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.     

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)     

Pharmacologic characterization of cirazoline-activated inositol phospholipid hydrolysis in rat brain cortical slices

Interaction of cirazoline, an imidazoline derivative, with alpha 1-adrenoceptor coupled inositol phospholipid hydrolysis was characterized in rat brain cortical slices. Norepinephrine, a full alpha 1-agonist, and phenylephrine, a partial alpha 1-agonist, on inositol phospholipid hydrolysis were included for comparison. Norepinephrine produced a fourfold stimulation of inositol phospholipid hydrolysis, whereas cirazoline and phenylephrine caused only submaximal responses (40-60%) when compared with norepinephrine. The stimulation of inositol phospholipid hydrolysis by cirazoline was completely blocked by the alpha 1-adrenoceptor antagonist prazosin, but not by selective alpha 2- or beta-adrenoceptor antagonists. Furthermore, the norepinephrine dose-response curve was shifted to the right in the presence of cirazoline, without affecting the maximal response. These results suggest that cirazoline behaves as a partial agonist at brain alpha 1-adrenoceptors linked to inositol phospholipid hydrolysis.     

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.     

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.     

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.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Tricyclic antidepressants,mianserin, and ouabain stimulate inositol phosphate formation in vitro in rat cortical slices

The ability of tricyclic antidepressants, monoamine oxidase inhibitors, mianserin and ouabain to stimulate hydrolysis of inositol phosphates was examined in rat cerebral cortex slices using a direct assay which involves labelling with [³H]inositol and assaying [³H]inositol phosphates in the presence of lithium. Desimipramine, imipramine, chlorimipramine, mianserin, and ouabain stimulated [³H]inositol phosphate accumulation in a concentration-dependent manner. The monoamine oxidase inhibitors, pargyline and nialamide were without effect. The stimulation of [³H]inositol phosphate accumulation caused by the various substances was not blocked by the antagonists prazosin, ketanserin, atropine, or mepyramine. In contrast, the antagonists prazosin, ketanserin, atropine and mepyramine selectively blocked stimulation of [³H]inositol phosphate accumulation caused by noradrenaline, serotonin, carbachol and histamine respectively. When desimipramine was substituted for lithium in the assay procedure, carbachol was ineffectual in stimulating [³H]inositol phosphate accumulation. In these experiments the control (unstimulated) values were much higher than in the normal (when lithium is present) assay procedure. Desimipramine is quite effective in stimulating [³H]inositol phosphate accumulation either in the presence or absence of lithium in the incubation medium. This is not the case for carbachol where it was essential to have lithium in the incubation medium in order to obtain a stimulation of [³H]inositol phosphate accumulation. Furthermore, in the case of carbachol stimulation, most of the radioactivity was associated with a peak corresponding to inositol monophosphate, while for desimipramine stimulation two clear peaks corresponding to inositol monophosphate and inositol bisphosphate were apparent.     

Adenosine Inhibition of Histamine-Stimulated Inositol Phospholipid Hydrolysis in Mouse Cerebral Cortex

The effects of adenosine on inositol phospholipid hydrolysis in mouse cerebrocortical slices were examined. Despite having no effect alone, adenosine and some structural analogues inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The responses to carbachol, noradrenaline, 5-hydroxytryptamine, and elevated KCl levels were unaffected. The effect of adenosine was on the maximal response to histamine rather than on its EC50. Several adenosine antagonists competitively blocked the inhibition due to adenosine. The results are discussed in relation to the previously reported enhancement of histamine-stimulated hydrolysis of inositol phospholipids in guinea pig brain.     

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.     

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.     

Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors

The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with L-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1-receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with Ki values of 1-6 X 10(-8) M. 11-[(2-[(Diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of approximately 2 X 10(-7) M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H- labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.     

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.     

Inositol Phospholipid Hydrolysis and Potentiation of Cyclic AMP Formation by Noradrenaline in Rat Cerebral Cortex Slices Are Not Mediated by the Same α-Adrenoceptor Subtypes

A pharmacological study was undertaken to determine whether the noradrenaline-stimulated breakdown of inositol phospholipids and the potentiation of isoprenaline-stimulated cyclic AMP by noradrenaline in rat cerebral cortex slices are mediated by the same alpha-receptor subtype. The rank order of potency of a range of alpha 1 and alpha 2 antagonists suggests that both responses may involve an alpha 1 receptor, but there were several differences between the pharmacological profiles for the two systems. Although in both cases, all selective alpha 1 antagonists were more potent than alpha 2 antagonists, the rank orders and the absolute potencies differed for the two responses. The inhibition of the inositol phosphate response was characterised by a high alpha 1/alpha 2 antagonist ratio, and in most cases, Hill slopes of inhibition were consistent with the involvement of a single receptor site. Inhibition of the cyclic AMP response had a much lower alpha 1/alpha 2 antagonist ratio and generally exhibited Hill slopes less than one. Evidence has been provided suggesting that adenosine is involved in the potentiation of cyclic AMP and that other, as yet unidentified, factors may also be involved. Even in the absence of an adenosine component, the results presented support the suggestion that the potentiation due to noradrenaline is mediated by a receptor whose identity does not easily fit with the currently accepted classification of alpha adrenoceptors.     

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.     

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.     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

Effects of Single and Repeated Electroconvulsive Shock Administration on Inositol Phosphate Accumulation in Rat Brain Slices

Accumulation of inositol-1-phosphate after labeling with [3H]inositol and stimulation with noradrenaline, carbachol, and serotonin was measured in rat cortical, caudate nucleus, and hippocampal slices. The response to noradrenaline was significantly increased in cortical slices from animals that had received either a single electroconvulsive shock (ECS) or a series of 10 daily ECS but was unchanged in caudate nucleus or hippocampal slices. The response to carbachol, a muscarinic cholinergic agonist, was unchanged in cortical or caudate nucleus slices but was significantly reduced in hippocampal slices from animals that had received chronic ECS. The response to serotonin in cortical slices was not affected by the treatment. The results are correlated with changes in receptor number, which have been demonstrated to occur after administration of ECS.     

The Na+, K(+)-ATPase inhibitor ouabain and the Na+ ionophore monensin have opposite effects upon carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms.

The effects of ouabain and monensin upon basal and carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms have been investigated. Basal inositol phospholipid breakdown was increased by both compounds at both 6 and 18 mM K+. Enhancement of the carbachol response at 6 mM, but not at 18 mM K+, was found with high concentrations of ouabain. On the other hand, monensin blocked the response to carbachol. Monensin also inhibited the specific binding of [3H]pirenzepine to cerebral cortical membranes, but this was found only at concentrations higher than required to affect the basal and carbachol-stimulated inositol phospholipid breakdown responses. Ouabain did not affect [3H] pirenzepine binding at any of the concentrations tested (6-600 muM). It is concluded that agents that increase the intracellular sodium ion concentration affect the inositol phospholipid breakdown response to carbachol, but that the modulation can be both to potentiate and to inhibit the response.     

Stimulation of Formation of Inositol Phosphates in Primary Cultures of Bovine Adrenal Chromaffin Cells by Angiotensin II, Histamine, Bradykinin, and Carbachol

Histamine, bradykinin, and angiotensin II stimulate release of catecholamines from adrenal medulla. Here we show, using bovine adrenal chromaffin cells in culture, that these agonists as well as carbachol (with hexamethonium) stimulate production of inositol phosphates. The histamine response was mepyramine sensitive, implicating an H1 receptor, whereas bradykinin had a lower EC50 than Met-Lys-bradykinin, and [Des-Arg9]-bradykinin was relatively inactive, implicating a BK-2 receptor. Total inositol phosphates formed in the presence of lithium were measured, with histamine giving the largest response. The relative contribution of chromaffin cells and nonchromaffin cells in the responses was assessed. In each case chromaffin cells were found to be responding to the agonists; in the case of histamine the response was solely on chromaffin cells. When the inositol phosphates accumulating over 2 or 5 min, with no lithium present, were separated on Dowex anion-exchange columns, bradykinin gave the greatest stimulation in the inositol trisphosphate fraction, whereas histamine gave a larger inositol monophosphate accumulation. On resolution of the isomers of stimulated inositol trisphosphate after 2 min of stimulation, the principal isomer present was inositol 1,3,4-trisphosphate in each case. Two hypotheses for the differential responses to histamine and bradykinin are discussed.