Effects of in vitro hypoxia on depolarization-stimulated accumulation of inositol phosphates in synaptosomes

The effects of potassium and in vitro histotoxic hypoxia (i.e. KCN) on phosphatidylinositol turnover in rat cortical synaptosomes were determined. [2-3H] Inositol prelabelled rat synaptosomes were prepared from cerebral cortex slices that had been incubated with [2-3H] inositol. Depolarization with 60 mM KCl increased [2-3H] inositol phosphates in a time dependent manner. Depolarization with 60 mM KCl increased [2-3H] inositol trisphosphate transiently at 5 s. K+ induced rapid formation of [2-3H]-inositol bisphosphate and maintained an elevated level for at least 5 min. K+ stimulated gradual formation of [2-3H] inositol monophosphate with time. One minute of hypoxia enhanced potassium-stimulated [2-3H] inositol bisphosphate formation. However, 30 min of hypoxia impaired potassium-stimulated accumulation of [2-3H] inositol phosphates. The effects of histotoxic hypoxia were all dependent upon calcium in the medium and on K+-depolarization. Thus, hypoxia altered the K+-induced accumulation of inositol phosphates in prelabelled synaptosomes in a time dependent, biphasic manner that was calcium dependent.     

Muscarinic Cholinergic Receptor-Mediated Phosphoinositide Metabolism in Peripheral Nerve

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.     

Sympathetic denervation impairs agonist-stimulated phosphatidylinositol metabolism in rat parotid glands.

Substance P, muscarinic and alpha-adrenoceptor agonists stimulated the incorporation of [3H]inositol into phosphatidylinositol in rat parotid gland slices. Surgical denervation of the sympathetic input to the rat parotid gland by superior cervical ganglionectomy produced marked reductions in these responses. The stimulated incorporation of radiolabelled precursors into phosphatidylinositol is a measure of its resynthesis after receptor-mediated breakdown of inositol phospholipids. We therefore examined the enzymic site of the lesion induced by sympathetic denervation using parotid gland slices labelled with either [3H]inositol or [32P]phosphate and stimulated with substance P. Receptor-activated phospholipase C attack upon [3H]inositol phospholipids was assayed by measuring the formation of [3H]inositol 1-phosphate in the presence of 10 mM-Li+ to inhibit further breakdown. It was not affected by denervation. Substance P elicited a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and this response was reduced in the denervated gland. The second step in stimulated phosphatidylinositol turnover, phosphorylation of diacylglycerol to phosphatidate was not affected by denervation. Sympathetic denervation appears to induce a specific enzymic lesion in the parotid gland that impairs receptor-stimulated resynthesis of phosphatidylinositol from phosphatidate. This change in membrane lipid metabolism may be related to a number of the effects of sympathetic denervation, such as agonist supersensitivity, reduced gland cell proliferation and induction of new surface receptors.     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

Effects of NMDA on Carbachol-Stimulated Phosphatidylinositol Resynthesis in Rat Brain Cortical Slices

N-methyl-D-aspartate (NMDA) inhibits carbachol-stimulated phosphoinositide breakdown in rat brain cortical slices but not in isolated membranes (1). To gain insight into the mechanisms, we examined the effects of NMDA on carbachol-stimulated [³H]inositol phosphate and intermediates of phosphatidylinositol cycle accumulation in rat cortical slices. The inhibition is primarily on the synthesis of inositol phospholipids subsequent to activation of muscarinic cholinergic receptors. In the absence of lithium, NMDA inhibited carbachol-stimulated [³²P]PtdIns but not [³²P]PtdOH synthesis. Carbachol-stimulated CDP-DAG formation required trace amount of Ca²⁺ and the response was inhibited by NMDA at low but not high extracellular Ca²⁺ concentrations. The inhibition due to NMDA was only seen at millimolar extracellular Mg²⁺. The inhibition of carbachol-stimulated CDP-DAG formation was not affected by adding tetrodotoxin or cobalt chloride suggesting the inhibitory effect was not due to releasing of neurotransmitters. The inhibitory effects of NMDA could be abolished by MK-801, the specific NMDA receptor associated channel antagonist. When cortical slices were preincubated with ligands and lithium to allow the build up of CDP-DAG, carbachol stimulated the incorporation of [³H]Ins into [³H]PtdIns. However, this response was not inhibited by NMDA. These results suggest that CDP-DAG synthesis is the primary site of regulation by NMDA. Because CDP-DAG cytidyltransferase requires Mg²⁺ as cofactor and is sensitive to Ca²⁺ it is possible that NMDA inhibits ligand-stimulated PtdIns breakdown by blocking the replenish of agonist-sensitive PtdIns pool through changes of divalent cation homeostasis.     

Increased Polyphosphoinositide Responsiveness in the Cerebral Cortex Induced by Cholinergic Denervation

Lesion of the nucleus basalis in the basal forebrain of the rat results in the degeneration of the large cholinergic neurones which innervate the cortex. Parameters of cholinergic function, namely, acetylcholinesterase activity, muscarinic acetylcholine receptor number, and the depolarisation-induced release of acetylcholine, fall in ipsilateral cortex subsequent to lesion. These deficits are likely to reflect the loss of the presynaptic input to the cortex. A reversal in these deficits is seen 1 month after lesion, and a full recovery is seen after 150 days. This is thought to be due to a process of "spared axon sprouting" followed by the reestablishment of synapses. To examine the integrity of the cortical muscarinic receptor response following denervation, an assay of the polyphosphoinositide response was carried out. Cortical tissue slices, prelabelled with [3H]inositol, were incubated for 40 min with carbachol in the presence of Li+; the accumulation of [3H]inositol monophosphate ([3H]IP1) was used as an index of this response. A 92% increase in the carbachol-stimulated production of [3H]IP1 was seen 5 days after lesion compared to normal cortex. Sham-operated animals showed no change in [3H]IP1 accumulation at this time point. Dose-response experiments showed that this increase was due to an increase in the maximal response to carbachol after lesion with no change in EC50 values. Two weeks after lesion, this increased response was much attenuated; tissue slices from denervated cortex showing a strong acetylcholinesterase decrease (36-66%) showed an increase of just 30% above normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of Muscarinic and of α1-Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.     

Inositol Lipid Metabolism in Rat Hippocampal Formation Slices: Basal Metabolism and Effects of Cholinergic Agonists

Incubation of rat hippocampal formation slices under steady-state conditions with [3H]inositol leads to only three phospholipids becoming labelled: phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. All three lipids incorporate [32P]Pi into their phosphodiester phosphate group with the polyphosphoinositides also incorporating this tracer into their monoester phosphate groups. As the concentrations of these lipids remain constant during these labelling processes we conclude that the phosphodiester phosphate, the inositol moiety, and the monoester phosphate groups undergo metabolic turnover in hippocampal formation slices incubated in vitro. The rate of incorporation of [3H]inositol into all three inositol phospholipids was stimulated by the addition of methacholine to the medium. Moreover, following steady-state labelling of the inositol lipids with [3H]inositol, methacholine in the presence of 10 mM LiCl caused a transient fall of 13% in the radiochemical concentration of phosphatidylinositol 4,5-bisphosphate after only 30 s stimulation and a fall of 15% in the radiochemical concentration of phosphatidylinositol after 30 min. Concomitantly, there was an approximately stoichiometric rise in the radiochemical concentration of inositol phosphates. Thus, we suggest that methacholine stimulates an inositol phospholipid phosphoinositidase C in rat hippocampal formation slices.     

Reduced inositol polyphosphate accumulation and inositol supply induced by lithium in stimulated cerebral cortex slices.

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.     

Age-Related Alterations In Pre-Synaptic and Receptor-Mediated Cholinergic Functions In Rat Brain

Fractional [³H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [³H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [³H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 M) significantly decreased stimulation-induced fractional [³H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [³H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [³H]ACh release from striatum of both age groups is modulated mainly by M₃ muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-³H]inositol decreased with aging, carbachol-stimulated [³H]myo inositol-1-fosfat (IP₁) accumulation was found similar in striatal, cortical and hippocampal slices.     

Lithium inhibits muscarinic-receptor-stimulated inositol tetrakisphosphate accumulation in rat cerebral cortex.

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.     

Muscarinic Receptors and Hydrolysis of Inositol Phospholipids in Rat Cerebral Cortex and Parotid Gland

Abstract: Exposure of rat brain or parotid gland slices to muscarinic receptor agonists stimulates a phospholipase C that degrades inositol phospholipids. When tissue slices were labelled in vitro with [³H]inositol, this response could be monitored by measuring the formation of [³H]inositol phosphates. Accumulation of inositol 1,4-biphosphate in stimulated brain slices suggests that polyphosphonositides are the primary targets for phospholipase C activity. Li⁺ (10 m M ) in the medium completely blocked the hydrolysis of inositol 1-phosphate, partially inhibited inositol 1,4bisphosphate hydrolysis, but had no effect on the hydrolysis of inositol 1,4,5-trisphosphate by endogenous phosphatases. Muscarinic receptor pharmacology was studied by measuring the accumulation of [³H]inositol 1-phosphate in the presence of 10 m M Li⁺. In experiments on brain slices, the response to carbachol was antagonised by atropine with an affinity constant of approximately 8.79 ± 0.12. Dose-response curves to several muscarinic agonists were constructed using brain and parotid gland slices. The results are consistent with relatively direct coupling of low-affinity muscarinic receptors to inositol phospholipid breakdown in brain slices; full agonists were relatively more potent in the parotid gland compared with the brain. Explanations for these differences are suggested.     

Time-, concentration-, and age-dependent inhibition of muscarinic receptor-stimulated phosphoinositide metabolism by ethanol in the developing rat brain

We have previously reported that administration of ethanol (EtOH; 4 g/Kg/day) to rats from postnatal day 4 to day 10 causes microencephaly and decreases muscarinic receptor-stimulated inositol metabolism on days 7 and 10 (1). An identical exposure to EtOH of adult rats, which resulted in similar blood EtOH concentrations, did not have any effect on the same system. Initial in vitro studies have shown the presence of a differential sensitivity to EtOH of the phosphoinositide system coupled to muscarinic receptors during development (2). In the present study we have expanded these findings by investigating the concentration-, time-, and age-dependent effects of EtOH on accumulation of [³H]inositol phosphates ([³H]InsPs) in brain slices. EtOH caused a dose-dependent inhibition of carbachol-stimulated phosphoinositide metabolism in cerebral cortex slices from 7 day-old rats. When the time of incubation with EtOH was increased to 90 minutes, concentrations as low as 50 mM, which are reached following in vivo administration of EtOH, significantly inhibited the muscarinic response. The effect of EtOH was rather specific for the muscarinic receptors, since, even with longer incubation times, the accumulation of [³H]InsPs induced by norepinephrine or serotonin was inhibited only at concentrations of 150–500 mM. The effect of EtOH was more pronounced in cerebral cortex, hippocampus and cerebellum, and less in the brainstem. The potency of EtOH in inhibiting carbachol-stimulated phosphoinositide metabolism was also dependent on the age of the animals. Its effect was maximal in the 7 day-old rat and less pronounced in younger and older animals. These results confirm that the phosphoinositide system coupled to muscarinic receptors might represent a relevant target for the developmental neurotoxicity of EtOH.Presented in part at the 29th Annual Meeting of the Society of Toxicology (Toxicologist 1990; 10: 273).     

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.     

Effects of Lithium on Inositol Phospholipid Hydrolysis and Inhibition of Dopamine D1 Receptor-Mediated Cyclic AMP Formation by Carbachol in Rat Brain Slices

The in vitro and ex vivo effects of lithium on muscarinic cholinergic inositol phospholipid hydrolysis and muscarinic cholinergic inhibition of dopamine D1-receptor-stimulated cyclic AMP formation were examined in rat brain slices. Following chronic lithium feeding, carbachol-stimulated inositol phosphate accumulation was reduced ex vivo in slices of cerebral cortex but not in striatal slices. Lithium (1 mM) in vitro had no direct effect on dopamine D1-receptor-stimulated cyclic AMP formation, but enhanced the inhibitory effect of carbachol on the D1 response, in striatal slices, and this was not significantly altered by prior lithium feeding. Lithium therefore has effects on two discrete muscarinic responses in rat brain which are apparently maintained after chronic exposure to the ion and might be relevant to its antimanic actions.     

Differential Effects of Lithium on Muscarinic Receptor Stimulation of Inositol Phosphates in Rat Cerebral Cortex Slices

The accumulation of labelled inositol mono-, bis-, and trisphosphate in rat cerebral cortex slices was examined following preincubation with [3H]inositol. The muscarinic receptor agonist carbachol produced a rapid and sustained increased accumulation of each labelled inositol phosphate both in the presence and absence of 5 mM lithium. Lithium potentiated carbachol-stimulated accumulation of inositol monophosphate (EC50 0.5 mM) and inositol bisphosphate (EC50 4 mM) in a concentration-dependent manner. However, exposure to lithium in the presence of the muscarinic agonist produced a concentration- and time-dependent inhibition of inositol trisphosphate accumulation that was not related to receptor desensitisation. Although the present data do suggest that polyphosphoinositides are substrates for agonist-stimulated phospholipase C in brain, these results may not be entirely consistent with the production of inositol mono- and bisphosphate through inositol trisphosphate dephosphorylation. Furthermore, these data suggest site(s) additional to inositol monophosphatase that are affected by lithium.     

Stimulatory and Inhibitory Effects of N-Methyl-D-Aspartate on 3H-Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.     

Increased Intracellular Calcium Stimulates 3H-Inositol Polyphosphate Accumulation in Rat Cerebral Cortical Slices

Agents that increase the intracellular Ca2+ concentration have been examined for their ability to stimulate 3H-inositol polyphosphate accumulation in rat cerebral cortex slices. Elevated extracellular K+ levels, the alkaloid sodium channel activator veratrine, the calcium ionophore ionomycin, and the marine toxin maitotoxin were all able to stimulate phosphoinositide metabolism. Certain features appear common to the agents studied. Thus, although [3H]inositol monophosphate, [3H]inositol bisphosphate ([3H]InsP2), and [3H]inositol trisphosphate were all stimulated, a proportionally greater effect was observed on [3H]InsP2 in comparison to stimulation by the muscarinic receptor agonist carbachol. However, only an elevated K+ level stimulated [3H]inositol tetrakisphosphate ([3H]InsP4) accumulation alone or produced marked synergy with carbachol on the formation of this polyphosphate. The results suggest that agents that elevate the cytoplasmic Ca2+ concentration in cerebral cells can increase the hydrolysis of membrane polyphosphoinositides. The pattern of the response differs from that produced by muscarinic receptor agonists and indicate that Ca2(+)-dependent hydrolysis may involve different pools of lipids, phosphoinositidase C enzymes, or both. However, clear differences in the ability of these agents to stimulate InsP4, alone or in the presence of muscarinic agonist, suggest that factors other than a simple elevated intracellular Ca2+ concentration are implicated.     

The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

Rapid increase in inositol pentakisphosphate accumulation by nicotine in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with nicotine (10 microM), a large and transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s, then declined to the basal level at 2 min. Nicotine also induced [3H]inositol tetrakisphosphate (InsP4) and [3H]inositol hexakisphosphate (InsP6) accumulation with a slower time course and a lesser magnitude than [3H]InsP5. The peaks of [3H]InsP4, [3H]InsP5 and [3H]InsP6 coincided with those of 32P radioactivity, when cells were doubly labeled with [3H]inositol and inorganic 32P. These results suggest that inositol pentakisphosphate is rapidly increased by nicotine, a cholinergic agonist, in cultured adrenal chromaffin cells.