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C C Felder

Publications and source records attributed to C C Felder.

At least 55 records · Page 3Linked to original sources
PubMed1995

Comparison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors.

The recently cloned CB2 cannabinoid receptor subtype was stably transfected into AtT-20 and Chinese hamster ovary cells to compare the binding and signal transduction properties of this receptor with those of the CB1 receptor subtype. The binding of [3H]CP 55,940 to both CB1 and CB2 was of similar high affinity (2.6 and 3.7 nM, respectively) and saturable. In competitive binding experiments, (-)-delta 9-tetrahydrocannabinol and CP 55,940 were equipotent at the CB1 and CB2 receptors, but WIN 55212-2 and cannabinol bound with higher affinity to the CB2 than the CB1 receptor. HU 210 had a higher affinity for the CB1 receptor. Anandamide, a recently identified endogenous cannabinoid agonist, was essentially equipotent at both receptor subtypes. The structurally related fatty acid ethanolamides dihomo-gamma-linolenylethanolamide and mead ethanolamide also bound with relatively equal affinity to both receptors, but adrenylethanolamide had a higher affinity for the CB1 receptor. The rank order of potency and efficacy for binding of the selected agonists to the CB1 and CB2 receptors was mimicked in functional inhibition of cAMP accumulation experiments for all compounds tested. Both CB1 and CB2 receptors couple to the inhibition of cAMP accumulation that was pertussis toxin sensitive. SR141716A, a CB1 receptor antagonist, was a poor antagonist at the CB2 receptor in both binding and functional inhibition of cAMP accumulation experiments. When expressed in AtT-20 cells, the CB1 receptor mediated an inhibition of Q-type calcium channels and an activation of inward rectifying potassium channels. In contrast, the CB2 receptor did not modulate the activity of either channel under identical assay conditions. Similar to results obtained for CB1 receptor, the CB2 receptor did not couple to the activation of phospholipases A2, C, or D or to the mobilization of intracellular Ca2+. Except for its inability to couple to the modulation of Q-type calcium channels or inwardly rectifying potassium channels, the CB1 and CB2 receptors display similar pharmacological and biochemical properties.

Adenylyl Cyclase Inhibitors↗
PubMed1995

Mead ethanolamide, a novel eicosanoid, is an agonist for the central (CB1) and peripheral (CB2) cannabinoid receptors.

The recently discovered endogenous agonist for the cannabinoid receptor, anandamide (arachidonylethanolamide), can be formed enzymatically by the condensation of arachidonic acid with ethanolamine. 5Z,8Z,11Z-Eicosatrienoic acid (mead acid) has been found to substitute for arachidonic acid in the sn-2 position of phospholipids and accumulate during periods of dietary fatty acid deprivation in rats. In the present study, the chemically synthesized ethanolamide of mead acid was evaluated as a potential agonist at the two known subtypes of cannabinoid receptor: CB1 (central) and CB2 (peripheral). This compound was equipotent to anandamide in competing with [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the human CB1 receptor and from ATt-20 cells expressing the human CB2 receptor. Mead ethanolamide was also equipotent to anandamide in inhibiting forskolin-stimulated cAMP accumulation in cells expressing the CB1 receptor. It inhibited N-type calcium currents with a lower potency than anandamide. Mead and arachidonic acid were equally efficacious as substrates for the enzymatic synthesis of their respective ethanolamides in rat and adult human hippocampal P2 membranes. Palmitic acid was not an effective substrate for the enzymatic synthesis of palmitoyl ethanolamide. Mead ethanolamide exhibits several characteristics of a novel agonist to CB1 and CB2 receptors and may represent another candidate endogenous ligand for the CB1 receptor. Due to the anticonvulsant properties of GABA and the positional similarity of L-serine to ethanolamine in membrane phospholipids, these compounds were synthetically coupled to arachidonic acid, and their resulting arachidonamides were tested as potential cannabinoid agonists. The arachidonamides of GABA and L-serine were inactive in both binding and functional assays at the CB1 receptor.

8,11,14-Eicosatrienoic Acid↗
PubMed1995

Muscarinic acetylcholine receptors: signal transduction through multiple effectors.

Muscarinic receptors regulate a number of important basic physiologic functions including heart rate and motor and sensory control as well as more complex behaviors including arousal, memory, and learning. Loss of muscarinic receptor number or function has been implicated in the etiology of several neurological disorders including Alzheimer's dementia, Down's syndrome, and Parkinson's disease. Muscarinic receptors transduce their signals by coupling with G-proteins, which then modulate the activity of a number of effector enzymes and ion channels. Five subtypes of muscarinic receptors (m1-m5) have been identified by molecular cloning and much has been learned about their distribution, pharmacology, and structure. Less is known about the molecular mechanisms of receptor-effector coupling and the biological role of each receptor subtype. The ectopic expression of genes encoding a single muscarinic receptor subtype in mammalian cell lines has provided an important model system in which to investigate receptor subtype-specific pharmacology and signal transduction. Expression models have revealed that single muscarinic receptor m1, m3, or m5 subtypes can activate multiple signaling effectors simultaneously including phospholipases A2, C, and D, as well as tyrosine kinase and a novel class of voltage-insensitive calcium channels. The m2 or m4 receptors have been shown to augment phospholipase A2 in addition to their established role as inhibitory receptors acting through the attenuation of adenylate cyclase. In addition to allowing investigations of the regulatory mechanisms of muscarinic receptors, expression models provide an excellent tool to investigate receptor-subtype specific physiology and pharmacology.

Adenylyl Cyclases↗
PubMed1994

Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI.

Evidence is accumulating that calcium homeostasis and calcium-regulated events may be selectively important in generation and maintenance of the malignant phenotype. CAI, a carboxyamido-triazole with a halogenated benzophenone tail, is a novel inhibitor of receptor-operated calcium influx and arachidonic acid release which inhibits malignant proliferation, invasion, and metastasis. The focus of this investigation was structural analysis of CAI and to determine if the inhibition of calcium influx and arachidonic acid release by CAI and its antiproliferative activity were mediated through the same chemical domains. Four families of molecular modifications of the CAI parent were synthesized: (I) modification or substitution of the triazole ring; (II) removal of the substituted benzophenone tail; (III) dehalogenation or partial truncation of the benzophenone moiety; and (IV) removal of the triazole and altered substitutions of the benzophenone tail. Compounds were tested for the inhibition of calcium influx and arachidonic acid release and inhibition of proliferation and colony formation in soft agar using the malignant CHO line transfected with the m5 muscarinic receptor and the A2058 human melanoma cell line. Only CAI and Group I compounds inhibited stimulated calcium influx, arachidonic acid release, and proliferation. Linear regression analysis of the relationship of the 50% inhibitory concentration values for all compounds in inhibition of calcium influx and arachidonate release was statistically significant (r2 = 0.993). Similarly, a linear relationship was demonstrated between inhibition of calcium influx and inhibition of tumor cell proliferation (r2 = 0.971). Groups II-IV had minimal or no signal or growth inhibitory activity. This investigation provides the first evidence for a coordinate link between calcium influx, calcium-mediated arachidonic acid release, and malignant proliferation and metastasis and constitutes the initial analysis of structurally important domains of the CAI molecule.

Aminoimidazole Carboxamide↗
PubMed1994

The cloned vasopressin V1a receptor stimulates phospholipase A2, phospholipase C, and phospholipase D through activation of receptor-operated calcium channels.

Arginine vasopressin mediates its effects through vasopressin receptor activation and second messenger production. Recent cloning of the V1a receptor provided the opportunity to investigate the possible signal transduction pathways associated with this single vasopressin receptor subtype. When stably expressed in CHO cells, vasopressin stimulated several signal transduction pathways simultaneously including calcium influx, phospholipase A2, phospholipase C, and phospholipase D. Vasopressin-stimulated release of arachidonic acid, IP3 formation, and phosphatidylethanol formation (in the presence of 1% ethanol) were used as indexes of phospholipase A2, phospholipase C, and phospholipase D activation, respectively. V1a receptor-activation stimulated a peak followed by a sustained plateau phase of intracellular calcium. The plateau phase was dependent on extracellular calcium, insensitive to blockers of voltage sensitive calcium channels, blocked by heavy metals, and quenched when MnCl2 was present in the extracellular media. Removal of extracellular calcium blunted the release of IP3, and blocked the release of arachidonic acid and phosphatidylethanol indicating that these responses were at least in part regulated by receptor-operated calcium influx. Vasopressin-stimulated release of arachidonic acid and phosphatidylethanol were augmented with the phorbol ester PMA, and this augmentation was blocked by inhibitors of protein kinase C and absent with long-term PMA treatment. Vasopressin-stimulated IP3 release was inhibited with PMA and the inhibition reversed with protein kinase C inhibitors.

Animals↗
PubMed1993

Cloning and characterization of the rat 5-HT5B receptor. Evidence that the 5-HT5B receptor couples to a G protein in mammalian cell membranes.

A gene encoding a novel G protein-coupled 5-hydroxytryptamine (5-HT) receptor, termed 5-HT5B, was cloned. The ligand binding profile of this receptor is distinct from that of other cloned 5-HT receptors. The 5-HT5B receptor couples to a G protein in COS1 cell membranes; however, activation of the 5-HT5B receptor does not appear to alter either cAMP accumulation or phosphoinositide turnover in a variety of fibroblast cell lines. In the rat brain, 5-HT5B gene expression occurs predominantly in the medial habenulae and hippocampal CA1 cells of the adult. Little expression is seen during embryonic development.

Amino Acid Sequence↗
PubMed1993

Muscarinic acetylcholine receptor subtypes associated with release of Alzheimer amyloid precursor derivatives activate multiple signal transduction pathways.

Five subtypes of muscarinic acetylcholine receptors have been identified and designated m1-m5. The m1 and m3 receptors have recently been shown to stimulate APP processing. The m1 and m3 receptors couple to a variety of signal transduction pathways in both tissue slices and a variety of cell lines endogenously expressing either or both subtypes. In contrast, the m2 and m4 receptors have been primarily associated with inhibition of adenylate cyclase. We have transfected all five subtypes of muscarinic receptors into a variety of mammalian cell lines in order to investigate the signaling associated with single receptor subtypes. The m1, m3, or m5 receptors stimulate phospholipase A2, C, and D, adenylate cyclase, receptor-operated calcium channels, and tyrosine kinase activity simultaneously. The m2 or m4 receptor inhibits cAMP accumulation and augments a previously stimulated release of arachidonic acid and calcium influx.

Alzheimer Disease↗
PubMed1993

Receptor-coupled amyloid precursor protein processing.

The family of beta-amyloid protein precursors (APP) can be processed via several alternative proteolytic pathways. Some generate potentially amyloidogenic APP derivatives, whereas others preclude the formation of such fragments. The cellular mechanisms regulating the relative activities of these pathways are thus important in determining the factors contributing to the formation of amyloidogenic APP derivatives. In order to investigate whether cell-surface receptor activity can regulate APP processing, HEK 293 cell lines stably expressing human muscarinic acetylcholine receptors (mAChR; subtypes m1, m2, m3, m4) were stimulated with the muscarinic agonist carbachol, and the release of APP derivatives was measured. Carbachol increased the release of large amino-terminal APP-fragments 4- to 6-fold in cell lines expressing the m1 or m3 receptors but not in those expressing m2 or m4 subtypes. This increase was blocked by various protein kinase inhibitors and mimicked by phorbol esters, indicating that it is mediated by protein kinase activation, presumably by protein kinase C (PKC). To determine whether additional cell-surface receptor types linked to this signal transduction pathway could also regulate APP processing, we stimulated differentiated PC-12 cells with bradykinin and found that this neuropeptide also increased the secretion of amino-terminal APP derivatives. We next investigated the possibility that neuronal depolarization might affect APP processing in mammalian brain. Electrically stimulated rat hippocampal slices released two times more amino-terminal APP derivatives than unstimulated control slices. This release increased with increasing stimulation frequencies in the physiological firing range of hippocampal pyramidal cells, and was blocked by tetrodotoxin. These results suggest that, in brain, APP processing is regulated by neuronal activity.

Amyloid beta-Protein Precursor↗
PubMed1993

Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.

Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.

Amides↗
PubMed1993

Muscarinic receptor-mediated tyrosine phosphorylation of phospholipase C-gamma. An alternative mechanism for cholinergic-induced phosphoinositide breakdown.

In Chinese hamster ovary cells transfected with m5 muscarinic receptors, carbachol stimulates both calcium influx and calcium release from intracellular stores. The marine toxin maitotoxin (MTX) elicits a similar response on calcium influx. Carbachol- and MTX-induced calcium influx can be inhibited by the proposed blockers of receptor-operated calcium channels (ROCC), CAI and SK&F 96365. Both carbachol and MTX induce a significant increase in total protein tyrosine phosphorylation, which is dependent on extracellular calcium and can be inhibited by CAI and SK&F 96365. Phospholipase C-gamma was identified as one of the substrates subject to calcium-dependent tyrosine phosphorylation following carbachol or MTX stimulation. Carbachol-induced [3H]inositol trisphosphate formation was partially inhibited by an inhibitor of tyrosine kinases, by removal of extracellular calcium, and by the inhibitor of receptor-operated calcium channels CAI suggesting that phosphorylation of phospholipase C-gamma plays a role in the muscarinic activation of phosphoinositide breakdown. Such an effect of carbachol is reminiscent of effects observed with peptide growth factors and represents a novel alternative signaling pathway for a muscarinic G protein-coupled receptor.

Animals↗
PubMed1993

Tumor-suppressor function of muscarinic acetylcholine receptors is associated with activation of receptor-operated calcium influx.

Several members of the family of guanine nucleotide-binding protein (G protein)-coupled receptors have recently been shown to induce agonist-dependent foci development in NIH 3T3 cells and tumors in nude mice. We selected the five subtypes of the muscarinic acetylcholine receptor family to investigate their role in tumor suppression. When transfected and expressed in CHO-K1 Chinese hamster ovary cells, m1, m3, and m5 muscarinic acetylcholine receptor activation resulted in a morphology change. Receptor activation did not slow or inhibit monolayer growth of CHOm5 cells in culture but markedly inhibited density-independent growth in soft agar and suppressed tumor formation in nude mice. Receptor-mediated tumor suppression was found to be agonist-dependent and reversible and was blocked with a muscarinic receptor antagonist. Of the five signaling pathways associated with the m1, m3, and m5 receptors, only receptor-operated, and inositol trisphosphate-independent, calcium influx was found to correlate with inhibition of tumorigenicity. These data suggest a pivotal role for inositol trisphosphate-independent receptor-regulated calcium homeostasis in CHO-K1 tumor suppression.

Animals↗
PubMed1993

Anandamide, an endogenous ligand of the cannabinoid receptor, induces hypomotility and hypothermia in vivo in rodents.

Anandamide (arachidonylethanolamide), an arachidonic acid derivative isolated from the porcine brain, displays binding characteristics indicative of an endogenous ligand for the cannabinoid receptor. The functional activity of anandamide was tested in vivo using behavioral and physiological paradigms in laboratory rodents. At IP doses from 2 to 20 mg/kg in mice, anandamide significantly decreased spontaneous motor activity in a Digiscan open field. Rectal body temperature significantly decreased at doses of 10 and 20 mg/kg in rats. At doses from 0.03 to 30 mg/kg, anandamide had no significant effect on chow consumption in ad lib fed rats. Over the dose range of 2-20 mg/kg, anandamide did not show anxiolytic properties in the mouse light<-->dark exploration model of anxiety. Over the dose range of 0.3-3 mg/kg, anandamide had no effect on choice accuracy or session duration in the delayed nonmatching to sample memory task (DNMTS) in rats. These results demonstrate that anandamide has biological and behavioral effects in awake rodents, some of which are similar to the reported actions of THC.

Amides↗
PubMed1993

cAMP-independent, G protein-linked inhibition of Na+/H+ exchange in renal brush border by D1 dopamine agonists.

When D1 dopamine agonists are incubated with renal cortical tissue, Na+/H+ exchange activity is inhibited, presumably due to D1 receptor-mediated stimulation of adenylyl cyclase and subsequent increase in protein kinase A activity. Although the role of adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase in the regulation of Na+/H+ exchange activity is well established, receptors functionally coupled to adenylyl cyclase can regulate Na+/H+ exchange activity independently of changes of cAMP accumulation. The current studies were designed to determine whether D1 agonists can inhibit Na+/H+ exchange activity independently of changes of cAMP accumulation and also to determine the role of G proteins in this process. The D1 agonist, fenoldopam, inhibited Na+/H+ exchange activity in a time-related and concentration-dependent manner. The 50% inhibitory concentration was 5-34 microM. Occupation of the renal D1 receptor mediates this action, since the D1 antagonist, SKF 83742, partially blocks the effect. This action, however, was independent of adenylyl cyclase, protein kinase A, and protein kinase C activity. Inhibition of adenylyl cyclase with dideoxyadenosine or inhibition of protein kinase A and C with the isoquinolines N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (H-4) and 1-(5-isoquinolinesfulfonyl)-2-methylpiperazine (H-7) did not block the effect of fenoldopam on the exchanger. The action of fenoldopam is not due to an amiloride-like action on the exchanger, because kinetic analysis of the inhibitory action was noncompetitive and the effect of fenoldopam was time dependent. The process involved G proteins, since guanosine 5'-O-(2-thiodiphosphate) prevented while guanosine 5'-O-(3-thiotriphosphate) increased the inhibitory effect of fenoldopam.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1992

Muscarinic receptor-operated Ca2+ influx in transfected fibroblast cells is independent of inositol phosphates and release of intracellular Ca2+.

Receptor-mediated changes in cytoplasmic calcium concentrations occur either through release from intracellular calcium stores or by the opening of channels in the plasma membrane, allowing influx of calcium from the extracellular fluid. Carbachol, a muscarinic receptor agonist, stimulated both calcium influx and inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular calcium release in A9 fibroblast cells expressing a m3 muscarinic receptor clone. The calcium influx persisted even after pretreatment of cells with phorbol 12-myristate 13-acetate, which completely prevented the rise in inositol phosphates and intracellular calcium levels. The calcium influx was blocked by divalent cations but was not affected by inhibitors of voltage-dependent calcium channels or high potassium depolarization, indicating the presence of a receptor-operated and voltage-insensitive calcium channel in these cells. Calcium influx was not stimulated by the addition of cAMP analogs or arachidonic acid. To examine the possible involvement of G proteins in m3 receptor-activated calcium influx, two chimeric m2 and m3 muscarinic receptors were expressed in A9 cells in which the third cytoplasmic loop (the primary structural determinant in G protein coupling selectivity of muscarinic receptors) had been exchanged between the m2 receptor, which has no effect on calcium influx, and the m3 receptor. Calcium influx was found to be associated with a structural component of the m3 muscarinic receptor other than the third cytoplasmic loop.

Animals↗
PubMed1992

[3H]-MK 912 binding delineates two alpha 2-adrenoceptor subtypes in rat CNS one of which is identical with the cloned pA2d alpha 2-adrenoceptor.

1. Simultaneous computer modelling of control and guanfacine-masked [3H]-MK 912 saturation curves as well as guanfacine competition curves revealed that the drugs bound to two alpha 2-adrenoceptor subtypes in the rat cerebral cortex with very different selectivities. These alpha 2-adrenoceptor subtypes were designated alpha 2A and alpha 2C. The Kd value of [3H]-MK 912 for the alpha 2A-subtype was 1.77 nM and for the alpha 2C-subtype 0.075 nM; the receptor sites showing capacities 296 and 33 fmol mg-1 protein, respectively. The Kds of guanfacine were 19.9 and 344 nM, respectively. 2. Binding constants of 26 compounds for the two rat cerebral cortex alpha 2-adrenoceptor subtypes were determined by simultaneous computer modelling of control and guanfacine-masked drug competition curves as well as plain guanfacine competition curves using [3H]-MK912 as labelled ligand (i.e. a '3-curve assay'). Of the tested drugs WB4101, corynanthine, rauwolscine, yohimbine, ARC 239 and prazosin were found to be clearly alpha 2C-selective with selectivities ranging from 16 to 30 fold whereas guanfacine, oxymetazoline, BRL 44408 and BRL 41992 were found to be alpha 2A-selective with selectivities ranging from 9 to 22 fold. 3. The Kds of compounds obtained for the cerebral cortex alpha 2C-adrenoceptors showed an almost 1:1 correlation with the corresponding Kds for alpha 2-adrenoceptors expressed by the pA2d-gene (the rat 'alpha 2-C4' adrenoceptor) in CHO-cells. The cerebral cortex alpha 2A-adrenoceptors did not correlate well with the pA2d alpha 2-adrenoceptor Kds. 4. In the rat spinal cord [3H]-MK 912 bound to alpha 2A- and alpha 2C-adrenoceptor sites with similar affinities as in the cerebral cortex and with densities 172 and 7.4 fmol mg-1 protein, respectively. Drug affinities for some compounds showing major selectivity for alpha 2A- and alpha 2C-adrenoceptors were fully compatible with the notion that the spinal cord sites were alpha 2A- and alpha 2C-adrenoceptors.

Adrenergic alpha-Antagonists↗
PubMed1992

Cannabinoid agonists stimulate both receptor- and non-receptor-mediated signal transduction pathways in cells transfected with and expressing cannabinoid receptor clones.

The physiologic activity of (-)-delta 9-tetrahydrocannabinol, the most active component of marijuana, and of many synthetic cannabimimetics may be mediated either through receptor binding and functional coupling to specific signal transduction pathways or through nonspecific interaction with cell membrane components. The cloning of the human and rat cannabinoid receptors has provided the opportunity to investigate the binding properties and signal transduction pathways directly associated with these receptors. Cannabinoid receptor cDNA was transfected into and stably expressed in fibroblast cell lines that do not contain native cannabinoid receptors, thus allowing comparison with untransfected cells. Binding constants measured using [3H]CP55,940 indicated that the rat and human cloned cannabinoid receptors were similar to native cannabinoid receptors measured in brain and neural cell lines. The cloned receptors coupled to the inhibition of cAMP accumulation, as previously demonstrated. CP55,940 binding and inhibition of cAMP accumulation were absent in untransfected cells. Cannabinoid agonist-stimulated release of arachidonic acid and increase in intracellular calcium were observed in both transfected and untransfected cells. Stereoselectivity of cannabinoid agonists was demonstrated for binding and functional inhibition of cAMP accumulation, but not for the release of arachidonic acid and intracellular calcium. Therefore, cannabinoid agonists can stimulate signaling pathways through both receptor- and non-receptor-mediated pathways in the same cell.

Animals↗
PubMed1991

A transduction pathway associated with receptors coupled to the inhibitory guanine nucleotide binding protein Gi that amplifies ATP-mediated arachidonic acid release.

ATP is copackaged and coreleased with adrenergic, serotonergic, and cholinergic neurotransmitters, suggesting a possible interaction between the signaling pathways for ATP and these coreleased neurotransmitters. Muscarinic m2 and m4, alpha 2-adrenergic, and D2-dopaminergic neurotransmitter receptors, which have in common their ability to inhibit adenylate cyclase through the inhibitory guanine nucleotide binding protein Gi, were transfected and expressed in Chinese hamster ovary (CHO) cells that contain endogenous ATP receptors coupled to the release of arachidonic acid. Normal functional coupling of m2, m4, alpha 2, and D2 receptors was demonstrated by their ability to inhibit forskolin-stimulated cAMP accumulation with dose-response activities consistent with previous reports for these Gi-coupled receptors. Stimulation of m2, m4, alpha 2, and D2 receptors resulted in an augmentation of ATP-stimulated arachidonic acid release. With the exception of the m4 receptor, none of the receptors tested was able to stimulate arachidonic acid release in the absence of ATP. Potentiation of ATP-stimulated arachidonic acid release was independent of changes in cAMP. The augmentation of ATP-stimulated arachidonic acid release and the inhibition of cAMP accumulation were both blocked by pertussis toxin, an inhibitor of Gi, but with different dose-response characteristics. Inhibition of protein kinase C with staurosporine or long-term pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the augmentation response. This demonstrates that Gi-coupled inhibitory receptors can amplify ATP-receptor-stimulated arachidonic acid release through a pertussis-toxin-sensitive G protein, independent of their ability to inhibit adenylate cyclase activity.

Adenosine Triphosphate↗