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PubMed1992

An inhibitor of sweet taste response modulates glucose-induced phosphoinositide breakdown in rat pancreatic islets.

We studied the effect of a specific-competitive inhibitor of the sucrose taste response, p-nitrophenyl-D-glucopyranoside (PNP-Glu) on insulin release and phosphoinositide metabolism in rat pancreatic islets. The alpha-anomer, but not the beta-anomer, of PNP-Glu at a concentration of 5 mM inhibited insulin release induced by 10 mM glucose. Islets were labeled by exposure for 2 h to 10 uCi of myo-[2-3H] inositol solution supplemented with 2.8 mM glucose. Forty islets were then incubated in the presence of 10 mM LiCl, 1 mM inositol and 10 mM glucose with or without the anomers of PNP-Glu. [3H] radioactivity in the incubation medium remained significantly greater in the presence of the alpha-anomer of PNP-Glu than in the presence of glucose alone after 5- and 20-min incubation. The inositol monophosphate levels in the islets incubated with glucose alone were increased more than in the islets with alpha-anomer. The beta-anomer of PNP-Glu did not change either glucose-induced insulin release or phosphoinositide breakdown. A patch-clamp study revealed that neither anomer affected the glucose-dependent ATP-sensitive K(+)-channels. These results indicate that the anomeric preference for glucose in insulin release in the pancreatic islets is closely associated with phosphoinositide breakdown.

Animals↗

PubMed1995

Endothelin-induced phosphoinositide hydrolysis in the muscular layer of stem villi vessels of human term placenta.

In the present study, we examined the relationship between endothelin receptors and phosphoinositide breakdown in muscle explants of placental stem villi vessels. All peptides examined, i.e. endothelin-1 (ET-1), ET-3, sarafotoxin 6b (S6b) and S6c, were able to induce phosphoinositide hydrolysis in a dose-dependent manner: ET-1 was more potent than S6b and ET-3, with corresponding EC50 values of 44 +/- 16 pmol/l, 18 +/- 13 nmol/l and 33 +/- 24 nmol/l, respectively. Sarafotoxin induced only moderate stimulation of inositol phosphate accumulation. Both ET-1- and S6b-induced accumulation of inositol phosphate was almost totally (90%) inhibited by 100 mumol/l BQ 123, while the S6c response was not affected by the ETA receptor antagonist. In contrast, the ETB receptor antagonist IRL 1038 inhibited S6c-induced inositol phosphate accumulation by more than 80%, whereas inhibition was only about 30% for ET-1 and S6b stimulations. This indicates that both ETA and ETB receptors were coupled to the phospholipase C transducing system in the muscular layer of placental stem villi vessels, and there is evidence that the phosphoinositide hydrolysis response is obtained predominantly via ETA receptor activation.

Amino Acid Sequence↗

PubMed2006

Phosphoinositide-derived messengers in endocrine signaling.

One of the fundamental questions in endocrinology is how circulating or locally produced hormones affect target cell functions by activating specific receptors linked to numerous signal-transduction pathways. An important subset of G protein-coupled cell-surface receptors can activate phospholipase C enzymes to hydrolyze a small but critically important class of phospholipids, the phosphoinositides. Although this signaling pathway has been extensively explored over the last 20 years, this has proven to be only the tip of the iceberg, and the multiplicity and diversity of the cellular functions controlled by phosphoinositides have surpassed any imagination. Phosphoinositides have been found to be key regulators of ion channels and transporters, and controllers of vesicular trafficking and the transport of lipids between intracellular membranes. Essentially, they organize the recruitment and regulation of signaling protein complexes in specific membrane compartments. While many of these processes have been classically studied by cell biologists, molecular endocrinology cannot ignore these recent advances, and now needs to integrate the cell biologist's views in the modern concept of how hormones affect cell functions and how derailment of simple molecular events can lead to complex endocrine and metabolic disorders.

Calcium↗

PubMed2006

Phosphoinositides in FCgamma receptor signaling.

Fcgamma receptors mediate a variety of immune responses to IgG-containing complexes. Virtually all of these responses appear to be regulated by phosphoinositides, in particular phosphatidylinositol (4,5)P2 and phosphatidylinositol (3,4,5)P3. Possible downstream effectors of phosphoinositides involved in multiple FcgammaR-mediated events are discussed, as are elements of the signaling pathways that may couple Fcgamma receptors to the enzymes of phosphoinositide metabolism.

Animals↗

PubMed1992

Phorbol esters inhibit ionomycin-induced hydrolysis of phosphoinositides and phosphatidylcholine in bovine corneal epithelial cells.

The effects of phorbol esters on phospholipase C (PLC) activity towards phosphoinositides and phosphatidylcholine (PC) in bovine corneal epithelial cells were examined. The cells were labeled with 32Pi, myo[3H]inositol or methyl[14C]choline, and PLC stimulated by incubation of the cells with Ca2+ ionophore, ionomycin. The PLC activity was assessed by monitoring the loss of radioactivity from the labeled phospholipids or the accumulation of their radioactive metabolites. The data from this study can be summarized as follows: Addition of 20 microM ionomycin to the prelabeled cells resulted in a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and somewhat slower hydrolysis of phosphatidylinositol (PI) and phosphatidylcholine (PC) with concomitant several-fold increase in phosphatidic acid (PA). The effects of the ionophore were time- and dose-dependent. Incubation of the cells with phorbol 12,13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) caused increased radioactivity in PC and PA, whereas the radioactivity in PI and PIP2 remained unchanged. The effects of PDBu were inhibited by staurosporine and H-7, and inactive derivatives of phorbol esters failed to exert any effect on phospholipid metabolism. Pretreatment of the corneal epithelial cells with PDBu or PMA abolished the ionomycin-induced hydrolysis of phosphoinositides and PC. The data suggest that activation of protein kinase C by phorbol esters in corneal epithelial cells results in inhibition of PLC activity towards phosphoinositides and PC through a mechanism probably involving phosphorylation of the enzyme.

Animals↗

PubMed1994

Dibutyltin dilaurate induced thymic atrophy and modulation of phosphoinositide pathway of cell signalling in thymocytes of rats.

A marked dose dependent reduction in thymus weight and its nucleated cell counts with histological alterations was observed in rats exposed to oral dibutyltin dilaurate (DBTL) for 2 weeks at 2, 4, 8 or 16 mg/kg body weight. The incorporation of [3H]-inositol into all the three major phosphoinositides was drastically reduced in thymocytes in a dose dependent manner. Furthermore, the basal and the mitogen (Con A) stimulated [3H]-inositol phosphates generation was diminished significantly in 8 mg DBTL group. However, in vitro incubation of DBTL with thymocytes failed to evoke any change in phosphoinositide hydrolysis. Similarly, a time and dose dependent inhibition in phosphoinositide synthesis with as high as 80% by 10 microM DBTL was exhibited under in vitro conditions. A 130% and 600% enhancement of protein kinase C (PKC) activity in thymocytes was seen in 4 mg and 8 mg DBTL group, respectively. Addition of DBTL to the cell free assay system of thymocytes resulted in a concentration dependent activation of the enzyme activity. A dose dependent increase in intracellular calcium was also evident when DBTL was added to thymocytes under in vitro conditions. These results are of significance and may bear close relationship to the observed thymic atrophy by DBTL.

Animals↗

PubMedDate not supplied

Phosphoinositide metabolism and shape control in sheep red blood cells.

Metabolic depletion of sheep red blood cells leads to decreased intracellular concentrations of ATP and reduced glutathione as well as degradation of phosphoinositides. In sheep red blood cells, depletion of ATP induced two types of shape transformation: one early phase involving formation of protrusions on the cell surface similar to those observed upon depletion of human red blood cells; and one late phase, in which the sheep red blood cells develop long, rod-shaped projections. During the initial stages of shape changes, degradation of the phosphoinositides parallels the discocyte-echinocyte transformation, thus giving further support to a shape-controlling mechanism based on the bilayer-couple hypothesis. However, formation of the long projections does not coincide with turnover of the phosphoinositides but rather with the level of reduced glutathione. This indicates that development of these rod-like extensions on the cell surface is induced by oxidative processes that may well involve cross-linking of membrane skeleton proteins.

Adenosine Triphosphate↗

PubMed1984

The application of HPLC methodology for the analysis of receptor mediated changes in phosphoinositide metabolism.

HPLC methodology has found wide application in analytical problems in biochemistry. To study the metabolism of phosphatidylinositol and its regulation by receptor mediated events, HPLC could be a valuable technique. It has been recently demonstrated that a variety of hormones and neurotransmittors act to stimulate hydrolysis of phosphoinositides by a phospholipase C. To monitor this reaction, we have analysed the formation of radiolabelled inositol phosphates from phosphoinositides. The present paper describes a rapid HPLC procedure, to separate inositol phosphates from myo-inositol, which could be used in pharmacological studies of receptors linked to phosphoinositide hydrolysis. The potential of the application of HPLC to the analysis of the phospholipids involved is discussed.

Animals↗

PubMed2004

Point mutations in the split PLC-gamma1 PH domain modulate phosphoinositide binding.

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal (PH(1)) and a split PH domain (nPH(2) and cPH(2)). We previously reported that the split PH domain of PLC-gamma1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P(2), we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-gamma1 nPH(2) domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-gamma1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-gamma1 molecules showed reduced PI(4,5)P(2) hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH(1) and nPH(2) domains are responsible for membrane-targeted translocation of PLC-gamma1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro(500) and His(503) are critical for binding of PLC-gamma1 to one of its substrates, PI(4,5)P(2) in the membrane.

Amino Acid Sequence↗

PubMed1992

Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.

The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).

Adenylyl Cyclases↗

PubMed1992

A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix.

Several enzymes involved in the phosphoinositide metabolism have been shown to be present in nuclei of rat liver and Friend cells. In this paper we demonstrate that nuclear matrices of mouse NIH 3T3-fibroblasts and rat liver cells, isolated by nuclease treatment and high salt extraction, contain phosphatidylinositol 4-kinase (PdtIns 4-kinase), phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), diacylglycerol kinase, and phospholipase C. By a selective extraction the nucleus can be dissected in the peripheral matrix (lamina-pore complex) and the internal matrix as shown by using marker antibodies. Surprisingly, PtdIns 4-kinase was found exclusively in the peripheral nuclear matrix, whereas PtdIns(4)P 5-kinase was found to be associated to internal matrix structures. Diacylglycerol kinase and phospholipase C activities were also preferentially detected in the internal matrix. These data demonstrate a differential localization of the phosphoinositide kinases in the nucleus and suggest that the phosphoinositide metabolism may play a specific role in the nucleus.

1-Phosphatidylinositol 4-Kinase↗

PubMed1992

EBV increases phosphoinositide kinase activities in human B cells.

To determine whether EBV affects phosphoinositide kinase activities of human B cells, we compared the activities between EBV- and EBV+ human B cell lymphoma lines. The two types of human B cells contained both phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4-phosphate (PtdIns(4)P) kinase activities irrespective of the presence of EBV. However, both activities were increased in EBV+ cells compared to EBV- cells. The increases were associated with neither altered Km values for substrates nor altered elution profiles in DEAE-cellulose chromatography. Furthermore, expression of a latent EBV protein, EBV nuclear Ag1 (EBNA1) in BHK cells by the transfection of EBNA1 DNA was accompanied by increased PtdIns 4-kinase and PtdIns(4)P kinase activities. These increases also were not associated with altered Km values for substrates. However, phospholipase C activity was altered in neither EBV+ cells nor in EBNA1-expressing cells. These results indicate that EBV selectively increases the two phosphoinositide kinase activities in human B cells, although the viral gene product has no intrinsic phosphoinositide kinase activity. PtdIns 4-kinase and PtdIns(4)P kinase cooperatively synthesize PtdIns 4,5-bisphosphate, the major source of 1,2-diacylglycerol and inositol 1,4,5-triphosphate, the two second messengers in transducing signals for cell activation. Such increase therefore may play a role in EBV-induced human B cell activation.

1-Phosphatidylinositol 4-Kinase↗

PubMed1992

Protein degradation by the phosphoinositide-specific phospholipase C-alpha family from rat liver endoplasmic reticulum.

A 60-kDa protein homologous to phosphoinositide-specific phospholipase C-alpha was purified to apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW. The purified protein was monomeric, with an M(r) of 60,000. Eight types of protein were further separated from the 60-kDa protein and named ER60A-ER60H according to the order of their elution from a TSK gel DEAE-5PW column. They were essentially identical in terms of immunochemical properties and the NH2-terminal amino acid sequence. The partial amino acid sequence of ER60F showed homology to that of phosphoinositide-specific phospholipase C-alpha. ER60A-ER60H showed no phosphoinositide-specific phospholipase C activity. However, ER60A-ER60H catalyzed cleavage of themselves and the endoplasmic reticulum proteins protein disulfide-isomerase and calreticulin. Proteolytic degradation was inhibited by p-chloromercuribenzoate. These results indicate that ER60A-ER60H comprise a group of endoplasmic reticulum resident proteins and show thiol group-related proteolytic activity.

Amino Acid Sequence↗

PubMedDate not supplied

[Dynamic features of rapid changes of blood phosphoinositides in patients with chronic alcoholism].

The time-course of rapid changes in blood phosphoinositide content was studied in 48 patients with Stage II chronic alcoholism of and in healthy volunteers. Content of phosphoinositides was estimated within short intervals 30, 60, 120 and 180 sec after the samples were obtained. In the blood of the patients with alcoholism as compared with healthy volunteers the time-course of rapid changes in phosphoinositide content exhibited a higher amplitude and failed to return to the initial level within 3 min. Alterations in content of the phospholipid correlated with duration of alcohol abuse. The data obtained suggest that homeostasis of main biochemical regulators is impaired after alcohol consumption.

Alcoholism↗

PubMed1992

Organophosphate-induced alterations in muscarinic receptor binding and phosphoinositide hydrolysis in the human SK-N-SH cell line.

Low level, chronic exposure (0.1 nM, 24 hrs) to the organophosphate paraoxon significantly inhibits N-[3H]methylscopolamine ([3H]NMS) binding to muscarinic receptors in a noncompetitive manner in the SK-N-SH neuroblastoma cell line. The effect of paraoxon on muscarinic receptor-mediated phosphoinositide hydrolysis was also investigated. At 0.1 nM, paraoxon caused a time-dependent increase in the accumulation of inositol phosphates, while the classical muscarinic receptor agonist carbachol was virtually ineffective at low concentrations. In contrast, carbachol-induced increases in phosphoinositide hydrolysis at higher concentrations (1 mM) were markedly larger (five-fold) than the increases induced by PX. Approximately 50% of the maximal increase in the accumulation of inositol phosphates due to paraoxon treatment was antagonized by saturating concentrations of muscarinic receptor antagonists, while the response to carbachol was completely antagonized. In addition, chronic treatment (24 hrs) of SK-N-SH cells with pertussis toxin blocked 75% of the stimulatory effects of carbachol, but inhibited only 38% of the paraoxon-induced response. Neomycin, a phospholipase C inhibitor, completely blocked both the paraoxon and carbachol-induced stimulation of phosphoinositide hydrolysis. The results suggest that paraoxon can modulate signal transduction by indirect activation of muscarinic receptors as well as by acting at a distal site along the pathway.

Acetylcholinesterase↗

PubMed1992

Chagasic IgG stimulates phosphoinositide hydrolysis via neurotransmitter receptor activation: role of calcium.

Induction of polyphosphoinositide hydrolysis in cardiac tissue by IgG from chagasic mice was assayed. BALB/c mice auricles were labelled with myo-[3H]inositol precursor and inositol phosphate production in the presence or absence of chagasic IgG and the corresponding F(ab')2 was measured. Both chagasic IgG and F(ab')2 but not the normal forms specifically increased phosphoinositide turnover. This increment was blocked by muscarinic cholinergic antagonists and to an even greater extent by the phospholipase C inhibitor NCDC. Moreover, calcium channel blocking agents such as diltiazem, verapamil and D-600 also exerted an inhibitory action. A muscarinic cholinergic agonist, carbachol, and the ionophore A-23187, mimicked the action of the chagasic IgG upon phosphoinositide turnover. It is concluded that murine chagasic IgG and its F(ab')2 fragments result in stimulation of phospholipase C-mediated phosphoinositide hydrolysis through the interaction with muscarinic cholinergic receptors requiring the cytosolic calcium concentration to be raised.

Animals↗

PubMed1992

Phosphoinositide fatty acid composition of peripheral blood lymphocytes from aging humans.

The interaction of the T-cell receptor complex with the ligands is associated with early molecular events involved in the process of signal transduction implicating phosphoinositide breakdown. In elderly people, abnormalities in membrane signal transduction pathways are the basis of the immune deficiency associated with aging. Peripheral blood lymphocytes from aging humans and young subjects were stimulated with anti-CD3 monoclonal antibody and the phosphoinositide fractions were analyzed in order to determine the fatty acid composition in resting and stimulated conditions. In aging humans, in resting conditions, the all three phosphoinositide fractions appeared more saturated than the corresponding fractions in young subjects. Following anti-CD3 stimulation a decrease in arachidonic acid relative molar content was detected in both young and old subjects, but the arachidonic acid content in resting conditions greatly differed between the two groups, suggesting a different modulation of the microenvironment of the T-cell receptor complex in elderly people, so determining alterations in the early activation steps of lymphocytes.

Adult↗

PubMed1992

The phosphoinositide pathway of lymphoid cells: labeling after permeabilization by alveolysin, a bacterial sulfhydryl-activated cytolysin.

An improved method allowing incorporation of [3H]myo-inositol into the phosphoinositide pool of human lymphoid cells is described. The procedure devised involves cell permeabilization with a thiol-activated membranolytic toxin, alveolysin, and optimization of the phosphoinositide labeling and extraction. In these conditions 4 to 10% of the added [3H]myo-inositol is found intracellularly and half of this amount (2-5%) is incorporated into the phosphoinositide pool in only 1 h as compared to the classical 0.2 to 0.3% incorporation obtained after 10 to 20 h. The integrity of coupling between receptors and phospholipase C was assessed by the inositol phosphate production after cell stimulation by various agonists.

Burkitt Lymphoma↗