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PubMed2001

Signaling with phosphoinositides: better than binary.

When the outside of cell is stimulated,the inside generates a flurry of signals. Phosphates are sprinkled over lipids and proteins,where they are recognized within diverse signaling pathways. The kinases that congregate beneath the cell surface to provide the phosphate tags that mediate signaling have become major targets of new wave of drug design. Phosphoinositide signaling presents a particularly intriguing network whose many mysteries are now being unlocked. Research into protein domains that specifically recognize phosphoinositides have established the ENTH, FYVE,Phox,and pleckstrin homology domains s four cornerstones of phosphoinositide signaling.

1-Phosphatidylinositol 4-Kinase↗

PubMed2006

Phosphate number and acyl chain length determine the subcellular location and lateral mobility of phosphoinositides.

Phosphoinositides are critical regulators of ion channel and transporter activity. There are multiple isomers of biologically active phosphoinositides in the plasma membrane and the different lipid species are non-randomly distributed. However, the mechanism by which cells impose selectivity and directionality on lipid movements and so generate a non-random lipid distribution remains unclear. In the present study we investigated which structural elements of phosphoinositides are responsible for their subcellular location and movement. We incubated phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) with short or long acyl chains in CHO and HEK cells. We show that phosphate number and acyl chain length determine cellular location and translocation movement. In CHO cells, PI(4,5)P2 with a long acyl chain was released into the cytosol easily because of a low partition coefficient whereas long chain PI was released more slowly because of a high partition coefficient. In HEK cells, the cellular location and translocation movement of PI were similar to those of PI in CHO cells, whereas those of PI(4,5)P2 were different; some mechanism restricted the translocation movement of PI(4,5)P2, and this is in good agreement with the extremely low lateral diffusion of PI(4,5)P2. In contrast to the dependence on the number of phosphates of the phospholipid head group of long acyl chain analogs, short acyl chain phospholipids easily undergo translocation movement regardless of cell type and number of phosphates in the lipid headgroup.

4-Chloro-7-nitrobenzofurazan↗

PubMed1991

Interaction between the epidermal growth factor receptor and phosphoinositide kinases.

Ligand-activated epidermal growth factor (EGF) receptors are coupled to the phosphatidylinositol (PtdIns) pathway to stimulate formation of two second messengers, inositol trisphosphate and diacylglycerol. Investigation of the interaction between EGF receptors and phosphoinositide kinases identified PtdIns and PtdIns(4)P kinase activities in extensively washed EGF receptor immunoprecipitates. Studies using COOH-terminal truncation mutant EGF receptors and immunoisolation by an EGF receptor peptide anti-serum in the presence of peptide (residues 644-666) indicated that the phosphoinositide kinases were associated with the region located between the inner membrane boundary and the kinase domain of the EGF receptor. In vivo cross-linking identified four tyrosine phosphorylated proteins of approximately 135, 62, 55, and 47 kDa associated with the EGF receptor. After EGF stimulation, PtdIns and PtdIns(4)P kinase activities were markedly increased among proteins isolated by monoclonal antiphosphotyrosine antibodies. The activities associated with the EGF receptor and with tyrosine-phosphorylated proteins were identified as PtdIns4-and PtdIns(4)P 5-kinase. Tyrosine dephosphorylation did not alter the activity of the prominent PtdIns(4)P 5-kinase activity. These results indicate that the phosphoinositide kinases are associated with and tyrosine phosphorylated by the EGF receptor as part of the mechanism coordinating responses between signal transduction pathways but do not demonstrate that tyrosine phosphorylation of PtdIns(4)P 5-kinase is sufficient to activate the enzyme.

1-Phosphatidylinositol 4-Kinase↗

PubMed1991

Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2.

Several components of the phosphoinositide cycle have been found to interact specifically and at physiological concentrations with the plasma membrane-associated clathrin assembly (adaptor) protein AP-2. These include phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, which are present at the plasma membrane, as well as other polyphosphoinositols. ATP and other polyphosphate molecules complete with the polyphosphoinositols, however, they are at least 80-fold less potent. Also, the effect of ATP, unlike the polyphosphoinositols, is blocked by physiological concentrations of Mg2+. Photoaffinity labeling of AP-2 by [alpha-32P]8-azidoadenosine 5'-triphosphate and its competition by polyphosphoinositols has been used to identify the alpha subunit of the AP-2 complex as the site of specific interaction with the polyphosphoinositols and to confirm direct ultrafiltration binding experiments. Proteolytic dissection of the labeled AP-2 demonstrated that binding occurred exclusively on the N-terminal portion of the alpha subunit. Interaction of purified AP-2 with sub-microM concentrations of polyphosphoinositols has inhibitory effects on a novel AP-2 self-association described in the accompanying paper (Beck, K. A., and Keen, J. H., J. Biol. Chem. 266, 4437-4441), and at higher concentrations on the binding of AP-2 to dissociated clathrin trimers as well as AP-2-mediated clathrin coat assembly. Review of the literature shows that several physiological stimuli that are known to result in increased coat pit formation in intact cells correlate with increased phosphoinositide turnover. These in vivo correlations and the in vitro observations reported here suggest that coated membrane and phosphoinositide cycles may be interdependent within cells.

Adaptor Proteins, Vesicular Transport↗

PubMed1991

Mutational analysis of antigen receptor regulation of B lymphocyte growth. Evidence for involvement of the phosphoinositide signaling pathway.

Stimulation of the antigen receptor of WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-IgM) induces irreversible growth arrest. Anti-IgM stimulates two kinds of transmembrane signaling events, phosphorylation of proteins on tyrosyl residues and breakdown of inositol phospholipids, which results in increases of inositol phosphates, diacylglycerol, and calcium. The roles of these reactions in mediating the growth arrest of the B lymphoma cells have not been established. To examine this issue, we took a genetic approach. Mutants of WEHI-231 cells were isolated that were resistant to anti-IgM-induced growth arrest. Five out of seven independent mutants analyzed had normal cell-surface expression of antigen receptors. Although each of these five mutants had tyrosine protein phosphorylation patterns comparable to wild-type cells, they exhibited alterations in the phosphoinositide signaling pathway. Four of the mutants had decreased phosphoinositide breakdown, probably due to an alteration in phospholipase C. Decreased second messenger production may be responsible for the growth-resistant phenotype. Full growth arrest was restored upon addition of the calcium ionophore ionomycin, suggesting that the limiting second messenger was intracellular free calcium. The final mutant appeared to be altered in a component(s) that responds to diacylglycerol and calcium. Taken together, these results provide further evidence that the phosphoinositide pathway is at least partly responsible for mediating antigen receptor regulation of B lymphoma cell growth.

Animals↗

PubMedDate not supplied

Signal transduction in rat adrenal fasciculata cells stimulated by ACTH1-39 and ACTH5-24: role of the phosphoinositide response in steroidogenesis.

Changes in the soluble inositol-containing metabolites of phosphoinositides have been measured in rat adrenal fasciculata reticularis cells stimulated by ACTH1-39 and ACTH5-24 (an analogue which is able to elicit maximal steroidogenesis in the absence of any discernible increase in cyclic AMP output). Small but significant increases in the total inositol-containing metabolites were found in response to stimulation with both ACTH analogues. For ACTH5-24 this effect on phosphoinositide (PI) metabolism occurred over the same dose range as that for the stimulation of steroidogenesis (with an ED50 of 10(-8) M ACTH5-24 for both effects). It is concluded that in the absence of cyclic AMP generation, ACTH5-24 increased the total inositol-containing metabolites in direct accordance with its steroidogenic capabilities. For ACTH1-39, the effect on PI metabolism reached a maximum with 10(-12) M ACTH1-39 and declined at higher concentrations, i.e., precisely those associated with increased cyclic AMP accumulation. These and the previous observations suggest that the cyclic AMP generated by ACTH1-39 may influence and inhibit the ACTH1-39 effect on PI metabolism. HPLC identification of the phosphoinositide metabolites of the adrenal cells showed that they include glycerophosphoinositol and glycerophosphoinositol phosphate (which would arise by sequential action of phospholipase A1 or A2 and lysophospholipase). Stimulation with ACTH1-39 elicited small but significant increases in glycerophosphoinositol levels, indicating that a phospholipase A may have been activated.

Adrenocorticotropic Hormone↗

PubMed1990

Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats.

Hydrolysis of phosphoinositides elicited by stimulation of cholinergic muscarinic receptors has been studied in brain from neonatal (7-day-old) rats in order to determine: 1) whether the neonatal rat could provide a good model system to study this signal-transduction pathway; and 2) whether potential differences with adult nerve tissue would explain the differential, age-related effects of cholinergic agonists. Accumulation of [3H] inositol phosphates in [3H]inositol prelabeled slices from neonatal and adult rats was measured as an index of phosphoinositide metabolism. Full (acetylcholine, methacholine, carbachol) and partial (oxotremorine, bethanechol) agonists had qualitatively similar, albeit quantitatively different, effects in neonatal and adult rats. Atropine and pirenzepine effectively blocked the carbachol-induced response with inhibition constants of 1.2 and 20.7 nM, respectively. In all brain areas, response to all agonists was higher in neonatal than adult rats, and in hippocampus and cerebral cortex the response was higher than in cerebellum or brainstem. The relative intrinsic activity of partial agonists was higher in the latter two areas (0.6-0.7) than in the former two (0.3-0.4). Carbachol-stimulated phosphoinositide metabolism in brain areas correlated well with the binding of [3H]QNB (r2 = 0.627) and, particularly, with [3H]pirenzepine (r2 = 0.911). In cerebral cortex the effect of carbachol was additive to that of norepinephrine and glutamate. The presence of calcium (250-500 microM) was necessary for maximal response to carbachol to be elicited; the EC50 value for Ca2+ was 65.4 microM. Addition of EDTA completely abolished the response. Removal of sodium ions from the incubation medium reduced the response to carbachol by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1990

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells.

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.

Animals↗

PubMed1990

Possible mechanism for preterm labor associated with bacterial infection. II: Enhancement of endotoxin-stimulated phosphoinositide metabolism by sex steroids in human endometrial fibroblasts.

We have previously reported that bacterial endotoxin stimulated membrane phosphoinositide metabolism, which resulted in liberation of arachidonic acid and preterm initiation of parturition. The aim of this study was to explore whether ovarian steroids alter the stimulation of phosphoinositide turnover by endotoxin. The phosphoinositide turnover was evaluated by [32P] phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI) in human endometrial fibroblasts. When the cells were exposed to endotoxin from Escherichia colli, 32P-labeling of PA and PI was increased in a dose-dependent manner; EC50 was 10 ng/ml. Preincubation with estradiol-17 beta (1 microM) and progesterone (1 microM) for 24 hours, but not for a few hours, enhanced the rate of the endotoxin-stimulated phosphorylation of the lipids in the fibroblasts by decreasing EC50 value of endotoxin to 1 ng/ml. Estradiol or progesterone alone failed to show any effects. These findings demonstrated that the sex steroids may increase the sensitivity of host cells to endotoxin, suggesting the mechanism(s) by which the preterm labor associated with intra-amniotic infection is initiated.

Bacterial Infections↗

PubMed1990

Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum.

The binding affinities of selective muscarinic antagonists were compared with their ability to block receptor-mediated inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum. When measured by competitive inhibition of the binding of N-[3H]methylscopolamine, the binding properties of selective muscarinic antagonists were consistent with a two-site model. Approximately 84% of the binding sites (major sites) had high affinity for the M2-selective antagonists methoctramine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one), whereas the remainder of the sites (minor sites) had high affinity for hexahydrosiladifenidol and its para-fluoro derivative. There was good agreement between the estimates of the dissociation constants of muscarinic antagonists for the major binding site and those measured by antagonism of the adenylate cyclase response. There was also good agreement between the dissociation constants of muscarinic antagonists for the minor binding site and those measured by antagonism of the phosphoinositide response and the contractile response. Our data indicate that there are at least two types of muscarinic receptors in the longitudinal muscle of the ileum, the more abundant being an M2 receptor, which mediates an inhibition of adenylate cyclase activity, and the less abundant being an M3 receptor, which triggers contraction and phosphoinositide hydrolysis.

Adenylyl Cyclases↗

PubMed1990

Purification and characterization of phosphoinositide 3-kinase from rat liver.

Phosphoinositide 3-kinase was purified 27,000-fold from rat liver. The enzyme was purified by acid precipitation of the cytosol followed by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, Mono-Q, and Mono-S columns. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified phosphoinositide 3-kinase preparation contained an 85-kDa protein and a protein doublet of approximately 110 kDa. The 85- and 110-kDa proteins focus together on native isoelectric focusing gels and are cross-linked by dithiobis(succinylamide propionate), showing that the 110- and 85-kDa proteins are a complex. The apparent size of the native enzyme, as determined by gel filtration, is 190 kDa. The 85-kDa subunit is the same protein previously shown to associate with polyoma virus middle T antigen and the platelet-derived growth factor receptor (Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029). The two proteins co-migrate on two-dimensional gels; and, using a Western blotting procedure, 32P-labeled middle T antigen specifically blots the 85-kDa protein. The purified enzyme phosphorylates phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. The apparent Km values for ATP were found to be 60 microM with phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate as the substrate. The apparent Km for phosphatidyinositol is 60 microM, for phosphatidylinositol 4-phosphate is 9 microM, and for phosphatidylinositol 4,5-bisphosphate is 4 microM. The maximum specific activity using phosphatidylinositol as the substrate is 0.8 mumol/mg/min. The enzyme requires Mg2+ with an optimum of 5 mM. Substitution of Mn2+ for Mg2+ results in only approximately 10% of the Mg2(+)-dependent activity. Physiological calcium concentrations have no effect on the enzyme activity. Phosphoinositide 3-kinase has a broad pH optimum around 7.

Animals↗

PubMed1990

Chimeric m2/m3 muscarinic receptors: role of carboxyl terminal receptor domains in selectivity of ligand binding and coupling to phosphoinositide hydrolysis.

The cloning and expression of five mammalian muscarinic receptor genes (m1-m5) have shown that the individual receptor subtypes differ in their functional and ligand-binding properties. To study the role of the carboxyl terminal receptor domains in this pharmacological diversity, we constructed chimeric m2/m3 receptors in which a region comprising part of transmembrane domain VI, the third extracellular loop, transmembrane region VII, and the cytoplasmic tail (collectively referred to as C-terminal domains) was exchanged between the human m2 and the rat m3 receptor. The ability of the cloned receptors to mediate stimulation of phosphoinositide hydrolysis and to bind subtype-selective muscarinic ligands was studied after their transient expression in COS-7 cells. Whereas wild-type m3 strongly stimulated phosphoinositide breakdown, wild-type m2 gave only a poor response. Exchange of the C-terminal domains between m2 and m3 had no significant effect on the magnitude of these responses. In N-[3H]methylscopolamine competition binding studies, the muscarinic antagonists AF-DX 116 and methoctramine showed 11- and 23-fold higher affinities, respectively, for m2 than for m3, whereas hexahydro-silad-ifenidol (HHSiD) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) displayed the reverse selectivity profile, having approximately 10-fold higher affinities for m3. In comparison with wild-type m3, the mutant m3 receptor containing the C-terminal domains of m2 displayed 2.5- and 8-fold higher affinities for AF-DX 116 and methoctramine but 7- and 3-fold lower affinities for HHSiD and 4-DAMP, respectively. The mutant m2 receptor with the C-terminal domains of m3 showed 2-3-fold lower affinities for AF-DX 116 and methoctramine but 2-3-fold higher affinities for HHSiD and 4-DAMP, as compared with wild-type m2. These data suggest that the C-terminal domains of the muscarinic receptors are not involved in conferring selectivity of coupling to phosphoinositide hydrolysis but contain major structural determinants of antagonist binding selectivity.

Animals↗

PubMed1990

Stimulatory effects of maitotoxin on insulin release in insulinoma HIT cells: role of calcium uptake and phosphoinositide breakdown.

In hamster insulinoma (HIT) cells, maitotoxin (MTX) induces a time-dependent and concentration-dependent release of insulin that requires the presence of extracellular calcium. The response is nearly completely blocked by cinnarizine and cadmium, but is not inhibited by the L-type calcium channel blocker nifedipine or by manganese. MTX induces 45Ca+ uptake in these cells in a dose-dependent mode, and the uptake is blocked with cinnarizine, nifedipine and cadmium, and is partially inhibited by manganese. MTX induces phosphoinositide breakdown in HIT cells, and the response is partially blocked by cadmium, but is not affected by nifedipine, cinnarizine or manganese. High concentrations of potassium ions also induce insulin release and calcium uptake in HIT cells. Both effects of potassium are blocked partially by nifedipine, cadmium and cinnarizine. High concentrations of potassium do not induce phosphoinositide breakdown in HIT cells. The results suggest that MTX-elicited release of insulin is attained by two mechanisms: 1) a nifedipine-sensitive action, which results from MTX-induced activation of L-type calcium channels, which can be mimicked with high potassium concentrations; and 2) a nifedipine-insensitive action, which may be initiated by the activation of phosphoinositide breakdown by MTX. Such an activation of phospholipase C would result in the formation of 1,4,5-inositol trisphosphate, a release of intracellular calcium and then release of insulin to the extracellular space. Cinnarizine is proposed to block both MTX-elicited mechanisms, the first by blockade of calcium channels and the second by blocking 1,4,5-inositol trisphosphate-induced release of internal calcium. Either mechanism alone appears capable of eliciting release of insulin.

Animals↗

PubMed1988

Relationship between enhanced phosphoinositide turnover and cellular responses in platelets from spontaneously hypertensive rats.

Thrombin-induced aggregation and serotonin release were markedly enhanced in platelets from spontaneously hypertensive rats (SHR) when compared to normotensive Wistar-Kyoto (WKY) controls. Since phosphoinositides are involved in calcium-mediated platelet responses, the metabolism of these lipids was investigated in SHR and WKY rats by using 32P-labeled quiescent platelets. In unstimulated cells, both the rate and extent of 32P incorporation into individual inositol-containing phospholipids and phosphatidic acid (PA) were identical in SHR and WKY rats. This suggests that the pool size and basal turnover of phosphoinositides did not differ between the two strains. In contrast, early thrombin-induced phosphoinositide metabolism, when monitored as changes in 32P-PA, was significantly higher in SHR than in WKY rats. Following thrombin stimulation, 32P-PA formation likely reflects the initial agonist-receptor interaction; therefore, our results suggest that phospholipase C activity is enhanced in SHR platelets. Thus, it can be postulated that the observed hypersensitivity of SHR phospholipase C may play a role in the overall alteration of cell calcium handling and hence in the SHR platelet response.

Animals↗

PubMed1988

Relationship between delta-9-tetrahydrocannabinol-induced arachidonic acid release and secretagogue-evoked phosphoinositide breakdown and Ca2+ mobilization of exocrine pancreas.

We have previously shown that addition of exogenous arachidonic acid to pancreatic acinar cells inhibits the incorporation of myo-[3H]inositol into membrane phosphoinositides and causes a reduction in the steady state levels of [32P]phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). In the present study, delta-9-tetrahydrocannabinol (THC) was utilized to raise endogenous levels of arachidonic acid. In acinar cells simultaneously prelabeled with [3H]arachidonic acid and [32P]Pi, THC (1-20 microM) produced a concentration-dependent increase in free [3H]arachidonic acid release and a reduction in the steady state levels of [32P]Ptd-Ins4,5P2. THC (1-20 microM) also caused a concentration-dependent inhibition of myo-[3H]inositol trisphosphate accumulation, cytoplasmic Ca2+ level, and amylase secretion elicited by 0.1 microM caerulein. The findings that THC (20 microM) was unable to inhibit either the rise in [Ca2+]i elicited by ionomycin, or the secretory response to phorbol myristic acid or ionomycin, indicate that THC exerts a selective inhibitory effect on the phosphoinositide messenger system. These results support the postulate that endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover in exocrine pancreas.

Amylases↗

PubMed1989

Changes in the concentration and fatty acid composition of phosphoinositides induced by hormones in hepatocytes.

The hormonal regulation of phosphoinositide levels in isolated hepatocytes was studied using chemical means. Extracted inositol phospholipids were adsorbed to neomycin-coated glass beads and then eluted and quantitated by charring after separation by thin layer chromatography on silica gel. The amounts (in nanograms/mg wet weight) of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) were 20 +/- 1, 16 +/- 1, and 1790 +/- 140, respectively). Incubation of the cells with 100 nM vasopressin decreased the value for PIP2 to 10 +/- 0.2 at 15 s, 12 +/- 1.5 at 1 min, and 14 +/- 2.1 at 5 and 30 min. In contrast, the hormone increased 1,2-diacylglycerol plus phosphatidate by over 200 ng/mg wet weight at 5 min under similar conditions (Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chem. 262, 15309-15315). PIP2 was also significantly decreased at 15 s by angiotensin II (100 nM), ATP (100 microM), and epinephrine (1 microM). In contrast, PIP was not significantly changed, and PI was significantly decreased (by approximately 15%) at later times (15 and 30 min). The changes in phosphoinositide mass were well correlated with changes in labeled phosphoinositides in hepatocytes previously incubated with [3H]inositol for 90 min. The amounts of inositol phospholipids in liver plasma membranes (in micrograms/mg protein) were 2.1 +/- 0.2 for PIP2, 0.24 +/- 0.03 for PIP, and 23 +/- 4 for PI. Comparison of these values with those for whole cells suggests that PIP2 is enriched in the plasma membrane, whereas PIP is present elsewhere in the cell. The fatty acid composition of whole cell PIP2 showed significant differences from that of PI. The percentages of palmitic, stearic, linoleic, and arachidonic acids were, respectively, 14, 41, 10, and 25 for PIP2 and 10, 34, 7, and 37 for PI. Vasopressin treatment for 15 s did not alter the fatty acid composition of PIP2. The corresponding fatty acid percentages for liver plasma membranes were 13, 41, 11, and 21 for PIP2 and 8, 34, 0, and 40 for PI. The fatty acid composition of PIP in whole cells and plasma membranes resembled that of PIP2.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1989

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.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner.

Prostaglandin E (PGE) receptor is coupled to a pertussis toxin-insensitive GTP-binding protein in bovine adrenal medulla, but PGE receptor partially purified from bovine adrenal medulla was functionally reconstituted with Gi into phospholipid vesicles (Negishi, M., Ito, S., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1988) J. Biol. Chem. 263, 6893-6900). We demonstrate here that PGE2 inhibited forskolin-induced accumulation of cAMP in cultured bovine chromaffin cells. In plasma membranes prepared from bovine adrenal medulla, PGE2 inhibited forskolin-stimulated adenylate cyclase activity in a GTP-dependent manner. This inhibitory action of PGE2 was abolished by treatment of the membrane with pertussis toxin. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi purified from bovine brain restored the potency of PGE2 to inhibit the adenylate cyclase activity. Inhibition of forskolin-induced cAMP accumulation by PGE2 was also abolished by exposure to the toxin in the cells, indicating that PGE receptors are coupled to Gi. In contrast, PGE2 stimulated the formation of inositol phosphates in chromaffin cells, but this effect was not affected by treatment of the cells with pertussis toxin, suggesting that the PGE receptors are coupled to phosphoinositide metabolism via a pertussis toxin-insensitive G-protein. Both the inhibitory action of cAMP accumulation and stimulation of phosphoinositide metabolism were specific for PGE1 and PGE2, and the Scatchard plot analysis of PGE2 binding to the membrane showed a single high-affinity binding site (Kd = 2 nM). In bovine adrenal chromaffin cells PGE2 enhanced catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). We further examined the modulation of catecholamine release by PGE2 through its inhibitory coupling to the adenylate cyclase system. Prior exposure of chromaffin cells to forskolin or dibutyryl-cAMP reduced nicotine-stimulated catecholamine release, and PGE2 attenuated forskolin-induced inhibition of catecholamine release stimulated by nicotine, but not dibutyryl-cAMP-induced inhibition. In the absence of evidence that PGE receptor subtypes exist, these results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.

Adenosine Diphosphate Ribose↗