Widespread distribution of Gq alpha/G11 alpha detected immunologically by an antipeptide antiserum directed against the predicted C-terminal decapeptide.

Antisera were raised to a synthetic peptide which represents the predicted C-terminal decapeptide of the alpha subunit of the G-proteins Gq and G11. Competitive ELISA indicated that antiserum CQ2 displayed strong reactivity against this peptide. Antiserum CQ2 identified an apparently single polypeptide of 42 kDa which was expressed widely. The mobility of this polypeptide in SDS-PAGE was not modified by pretreatment of cells with pertussis toxin, indicating that it was not a substrate for this toxin. Furthermore, the levels and mobility of this polypeptide were unaltered by treatment of cells with cholera toxin, defining that it was not related to Gs alpha.

The inhibition of agonist- or depolarisation-evoked formation of inositol phosphate by excitatory amino acids in rat cerebral cortex is due to the neurotoxic action of this class of neurotransmitter and is mediated by sodium influx.

Previous work has shown that excitatory amino acids inhibit agonist or depolarisation evoked formation of inositol phosphate in brain. In this paper, possible mechanisms by which this may be occurring have been investigated. The inhibition of carbachol-stimulated formation of inositol phosphate by kainic acid (KA) was abolished if the tissue was incubated in a sodium-free medium. The sodium channel activator, veratridine (10 microM) and the sodium ionophore, monensin (3 microM), also inhibited the response of inositol phosphate to carbachol; tetrodotoxin (300 nM) reversed the effect of veratridine but not monensin or KA. Incubation with cadmium (0.3 mM) or removal of extracellular calcium did not alter the effects of KA, monensin or veratridine. The effects of KA were significantly reduced with the Na+/K(+)-ATPase inhibitor, ouabain (10-100 microM). Inhibition by KA was still observed in tissue that had been prestimulated with KA and then washed to remove the agonist. Incorporation of [3H]inositol into inositol lipids was significantly reduced by KA, in the absence or presence of carbachol. It is suggested that the inhibition of the turnover of stimulated phosphoinositide, by excitatory amino acids, is related to the neurotoxic actions of these transmitters and is mediated by Na+ influx, with a consequent activation of Na+/K(+)-ATPase, depletion of cellular ATP and reduction in synthesis of inositol lipid.

Stimulatory and inhibitory actions of excitatory amino acids on inositol phospholipid metabolism in rat cerebral cortex.

1. The effects of excitatory amino acids on [3H]-inositol phosphate levels have been examined in rat cortical slices under basal conditions or following agonist stimulation. 2. Ibotenate and quisqualate provoked a substantial dose-dependent (EC50, 30 microM and 20 microM respectively) increase in inositol phosphates; these responses were not additive suggesting a common site of action for the two amino acids. The responses to maximally effective concentrations of ibotenate and quisqualate were not blocked by verapamil, tetrodotoxin or Cd2+, indicating that these effects are not indirect. Small, but significant, increases in inositol phosphates were also seen with glutamate and N-methyl-DL-aspartate (NMDLA); kainate and aspartate were ineffective. 3. Each excitatory amino acid tested reduced carbachol (1 mM) stimulated inositol phosphate formation. Kainate (IC50, 20 microM) and NMDLA (IC50, 20 microM) were the most effective inhibitors. Kainate also reduced the responses to noradrenaline, 5-hydroxytryptamine and 20 mM K+. 4. The inhibitory action of NMDLA, but not kainate, could be reversed with the NMDA antagonists, DL-2-amino-5-phosphonovalerate (APV) and MK-801; DL-2-amino-4-phosphonobutyrate (APB) was without effect. Since MK-801 blocks the ion channels associated with the NMDA receptor, it appears that inhibition requires the entry of ions into the cell. 5. APV and MK-801 potentiated the stimulatory response to ibotenate but had no effect on the response to quisqualate. Potentiation was presumably the result of blocking the inhibition by ibotenate mediated through NMDA receptors. 6. In conclusion, excitatory amino acids appear to reduce agonist-mediated inositol phosphate formation in rat cerebral cortex by a non-specific action, possibly including the influx of Na+ ions. In addition ibotenate and quisqualate substantially enhance inositol phosphate production: the pharmacology of the response suggests that it is mediated by a receptor distinct from previously defined excitatory amino acid receptor subtypes.

Chronic carbamazepine down-regulates adenosine A2 receptors: studies with the putative selective adenosine antagonists PD115,199 and PD116,948.

Carbamazepine (CBZ), an anticonvulsant with psychotropic and anti-pain properties, has been reported to displace ligands at adenosine binding sites. This paper describes biochemical and behavioural studies in rodents comparing CBZ to the adenosine agonists L-phenylisopropyl-adenosine (L-PIA) and N-ethylcarboxamido-adenosine (NECA), the new antagonists PD116,948 and PD115,199 which are also relatively A1 and A2 specific respectively, and the mixed antagonists theophylline and caffeine, attempting to determine functional correlates of the binding studies. Changes in cAMP synthesis and behavioural syndromes produced by the drugs, alone and in combination, were monitored. Classification of the observed effects in terms of A1 and A2 activity was complex, probably due to functional interactions between A1 and A2 subtypes. Nevertheless, it was found that chronic CBZ administration (0.25% in food for 3 days, followed by 0.5% for 11 days) produced a pattern of interaction identical to that of PD115,199 (10-100 mg/kg IP). Thus, both treatments attenuated the behavioural syndrome produced by L-PIA (0.1 or 0.5 mg/kg SC), but did not affect that produced by NECA (0.03 mg/kg SC). CBZ mildly increased hypoactivity after clonidine (0.2 mg/kg IP) which was used as a control. By contrast, the A1 antagonist PD116,948 (0.1-10 mg/kg IP) antagonised both behavioural syndromes. Similarly in the biochemical experiments both chronic CBZ and PD115,199 (10-100 microM) reduced stimulation of cAMP synthesis by L-PIA (confirming that this is mediated by A2 receptors), while only basal cAMP synthesis was affected by PD116,948 (10 microM) and theophylline (60 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of non-NMDA antagonists and phorbol esters on excitatory amino acid stimulated inositol phosphate formation in rat cerebral cortex.

The effects of various modulators of excitatory amino acid stimulated inositol phosphate (IP) formation were investigated in slices of rat cerebral cortex. Both quisqualic acid (Quis) and ibotenic acid (Ibo) stimulated IP formation in a dose-dependent manner. Quis (0.3mM) stimulated the rapid formation of inositol bis, tris and tetrakis-phosphates, with a slower linear rise in the monophosphate which plateaued after 20 min. The responses to both Ibo (0.3 mM) and Quis (0.3 mM) were dose-dependently inhibited by phorbol dibutyrate (PDBu); the Ibo response was particularly sensitive to PDBu, with a 90% inhibition of the response at 1 ?M and an IC(50) of 200 nM; Quis stimulated IP formation was less sensitive with a 50% inhibition observed at 10 ?M PDBu. PMA (1 ?M) and dioctanoylglycerol (30 ?M) also inhibited Ibo-stimulated IP formation though 4-?-phorbol (10 ?M) was ineffective. The inhibition by PDBu was reversed with staurosporine (10 ?M) and polymyxin (10 ?M), and both protein kinase C inhibitors potentiated the Ibo response in the absence of PDBu. The putative Quis-kainate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and 6-cyano-7-nitroquinoxaline-2,3-dione did not inhibit the responses to either Quis or Ibo at a concentration of 100 ?M. Both responses were, however, inhibited by dl-2-amino-4-phosphonobutyric acid and O- phospho- l -serine (IC(50)s were 1-2 mM), and also by the putative Quis receptor agonist dl-?-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA; IC(50) 10?M). Our data suggest that excitatory amino acid stimulated IP formation in rat cerebral cortex is via a site distinct from previously defined amino acid receptors.

Differential effects of lithium on agonist-stimulated inositol polyphosphate formation in rat cerebral cortex slices: Selective actions on muscarinic cholinergic responses.

The effect of lithium ions on agonist-stimulated inositol polyphosphate production was investigated in slices of rat cerebral cortex. LiCl potentiated the formation of inositol monophosphate and inositol bisphosphate following stimulation with a variety of agonists including carbachol (1 mM), noradrenaline (NA, 300 ?M), 5-hydroxytryptamine (5-HT, 100 ?M) and quisqualic acid (Quis, 100 ?M), the EC(50) for these effects was in the range 0.5-5 mM. The production of inositol trisphosphate and inositol tetrakisphosphate following NA, 5-HT or Quis stimulation was not significantly affected by LiCl, though there was a delayed but striking inhibition of both the inositol 1,4,5-trisphosphate and, particularly, inositol tetrakisphosphate responses to carbachol, which was even greater in buffer containing elevated (20 mM) KCl. The possible mechanisms underlying this effect of LiCl are discussed in relation to previously observed effects of this ion on phosphoinositide metabolism.

Lithium potentiates agonist formation of [3H]CDP-diacylglycerol in human platelets.

Thrombin stimulated a rapid formation of [3H]CDP-diacylglycerol in platelets prelabelled with [3H]cytidine. This response was increased in the presence of LiCl after a delay of 5 min; potentiation could be prevented by myo-inositol. Since Li+ inhibits the liberation of inositol from inositol phosphates, the conversion of diacylglycerol to phosphatidylinositol via CDP-diacylglycerol may be dependent on the regeneration of inositol from this pathway.

GABAB receptor activation inhibits 5-hydroxytryptamine-stimulated inositol phospholipid turnover in mouse cerebral cortex.

The effects of GABA agonists on inositol phospholipid metabolism were investigated in mouse cerebral cortex. GABA (in the presence of 1 mM nipecotic acid) dose dependently inhibited 5-hydroxytryptamine (5-HT)-stimulated [3H]inositol phosphate formation; maximal inhibition 60.4 +/- 8.8%, ED50 200 nM. The GABAB agonist baclofen had a similar effect; maximal inhibition 41.7 +/- 4.5%, ED50 100 nM. The inhibitory action of GABA was not blocked by bicucculine. The results suggest that GABAB receptors may modulate 5-HT2 receptor function at the level of signal transduction.

Alteration of inositol phospholipid metabolism in rat cortex by lithium but not carbamazepine.

The effect of carbamazepine on inositol phospholipid metabolism was investigated in rat cerebral cortex. The stimulation of inositol phosphate formation by 5-HT (10 microM), carbachol (100 microM) and noradrenaline (30 microM) was not significantly altered by carbamazepine in vitro. 14 days treatment of rats in vivo with carbamazepine was similarly without effect on these parameters. In contrast 14 days treatment with LiCl reduced the agonist responses by 25-50%. The results are discussed in relation to the therapeutic actions of these drugs.

Serotonergic agonists stimulate inositol lipid metabolism in rabbit platelets.

The metabolism of inositol phospholipids in response to serotonergic agonists was investigated in rabbit platelets. In platelets prelabelled with [3H]-inositol, in a medium containing 10 mM LiCl which blocks the enzyme inositol-1-phosphatase, 5-hydroxytryptamine (5-HT) caused a dose-dependent accumulation of inositol phosphates (IP). This suggests a phospholipase-C-mediated breakdown of phosphoinositides. Ketanserin, a selective 5-HT2 antagonist, was a potent inhibitor of the 5-HT response, with a Ki of 28 nM, indicating that 5-HT is activating receptors of the 5-HT2 type in the platelet. Lysergic acid diethylamide (LSD) and quipazine also caused dose-related increases in inositol phosphate levels, though these were considerably less than those produced by 5-HT. These results show that relatively small changes in phosphoinositide metabolism induced by serotonergic agonists can be investigated in the rabbit platelet, and this cell may therefore be a useful model for the study of some 5-HT receptors.

Potentiation by lithium of CMP-phosphatidate formation in carbachol-stimulated rat cerebral-cortical slices and its reversal by myo-inositol.

This paper describes a rapid and simple method for measuring CMP-phosphatidate (CMP-PA; CDP-diacylglycerol), providing a novel assay for inositol phospholipid metabolism. Rat cerebral-cortical slices labelled with [14C]cytidine were incubated with the muscarinic cholinergic agonist carbachol in the presence of various concentrations of LiCl; 10 mM-LiCl greatly enhanced the carbachol-stimulated formation of [14C]CMP-PA over a 60 min incubation period. The potentiation by Li+ was concentration-dependent, with a maximal enhancement at 3 mM and half-maximal enhancement at 0.6 mM-LiCl. The enhancement by Li+ could be reversed by incubation with myo-inositol; a maximal effect was observed with 10 mM-inositol. A similar, though smaller, enhancement of CMP-PA concentrations in the presence of LiCl was observed in slices stimulated with noradrenaline, 5-hydroxytryptamine and K+. The results are discussed in relation to previously observed effects of Li+ on inositol phospholipid metabolism.

Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells.

The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.

Fluoride inhibits agonist-induced formation of inositol phosphates in rat cortex.

Sodium fluoride inhibited carbachol, 5-hydroxytryptamine and noradrenaline stimulated formation of inositol phosphates in rat cerebral cortex. For example, carbachol (1 mM) induced a 337% increase of inositol phosphates above basal in 30 min which was reduced to 69% in the presence of NaF (10 mM). The IC50 for NaF was approximately 1.5 mM and inhibition was mediated by a decrease in maxima of the carbachol dose response curve rather than a shift to the right. This inhibitory action was not mimicked by NaBr or NaI, or by agents which increase cAMP. Inhibition did not appear to result from a toxic action of NaF since it had no effect on the formation of inositol phosphates by high K+; moreover, in higher concentrations NaF stimulated phospholipase C activity. Since fluoride ions are known to activate G-proteins in the concentrations used in this study, these results may indicate the existence of a novel G-protein linked to receptor inhibition of phospholipase C.

Dose-dependent inhibition of phosphoinositide metabolism in human platelets by aspirin in vitro and in vivo.

The effects of aspirin on the metabolism of phosphoinositides in human platelets were studied in vitro and in vivo. Eight volunteers received, at two-weekly intervals, a single dose of 10, 30, 100 or 600 mg aspirin. Before the first dose platelets were taken and incubated in vitro with a range of concentrations (10 nM-100 microM) of aspirin. Formation of inositol phosphates (IP) was measured in [3H]-inositol labelled platelets after incubation with collagen and thrombin for 30 min, a time at which a maximal increase in [3H]-IP was observed. The in vitro IC50 for inhibition of the response to collagen by aspirin was approximately 1 microM; the in vivo ID50 was 40-50 mg. Aspirin did not fully inhibit the collagen stimulated IP formation either in vitro or in vivo, and the response to thrombin was unaffected. The ID50 and IC50 of aspirin is thus in accord with the doses of aspirin associated with inhibition of platelet aggregation and thromboxane production in other studies. The possible relevance of these data to the clinical uses of aspirin is discussed.

Neomycin cannot be used as a selective inhibitor of inositol phospholipid hydrolysis in intact or semi-permeabilized human platelets. Aminoglycosides activate semi-permeabilized platelets.

High concentrations of neomycin (2-10 mM) inhibited aggregation, but not shape change, of intact platelets by collagen, ADP and the Ca2+ ionophore, A23187, the last two studies being carried out in the presence of the cyclo-oxygenase inhibitor indomethacin. In contrast, over the same range of concentrations neomycin inhibited both aggregation and shape change induced by thrombin. Under these conditions activation of platelets by collagen and by thrombin, but not by A23187 or by ADP, is believed to be dependent on the hydrolysis of membrane inositol phospholipids. These data therefore suggest that the inhibitory action of neomycin on intact platelets is not related to its previously reported inhibitory effect on phosphoinositide metabolism. The selective inhibition of thrombin-induced shape change indicates a second site of action of neomycin on intact platelets. On platelets rendered semi-permeable with saponin, neomycin and a second aminoglycoside antibiotic, streptomycin (each 0.06-2 mM), stimulated secretion and aggregation responses. These effects were inhibited by indomethacin and by EGTA. Activation of semi-permeabilized platelets by neomycin is associated with the formation of inositol phosphates and phosphatidic acid, indicating activation by phospholipase C. This effect is also inhibited by indomethacin, implying that it is secondary to the formation of prostaglandins and endoperoxides. These results are discussed in the context of the use of neomycin as a selective inhibitor of polyphosphoinositide metabolism.

Effects of colchicine and vinblastine on output of proteins into bile.

The microtubule poisons colchicine and vinblastine caused a reduction in the biliary output of total protein, compared with controls, in bile-fistula rats. The various protein components of bile showed patterns of change in output differing from one another. Alkaline phosphodiesterase I, a 'plasma-membrane' enzyme, showed a decline in output during the first hour after drug administration. Immunoglobulin A output did not decline until after the first hour. In contrast with these reductions, the biliary output of (rat) plasma albumin into bile was increased. At no time was there any evidence (as measured by lactate dehydrogenase release into bile) for any cytolytic damage. These results are discussed in relation to current theories on the output of proteins into bile and the repair of the plasma membrane in the bile canaliculus.

Enzymes and proteins in bile. Variations in output in rat cannula bile during and after depletion of the bile-salt pool.

The protein concentration in bile from several species is reported. The changes in output of protein, bile salts and several enzymes have been followed in rat bile over a 48 h cannulation period. Bile-salt concentration dropped rapidly owing to interruption of the enterohepatic circulation but the output of protein, lysosomal enzymes [acid phosphatase (EC 3.1.3.2) and beta-D-glucuronidase (EC 3.2.1.31)] and plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I (EC 3.1.4.1)] was maintained. Liver cell damage, monitored by output of lactate dehydrogenase, was very low throughout. Protein, lysosomal enzymes and plasma-membrane enzymes showed different patterns of output with time, but all showed a net increase between 12 and 24 h. The output of lysosomal and plasma-membrane enzymes was between 1 and 5% of the total liver complement over the first 24 h; if inhibition by biliary components is taken into account the output of some of these enzymes, particularly acid phosphatase, may be greater. Ultracentrifugation of bile showed that as the concentration of bile salts decreases the proportion of plasma-membrane enzymes in a sedimentable form increases. The results are discussed in relation to other studies of biliary proteins and to studies of the perturbation of membranes and cells with bile salts.

Membranes and bile formation. Composition of several mammalian biles and their membrane-damaging properties.

The total content and profile of bile salts and phospholipids are reported for several mammalian biles. Rabbit and guinea-pig biles are characterized by high proportions of conjugated dihydroxy bile salts with respect to trihydroxy bile salts, but contain relatively little phospholipid. Both rabbit and guinea-pig biles exhibit little evidence of hepatic cell damage, even though they are able to cause membrane damage (as evidenced by lysis of human erythrocytes) at low (2--3 mM) concentrations of bile salts; this lytic behaviour is also a property of their predominant bile salts. Addition of phosphatidylcholine to the bile or bile salt is able to decrease the lytic behaviour. Perhaps the most significant observation is that these biles, and their predominant bile salts, are dramatically less lytic towards sheep erythrocytes, indicating that some factor(s) in membrane composition and structure may partly explain the resistance of membranes of the biliary tract to the presence of high concentrations of potentially membrane-damaging bile salts.

5-Hydroxytryptamine-stimulated inositol phospholipid hydrolysis in the mouse cortex has pharmacological characteristics compatible with mediation via 5-HT2 receptors but this response does not reflect altered 5-HT2 function after 5,7-dihydroxytryptamine lesioning or repeated antidepressant treatments.

5-Hydroxytryptamine (5-HT; 3 x 10(-8)-1 x 10(-5)M) produced a dose-dependent increase in phosphatidylinositol/polyphosphoinositide (PI) turnover in mouse cortical slices, as measured by following production of 3H-labelled inositol phosphates (IPs) in the presence of 10 mM LiCl. Analysis of individual IPs, in slices stimulated for 45 min, indicated substantial increases in inositol monophosphate (IP1; 140%) and inositol bisphosphate (IP2; 95%) contents with smaller increases in inositol trisphosphate (IP3; 51%) and inositol tetrakisphosphate (IP4; 48%) contents. The increase in IP3 level was solely in the 1,3,4-isomer. This response was inhibited by the nonselective 5-HT antagonists methysergide, metergoline, and spiperone. It was also inhibited by the selective 5-HT2 antagonists ketanserin and ritanserin but not by the 5-HT1 antagonists isapirone, (-)-propranolol, or pindolol. 5-HT-stimulated IP formation was also unaltered by atropine, prazosin, and mepyramine. Lesioning brain 5-HT neurones using 5,7-dihydroxytryptamine (5,7-DHT; 50 micrograms i.c.v.) produced a 210% (p less than 0.01) increase in the number of 5-HT2-mediated head-twitches induced by 5-methoxy-N,N-dimethyltryptamine (2 mg/kg). However, 5,7-DHT lesioning had no effect on 5-HT-stimulated PI turnover in these mice. Similarly, an electroconvulsive shock (90 V, 1 s) given five times over a 10-day period caused an 85% (p less than 0.01) increase in head-twitch responses but no change in 5-HT-stimulated PI turnover. Decreasing 5-HT2 function by twice-a-day injection of 5 mg/kg of zimeldine or desipramine (DMI) produced 50% (p less than 0.01) and 56% (p less than 0.01), respectively, reductions in head-twitch behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)