Maitotoxin, a potent, general activator of phosphoinositide breakdown

Maitotoxin (MTX), a potent marine toxin, elicits a calcium-dependent activation of cells that can be inhibited by calcium channel blockers like nifedipine. MTX also stimulates phosphoinositide breakdown in smooth muscle cells, NCB-20 cells and PC12 cells through a nifedipine-insensitive mechanism. We now report that MTX stimulates phosphoinositide breakdown in a wide variety of cells, and appears to represent the first general activator of this second messenger-generating system. MTX-induced stimulation of phosphoinositide breakdown is dependent in every cell line on the presence of extracellular calcium. In differentiated HL60 cells, in which a chemotactic peptide (fMLP) activates phosphoinositide breakdown via a pertussis toxin-sensitive mechanism, MTX-induced stimulation is not affected by pertussis toxin treatment. A phorbol ester has no effect on the response to MTX. Thus, MTX stimulates phosphoinositide breakdown through a calcium-dependent mechanism that at least in three cell lines (PC12, NCB20 and HL60) is not mediated by a pathway that involves a pertussis toxin-sensitive guanine nucleotide-binding protein.     

Characterization of Bradykinin-Induced Phosphoinositide Turnover in Neurohybrid NCB-20 Cells

Phosphoinositide hydrolysis was studied in neurohybrid NCB-20 cells prelabeled with myo-[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IP1) in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 microM. The bradykinin response was robust (10-fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D-Arg-(Hyp3, Thi, D-Phe)-bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine H1 receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin-induced [3H]IP1 accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carbachol-induced accumulation in the same cell line. In contrast, using related neurohybrid NG108-15 cells, bradykinin-induced [3H]IP1 accumulation was found to require much less lithium. IN the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]-inositol bis- and trisphosphate. Basal and bradykinin-induced phosphoinositide breakdown was inhibited by 4 beta-phorbol 12,13-dibutyrate, but was unaffected by the biologically inactive 4 beta-phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the bradykinin-induced [3H]IP1 accumulation, without affecting basal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.     

Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C

Phosphoinositide hydrolysis was studied in a washed membrane preparation of 1321N1 astrocytoma cells prelabeled with [3H]inositol. GTP gamma S stimulated the formation of [3H]inositol mono-, bis-, and trisphosphate ([3H]InsP, [3H]InsP2, and [3H]InsP3) with a half-maximal effect on [3H]InsP formation at 5 microM. Carbachol increased the accumulation of [3H]inositol phosphates only in the presence of added guanine nucleotide. Calcium increased [3H]InsP3 accumulation over a range of concentrations (10 nM-3 mM free calcium). When 1321N1 cells were treated with phorbol ester (100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)) prior to preparation of the membranes, the maximal [3H]InsP formation induced by GTP gamma S or GTP gamma S plus carbachol was decreased by 50-75%. In contrast, the response to a maximal calcium concentration presumed to activate phospholipase C directly was minimally inhibited (approximately 15%). PMA treatment did not affect muscarinic receptor affinity for carbachol or the effect of GTP on agonist binding. PMA treatment was also without effect on the breakdown of exogenous [3H]InsP3 in homogenates, permeabilized cells, and membranes, indicating that the InsP3-phosphatase was not the site of phorbol ester action. PMA treatment inhibited [3H] InsP3 formation only in membranes and not in cytosol prepared from the same cells, suggesting a membrane site of PMA action. Membranes were also required to demonstrate GTP gamma S-stimulated [3H]InsP3 formation although calcium-stimulated [3H]InsP3 formation was demonstrable in both membranes and cytosol. The addition of purified protein kinase C to the membranes mimicked the effect of PMA treatment to decrease GTP gamma S-stimulated [3H]InsP3 production. These data indicate that the effect of PMA on phosphoinositide metabolism is demonstrable in a cell-free system and that it can be mimicked by protein kinase C. We suggest that the ability of PMA to block GTP gamma S-stimulated formation of [3H]InsP3 results from inhibition of the G protein interaction with phospholipase C.     

Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.     

Metabolism of inositol phosphates in parotid cells: implications for the pathway of the phosphoinositide effect and for the possible messenger role of inositol trisphosphate

Rat parotid acinar cells were used to investigate the time course of formation and breakdown of inositol phosphates in response to receptor-active agents. In cells preincubated with [3H]inositol and in the presence of 10 mM LiCl (which blocks hydrolysis of inositol phosphate), methacholine (10(-4)M) caused a substantial increase in cellular content of [3H]inositol phosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Subsequent addition of atropine (10(-4) M) caused breakdown of [3H]inositol trisphosphate and [3H]inositol bisphosphate and little change in accumulated [3H]inositol phosphate. The data could be fit to a model whereby inositol trisphosphate and inositol bisphosphate are formed from phosphodiesteratic breakdown of phosphatidylinositol bisphosphate and phosphatidylinositol phosphate respectively, and inositol phosphate is formed from hydrolysis of inositol bisphosphate rather than from phosphatidyl-inositol. Consistent with this model was the finding that [3H]inositol trisphosphate and [3H]inositol bisphosphate levels were substantially increased in 5 sec while an increase in [3H]inositol phosphate was barely detectable at 60 sec. These results indicate that in the parotid gland the phosphoinositide cycle is activated primarily by phosphodiesteratic breakdown of the polyphosphoinositides rather than phosphatidyl-inositol. Also, the results show that formation of inositol trisphosphate is probably sufficiently rapid for it to act as a second messenger signalling internal Ca2+ release in this tissue.     

Thyrotropin-releasing hormone stimulates inositol phosphate production in normal anterior pituitary cells and GH3 tumour cells in the presence of lithium

Phosphatidylinositol (Ptd Ins) breakdown in response to thyrotropin-releasing hormone (TRH) was measured after preincubation of both normal rat anterior pituitary cells and GH₃ turnout cells with [³H]inositol by the determination of [³H]inositol phosphate accumulation in the presence of lithium (which inhibits myo-inositol phosphatase). The method employed, which was originally developed for use with tissue slices, was adapted for isolated cells in monolayer culture. In GH₃ cells, TRH stimulated the breakdown of phosphoinositide in a manner similar to that reported previously using alternative methods. Furthermore, in normal male anterior pituitary cells the dose-response profile for TRH stimulation of inositol phosphate accumuJation was found to correlate well with the dose-response profile for TRH stimulation of prolactin secretion. As this response was maintained in the absence of added calcium, the breakdown of phosphoinositide would appear to be implicated as an event preceding calcium mobilization.     

Bradykinin induces a rapid release of inositol trisphosphate from a neuroblastoma hybrid cell line NCB-20 that is not antagonized by enkephalin

In mouse neuroblastoma x Chinese hamster brain clonal cell line NCB-20, bradykinin (BK) receptor stimulation causes phosphoinositide hydrolysis and release of inositol phosphates. Maximum stimulation (4-fold) of [2-3H]inositol trisphosphate (IP3) release in the absence of Li+ from NCB-20's prelabelled for 20-24 hours with [2-3H]myo-inositol (15 microCi/confluent 60mm dish) occurred after 5-10 seconds of bradykinin exposure, with an EC50 of approximately 100nM. Inositol bisphosphate (IP2) and inositol monophosphate (IP1) also showed increases (2.9-fold and 1.5 fold, respectively), with peaks at 15-20 seconds and 50 seconds, respectively. Under these same conditions, D-Ala2-D-Leu5 enkephalin (DADLE) (10 microM), an opiate agonist with 2nM affinity, gave no stimulation of IP3 release. Furthermore, it did not block BK-initiated release, both when applied simultaneously with BK and when cells were preincubated with DADLE for 100 minutes to lower cyclic AMP levels. These results show that pain-inducing BK has a major acute stimulatory effect on receptor-phospholipase C-coupled IP3 release, the opioid peptide DADLE has no such effect and, DADLE does not block the IP3 release induced by BK.     

Calcitonin gene-related peptide and cyclic AMP stimulate phosphoinositide turnover in skeletal muscle cells. Interaction between two second messenger systems

Calcitonin gene-related peptide (CGRP) has previously been shown to coexist with acetylcholine in spinal cord motoneurons and to stimulate adenylate cyclase in skeletal muscle cells. We now demonstrate that in cultured chick myotubes whose phosphoinositides have been labeled with [3H]inositol, CGRP enhanced the accumulation of [3H]inositol mono-, bis-, and trisphosphates. Rat CGRP-I (rCGRP) (0.1 microM) elicited a transient increase in [3H]inositol 1,4,5-trisphosphate, as well as a more sustained elevation of [3H]inositol 1,3,4-trisphosphate levels. In the presence of Li+, rCGRP evoked an approximately 3-fold increase of [3H]inositol monophosphate levels, which persisted for up to 1 h. This effect of rCGRP was concentration-dependent, the half-maximal response being obtained at 1 nM. Since rCGRP also accelerated the rate of synthesis of [3H]inositol-containing lipids, it appears that the peptide acts by stimulating phosphoinositide turnover in chick myotubes. Agents that either mimic or elevate intracellular cyclic AMP also enhanced the synthesis of [3H]inositol-containing lipids, and the accumulation of inositol phosphates, suggesting that the effects of rCGRP are mediated, at least in part, via the activation of adenylate cyclase. This hypothesis was strengthened by the non-additivity of the inositol phosphate responses elicited by rCGRP and other cAMP-mobilizing agents, and by the sensitivity of these responses to various pharmacological treatments. The present results provide an example of positive interaction between cAMP and the phosphoinositide signaling system. They further suggest that a coexisting neuropeptide may exert pleiotropic actions upon its target cell by stimulating multiple signal transduction pathways.     

Lithium-induced accumulation of inositol 1-phosphate during cholecystokinin octapeptide- and acetylcholine-stimulated phosphatidylinositol breakdown in dispersed mouse pancreas acinar cells

Dispersed mouse pancreas acinar cells were prepared in which phosphatidylinositol had been labeled with myo[2-3H]inositol. During incubation with 0.3 microM cholecystokinin octapeptide (CCK-8) for 15 min, there was a loss of [3H]phosphatidylinositol radioactivity (23%) and a 3-fold gain in trichloroacetic acid-soluble radioactivity. Replacement of NaCl by up to 58 mM LiCl did not significantly affect the amount of CCK-8-stimulated [3H]phosphatidylinositol breakdown or the gain in acid-soluble radioactivity. However, in normal medium, the product of phosphatidylinositol breakdown was almost all inositol, whereas in Li+-containing medium, the product was almost all inositol 1-phosphate. Similar results were obtained with acetylcholine which, in the presence of Li+, gave a dose-responsive increase in inositol 1-phosphate over the concentration range of 0.1 to 10 microM. No increased accumulation of [3H]inositol diphosphate or [3H]inositol triphosphate was detected in stimulated cells. Time courses in the presence of Li+ indicated that the formation of inositol 1-phosphate preceded the formation of inositol. Addition of up to 50 mM myoinositol to the incubation medium showed no diluting effect on the amount of [3H]inositol 1-phosphate found. The accumulation of inositol 1-phosphate is presumably due to the known ability of Li+ to inhibit myoinositol 1-phosphatase. The results provide clear evidence that stimulated phosphatidylinositol breakdown involves a phospholipase C type of phosphodiesterase activity. 1.25 mM Li+ gave half-maximal inositol 1-phosphate accumulation. This is close to the range of plasma Li+ levels which is used therapeutically in psychiatric disorders. In unstimulated cells, [3H]inositol 1-phosphate accumulation in the presence of Li+ corresponded to a breakdown rate for [3H]phosphatidylinositol of 2 to 3%/h.     

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.     

Insulin-stimulated phosphoinositide metabolism in isolated fat cells

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.     

Enhanced arterial contractility to noradrenaline in diabetic rats is associated with increased phosphoinositide metabolism.

The purpose of this study was to investigate whether the increased contractile responsiveness of aortae from male rats with 12-14 week streptozotocin-induced diabetes to noradrenaline is associated with alterations in phosphoinositide metabolism. The contractile response to noradrenaline (10 microM) in both the presence and absence of extracellular calcium was significantly enhanced in aortae from diabetic rats. No significant differences were found between control and diabetic arteries in the basal incorporation of 32P and [3H]myo-inositol into phosphoinositides, or in the basal accumulation of [32P]phosphatidic acid and [3H]inositol phosphates. However, noradrenaline (10 microM) caused significantly greater breakdown of [32P]phosphatidylinositol 4,5-bisphosphate and formation of [32P]phosphatidic acid and [3H]inositol phosphates in diabetic aortae than in control preparations. The production of [3H]inositol phosphates induced by noradrenaline was selectively reduced by the alpha 1-adrenoceptor antagonist, prazosin, in both control and diabetic tissues. These results indicate that phosphoinositide metabolism in response to noradrenaline via stimulation of alpha 1-adrenoceptors is enhanced in aortae from chronic streptozotocin-diabetic rats. The increase in inositol 1,4,5-trisphosphate and 1,2-diacylglycerol production that presumably results could be responsible, at least in part, for the enhanced contractile response of aortae from diabetic rats to noradrenaline.     

Source of 3H-labeled inositol bis- and monophosphates in agonist-activated rat parotid acinar cells

The kinetics of [3H]inositol phosphate metabolism in agonist-activated rat parotid acinar cells were characterized in order to determine the sources of [3H]inositol monophosphates and [3H]inositol bisphosphates. The turnover rates of D-myo-inositol 1,4,5-trisphosphate and its metabolites, D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate, were examined following the addition of the muscarinic receptor antagonist, atropine, to cholinergically stimulated parotid cells. D-myo-Inositol 1,4,5-trisphosphate declined with a t1/2 of 7.6 +/- 0.7 s, D-myo-inositol 1,3,4-trisphosphate declined with a t1/2 of 8.6 +/- 1.2 min, and D-myo-inositol 1,4-bisphosphate was metabolized with a t1/2 of 6.0 +/- 0.7 min. The sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate (2.54% phosphatidylinositol/min) did not exceed the calculated rate of breakdown of D-myo-inositol 1,4,5-trisphosphate (2.76% phosphatidylinositol/min). Thus, there is no evidence for the direct hydrolysis of phosphatidylinositol 4-phosphate in intact cells since D-myo-inositol 1,4-bisphosphate formation can be attributed to the dephosphorylation of D-myo-inositol 1,4,5-trisphosphate. The source of the [3H]inositol monophosphates also was examined in cholinergically stimulated parotid cells. When parotid cells were stimulated with methacholine, D-myo-inositol 1,4,5-trisphosphate, D-myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1,4-bisphosphate, and D-myo-inositol 4-monophosphate levels increased within 2 s, whereas D-myo-inositol 1-monophosphate accumulation was delayed by several seconds. Rates of [3H]inositol monophosphate accumulation also were examined by the addition of LiCl to cells stimulated to steady state levels of [3H]inositol phosphates. The sum of the rates of accumulation of D-myo-inositol 1-monophosphate and D-myo-inositol 4-monophosphate did not exceed the rate of breakdown of D-myo-inositol 1,4,5-trisphosphate or the sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate. These kinetic analyses suggest that agonist-stimulated [3H]inositol bis- and monophosphate formation in intact rat parotid acinar cells can be accounted for by the metabolism of D-myo-[3H]inositol 1,4,5-trisphosphate rather than by phospholipase C-catalyzed hydrolysis of phosphatidylinositol or phosphatidylinositol 4-phosphate.     

Effects of maitotoxin on atrial natriuretic factor-mediated accumulation of cyclic GMP in PC12 cells

Maitotoxin (MTX) activates calcium channels and stimulates phosphoinositide breakdown in pheochromocytoma PC12 cells, while having no effect on basal levels of the cyclic nucleotides cAMP and cGMP. Atrial natriuretic factor (ANF) induces a dose-dependent accumulation of cGMP in PC12 cells through the activation of a membrane bound guanylate cyclase. Effects of ANF on cGMP are independent of extracellular concentrations of calcium. Since agents that activate phosphoinositide breakdown can indirectly affect cyclic nucleotide formation, the effects of MTX on ANF-mediated accumulation of cGMP was studied. MTX induces a dose-dependent inhibition of ANF-mediated accumulation of cGMP. The inhibition by MTX requires the presence of extracellular calcium, but is unaffected by the calcium channel blocker nifedipine. The inhibitory effect of MTX is not mimicked by the calcium ionophore ionomycin. A phorbol ester, PMA, which stimulates protein kinase C, also inhibits ANF-mediated accumulation of cGMP. Sodium nitroprusside induces large accumulations of cGMP in PC12 cells through the stimulation of a soluble guanylate cyclase. Neither MTX nor PMA inhibit nitroprusside-mediated accumulation of cGMP. The results indicate that in PC12 cells, protein kinase C activation, either directly with PMA, and indirectly with MTX through phosphoinositide breakdown and formation of diacylglycerol, leads to inhibition of ANF-mediated, but not nitroprusside-mediated accumulation of cGMP.     

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.     

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.     

Modulation of Phosphoinositide Hydrolysis by NaF and Aluminum in Rat Cortical Slices

NaF stimulated phosphoinositide hydrolysis in rat cortical slices. The production of [3H]inositol monophosphate was rapid for the first 15 min of incubation with NaF, followed by a plateau. The major product detected was [3H]inositol monophosphate, although significant amounts of [3H]inositol bisphosphate and [3H]inositol trisphosphate were also produced. The stimulation of [3H]inositol monophosphate production by NaF was concentration dependent between 2 and 20 mM NaF. Addition of 10 or 100 microM AlCl3 or aluminum maltol did not alter the effect of NaF, whereas at 500 microM, these aluminum preparations resulted in significant inhibition. Increasing the concentration of K+ from 5 to 20 mM potentiated [3H]inositol monophosphate production induced by carbachol but not by NaF. Incubation with 1 microM phorbol 12-myristate 13-acetate, a phorbol ester, inhibited carbachol-induced, but not NaF-induced, [3H]inositol monophosphate production. These results further support the hypothesis that a guanine nucleotide binding protein that can be activated by NaF is involved in phosphoinositide hydrolysis in brain. The use of NaF provides a means to bypass receptors to study intracellular regulatory sites of phosphoinositide metabolism without disrupting cells.     

Thyrotropin-releasing hormone stimulation of polyphosphoinositide hydrolysis in GH3 cell membranes is GTP dependent but insensitive to cholera or pertussis toxin

Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP).     

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.