Rapid accumulation and sustained turnover of inositol phosphates in cerebral-cortex slices after muscarinic-receptor stimulation.
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ABSTRACT:
SUBMITTER: Batty IH
PROVIDER: S-EPMC1138651 | biostudies-other | 1989 May
REPOSITORIES: biostudies-other
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Project description:Stimulation of [3H]inositol-prelabelled rat cerebral-cortex slices with carbachol results in the accumulation of four [3H]inositol bisphosphate isomeric species, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P2 and Ins(4,5)P2. Although the last isomer ran as a minor peak on h.p.l.c., its accumulation was dramatically enhanced in the presence of Li+ (1 mM), such that at 30 min it represented almost 35% of the total bisphosphate fraction. The accumulation of Ins(4,5)P2 appeared to be very sensitive to Li+ (EC50 = 94 +/- 3 microM), strongly implicating a Li(+)-sensitive metabolism. Evidence for this is provided from the rapid but Li(+)-sensitive decay of Ins(4,5)P2 when muscarinic-receptor stimulation is antagonized by atropine at a time when accumulations have reached a new steady state. Manipulation of phospholipase D by activators and inhibitors of protein kinase C did not suggest a role for phospholipase D hydrolysis of PtdInsP2 in the formation of Ins(4,5)P2. Attempts to reveal Ins(4,5)P2 metabolism, or indeed its synthesis from Ins(1,4,5)P3, were not successful with broken cell preparations and strongly suggest discrete compartmentation of inositol phosphate metabolism in the intact cell.
| S-EPMC1133184 | biostudies-other
Project description:Muscarinic-receptor stimulation or depolarization by elevated K+ leads to increased accumulation of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4 and several degradation products of these polyphosphates separated by h.p.l.c. On the other hand, agents such as ionomycin and maitotoxin, which increase intracellular Ca2+ directly, produce a small accumulation of [3H]Ins(1,4,5)P3 and markedly increase [3H]Ins(1,4)P2, but [3H]Ins(1,3,4,5)P4, [3H]Ins(1,3,4)P3 and [3H]Ins(1,3)P2 are virtually unaffected. Ca2(+)-dependent [3H]inositol polyphosphate metabolism may involve different pools of lipids and/or phosphoinositidases.
| S-EPMC1131374 | biostudies-other
Project description:The effects of Li+ on carbachol-stimulated phosphoinositide metabolism were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. The muscarinic agonist carbachol evoked an enhanced steady-state accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2), [3H]inositol 1,3,4-trisphosphate ([3H]Ins(1,3,4)P3), [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and [3H]inositol tetrakisphosphate ([3H]InsP4). Li+ (5 mM), after a 10 min lag, severely attenuated carbachol-stimulated [3H]InsP4 accumulation while simultaneously potentiating accumulation of both [3H]InsP1 and [3H]InsP2 and, at least initially, of [3H]Ins(1,3,4)P3. These data are consistent with inhibition of inositol mono-, bis- and 1,3,4-tris-phosphate phosphatases to different degrees by Li+ in brain, but are not considered to be completely accounted for in this way. Potential direct and indirect mechanisms of the inhibitory action of Li+ on [3H]InsP4 accumulation are considered. The present results stress the complex action of Li+ on cerebral inositol metabolism and indicate that more complex mechanisms than are yet evident may regulate this process.
| S-EPMC1148483 | biostudies-other
Project description:The ability of lithium to interfere with phosphoinositide metabolism in rat cerebral cortex slices has been examined by monitoring the accumulation of CMP-phosphatidate (CMP-PtdOH) and the reduction in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels. A small accumulation of [14C]CMP-PtdOH was seen in slices prelabelled with [14C]cytidine and stimulated with carbachol (1 mM) or Li+ (1 mM). However, simultaneous addition of both agents for 30 min produced a 22-fold accumulation, with Li+ producing a half-maximal effect at a concentration of 0.61 +/- 0.19 mM. Kinetic studies revealed that the effects of carbachol and Li+ on CMP-PtdOH accumulation occurred with no initial lag apparent under these conditions and that preincubation with myo-inositol (10 or 30 mM) dramatically attenuated CMP-PtdOH accumulation. myo-Inositol could also attenuate the rate of accumulation of CMP-PtdOH when added 20 min after carbachol and Li+; these effects were not observed when equimolar concentrations of scyllo-inositol were added. Use of specific radioreceptor assays allowed the mass accumulations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 to be monitored. Following a lag of 5-10 min, Li+ resulted in a marked reduction in the accumulation of both inositol polyphosphates resulting from muscarinic-cholinergic stimulation. Preincubation of cerebral cortex slices with myo- (but not scyllo-) inositol delayed, but did not prevent, the reduction in the accumulation of Ins(1,4,5)P3 or Ins(1,3,4,5)P4. The results suggest that cerebral cortex, at least in vitro, is very sensitive to myo-inositol depletion under conditions of muscarinic receptor stimulation. The relationship of such depletion to the generation of inositol polyphosphate second messengers is discussed.
| S-EPMC1131366 | biostudies-other
Project description:Carbachol stimulation of muscarinic receptors in rat cortical slices prelabelled with myo-[2-3H]inositol caused the rapid formation of a novel inositol polyphosphate. Evidence derived from its chromatographic behaviour, and from the structure of the products formed in partial dephosphorylation experiments, suggests that it is probably D-myo-inositol 1,3,4,5-tetrakisphosphate. An enzyme in human red cell membranes specifically removes the 5-phosphate from it to form inositol 1,3,4-trisphosphate. It is suggested that inositol 1,3,4,5-tetrakisphosphate is likely to be a second messenger, and that it is the precursor of inositol 1,3,4-trisphosphate and possibly of inositol 1,4,5-trisphosphate.
| S-EPMC1152860 | biostudies-other
Project description:An accumulation of 3H-labelled inositol phosphates is observed when prelabelled rat superior cervical sympathetic ganglia are exposed to [8-arginine]vasopressin or to muscarinic cholinergic stimuli. The response to vasopressin is much greater than the response to cholinergic stimuli. The response to vasopressin is blocked by a V1-vasopressin antagonist, and oxytocin is a much less potent agonist than vasopressin. Vasopressin causes no increase in the cyclic AMP content of ganglia. These ganglia therefore appear to have functional V1-vasopressin receptors that are capable of activating inositol lipid breakdown, but no V2-receptors coupled to adenylate cyclase. The first [3H]inositol-labelled products to accumulate in stimulated ganglia are inositol trisphosphate and inositol bisphosphate, suggesting that the initiating reaction in stimulated inositol lipid metabolism is a phosphodiesterase-catalysed hydrolysis of phosphatidylinositol 4,5-bisphosphate (and possibly also phosphatidylinositol 4-phosphate). This response to exogenous vasopressin occurs in ganglia incubated in media of reduced Ca2+ concentration. The physiological functions of the V1-vasopressin receptors of these ganglia remain unknown.
| S-EPMC1144110 | biostudies-other
Project description:1. Basal and carbachol-stimulated accumulations of isomeric [3H]inositol mono-, bis-, tris- and tetrakis-phosphates were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. 2. In control samples the major [3H]inositol phosphates detected were co-eluted on h.p.l.c. with Ins(1)P, Ins(4)P (inositol 1- and 4-monophosphate respectively), Ins(1,4)P2 (inositol 1,4-bisphosphate), Ins(1,4,5)P3 (inositol 1,4,5-tris-phosphate) and Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate). 3. After stimulation to steady state with carbachol, accumulation of each of these products was markedly increased. 4. Agonist stimulation, however, also evoked much more dramatic increased accumulations of a second [3H]inositol trisphosphate, which was co-eluted on h.p.l.c. with authentic Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) and of three further [3H]inositol bisphosphates ([3H]InsP2(s]. 5. Examination of the latter by chemical degradation by periodate oxidation and/or h.p.l.c. allowed identification of these as [3H]Ins(1,3)P2, [3H]Ins(3,4)P2 and [3H]Ins(4,5)P2 (inositol 1,3-, 3,4- and 4,5-bisphosphates respectively), which respectively accounted for about 22%, 8% and 3% of total [3H]InsP2 in extracts from stimulated tissue slices. 6. By using a h.p.l.c. method which clearly resolves Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (inositol 1,3,4,6-tetrakisphosphate), only the former isomer could be detected in extracts from either control or stimulated tissue slices. Similarly, [3H]inositol pentakis- and hexakis-phosphates were not detectable either in the presence or absence of carbachol under the radiolabelling conditions described. 7. The catabolism of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 by cell-free preparations from cerebral cortex was also studied. 8. In the presence of Mg2+, [3H]Ins(1,4,5)P3 was specifically dephosphorylated via [3H]Ins(1,4)P2 and [3H]Ins(4)P to free [3H]inositol, whereas [3H]Ins(1,3,4)P3 was degraded via [3H]Ins(3,4)P2 and, to a lesser extent, via [3H]Ins(1,3)P2 to D- and/or L-[3H]Ins(1)P and [3H]inositol. 9. In the presence of EDTA, hydrolysis of [3H]Ins(1,4,5)P3 was greater than or equal to 95% inhibited, whereas [3H]Ins(1,3,4)P3 was still degraded, but yielded only a single [3H]InsP2 identified as [3H]Ins(1,3)P2. 10. The significance of these observations with cell-free preparations is discussed in relation to the proportions of the separate isomeric [3H]inositol phosphates measured in stimulated tissue slices.
| S-EPMC1138319 | biostudies-other
Project description:Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)
| S-EPMC1144426 | biostudies-other
Project description:1. Intracellular concentrations of intermediates and cofactors of glycolysis were measured in guinea-pig cerebral cortex slices incubated under varying conditions. 2. Comparison of mass-action ratios with apparent equilibrium constants for the reactions of glycolysis showed that hexokinase, phosphofructokinase and pyruvate kinase catalyse reactions generally far from equilibrium, whereas phosphoglucose isomerase, aldolase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, adenlyate kinase and creatine phosphokinase are generally close to equilibrium. The possibility that glyceraldehyde 3-phosphate dehydrogenase may catalyse a ;non-equilibrium' reaction is discussed. 3. Correlation of changes in concentrations of substrates for enzymes catalysing ;non-equilibrium' reactions with changes in rates of glycolysis caused by alteration of the conditions of incubation showed that hexokinase, phosphofructokinase, pyruvate kinase and possibly glyceraldehyde 3-phosphate dehydrogenase are subject to metabolic control in cerebral cortex slices. 4. It is suggested that the glycolysis is controlled by two regulatory systems, the hexokinase-phosphofructokinase system and the glyceraldehyde 3-phosphate dehydrogenase-pyruvate kinase system. These are discussed. 5. It is concluded that the rate of glycolysis in guinea-pig cerebral cortex slices is limited either by the rate of glucose entry into the slices or by the hexokinase-phosphofructokinase system. 6. It is concluded that addition of 0.1mm-ouabain to guinea-pig cerebral cortex slices causes inhibition of either glyceraldehyde 3-phosphate dehydrogenase or phosphoglycerate kinase or both, in a manner independent of the known action of ouabain on the sodium- and potassium-activated adenosine triphosphatase.
| S-EPMC1270615 | biostudies-other
Project description:Activation of muscarinic cholinergic receptors on pyramidal cells of the cerebral cortex induces the appearance of a slow afterdepolarization that can sustain autonomous spiking after a brief excitatory stimulus. Accordingly, this phenomenon has been hypothesized to allow for the transient storage of memory traces in neuronal networks. Here we investigated the molecular basis underlying the muscarinic receptor-induced afterdepolarization using molecular biological and electrophysiological strategies. We find that the ability of muscarinic receptors to induce the inward aftercurrent underlying the slow afterdepolarization is inhibited by expression of a Galpha(q-11) dominant negative and is also markedly reduced in a phospholipase C beta1 (PLCbeta1) knock-out mouse. Furthermore, we show, using a genetically encoded biosensor, that activation of muscarinic receptor induces the breakdown of phosphatidylinositol 4,5-bisphosphate in pyramidal cells. These results indicate that the Galpha(q-11)/PLCbeta1 cascade plays a key role in the ability of muscarinic receptors to signal the inward aftercurrent. We have shown previously that the muscarinic afterdepolarization is mediated by a calcium-activated nonselective cation current, suggesting the possible involvement of TRPC channels. We find that expression of a TRPC dominant negative inhibits, and overexpression of wild-type TRPC5 or TRPC6 enhances, the amplitude of the muscarinic receptor-induced inward aftercurrent. Furthermore, we find that coexpression of TRPC5 and T-type calcium channels is sufficient to reconstitute a muscarinic receptor-activated inward aftercurrent in human embryonic kidney HEK-293 cells. These results indicate that TRPC channels mediate the muscarinic receptor-induced slow afterdepolarization seen in pyramidal cells of the cerebral cortex and suggest a possible role for TRPC channels in mnemonic processes.
| S-EPMC2747319 | biostudies-literature