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At least 289 records · Page 16Linked to original sources

PubMed1984

alpha Latrotoxin of black widow spider venom binds to a specific receptor coupled to phosphoinositide breakdown in PC12 cells.

alpha Latrotoxin of black widow spider is known to bind with high affinity to surface sites of rat pheochromocytoma (PC12) cells, thereby causing depolarization, calcium influx and massive neurotransmitter release. We show here that the toxin causes the accumulation of inositol phosphates, the products of phosphoinositide breakdown. Inositol 1,4,5, trisphosphate was predominantly accumulated shortly after toxin application. Phosphoinositide breakdown appears to be a direct consequence of toxin binding because high K+ and ionophores (which induce depolarization, calcium influx and transmitter release by different mechanisms) were without such effect. Phosphoinositide breakdown is known as an event coupled to the activation of receptors of various hormones and transmitters. We suggest therefore that the alpha latrotoxin binding site is a receptor coupled across the membrane to the phosphoinositide hydrolysing system.

Adrenal Gland Neoplasms↗

PubMed1989

Pharmacological differences between muscarinic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase inhibition.

Pharmacological differences between muscarinic cholinergic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase were studied. Stimulation of muscarinic receptors from SK-N-SH human neuroblastoma cells resulted in phosphoinositide hydrolysis, but not in inhibition of cAMP formation. As has been shown previously, stimulation of muscarinic receptors from NG108-15 neuroblastoma x glioma cells, on the other hand, resulted in inhibition of cAMP formation without any observable phosphoinositide hydrolysis. These two cell lines provide a useful model system in which to study differential coupling of muscarinic cholinergic receptors. Inhibition of [3H]N-methyl scopolamine [( 3H]NMS) binding and inhibition of carbachol-stimulated function by the antagonists pirenzepine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were studied in this system. Pirenzepine inhibited [3H]NMS binding in both cell lines with low affinity (Ki of 130 and 160 nM in NG108-15 and SK-N-SH cells respectively), indicating that both cell lines express M2 receptors. None of the three antagonists studied exhibited any clear selectivity for the receptors in one cell line over those of the other. In contrast, several agonists including acetylcholine, bethanechol and carbachol exhibited pronounced selectivity. These agonists inhibited [3H]NMS binding to membranes from SK-N-SH cells with IC50 values that were 17-, 3- and 38-fold higher, respectively, than those of NG108-15 cells. This selectivity was still observed when whole cells rather than membranes were studied. These findings indicate that pharmacological differences between receptors coupled to phosphoinositide turnover and those coupled to cAMP inhibition can be detected with certain agonists, but not with the antagonists pirenzepine, AF-DX 116 or 4-DAMP.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Developmental neurotoxicity of ethanol: in vitro inhibition of muscarinic receptor-stimulated phosphoinositide metabolism in brain from neonatal but not adult rats.

The in vitro effects of ethanol (EtOH) on muscarinic receptor-stimulated phosphoinositide metabolism were measured in cerebral cortex slices of adult and 7-day-old rats. EtOH (500 mM) caused a significant decrease (32-43%) of maximal accumulation of [3H]inositol phosphates (InsPs) induced by carbachol, and a 2-fold increase in its EC50 in 7-day-old rats, but had no effect in adult rats. The effect of EtOH on [3H]InsPs accumulation in neonatal rats was significant at a concentration as low as 150 mM. The inhibitory effect of EtOH was maximal in cerebral cortex and hippocampus and lower in cerebellum, while no effect was observed in the brainstem. While carbachol- and acetylcholine-stimulated phosphoinositide metabolism were inhibited by EtOH, EtOH had no effect on norepinephrine-, histamine-, and serotonin-stimulated phosphoinositide hydrolysis. These results are qualitatively and quantitively similar to those previously found following in vivo administration of EtOH to developing and to adult rats, suggesting that the muscarinic receptor-stimulated phosphoinositide metabolism might represent a target for EtOH-induced developmental neurotoxicity.

Aging↗

PubMed1992

Trans-ACPD-induced phosphoinositide hydrolysis and modulation of hippocampal pyramidal cell excitability do not undergo parallel developmental regulation.

The selective metabotropic glutamate receptor agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), stimulates phosphoinositide hydrolysis and elicits a number of electrophysiological responses in the hippocampus. If these effects are mediated by the same receptor subtype, they should undergo parallel developmental regulation. Therefore, we compared the phosphoinositide hydrolysis response and the electrophysiological responses to trans-ACPD at two different developmental stages. Trans-ACPD-stimulated phosphoinositide hydrolysis was significantly greater in hippocampal slices from immature (6-11-day-old) rats than from adults. In contrast, trans-ACPD elicited decreases in spike frequency adaptation and in the amplitude of the slow afterhyperpolarization in roughly equal percentages of immature and adult CA1 pyramidal cells. Similar results were obtained using the putative endogenous agonist, glutamate. These data support the hypothesis that certain electrophysiological effects of trans-ACPD are mediated by a metabotropic glutamate receptor that is distinct from the phosphoinositide hydrolysis-linked glutamate receptor.

Action Potentials↗

PubMed1984

Phosphoinositide breakdown in isolated rat parotid membranes. Stimulation by cholinergic and alpha-adrenergic agonists.

Parotid gland membranes labelled with [3H]inositol were challenged with the cholinergic agonist, carbamylcholine, or with epinephrine in the presence of propranolol. Both agonists caused a significant breakdown of labelled phosphoinositides (17.5%) in membranes suspended in Krebs-Ringer bicarbonate buffer. This effect was abolished by the respective antagonists, atropine or phentolamine. The carbamylcholine-induced breakdown of labelled phosphoinositides did not require cytosol. The addition of cytosol alone, or the exposure of membranes to a medium of low ionic strength caused a significant breakdown of phosphoinositides (10-40%). No further breakdown due to the addition of carbamylcholine was observed under these conditions. It is suggested that neurotransmitter-induced breakdown of phosphoinositides is effected by membrane-associated enzyme(s) and can be observed only in a medium of high ionic strength.

Animals↗

PubMed1986

Biochemical characterization of serotonin stimulated phosphoinositide turnover.

Serotonin (5HT) stimulates phosphoinositide turnover in a number of tissues, but it is not known whether this effect is due to activation of a 5HT receptor which is coupled to phosphoinositide hydrolysis or if the effect is secondary to 5HT stimulated arachidonate metabolism or to the release of another neurotransmitter. In the present study we show that neither indomethacin nor BW 755C inhibits 5HT stimulated phosphoinositide hydrolysis in rat cerebral cortex, suggesting that neither cyclooxygenase nor lipoxygenase activity is required for the response to 5HT. Proteinase inhibitors do not potentiate the response to 5HT, suggesting that 5HT's effect is not due to stimulation of release of a peptide neurotransmitter. Tetrodotoxin does not inhibit the effect of 5HT and 5HT's effect is additive with that of KCl and veratrine. These data suggest that 5HT stimulated phosphoinositide hydrolysis is not dependent upon release of another neurotransmitter.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1986

Regulation of phosphoinositide breakdown by guanine nucleotides.

Phosphoinositide hydrolysis is coupled to receptor systems involved in the elevation of cytosolic Ca2+ and activation of protein kinase C. In cell-free systems, guanine nucleotides are required to transduce the effects of receptor activation to phosphoinositide breakdown. Non-hydrolyzable guanine nucleotides stimulate phosphoinositide breakdown in permeabilized cells as well as membranes prepared from salivary glands, GH3 cells, neutrophils, hepatocytes and cerebral cortical tissue. In blowfly salivary gland membranes, 5-hydroxytryptamine stimulates a guanine-nucleotide dependent breakdown of both endogenous and exogenous phosphoinositide substrate through activation of phospholipase C. These data suggest that a GTP-binding protein modulates phospholipase C activity. The identity of this GTP-binding protein has not been established but may resemble other regulatory GTP-binding proteins which have been identified as transducing proteins in a variety of receptor systems.

Adenylyl Cyclases↗

PubMed1993

Melatonin binding sites are functionally coupled to phosphoinositide hydrolysis in Syrian hamster RPMI 1846 melanoma cells.

Recent pharmacological studies demonstrate that Syrian hamster melanoma (RPMI 1846) cells possess a nanomolar-affinity binding site for melatonin. Inhibition of 2-[125I]-iodomelatonin binding to RPMI membranes by melatonergic ligands exhibit a rank order relationship similar to that observed in hamster hypothalamus. Biochemical studies indicate that the melatonin binding site in RPMI 1846 cells is not coupled in either a stimulatory or inhibitory fashion to adenylate cyclase as a second messenger. We now report that stimulation of RPMI 1846 melanoma cells with melatonergic agonists induces phosphoinositide hydrolysis in a concentration-dependent manner (EC50: N-acetylserotonin = 0.29 microM; 2-I-Melatonin = 0.39 microM; 6-Cl-Melatonin = 0.38 microM; Melatonin = 0.45 microM). Further, phosphoinositide hydrolysis induced by 2-I-melatonin and N-acetylserotonin was blocked by pre-incubation with the melatonin antagonist N-acetyltryptamine and prazosin, an antagonist which exhibits potency at the nanomolar affinity melatonin binding site. 2-I-melatonin and N-acetylserotonin-induced phosphoinositide hydrolysis were not blocked by the serotonin type 2 antagonist ketanserin or alpha-adrenergic antagonist, phentolamine. These data suggest that melatonin binding sites on RPMI 1846 cells are linked to phosphoinositide hydrolysis as a second messenger.

Animals↗

PubMed1996

Inhibition of muscarinic stimulated phosphoinositide hydrolysis in the rat parotid gland by cAMP.

The ability of agents that increase or mimic cAMP to affect muscarinic receptor mediated phosphoinositide hydrolysis was investigated in the rat parotid gland. Forskolin (10 microM) and isoproterenol (10 microM) elevated cAMP in the parotid gland by 2-fold and 7-fold, respectively, and these agents also inhibited oxotremorine-M (3 microM) mediated phosphoinositide hydrolysis by 14% and 26%, respectively. Forskolin (1, 4.3, 18, and 75 microM) increased cAMP accumulation and inhibited PIP2 hydrolysis in a concentration-dependent manner. Forskolin (75 micrometers) shifted the concentration-response curve for the full agonist oxotremorine-M rightward by 4.2-fold. Pre-treatment with the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) reduced the maximum effect of oxotremorine-M by 31%. The inhibitory effect of isoproterenol and forskolin on muscarinic receptor-mediated phosphoinositide hydrolysis was unaffected by the removal of extracellular Ca2+. Moreover, isoproterenol and forskolin dampened sodium fluoride and oxotremorine-M mediated phosphoinositide hydrolysis to the same extent suggesting that the inhibitory effect of cAMP is downstream from the muscarinic receptor.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Inhibition of muscarinic receptor-stimulated phosphoinositide metabolism by cocaine, norcocaine and cocaethylene in rat brain.

The interaction of cocaine, its metabolites norcocaine and benzoylecgonine, and cocaethylene, which is formed following a combined cocaine and ethanol exposure, with muscarinic receptor binding and phosphoinositide metabolism was investigated in brain from immature rats. Cocaine and norcocaine inhibited binding of [3H]telenzepine and carbachol-stimulated phosphoinositide metabolism in cerebral cortex, while benzoylecgonine was devoid of any inhibitory activity. Cocaethylene was the most potent inhibitor of both binding and phosphoinositide metabolism. The effect of cocaine was more pronounced at the muscarinic receptors, but a small inhibition of histamine--and serotonin--stimulated phosphoinositide metabolism was also observed.

Animals↗

PubMed1991

Selective time-dependent effects of insulin on brain phosphoinositide metabolism.

The effect of insulin on phosphoinositide metabolism in the cerebral cortex was examined using 32P as precursor. A maximal increase was detected as early as 15 s; phospholipid labeling declined after this initial peak but then increased to another maximum at 30 min. The levels of these phospholipids were unchanged at the earliest time examined, but at 30 min insulin caused an increase in the content of all phospholipids tested. In pulse-chase experiments, insulin stimulated depletion of 32P-labeled phosphoinositides only at 15 s. On the other hand, insulin treatment caused a biphasic diacyglycerol (DAG) production. We conclude that in cerebral cortex, insulin has a dual mechanism of action on phosphoinositide metabolism. First, insulin causes a rapid but transient hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism, followed by subsequent resynthesis; thereafter, insulin increases de novo phospholipid synthesis.

Animals↗

PubMed1994

Phosphatidic acid promotes phosphoinositide metabolism and DNA synthesis in cultured cortical astrocytes.

The addition of exogenous phosphatidic acid (PA) to cultured cortical astrocytes prelabelled with [3H]inositol resulted in the accumulation of intracellular [3H]inositol phosphates (IP) in a concentration-dependent (EC50 = 20 microM) manner. Analysis of the individual IPs formed following a PA challenge revealed a rapid but transient generation of [3H]inositol trisphosphate (IP3) indicating the involvement of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown in this response a fact which was confirmed when the recovery of radiolabel in membrane phosphoinositides was assessed. PA's ability to stimulate IP3 accumulation was found to be dependent upon its acyl-chain length. Dioleoyl-PA (C18:1) was equally as effective as PA from egg yolk lecithin in this respect whilst dipalmitoyl-PA (C16:0) was less so and dimyristoyl-PA (C14:0) and dilauroyl-PA (C12:0) were without effect. In subconfluent, serum-deprived cultures, PA was found to increase DNA synthesis following a 48 h exposure period. This effect was observed over the same concentration range used to measure phosphoinositide breakdown and was found to be mediated by the activation of protein kinase C. As with its effect on phosphoinositide metabolism, PA's ability to promote DNA synthesis was correlated with its acyl-chain length. These data show that PA is capable of stimulating both phosphoinositide metabolism and DNA synthesis in cultured astrocytes possibly via the activation of specific membrane receptors. However, the precise relationship between these events remains to be elucidated.

Animals↗

PubMed1990

Indications for selective coupling to phosphoinositide hydrolysis or to adenylate cyclase inhibition by endogenous muscarinic receptor subtypes M3 and M4 but not by M2 in tumor cell lines.

The muscarinic receptor subtype mRNAs expressed in cell lines were determined by Northern blot analysis. The biochemical responses of the muscarinic receptors in these cell lines (phosphoinositide hydrolysis and cAMP levels) were studied and correlated to the corresponding muscarinic receptor subtype as determined by mRNA expression. PC12 cells that expressed M4 subtype mRNA exhibited muscarinically dependent adenylate cyclase inhibition, whereas C6 and SK-N-SH cells expressing M3 subtype mRNA exhibited muscarinically dependent phosphoinositide hydrolysis. IMR-32 cells (M2 subtype mRNA) exhibited both muscarinically dependent phosphoinositide hydrolysis and adenylate cyclase inhibition. These results suggest that endogenous M3 and M4 receptor subtypes are selectively coupled to phosphoinositide hydrolysis and adenylate cyclase inhibition, respectively, whereas the M2 receptor subtype is coupled to both responses.

Adenylyl Cyclases↗

PubMed1991

Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells.

Two cell lines transformed by the k-ras oncogene (KiKi and KiMol cells) and a temperature sensitive clone (Ts), all originated from a normal rat thyroid line (FRTL5 cells), have been employed to analyse the intracellular mechanisms affected by the ras p21. In k-ras transformed cells two phosphoinositide derivatives, glycerophosphoinositol and inositol monophosphate, were markedly increased, whereas inositol bisphosphate and trisphosphate maintained the same level as in normal cells. Cytosolic Ca2+ was also unaffected. This indicates that in epithelial cells the phospholipase C activity is not altered upon ras transformation. The formation of glycerophosphoinositol involved the activation of a phosphoinositide specific phospholipase A2. The higher phospholipase A2 activity in ras transformed cells could be further demonstrated by the increase in total arachidonic acid release. In the Ts clone the increase in glycerophosphoinositol and inositol monophosphate was evident only at the permissive temperature (33 degrees C), whereas it disappeared at 39 degrees C. At 33 degrees C the cells were also characterized by an enriched membrane pool of phosphoinositides. All these changes occurred in parallel with morphological transformation. We propose that cell transformation by the k-ras oncogene affects different steps of the membrane lipid metabolism, among which the most prominent one is the activation of a phosphoinositide specific phospholipase A2. These effects could originate mitogenic metabolites. Moreover, they correlate well with the induction of the malignant phenotype.

Animals↗

PubMed1993

Endothelin receptors in rat cerebellum: activation of phosphoinositide hydrolysis is transduced by multiple G-proteins.

Induction of phosphoinositide hydrolysis in rat cerebellar slices by endothelins (ET-1 and ET-3) and sarafotoxins (SRTX-b and SRTX-c) was demonstrated by measurement of labelled inositol phosphate generation. Pertussis toxin (PT) enhanced the induction of phosphoinositide hydrolysis by all four peptides. The process seems to be mediated by at least two heterotrimeric G-proteins, the one sensitive and the other insensitive to PT. Measurement of the GTPase activity induced in this preparation indicated that phosphoinositide hydrolysis is stimulated via a functional coupling between the endothelin receptor of the ETB-R subtype and a PT-insensitive G-protein family, i.e. Gq/11. The involvement of PT-sensitive G-proteins, i.e. Gi-like and/or Go-like proteins, in the stimulation of phosphoinositide hydrolysis by ETs and SRTXs is discussed.

Adenosine Diphosphate Ribose↗

PubMed1996

Regulation of phosphoinositide cycle by intracellular sodium in the blood-brain barrier.

In the present study of cerebral microvessels, we report that monensin, a Na+ ionophore, elicits a decrease in 32P radioactivity incorporation into phosphoinositides in cerebral microvessels. In addition, monensin evokes enhanced production of inositol-1-monophosphate (IP) and inositol-1,4-bisphosphate (IP2), together with an increase in the diacylglycerol (DAG) mass. These results indicate that monensin evokes a phosphoinositide hydrolysis by phospholipase C (PLC). The absence of inositol-1,4,5-trisphosphate (IP3) production leads us to think that although phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis occurs in this process, there is a very rapid disappearance of IP3. The net decrease in 32P radioactivity incorporated into phosphoinositides suggests that a partial inhibition of their re-synthesis is also evoked. Experimental evidence with pharmacological tools suggests that: (1) these effects are secondary to an increase in Ca2+ through the Na+/Ca2+ exchanger; and (2) the intracellular Ca2+ release is not involved in these effects of monensin. Since some neuropeptide receptors in cerebral microvessels have been reported to be coupled to either the Na+/H+ exchanger or to PLC, we discuss the possibility that cross-talk exists between these intracellular signalling pathways (phosphoinositide metabolism and Na+ transport) in the blood-brain barrier (BBB).

Animals↗

PubMed1992

Thrombin is the major serum factor stimulating phosphoinositide turnover, but not DNA synthesis in human neuroblastoma SH-EP cells.

Fetal calf serum stimulates both phosphoinositide turnover and DNA synthesis in SH-EP cells. The phosphoinositide turnover-stimulating activity of serum is largely (70%) reduced in the presence of hirudin, a blocker of thrombin activity. Yet, hirudin does not alter the ability of serum to stimulate DNA synthesis. Purified alpha-thrombin is a potent (EC50, 35 pM) stimulator of phosphoinositide turnover in SH-EP cells, but induces DNA synthesis only at much higher concentrations (10 nM-1 microM). Thus, serum thrombin accounts for most of the ability of serum to stimulate phosphoinositide hydrolysis, but not for the effect of serum on cell division, since the concentration of thrombin in serum is not sufficient to induce DNA synthesis. These data suggest that hydrolysis of inositol lipids may not be the main signalling event mediating the mitogenic effects of alpha-thrombin.

Blood↗

PubMed1995

Phosphoinositide metabolism triggered by wheat germ agglutinin in human platelets.

The lectin wheat germ agglutinin (WGA) has been shown to elicit platelet functional responses such as aggregation and release reaction as a consequence of binding several cell surface glycoproteins in platelets, whereas physiological activators act on their specific receptors. To examine the mechanism of WGA-induced platelet activation, we analyzed phosphoinositide metabolism triggered by this lectin. The binding of WGA to human platelets induced a rapid and dose-dependent surge in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis, as monitored by an increase in [Ca2+]i and phosphatidic acid production. Furthermore, it similarly elicited the synthesis of PtdIns(3,4)P2, which is a major 3-phosphorylated phosphoinositide in platelets. This 3-phosphorylated lipid generation induced by WGA was very strong and did not appear to be a consequence of the classical PtdIns(4,5)P2 hydrolysis. WGA also induced a marked increase in the degree of tyrosine phosphorylation of proteins in platelets, and this increase was correlated with the phosphoinositide metabolism, i.e. PtdIns(4,5)P2 breakdown and PtdIns(3,4)P2 synthesis. The non-selective inhibitor of protein kinases, staurosporine, inhibited similarly both the increase in tyrosine phosphorylation of several platelet proteins and the phosphoinositide changes in a dose-dependent manner. These findings suggest that both PtdIns(4,5)P2 hydrolysis and PtdIns(3,4)P2 synthesis are related to protein-tyrosine phosphorylation in platelets stimulated with WGA.

Blood Platelets↗