Basal Inositol Phosphate Accumulation Research Articles

GPR80/99, proposed to be the P2Y15 receptor activated by adenosine and AMP, is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y15 receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca2+ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9[12]). However, the cell line (HEK293) used to carry out those studies endogenously expresses A2A and A2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93 [15]) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, α-ketoglutarate. Consistent with this report, α-ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts α-ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by α-ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.

Activation of cyclic AMP-dependent protein kinase inhibits the desensitization and internalization of metabotropic glutamate receptors 1a and 1b.

In this study, we characterized the effects of activation of cyclic AMP-dependent protein kinase (PKA) on the internalization and functional coupling of the metabotropic glutamate receptor (mGluR1) splice variants mGluR1a and mGluR1b. Using an enzyme-linked immunosorbent assay technique to assess receptor internalization, we found that the glutamate-induced internalization of mGluR1a or mGluR1b transiently expressed in human embryonic kidney (HEK) 293 cells was inhibited by coactivation of endogenous beta2-adrenoceptors with isoprenaline or by direct activation of adenylyl cyclase with forskolin. The PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked the effects of both isoprenaline and forskolin. The heterologous internalization of the mGluR1 splice variants triggered by carbachol was also inhibited by isoprenaline and forskolin in a PKA-sensitive fashion, whereas the constitutive (agonist-independent) internalization of mGluR1a was inhibited only modestly by PKA activation. Using inositol phosphate (IP) accumulation in cells prelabeled with [3H]inositol to assess receptor coupling, PKA activation increased basal IP accumulation in mGluR1a receptor-expressing cells and also increased glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells, but only at short times of glutamate addition. Furthermore, PKA activation completely blocked the carbachol-induced heterologous desensitization of glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells. In coimmunoprecipitation experiments, the ability of glutamate to increase association of GRK2 and arrestin-2 with mGluR1a and mGluR1b was inhibited by PKA activation with forskolin. Together, these results indicate that PKA activation inhibits the agonist-induced internalization and desensitization of mGluR1a and mGluR1b, probably by reducing their interaction with GRK2 and nonvisual arrestins.

Agonist-independent internalization of metabotropic glutamate receptor 1a is arrestin- and clathrin-dependent and is suppressed by receptor inverse agonists.

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.

Constitutive activity of the serotonin2C receptor inhibits in vivo dopamine release in the rat striatum and nucleus accumbens.

Numerous research has pointed out that serotonin2c (5-HT2C) receptor, a subtype of 5-HT receptors belonging to the G-protein-coupled receptor superfamily, modulates the activity of mesencephalic dopamine (DA) neurons, the dysfunction of which is involved in devastating diseases such as schizophrenia, Parkinson's disease, and drug addiction. In the present study, using in vivo intracerebral microdialysis and Chinese hamster ovary (CHO) cells expressing 5-HT2C receptors to identify appropriate 5-HT2C receptor ligands, we sought to determine whether the property of 5-HT2C receptors to spontaneously activate intracellular signaling pathways in vitro (constitutive activity) participates in the tonic inhibitory control that they exert on DA release in the rat striatum and nucleus accumbens in vivo. In CHO cells, the purported antagonist 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f] indole hydrochloride (SB 206553), but not 6-chloro-5-methyl-1-[6-(2-methylpiridin-3-yloxy)pyridin-3-yl carbamoyl] indoline (SB 242084), decreased basal inositol phosphate accumulation, thus behaving as a 5-HT2C inverse agonist. Its effect was prevented by SB 242084. In vivo, SB 206553 (1-10 mg/kg) elicited a dose-dependent and clear-cut increase in accumbal and striatal DA release compared with SB 242084 (1-10 mg/kg), and the 5-HT2C agonist S-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine hydrochloride (Ro-60-0175) (0.3-3 mg/kg) inhibited DA release. Pretreatment by SB 242084 reversed the change in DA release elicited by Ro-60-0175 and SB 206553. Furthermore, SB 206553-stimulated DA release was insensitive to reduction of 5-HT neuronal function induced by the 5-HT1A agonist (+/-)-8-hydroxy-2-dipropylaminotetralin or intra-raphe injections of 5,7-dihydroxytryptamine neurotoxin. The obtained results provide the first in vivo evidence that constitutive activity of the 5-HT2C receptor tonically inhibits mesencephalic DA neurons and underscore the need for a better understanding of the pathophysiological role of constitutive receptor activity.

Peptide and nonpeptide antagonist interaction with constitutively active human AT 1 receptors

Wild type human AT 1 receptors (WT-AT 1) and mutant receptors, in which Asn 111 was replaced by glycine (N111G), alanine (N111A) and serine (N111S), or in which Asp 281 was replaced by alanine (D281A) or in which N111G and D281A replacements were combined, were transiently expressed in CHO-K1 cells. While the biphenyltetrazole compound candesartan dissociated slowly and behaved as an insurmountable antagonist for WT-AT 1, it dissociated swiftly and only produced a rightward shift of the angiotensin Ang II- and -IV dose–response curves for inositol phosphate (IP) accumulation in cells expressing N111G. [ 3 H ]candesartan competition binding yielded the same potency order of the related biphenyltetrazoles for WT-AT 1 and mutated receptors, i.e. candesartan>EXP3174>irbesartan>losartan. Affinities were equal for WT-AT 1 and D281A and 40- to 400-fold lower for all Asn 111 mutants. Mutations did not affect the affinity of the peptide antagonist [Sar 1Ile 8]Ang II (SARILE). Basal IP accumulation in cells with WT-AT 1 was not affected by any biphenyltetrazole antagonists and was increased by SARILE to 19% of the maximal Ang II stimulation. Basal IP accumulation was higher for cells expressing the Asn 111-mutated receptors. For N111G, this accumulation was partially inhibited by all the biphenyltetrazoles upon long-term (18 hr) exposure. In these cells SARILE produced the same maximal stimulation as Ang II. Asn 111-mutated AT 1 receptors are thought to mimic the pre-activated state of the wild type receptor and comparing the efficacy and affinity of ligands for such mutated receptors facilitate the distinction of partial (SARILE) and inverse (biphenyltetrazoles) agonists from true antagonists.

A fusion protein of the human P2Y(1) receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling responses to endogenously released nucleotides.

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y(1) receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y(1) receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y(1)-NTPDase1 fusion protein. Stable expression of the P2Y(1)-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca(2+) response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPbetaS to stimulate inositol phosphate accumulation was similar, and the EC(50) of ADPbetaS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y(1) and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y(1) receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y(1) receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y(1) receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.

Adenosine A1 receptor activation inhibits basal accumulation of inositol phosphates in rat hippocampus.

The ability of the adenosine A1 receptor selective agonist, N6-cyclopentyladenosine, to modify basal accumulation of inositol phosphates in rat hippocampal slices, was investigated. Cyclopentyladenosine (10-300 nM) inhibited the basal accumulation of total [3H]inositol phosphates, with an EC50 of 10 nM and an Emax of 24%. This effect of cyclopentyladenosine was prevented by the adenosine A1 receptor selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (30 nM). Cyclopentyladenosine (100 nM) also inhibited histamine (300 nM)-stimulated accumulation of [3H]inositol phosphates, this effect being quantitatively similar to that observed on basal [3H]inositol phosphates accumulation. The results suggest that adenosine A1 receptor activation is able, per se, to inhibit the formation of phosphatidylinositol-derived second messengers in hippocampus.

Disruption of Inositol Phosphate Accumulation in Cerebellar Granule Cells by Polychlorinated Biphenyls: A Consequence of Altered Ca2+Homeostasis

The present study examined the activation of protein kinase C (PKC) and disruption of Ca2+homeostasis as potential mechanisms underlying effects of the polychlorinated biphenyl (PCB) congener 2,2′-dichlorobiphenyl (DCB) on inositol phosphate (IP) signaling in cerebellar granule cells. DCB (100 μM) increased basal IP accumulation in cerebellar granule cells when the extracellular free Ca2+concentration ([Ca2+]e) was 0.75 mMbut not when [Ca2+]ewas 1 μM. Ionomycin (0.1 to 30 μM), a Ca2+ionophore, also increased basal IP accumulation and [Ca2+]iin a concentration-dependent manner in cerebellar granule cells in the absence of DCB; increases in basal IP accumulation induced by 100 μMDCB were not additive with ionomycin. Ionomycin also disrupted carbachol (CARB, 1 mM)-stimulated IP accumulation. A 30-min preincubation with 0.3 or 1.0 μMionomycin decreased CARB-stimulated IP accumulation, whereas simultaneous addition of 1.0 and 10 μMionomycin with CARB increased and decreased, respectively, IP accumulation. DCB caused concentration-dependent increases in intracellular free Ca2+concentration ([Ca2+]i) in cerebellar granule cells under experimental conditions identical to those used to measure IP accumulation. Following a one-half hour exposure to DMSO, 50 or 100 μMDCB, the [Ca2+]iwas 36, 103, and 453 nM, respectively. We examined whether direct or indirect activation of PKC underlies DCB-induced inhibition of agonist-stimulated IP accumulation. DCB (100 μM) did not alter PKC activity in cytosolic or membrane fractions of granule cell homogenates. In intact cells, 50 nMphorbol 12-myristate, 13-acetate (PMA) inhibited CARB-stimulated IP accumulation by 80%, an effect which was blocked completely by the PKC inhibitor bisindolylmaleimide (2 μM; BIM). However, inhibition of CARB-stimulated IP accumulation (90%) induced by 100 μMDCB was not relieved by BIM. These results suggest that (1) perturbations of Ca2+homeostasis may underlie DCB effects on IP accumulation, (2) at a time which corresponds to addition of agonists in IP accumulation assays, [Ca2+]iis elevated in cerebellar granule cells exposed to DCB, and (3) activation of PKC is not a mechanism by which DCB inhibits agonist-stimulated IP accumulation.

Functional characteristics of three new germline mutations of the thyrotropin receptor gene causing autosomal dominant toxic thyroid hyperplasia.

We report three unrelated families in which hyperthyroidism associated with thyroid hyperplasia was transmitted in an autosomal dominant fashion, in the absence of signs of autoimmunity. Exon 10 of the TSH receptor gene was directly sequenced after PCR amplification from DNA of peripheral leukocytes. In one family, a C to A transversion resulted in an S505R substitution in the third transmembrane segment; in the second, an A to T transversion caused a N650Y substitution in the sixth transmembrane segment; and in the third family, an A to G transition resulted in an N670S substitution in the seventh transmembrane segment. When expressed by transfection in COS-7 cells, each mutated receptor displayed an increase in constitutive stimulation of cAMP production; no effect on basal accumulation of inositol phosphates (IP) could be detected. In binding studies, cells transfected with wild-type or mutated receptors showed similar levels of expression, with the mutated receptors displaying similar or slightly increased affinity for bovine TSH (bTSH) binding. Cells transfected with S505R and N650Y mutants showed a similar cAMP maximal TSH-stimulated accumulation over the cells transfected with the wild type, whereas N670S transfectants showed a blunted response with an increase in EC50. A higher IP response to 100 mU/mL bTSH over that obtained with the wild-type receptor was obtained in cells transfected with N650Y; in contrast, cells transfected with S505R showed a blunted IP production (50% less), and the N670S mutant completely lost the ability to stimulate IP accumulation in response to bTSH. The differential effects of individual mutations on stimulation by bTSH of cAMP or IP accumulation suggest that individual mutant receptors may achieve different active conformations with selective abilities to couple to Gs alpha and to Gq alpha.

Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown

The dependence on Ca 2+ of basal, glutamate- and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca 2+ concentrations were buffered by bis-(α-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid, in its tetra(acetoxymethyl)-ester form (BAPTA-AM) and in its free acid form (BAPTA), respectively. The effects of both forms of the calcium chelator intracellular and extracellular Ca 2+ buffering on intracellular and extracellular Ca 2+ concentration ([Ca 2+] i and [Ca 2+] e) were determined with fluorimetric assay using fura2, either in its acetoxymethyl ester form (fura2-AM) or in its free acid form. Intracellular chelation of Ca 2+ ions with BAPTA-AM induced a dose-dependent reduction of the [Ca 2+] i. Basal inositol phosphate (IP) formation was slightly affected by this [Ca 2+] i buffering, while glutamate and carbachol stimulations of PI hydrolysis were similarly diminished. Chelation of extracellular Ca 2+ ions with BAPTA produced a reduction of both [Ca 2+] e and [Ca 2+] i. Basal IP accumulation was maximally inhibited by 50%. The carbachol-induced PI hydrolysis was completely inhibited in the presence of 200 μM BAPTA, while a substantial residual glutamate-elicited IP response remained (40% of the control response). It is concluded that [Ca 2+] i of synaptoneurosomes is not critical for basal and neurotransmitter-stimulated IP formation, whilst [Ca 2+] e is critical. Glutamate may, in part, stimulate PI breakdown in a Ca 2+-insensitive way.

Glutamate-dependent mechanisms in the induction of a calcium long-term potentiation-like phenomenon

The electric synaptic efficacy, in terms of extracellular electrical potentials, and the intracellular postsynaptic efficacy, in terms of inositol phosphate (IP) accumulation, were evaluated in rat hippocampal slices exposed for a brief period (10 min) to a high concentration of calcium (+2.7 mM). In addition, the effects of N-methyl-D-asparate (NMDA) ionotropic and metabotropic glutamate receptor (mGluR) antagonists on the induction and the establishment or maintenance of enhanced synaptic efficacy of CA1 pyramidal neurons due to high-calcium exposure were also tested. Elevation of the calcium concentration from 1.3-4 mM in the medium bathing hippocampal slices produced a long-lasting (80 over 90 min) increase in the slope of the CA1 somatic excitatory postsynaptic potential and the amplitude of the population spike (PS). Slice perfusion with NMDA antagonists cyclazocine and cis-4-phosphonomethyl-2-piperidine-carboxylic acid (CGS 19755) or with mGluR antagonists L-2-amino-3-phosphonopropionic acid (AP3) or alpha-methyl-4-carboxyphenyl-glycine (all 0.1 mM), during the 10-min period of exposure to high-calcium prevented the induction of such changes. By contrast, slice perfusion with the same concentration of CGS 19755 or L-AP3 did not affect the already established long-lasting increase in amplitude of CA1 PS induced by high-calcium. Moreover, high-calcium failed to produce any significant modification of the basal IP accumulation or of the IP accumulation elicited by mGluR agonist 1S,3R-trans-amino cyclo-pentane-1,3-dicarboxylic acid (ACPD). In conclusion, the results confirm that high-calcium induces a long-lasting increase in synaptic efficacy in rat hippocampal slices. Both NMDA ionotropic and mGluR receptors are involved in the induction, but not in the maintenance, of this phenomenon. In line with these data no modifications of basal or ACPD-induced phosphoinositide hydrolysis have been found during the maintenance stage.

Signal transduction mechanisms of the vasoconstriction in hypertension

We tested the hypothesis that vascular smooth muscle in genetic hypertension is characterised by hypereactivity to vasoactive agonists, by abnormalities in Ca 2+ handling and the phosphoinositide signalling system. Activation of these signal transduction mechanisms by noradrenaline and endothelin-1 was compared in isolated perfused tail arteries from adult hypertensive and normotensive Wistar Kyoto rats. Basal cytosolic Ca 2+ was greater in arteries from hypertensive rats, but basal perfusion pressure and basal inositol phosphate accumulation were unchanged. Contractile responses and Ca 2+ mobilisation after noradrenaline, but not endothelin-1, were enhanced in arteries from hypertensive rats. Total inositol phosphates accumulation was similar in hypertensive and normotensive rats after either noradrenaline or endothelin-1 stimulation. In both hypertensive and normotensive rats, for a given Ca 2+ mobilisation, higher contractile responses and higher levels of inositol phosphates were observed after endothelin-1 than noradrenaline stimulation. In conclusion, changes in contractility associated with modifications in the Ca 2+ handling between hypertensive and normotensive rats suggested that alterations in the signal-transduction system occur with hypertension. The different effects of endothelin-1 and noradrenaline could be related to interactions with other signalling pathways.

Inhibition of dopamine agonist-induced phosphoinositide hydrolysis by concomitant stimulation of cyclic AMP formation in brain slices.

We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp-cyclic adenosine monophosphothioate (Sp-cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D1 receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp-cyclic adenosine monophosphothioate (Rp-cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 microM Rp-cAMPS, the dose-response curves of the dopamine D1 receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC50 values, however, were not significantly altered by protein kinase A inhibition. Neither Sp-cAMPS nor Rp-cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D1 receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.

Altered protein kinase C regulation of phosphoinositide-coupled receptors in deoxycorticosterone acetate-salt hypertensive rats.

This study examined the contribution of phosphatidylinositol metabolism and the efficacy of protein kinase C-mediated desensitization in the exaggerated alpha 1b-adrenergic receptor-mediated inositol phosphate response in the aorta of the deoxycorticosterone acetate (DOCA)-salt rat model of hypertension. The basal accumulation of inositol phosphates and the basal incorporation of [3H]myo-inositol in the phosphatidylinositol lipid pool were significantly higher in the aorta of these hypertensive rats. A positive correlation (r = .88, P < .01) was demonstrated between basal inositol phosphate levels and the [3H]myo-inositol-labeled phosphatidylinositol lipid pool. In hypertensive rats, alpha 1b-adrenergic receptor-mediated inositol phosphate production in response to phenylephrine was significantly higher compared with normotensive rats. Despite the normalization of phenylephrine-mediated inositol phosphate production to the [3H]myo-inositol-labeled phosphatidylinositol lipid pool, the alpha 1b-adrenergic response remained significantly higher in the hypertensive rats. Phorbol ester activation of protein kinase C attenuated to a lesser extent phenylephrine-mediated inositol phosphate production (40%) in the aorta of hypertensive rats compared with the 80% attenuation observed in the aorta of normotensive rats. This desensitization was inhibited in both groups by the protein kinase C inhibitor staurosporine. The blunted desensitization of the alpha 1b-adrenergic receptor by protein kinase C activation was not associated with a decrease in protein kinase C activity in the hypertensive rats, because aortic strips from these animals were more responsive to phorbol ester activation than aortic strips from normotensive animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the Endothelin-1 Transmembrane Signaling Pathway: the Potential Role of Agonist-Induced Desensitization in the Coronary Artery of the Rapid Ventricular Pacing-Overdrive Dog Model of Heart Failure

This study examined the potential role of ET-1 and the contribution of protein kinase C (PKC) in the desensitization of the ET-1 transmembrane signaling pathway in the left circumflex coronary artery (CCA) of a dog model of congestive heart failure (CHF). In the CCA of the rapid ventricular pacing-overdrive dog model of CHF, elevated plasma endothelin levels were associated with a decrease in the basal accumulation of inositol phosphates and ET-1 mediated activation of phosphatidylinositol (PI) turnover ( P < 0.05). To assess whether elevated plasma ET-1 levels may have contributed to the diminished ET-1 responsiveness in the heart failure dogs, ET-1 generation of inositol phosphates was measured following a one hour pretreatment of normal coronary artery rings with 0.1 nM ET-1. As compared to non-treated rings, ET-1 pretreatment resulted in a 33% decrease of ET-1 (10 nM) production of inositol phosphates. To evaluate the role of PKC in this process, normal coronary rings pretreated for a period of one hour with the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 μM), resulted in a similar attenuation (36%) of ET-1 production of inositol phosphates. In the presence of the protein kinase C inhibitor staurosporine, both the agonist and phorbol ester induced decreases in ET-1 mediated PI turnover were reversed. Staurosporine even potentiated (75%) ET-1 induced PI turnover despite ET-1 and PMA pretreatments. These results suggest that agonist-induced desensitization of ET-1 mediated PI turnover can occur and is at least one of the possible mechanisms contributing to the desensitization of the ET-1 transmembrane signaling pathway in the pacing-overdrive model of heart failure in the dog.

Enhanced receptor-dependent inositol phosphate accumulation in hypoxic myocytes.

The arrhythmogenic effects of alpha 1-adrenergic receptor agonists are enhanced in the ischemic myocardium. The present study was designed to determine whether hypoxia influences alpha 1-receptor subtype activation of phosphoinositide hydrolysis in neonatal rat ventricular myocyte cultures. Hypoxia did not alter basal inositol phosphate accumulation, but markedly increased norepinephrine-dependent inositol phosphate accumulation. This effect was apparent within 30 min and readily reversed on 30 min of reoxygenation. The response to norepinephrine reflected activation of a specific alpha 1-adrenergic receptor subtype; it was inhibited by prazosin and WB-4101 but not by chloroethylclonidine or propranolol. The density of alpha 1-adrenergic receptors identified by [125I]IBE-2254 was similar in normoxic and hypoxic myocytes. Consistent with this observation, the response to a maximal concentration of norepinephrine was enhanced by hypoxia, but the half-maximum effective dose for norepinephrine was not modified. The effects of isoproterenol to stimulate adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and of carbachol to inhibit isoproterenol-stimulated cAMP accumulation were not influenced by hypoxia. In contrast, inositol phosphate accumulation in response to carbachol or thrombin was markedly increased in hypoxic myocytes. These results demonstrate an effect of hypoxia to enhance phosphoinositide hydrolysis through a mechanism(s) distal to the receptor that may have important implications with respect to calcium overload and electrical abnormalities during myocardial ischemia.

A Specific Trarisduction Mechanism for the Glutamate Action on Phosphoinositide Metabolism via the Quisqualate Metabotropic Receptor in Rat Brain Synaptoneurosomes: I. External Na+ Requirement

The characteristics of the transduction mechanism(s) activated by glutamate (Glu) via the quisqualate metabotropic receptor, as well as by depolarizing agents, to trigger formation of inositol phosphates (IPs) were investigated in 8-day-old rat forebrain synaptoneurosomes. The replacement of external Na+ by various compounds (Li+, Tris+, N-methyl-D-glucamine+, and sucrose) induces an increase in basal accumulation of IPs and depolarizes synaptoneurosome membranes. Under these conditions, Glu- and K(+)-induced accumulations of IPs are inhibited, whereas the carbachol (Carb)-elicited response of IPs parallels the basal one. Agents increasing Na+ influx, such as veratridine and monensin, depolarize synaptoneurosomes and stimulate formation of IPs. These stimulations are not additive with responses of IPs elicited by Glu or K+. These data suggest that (a) Glu activates phosphoinositide metabolism via a specific mechanism (distinct from that of cholinergic agonists), (b) depolarizing agents and Glu share at least one common intermediate step in their mechanisms of activation of the metabolism of IPs, and (c) the depolarization may correspond to this common step. In addition, Na+ seems to be required for Glu stimulation of metabolism of IPs. The depolarization associated with the action of Glu on formation of IPs results neither from an influx via tetrodotoxin-sensitive voltage-dependent Na+ channels nor from an entry via the classically characterized Na+/Ca2+ or Na+/H+ exchangers. In fact, tetrodotoxin (2 microM) has no effect on the Glu- or K(+)-elicited response of IPs. Amiloride (greater than 50 microM) and some of its derivatives similarly inhibit not only Glu- and K(+)- but also Carb-evoked formation of IPs.

Further evidence against the coupling of dopamine receptors to phosphoinositide hydrolysis in rat striatum

The effects of D 1 and D 2 dopamine receptor agonists on phosphoinositide hydrolysis were studied by measuring the accumulation of radioactive inositol phosphates in slices of rat corpus striatum prelabelled with [ 3H]inositol. All assays were performed in the presence of lithium. Neither the D 1 receptor agonist SKF 38393 nor the D 2 receptor agonist quinpirole, alone or in combination, had an effect on basal accumulation of inositol phosphates. The muscarinic receptor agonist carbachol produced a robust increase in the accumulation of inositol monophosphate and a smaller increase in the accumulation of inositol bisphosphate. These effects were not altered by the presence of quinpirole. Additionally, quinpirole also had no effect when assays were conducted in the presence of the muscarinic receptor antagonist scopolamine, the glutamic acid receptor antagonist kynurenic acid, and the antioxidant glutathione. These results are discussed in relation to recent contradictory reports and lend support to the position that D 2 dopamine receptors are not coupled to phosphoinositide hydrolysis in rat striatum.

Effects of insulin on inositol phosphate production in cultured rat hepatocytes

Addition of vasopressin (100 nM) to rat hepatocytes prelabelled with [ 3H]inositol stimulated the production of inositol phosphates in the presence of 20 mM Li +. Preincubation of hepatocytes with insulin (50 nM) or glucagon (10 nM) had no significant effect alone but enhanced the effects of vasopressin after a lag period of at least 1 min. The effects of insulin and glucagon appeared additive in this respect. Insulin also enhanced the norepinephrine-mediated stimulation of inositol phosphate accumulation. The enhancement by insulin of the effects of vasopressin required at least 0.5–5 nM insulin and did not involve changes in 3H]inositol lipid labelling or IP 3 phosphatase activity. The effect of insulin appeared insensitive to prior treatment of hepatocytes with pertussis toxin (200 ng/ml for 18–24 h) or cholera toxin (100 ng/ml for 3–4 h). The glucagon enhancement of the effects of vasopressin was not affected by pertussis toxin but was mimicked by cholera toxin. The response of hepatocytes to vasopressin in the absence of Li + was smaller and more transient. Under these conditions a 5 min prior incubation with insulin inhibited the stimulation by vasopressin of inositol phosphate accumulation. A similar inhibitory effect of prior insulin exposure on the transient activation by vasopressin of exogenous phosphatidylinositol 4,5-bisphosphate breakdown by hepatocyte homogenates was also seen. These data indicate that insulin, although having no effect on basal inositol phosphate accumulation, can either enhance or antagonise the effects of vasopressin in primary rat liver hepatocyte cultures depending on the experimental conditions.

Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats

Recent studies suggest that serotonergic receptor activation is coupled to phospholipase C-mediated phosphoinositide hydrolysis, which results in the release of intracellular second messengers. The purpose of this study was to determine whether altered phosphoinositide metabolism is the basis for augmented vascular responsiveness to serotonin in genetic hypertension. Thoracic aortic segments isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) were labeled with myo-[3H]inositol and stimulated with serotonin in the presence of LiCl. Accumulation of [3H]inositol phosphates was then quantitated by column chromatography. Basal inositol phosphate accumulation and basal incorporation of myo-[3H]inositol into aortic cell membranes from SHRSP was not significantly different from WKY values. At 2.6 x 10(-7) to 2.6 x 10(-4) M serotonin, phosphoinositide metabolism was significantly augmented in aortae from SHRSP compared with WKY. Depolarization (100 mM KCl) did not increase phosphoinositide hydrolysis above basal levels in SHRSP or WKY. 2-Nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phospholipase C, prevented the serotonin-induced phosphoinositide metabolism. NCDC also partially inhibited phasic contractions (responses in calcium-free solution) to serotonin in aortas from SHRSP and WKY. In conclusion, abnormal phosphoinositide metabolism may be one mechanism responsible for the characteristic increase in vascular reactivity to serotonin in hypertension.