotor effects of human urotensin II and phosphoinositide turnover in isolated rabbit thoracic aorta. Human urotensin II produced a slowly developing increase in isometric contractile force ( pEC 50=9.0) that was endothelium-independent. The contractile effect of urotensin II was significantly inhibited by the phospholipase C inhibitor, 2-nitro-4-carboxyphenyl- N, N,-diphenylcarbamate (NCDC), but not by the cyclooxygenase inhibitor, indomethacin. In slices of rabbit thoracic aorta, human urotensin II increased phosphoinositide hydrolysis, and this effect was also inhibited by NCDC. The potency of urotensin II ( pEC 50=8.6) was similar to that found in the contractile studies. Thus, vasoconstrictor effects of human urotensin II appear to be mediated by a phospholipase C-dependent increase in inositol phosphates, suggesting that the peptide acts via a G q protein-coupled receptor.

Phorbol ester-induced phosphoinositide hydrolysis in rat aorta: Role of cyclooxygenase products

This study investigates whether phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and the mechanism underlying the hydrolysis. Phorbol myristate acetate induced time- and concentration-dependent increases in phosphoinositide hydrolysis, as demonstrated by elevated inositol monophosphate levels, in deendothelialized rat aorta. The phorbol ester-elevated inositol monophosphate levels were abolished by indomethacin, a cyclooxygenase inhibitor, but were only partially decreased by SQ29548, a thromboxane A 2/prostaglandin H 2 receptor antagonist. SQ29548 also only partially decreased elevated inositol monophosphate levels due to prostaglandin E 2, prostaglandin F 2α, prostaglandin I 2 and carbacyclin, a stable prostaglandin I 2 analog. SQ29548 abolished elevated inositol monophosphate levels due to U46619, a stable thromboxane A 2/prostaglandin H 2 receptor agonist. These studies demonstrate that phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and that the increase is due, at lease in part, to endogenously released prostaglandins other than prostaglandin H 2.

Intracellular signals coupled to muscarinic acetylcholine receptor activation in cerebral frontal cortex from hypoxic mice.

1. The aim of the present work was to determine hypoxia-induced modifications in the cascade of intracellular events coupled to muscarinic acetylcholine receptor (mAChR) activation in brain. For this purpose, enzymatic activities were measured on normoxically incubated frontal cortical slices from mice exposed to hypobaric hypoxia for 72 hr. 2. We found that hypoxia induced alterations in several cerebral enzymatic basal activities: it increased nitric oxide synthase (NOS), but it decreased both membrane protein kinase C (PKC) and phospholipase C activities. 3. The mAChR agonist carbachol was found to increase phosphoinositide hydrolysis to greater values in hypoxic tissues than those found in normoxic conditions. Furthermore, a greater translocation of PKC in response to carbachol was observed in hypoxic tissues than in normoxic ones. 4. Besides, carbachol induced a drastic reduction of NOS activity in hypoxic brains, at concentrations that stimulated this enzyme activity in normoxic preparations. In the latter, inhibition is obtained only with high concentrations of the cholinergic muscarinic agonist. 5. These results pointed to a carbachol-mediated mAChR hyperactivity induced by hypoxic insult. 6. The possibility that these effects would account for a compensatory mechanism to diminish NOS hyperactivity, probably protecting for NO neurotoxic action in hypoxic brain, is also discussed.

Functional pharmacology of cloned heterodimeric GABAB receptors expressed in mammalian cells.

1. In this study we report a new assay of heterodimeric gamma-amino-butanoic acid subtype B (GABAB) receptors where either GABABR1a or GABABR1b are co-expressed with GABABR2 and the chimeric G-protein Galphaq-z5 in tsA cells. In this manner we obtained a robust response to GABAB agonists measured as increase in phosphoinositide hydrolysis. 2. We used this assay to characterize a number of commonly used GABAB receptor ligands. Both splice variants displayed the same rank order of agonist potency; 3-aminopropyl(methyl)phosphinic acid (SKF-97541)>GABA>(R)-4-amino-3-(4-chlorophenyl)butanoic acid ((R)-baclofen)>(RS)-4-amino-3-(5-chloro-2-thienyl)butanoic acid (BCTG)>3-aminopropylphosphonic acid (3-APPA) and furthermore, the absolute agonist potency values were very close to each other. 3. 3-APPA was a partial agonist displaying maximal responses of 41 and 61% compared to GABA at GABABR1a and GABABR1b, respectively. The antagonist (RS)-3-amino-2-(4-chlorophenyl)-2-hydroxypropylsulphonic acid (2-OH-saclofen) displayed KB values of 15 and 7.8 microM at GABABR1a and GABABR1b, respectively. 4. The rank order of agonist potency as well as the absolute ligand potencies correspond very well with those previously reported in different tissues, and this study thus provides a functional assay of cloned GABAB receptors which should be a valuable tool for further characterization of GABAB ligands. Finally, we can conclude that the functional pharmacological profiles of the two GABABR1 splice variants are very similar.

The in vivo effects of olanzapine and other antipsychotic agents on receptor occupancy and antagonism of dopamine D1, D2, D3, 5HT2A and muscarinic receptors.

The atypical antipsychotic olanzapine was compared to other atypical as well as typical antipsychotic agents for in vivo occupancy of D1, D2, D3, 5HT2, and muscarinic receptors in rat brain. Blockade of D2 receptors was determined by measuring the levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). To assess the interaction with phosphoinositide (PI)-coupled 5HT2A and muscarinic receptors in vivo, we used a novel radiometric technique to measure in vivo PI hydrolysis. The antagonism of olanzapine and other antipsychotic agents on 5HT2A and muscarinic receptors was determined by in vivo blockade of PI hydrolysis, stimulated by the 5HT2 agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) or the muscarinic agonist pilocarpine. Olanzapine inhibited 5HT2, D2, and D3 in vivo binding with high potency (ID50=0.15, 0.6 and 1.2 mg/kg, IP, respectively), while inhibiting D1 and muscarinic in vivo binding with much less potency (ID50 > 10 mg/kg, IP). The binding of olanzapine to D2 receptors in neostriatum was well correlated with the increase of DOPAC (ED200 = 0.8 mg/kg, IP) in vivo, indicating dopamine D2 antagonism. In vivo PI hydrolysis was increased by DOI in frontal cortex and by pilocarpine in hippocampus up to 2- and 7-fold above the basal level, respectively. The agonist-induced increases in PI hydrolysis were fully blocked by the 5HT2A antagonist MDL100907 and the muscarinic antagonist scopolamine, indicating the mediation by 5HT2A receptors in frontal cortex and PI-coupled muscarinic receptors (ml, m3, and m5) in hippocampus, respectively. Olanzapine was about 8-fold more potent in vivo in blocking DOI-induced stimulation of PI hydrolysis (ID50 = 0.1 mg/kg, IP) than pilocarpine-induced stimulation of PI hydrolysis (ID50 = 0.8 mg/kg, IP). In conclusion, olanzapine is more potent in blocking the 5HT2A receptor than D1, D2, D3 and muscarinic receptors in vivo, consistent with its favorable clinical profiles. In addition, the novel in vivo PI hydrolysis assay proved to be a useful and reliable in vivo method to assess the functional efficacy of compounds that interact with the 5HT2 and muscarinic receptors.

Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F secretion by luminal epithelial, glandular epithelial, and stromal cells from pig endometrium. I. Response of cyclic pigs on day 16 postestrus.

Oxytocin (OT) is the physiological stimulus for pulsatile release of endometrial prostaglandin (PG) F2alpha during luteolysis in domestic ungulates, and the cellular mechanism for this appears to involve phosphoinositide (PI) hydrolysis. To determine which endometrial cell type(s) was responsive to OT during luteolysis in swine, luminal epithelial (LEC), glandular epithelial (GEC), and stromal cells (SC) were isolated from endometrium by differential enzymatic digestion and sieve filtration on Day 16 postestrus and cultured. For PI hydrolysis in experiment 1, SC were most responsive to 100 nM OT (p < 0.001), whereas LEC were least responsive and GEC had an intermediate response (p < 0.001). For PGF secretion in experiment 2, the response to OT was greatest for SC, least for LEC, and intermediate for GEC. In experiment 3, 100 nM OT increased PI hydrolysis in SC within 30 min (p < 0.05) and in GEC within 60 min (p < 0.05) but did not increase PI hydrolysis in LEC. In experiment 4, PI hydrolysis in SC was increased (p < 0.05) by 33-333 nM OT but was not increased by </= 333 nM OT in GEC or LEC after 30 min. However, PGF secretion from SC was increased (p < 0.05) by 10-333 nM OT, and from GEC by 10-333 nM OT, but was not increased from LEC by </= 333 nM OT. Results of this study indicate that 1) there was differential responsiveness to OT among endometrial cell types, and 2) within cell type, there generally was a similar response to OT for both PI hydrolysis and PGF secretion, further implicating PI hydrolysis as the signaling pathway for OT-stimulated PGF2alpha release. The differential response of endometrial cell types may have an important role in the pattern of PGF2alpha secretion during luteolysis in swine.

Muscarinic regulation of intracellular signaling and neurosecretion in gonadotropin-releasing hormone neurons.

Agonist activation of cholinergic receptors expressed in perifused hypothalamic and immortalized GnRH-producing (GT1-7) cells induced prominent peaks in GnRH release, each followed by a rapid decrease, a transient plateau, and a decline to below basal levels. The complex profile of GnRH release suggested that acetylcholine (ACh) acts through different cholinergic receptor subtypes to exert stimulatory and inhibitory effects on GnRH release. Whereas activation of nicotinic receptors caused a transient increase in GnRH release, activation of muscarinic receptors inhibited basal GnRH release. Nanomolar concentrations of ACh caused dose-dependent inhibition of cAMP production that was prevented by pertussis toxin (PTX), consistent with the activation of a plasma-membrane Gi protein. Micromolar concentrations of ACh also caused an increase in phosphoinositide hydrolysis that was inhibited by the M1 receptor antagonist, pirenzepine. In ACh-treated cells, immunoblot analysis revealed that membrane-associated G(alpha q/11) immunoreactivity was decreased after 5 min but was restored at later times. In contrast, immunoreactive G(alpha i3) was decreased for up to 120 min after ACh treatment. The agonist-induced changes in G protein alpha-subunits liberated during activation of muscarinic receptors were correlated with regulation of their respective transduction pathways. These results indicate that ACh modulates GnRH release from hypothalamic neurons through both M1 and M2 muscarinic receptors. These receptor subtypes are coupled to Gq and Gi proteins that respectively influence the activities of PLC and adenylyl cyclase/ion channels, with consequent effects on neurosecretion.