Enhancement by Potassium of Carbachol-Stimulated Inositol Phospholipid Breakdown in Rat Cerebral Cortical Miniprisms: Comparison with Other Depolarising Agents

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.     

Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by BAY K-8644 is dependent upon the assay calcium and potassium concentrations used.

The calcium and potassium ion dependency of the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 microM) potentiated the response to carbachol at 6 mM K+ when Ca2(+)-free, but not when 2.52 mM Ca2+ assay buffer was used. In Ca2(+)-free buffer, verapamil (10 microM) inhibited the response to carbachol at both 6 and 18 mM K+ but higher concentrations (30-300 microM) were needed when 2.52 mM Ca2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 microM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K+ at 1.3 mM Ca2+, but was without effect on the basal rate at other K+ and Ca2+ concentrations. In the presence of NMDA (100 microM) or quisqualate (100 microM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K+ and Ca2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.     

Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells.

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.     

Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [³H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [³H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [³H]inositol-l-phosphate. Incubation of slices withN-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [³H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4,5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.     

Lithium treatment of affective disorders: effects of lithium on the inositol phospholipid and cyclic AMP signalling pathways.

The effects of lithium (Li+) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li+ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC50 of approx. 20 mM. By contrast, half-maximal effects of Li+ on inositol monophosphate (InsP) accumulation in [3H]inositol labelled tissue slices occurred at about 1 mM. A similar IC50 for Li+ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [3H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated cortical slices using a novel mass assay for InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li+ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li+ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are compatible with the inositol depletion hypothesis of Li+ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.     

Effects of Muscarinic Agonists and Depolarizing Agents on Inositol Monophosphate Accumulation in the Rabbit Vagus Nerve

The effects of muscarinic agonists and depolarizing agents on inositol phospholipid hydrolysis in the rabbit vagus nerve were assessed by the measurement of [3H]inositol monophosphate production in nerves that had been preincubated with [3H]inositol. After 1 h of drug action, carbachol, oxotremorine, and arecoline increased the inositol monophosphate accumulation, though the maximal increase induced by these agonists differed. Addition of the muscarinic antagonists atropine or pirenzepine shifted the carbachol dose-response curves to the right, without decreasing the carbachol maximal stimulatory effects. The KB for pirenzepine was 35 nM, which is characteristic of muscarinic high-affinity binding sites coupled to phosphoinositide turnover and often associated with the M1 receptor subtype. On the other hand, agents known to depolarize or to increase the intracellular Ca2+ concentration, e.g., elevated extracellular K+, ouabain, Ca2+, and the Ca2+ ionophore A23187, also increased inositol monophosphate accumulation. These effects were not mediated by the release of acetylcholine, as suggested by the fact that they could not be potentiated by the addition of physostigmine nor inhibited by the addition of atropine. The Ca(2+)-channel antagonist Cd2+, also known to inhibit the Na+/Ca2+ exchanger, was able to block the effects of K+ and ouabain, but did not alter those of carbachol. These results suggest that depolarizing agents increase inositol monophosphate accumulation in part through elevation of the intracellular Ca2+ concentration and that muscarinic receptors coupled to phosphoinositide turnover are present along the trunk of the rabbit vagus nerve.     

Cholinergic- and Adrenergic-Stimulated Inositide Hydrolysis in Brain: Interaction, Regional Distribution, and Coupling Mechanisms

Carbachol and norepinephrine were used as agonists to compare and contrast cholinergic and adrenergic stimulation of inositide breakdown in rat brain slices. Carbachol acts through a muscarinic (possibly M1) receptor and norepinephrine acts through an alpha 1 adrenoceptor. Studies in cerebral cortical slices indicated that both agonists stimulated the production of inositol-1-phosphate and glycerophosphoinositol. Although the initial rates for the stimulation of inositol phosphate release were similar for the two ligands, the response to norepinephrine continued for 60 min and was larger compared with carbachol which plateaued at 30 min. The presence of carbachol did not affect the ED50 for norepinephrine. Concentrations of carbachol near the ED50 in combination with norepinephrine resulted in an additive response whereas maximal concentrations of carbachol and norepinephrine resulted in a less than additive response in the cortex. This negative interaction was also seen in the hippocampus and hypothalamus but not in the striatum, brainstem, spinal cord, olfactory bulb, or cerebellum. Norepinephrine had a larger response than carbachol in the hippocampus, striatum, and spinal cord, but the reverse was true in the olfactory bulb. Manganese (1 mM) stimulated the incorporation of [3H]inositol into phosphatidylinositol (PtdIns) four- to fivefold but not into polyphosphoinositides. The stimulation by manganese of PtdIns labelling increased the nonstimulated release of inositol phosphates but did not affect the stimulated release of inositol phosphates by carbachol or norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine Receptor Stimulation Does Not Affect Phosphoinositide Hydrolysis in Slices of Rat Striatum

The effect of dopamine receptor stimulation on the accumulation of labelled inositol phosphates in rat striatal slices under basal and stimulated conditions was examined following preincubation with [3H]inositol. Incubation of striatal slices with the selective D-1 agonist SKF 38393 or the selective D-2 agonist LY 171555 for 5 or 30 min did not affect the basal accumulation of labelled inositol mono-, bis-, tris-, and tetrakisphosphate. Resolution by HPLC of inositol trisphosphate into inositol-1,3,4-tris-phosphate and inositol-1,4,5-trisphosphate isomers revealed that under basal conditions dopamine did not influence the accumulation of inositol-1,4,5-trisphosphate. Depolarisation evoked by KCl, or addition of the muscarinic receptor agonist carbachol, produced a marked increase in the accumulation of labelled inositol phosphates in both the presence and absence of lithium. Addition of dopamine did not reduce the ability of KCl or carbachol to increase inositol phospholipid hydrolysis. In the presence of lithium, dopamine (100 microM) enhanced KCl-stimulated inositol phospholipid hydrolysis, but this effect appears to be mediated by alpha 1 adrenoceptors because it was blocked by prazosin. SKF 38393 (10 microM) or LY 171555 (10 microM) also did not affect carbachol-stimulated inositol phospholipid hydrolysis. These data, in contrast to recent reports, suggest that striatal dopamine receptors do not appear to be linked to inositol phospholipid hydrolysis.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: I. Receptor Characterisation

Characterisation of receptor-mediated breakdown of inositol phospholipids in rat cortical slices has been performed using a direct assay which involves prelabelling with [3H]inositol. When slices were preincubated with [3H]inositol, lithium was found to greatly amplify the capacity of receptor agonists such as carbachol, noradrenaline, and 5-hydroxytryptamine to increase the amount of radioactivity appearing in the inositol phosphates. Using a large variety of agonists and antagonists it could be shown that cholinergic muscarinic, alpha 1-adrenoceptor, and histamine H1 receptors appear to be linked to inositol phospholipid breakdown in cortex. The large responses produced by receptor agonists allowed a clear discrimination between full and partial agonists as well as quantitative analysis of competitive antagonists for each receptor. Whereas carbachol and acetylcholine (in the presence of a cholinesterase inhibitor) were full agonists, oxotremorine and arecoline were only partial agonists. Very low concentrations of atropine shifted the carbachol dose-response curve to the right and allowed inhibition constants for the antagonist to be easily calculated. The nicotinic antagonist, mecamylamine, was ineffective. Noradrenaline adrenaline were full agonists at alpha 1-adrenoceptors, but phenylephrine and probably methoxamine were partial agonists. Prazosin, but not yohimbine, potently and competitively antagonised the noradrenaline inositol phospholipid response. Mepyramine but not cimetidine competitively antagonised the histamine response. These data provide strong confirmation for the potentiating effect of lithium on neurotransmitter inositol phospholipid breakdown and emphasise the ease with which functional responses at a number of cortical receptors can be characterised.     

Long-term atropine treatment lowers the efficacy of carbachol to stimulate phosphatidylinositol breakdown in the cerebral cortex and hippocampus of rats.

The effect of long-term treatment with atropine, a muscarinic antagonist, known to cause up-regulation of receptor numbers, was examined on the muscarinic-receptor-mediated stimulation of phosphoinositide breakdown in the rat cerebral cortex and hippocampus. Although the numbers of both M1 muscarinic receptors, as measured by [3H]pirenzepine binding, and M1 and M2 receptors increased in both brain regions, the maximal breakdown of myo-[3H]inositol-labelled phosphoinositides was unaltered in the presence of carbachol at a saturating concentration (10(-2) M). In fact the efficacy of carbachol was decreased in slices from atropine-treated cerebral cortex [EC50 (concentration producing half-maximal effect) = 93 microM] as compared with the saline-treated control (EC50 = 23 microM)(P less than 0.005). Similarly the EC50 value (23 microM) in hippocampal slices from saline-treated rats increased in atropine-treated rats to 126 microM (P less than 0.005). This lowered efficacy of muscarinic stimulation could not be explained in terms of residual atropine in the tissue from treated rats. The noradrenaline- or serotonin (5-hydroxytryptamine)-stimulated breakdown or the K+ potentiation of the muscarinic-receptor-stimulated breakdown of [3H]phosphoinositides was not affected by the atropine treatment. Chromatography of the released [3H]inositol phosphates shows that atropine treatment did not cause any qualitative change in the pattern of [3H]inositol phosphates released by carbachol stimulation.     

Stimulation of inositol phospholipid breakdown in pig brain miniprisms by carbachol and monoamines: effect of K+

1. The effects of carbachol, monoamines and K+ upon the rate of inositol phospholipid breakdown in pig brain miniprisms have been investigated. 2. In the striatum, carbachol (EC50 approx. 1 microM) and noradrenaline (EC50 approx. 25 microM) stimulated inositol phospholipid breakdown, whereas 5-hydroxytryptamine (1-1000 microM) was without effect. 3. The rate of inositol phospholipid breakdown was increased by raising the assay [K+] to greater than or equal to 40 mM. In the hippocampus and hypothalamus, a synergistic effect between K+ and carbachol was noted, whereas in the striatum, the effects were additive. 4. In striatal and hippocampal miniprisms, dopamine also increased inositol phospholipid breakdown, albeit only at high (greater than or equal to 1 mM) concentrations. Dopamine (1 mM) reduced the stimulation produced by noradrenaline (1 mM), suggesting that the effect of dopamine is due to a weak noradrenergic action of this catecholamine.     

Selective interaction of tricyclic antidepressants with a subclass of rat brain cholinergic muscarinic receptors

Experiments were undertaken to determine whether the anticholinergic actions of tricyclic antidepressants are mediated by a selective interaction with a subclass of muscarinic receptors. To this end, the potencies of these antidepressants to inhibit [3H]-QNB binding to rat brain cerebral cortical membranes was compared to their potencies as antagonists of carbachol-stimulated inositol phosphate accumulation in cerebral cortical slices and carbachol-induced inhibition of GTP-stimulated adenylate cyclase in striatal membranes. Whereas amitriptyline was more potent than pirenzepine, a selective muscarinic M1 receptor antagonist, in competing for [3H]-QNB binding sites and as an antagonist of carbachol-induced inhibition of adenylate cyclase, pirenzepine was substantially more active (ten-fold) than amitriptyline in blocking carbachol-stimulated phosphatidyl inositol turnover. Atropine was more potent than all other agents in these assays, failing to display any significant degree of selectivity. The results suggest that the tricyclic antidepressants, in particular amitriptyline, appear to be selective antagonists for muscarinic receptors associated with adenylate cyclase in striatal membranes. Given the current classification of cholinergic receptors, these findings indicate that the tricyclic antidepressants may be useful for defining the properties of M2 receptors in brain.     

Characterization of agonist-stimulated incorporation of myo-[3H]inositol into inositol phospholipids and [3H]inositol phosphate formation in tracheal smooth muscle.

The effects of the muscarinic agonist carbachol, histamine and bradykinin on incorporation of [3H]inositol into the phosphoinositides and the formation of [3H]InsPs were examined in bovine tracheal smooth-muscle (BTSM) slices labelled with [3H]inositol. These agonists result in substantial and dose-related increases in the incorporation of [3H]inositol into the phospholipids. Carbachol and histamine stimulated the incorporation of [3H]inositol into the phospholipids to the same degree, despite histamine being only 35% as effective as carbachol on [3H]InsP accumulation. Histamine and carbachol, at maximal concentrations, were non-additive with respect to both the stimulated incorporation of [3H]inositol and [3H]InsP formation. For carbachol this effect on incorporation was found to occur to a similar extent in PtdInsP and PtdInsP2 as well as PtdIns. The initial effect of carbachol on [3H]inositol incorporation was rapid (maximal by 10 min); however, with prolonged stimulation large secondary declines in PtdInsP and PtdInsP2 labelling were observed, with depletion of the much larger PtdIns pool only evident in the presence of Li+. Lowering buffer [Ca2+] increased the incorporation of [3H]inositol under basal conditions, but did not attenuate the subsequent agonist-stimulated incorporation effect. The large changes in specific radioactivity of the phosphoinositides, and consequently the [3H]InsP products, after carbachol stimulation resulted in the apparent failure of atropine to reverse the [3H]InsP response completely. Labelling muscle slices with [3H]inositol in the presence of carbachol or labelling for longer periods (greater than 6 h) prevented subsequent carbachol-stimulated effects on incorporation without significantly altering the dose-response relationship for carbachol-stimulated [3H]InsP formation and resulted in steady-state labelling conditions confirmed by the ability of atropine to reverse fully the [3H]InsP response to carbachol. This study demonstrates the profound effects of a number of agonists on [3H]inositol incorporation into the phospho- and polyphosphoinositides in BTSM with important consequent changes in the specific radioactivity of these lipids and the resulting [3H]InsP products. In addition, a selective depletion of PtdInsP and PtdInsP2 over PtdIns has been demonstrated with prolonged muscarinic-receptor stimulation.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: II. Calcium Requirement

The calcium requirement for agonist-dependent breakdown of phosphatidylinositol and polyphosphoinositides has been examined in rat cerebral cortex. The omission of added Ca2+ from the incubation medium abolished [3H]inositol phosphate accumulation from prelabelled phospholipid induced by histamine, reduced that due to noradrenaline and 5-hydroxytryptamine, but did not affect carbachol-stimulated breakdown. EC50 values for agonists were unaltered in the absence of Ca2+. Removal of Ca2+ by preincubation with EGTA (0.5 mM) abolished all responses, but complete restoration was achieved by replacement of Ca2+. The EC50 for Ca2+ for histamine-stimulated [3H]inositol phosphate accumulation was 80 microM. Noradrenaline-stimulated breakdown was antagonised by manganese (IC50 1.7 mM), but not by the calcium channel blockers nitrendipine or nimodipine (30 microM). The calcium ionophore A23187 stimulated phosphatidylinositol/polyphosphoinositide hydrolysis with an EC50 of 2 microM, and this response was blocked by EGTA. Omission of Ca2+ or preincubation with EGTA or Mn2+ (EC50 = 230 microM) greatly enhanced the incorporation of [3H]inositol into phospholipids. The IC50 for Ca2+ in inhibiting incorporation was 25 microM. The results show that different receptors mediating phosphatidylinositol/polyphosphoinositide breakdown in rat cortex have quantitatively different Ca2+ requirements, and it is suggested that rigid opinions regarding phosphatidylinositol/polyphosphoinositide breakdown as either cause or effect of calcium mobilisation in rat cortex are inappropriate.     

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.     

Lithium treatment of affective disorders: effects of lithium on the inositol phospholipid and cyclic AMP signalling pathways

The effects of lithium (Li⁺) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li⁺ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC₅₀ of approx. 20 mM. By contrast, half-maximal effects of Li⁺ on inositol monophosphate (InsP) accumulation in [³H]inositol labelled tissue slices occurred at about 1 mM. A similar IC₅₀ for Li⁺ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [³H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated corticol slices using a novel mass assay fro InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li⁺ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li⁺ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are comptable with the inositol depletion hypothesis of Li⁺ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.     

Is Inositol Bisphosphate the Product of A23187 and Carbachol-Mediated Polyphosphoinositide Breakdown in Synaptosomes?

Synaptosomes have been isolated from rat cerebral cortex and labelled in vitro with [32P]orthophosphate and myo-[2-3H]inositol. Subsequent addition of the Ca2+ ionophore A23187 in the presence of 2 mM extrasynaptosomal Ca2+ raised intrasynaptosomal free [Ca2+] to greater than 2 microM from a resting level of 200 nM and led to rapid breakdown of polyphosphoinositides. This was accompanied by a small increase in the level of inositol monophosphate, greatly enhanced accumulation in inositol bisphosphate, but no detectable increase in inositol trisphosphate. Depolarising (25 mM) extrasynaptosomal K+ produced a smaller increase in intrasynaptosomal free [Ca2+] (to around 400 nM) and a proportional increase in inositol bisphosphate radioactivity. Carbachol (1 mM) alone elicited only limited polyphosphoinositide breakdown and inositol mono- and bisphosphate formation, but this was greatly increased in the presence of 25 mM K+. The effect of carbachol in the presence of depolarising K+ was time- and dose-dependent and was antagonised by atropine (10 microM). There was no detectable accumulation of inositol trisphosphate in the presence of carbachol, K+, or carbachol plus K+, even after short (30 s.) incubations. The lack of inositol trisphosphate accumulation does not appear to result from rapid formation of inositol tetrakisphosphate or from enhanced breakdown of the trisphosphate in synaptosomes.     

Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig.

Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.     

Subacute and Chronic In Vivo Lithium Treatment Inhibits Agonist- and Sodium Fluoride-Stimulated Inositol Phosphate Production in Rat Cortex

We have investigated the effects of in vivo lithium treatment on cerebral inositol phospholipid metabolism. Twice-daily treatment of rats with LiCl (3 mEq/kg) for 3 or 16 days resulted in a 25-40% reduction in agonist-stimulated inositol phosphate production, compared with NaCl-treated controls, in cortical slices prelabelled with [3H]inositol. A small effect was also seen with 5-hydroxytryptamine (5-HT) 24 h after a single dose of LiCl (10 mEq/kg). Dose-response curves to carbachol and 5-HT showed that lithium treatment reduced the maximal agonist response without altering the EC50 value. This inhibition was not affected by the concentration of LiCl in the assay buffer. Stimulation of inositol phosphate formation by 10 mM NaF in membranes prepared from cortex of 3-day lithium-treated rats was also inhibited, by 35% compared with NaCl-treated controls. Lithium treatment did not alter the kinetic profile of inositol polyphosphate formation in cortical slices stimulated with carbachol. Muscarinic cholinergic and 5-HT2 bindings were unaltered by lithium, as was cortical phospholipase C activity and isoproterenol-stimulated cyclic AMP formation. [3H]Inositol labelling of phosphatidylinositol 4,5-bisphosphate was significantly enhanced by 3-day lithium treatment. The results, therefore, indicate that subacute or chronic in vivo lithium treatment reduces agonist-stimulated inositol phospholipid metabolism in cerebral cortex; this persistent inhibition appears to be at the level of G-protein-phospholipase C coupling.     

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.