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Accumulation of inositol phosphates in sympathetic ganglia. Effects of depolarization and of amine and peptide neurotransmitters.

ABSTRACT: Depolarization of isolated [3H]inositol-labelled rat superior cervical sympathetic ganglia in a high-K+ medium stimulates an accumulation of labelled inositol phosphates. This accumulation occurs only when ganglia are incubated in a Ca2+-containing medium, suggesting that it represents a receptor-stimulated hydrolysis of inositol lipid(s) activated by an endogenously released neurotransmitter. A minor fraction of this accumulation appears to be activated by intraganglionically released acetylcholine, since it is slightly reduced by atropine. The accumulation of inositol phosphates is unaffected by blockade of appropriate catecholamine, histamine and 5-hydroxytryptamine receptors and also by aspirin and indomethacin. This response to depolarization is potentiated by incubation with proteinase inhibitors, suggesting that it might be caused by an endogenously released peptide neutrotransmitter. However, it is not prevented by a V1-vasopressin receptor antagonist, and none of the peptides tested so far fully reproduces the response: these include a stable substance P analogue, physalaemin, neurokinin alpha, bradykinin, angiotensin, pancreozymin, bombesin and luteinizing-hormone-releasing hormone. Stimulated inositol lipid breakdown in depolarized sympathetic ganglia seems likely to be activated by an as-yet-unidentified peptide neurotransmitter: this might serve as an intraganglionic mediator of postsynaptic excitation by employing the same signalling mechanism as muscarinic cholinergic and V1-vasopressin receptors.

SUBMITTER: Bone EA

PROVIDER: S-EPMC1144835 | biostudies-other | 1985 Apr

REPOSITORIES: biostudies-other

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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)

Project description:Previous studies showed that lithium, beginning at therapeutic plasma concentrations in the treatment of manic depression, increased the accumulation of second-messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in cerebral cortex slices of guinea pig and rhesus monkey [Lee, Dixon, Reichman, Moummi, Los and Hokin (1992) Biochem. J. 282, 377-385; Dixon, Lee, Los and Hokin (1992) J. Neurochem. 59, 2332-2335; Dixon, Los and Hokin (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 8358-8362]. These studies have now been extended to a peripheral tissue, mouse pancreatic minilobules. In the presence of carbachol, concentrations of lithium from 1 to 20 mM sharply and progressively increased the accumulation of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate, followed by a decrease. Assay of these inositol polyphosphates by either the prelabelling technique or mass assay gave similar results. Atropine quenching of cholinergically stimulated pancreatic minilobules led to a rapid disappearance of Ins(1,4,5)P3. This disappearance was impeded by lithium. This suggested that the lithium-induced elevation in Ins(1,4,5)P3 was due to inhibition of the 5-phosphatase and, on the basis of the markedly elevated concentrations of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and inositol 1,4-bisphosphate in the presence of lithium, probably by feedback inhibition by these latter two compounds. An additional mechanism, i.e. a stimulatory effect of lithium on phospholipase C, cannot, however, be ruled out. The other reaction product of phospholipase C, inositol cyclic 1:2,4,5-trisphosphate, also increased in the presence of lithium. This may also be due to inhibition of the 5-phosphatase, which is the exclusive mechanism for removal of this compound. The effects of lithium on the accumulation of other inositol phosphates paralleled that of Ins(1,4,5)P3, with the exception of inositol 3,4-bisphosphate, which decreased. This was presumably due to the inhibition of Ins(1,3,4)P3 1-phosphatase by lithium. Unlike mouse cerebral cortex slices [Lee, Dixon, Reichman, Moummi, Los and Hokin (1992) Biochem. J. 282, 377-385], inositol supplementation was not required to demonstrate lithium-stimulated Ins(1,4,5)P3 accumulation in mouse pancreatic minilobules. This indicates that inositol depletion sufficient to impair lithium-stimulated Ins(1,4,5)P3 accumulation does not occur in mouse pancreatic minilobules, even though an elevation of cytidine diphosphodiacylglycerol occurred, indicating some inositol depletion due to lithium. Elevation of Ins(1,4,5)P3 by lithium may be a general phenomenon in the central nervous system and peripheral tissues under non-rate-limiting concentrations of inositol.

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Accumulation of inositol phosphates in sympathetic ganglia. Effects of depolarization and of amine and peptide neurotransmitters.

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