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PubMed1991

The role of protein kinase C activation and inositol phosphate production in the regulation of cell-surface expression of Mac-1 by complement fragment C5a.

The adhesion of leukocytes to endothelial and other cell types is an essential part of the acute inflammatory response. One means by which adherence can be increased is by activation of the CD11/CD18 family of leukocyte glycoproteins. Chemotactic peptides, lipid mediators, phorbol esters and tumour necrosis factor are all able to increase the cell surface expression of one member of this family, CD11b/CD18 or Mac-1, by an unknown signal transduction mechanism. In this report, regulation of Mac-1 expression by C5a is shown to be independent of protein kinase C (PK-C) activation. The inhibitor of PK-C, H-7, has no effect on the action of C5a and only a slight effect on phorbol ester-induced up- and down-regulation of Mac-1, at a concentration that inhibits superoxide production in response to both factors by 40%. Inositol phospholipid hydrolysis, an important pathway leading to PK-C activation, and the transient increases in cytosolic Ca2+ associated with inositol phosphate production, are also shown to be not essential processes in C5a-stimulation of Mac-1 expression.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2003

Ca2+ channel blockers enhance neurotensin (NT) binding and inhibit NT-induced inositol phosphate formation in prostate cancer PC3 cells.

Neurotensin (NT) stimulates Ca2+ release and Ca2+ influx in many cells. Its contractile effects in smooth muscle are inhibited by removal of Ca2+ and by Ca2+ channel blockers (CCBs). To better understand NT signaling in prostate cancer PC3 cells, blockers of voltage-gated and store-operated Ca2+ channels (VGCC and SOCC) were tested for effects on NT-binding and signaling. Eight chemical types of agents, including VGCC-blocker nifedipine and SOCC-blocker SKF-96365 (1-[beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenyl]-1H-imidazole), enhanced cellular NT binding up to 3-fold, while inhibiting (by congruent with 70%) NT-induced inositol phosphate (IP) formation. The ability to enhance NT binding correlated with the ability to inhibit NT-induced IP formation, and both effects were relatively specific for NT. Although cellular binding for beta2-adrenergic, V1a-vasopressin, and epidermal growth factor receptors was not enhanced by these drugs, bombesin receptor binding was increased approximately equal to 19% and bombesin-induced IP formation was inhibited approximately equal to 15%. One difference was that the effect on NT binding was Ca2+-independent, whereas the effect on IP formation was Ca2+-dependent (in part). The Ca2+-dependent part of the IP response seemed to involve SOCC-mediated Ca2+ influx to activate phospholipase C (PLC)delta, while the Ca2+-independent part probably involved PLCbeta. Photoaffinity labeling of the NT receptor NTR1 was enhanced in CCB-treated cells. NTR1 affinity was increased but NTR1 number and internalization were unchanged. Since CCBs did not alter NT binding to isolated cell membranes, the effects in live cells were indirect. These results suggest that CCBs exert two effects: 1) they inhibit NT-induced IP formation, perhaps by preventing Ca2+ influx-dependent activation of PLCdelta; and 2) they enhance NTR1 affinity by an unexplained Ca2+-independent mechanism.

Binding Sites↗
PubMed1985

Accumulation of inositol phosphates and cyclic AMP in guinea-pig cerebral cortical preparations. Effects of norepinephrine, histamine, carbamylcholine and 2-chloroadenosine.

Norepinephrine and serotonin augment by about 2-fold the accumulation of cyclic [3H]AMP elicited by 2-chloroadenosine in [3H]adenine-labeled guinea-pig cerebral cortical slices. Histamine causes a 3-fold augmentation. The first two agents have no effect on cyclic AMP alone, while histamine has only a small effect alone. The augmentation of the 2-chloroadenosine response appears to be mediated by alpha 1-adrenergic, 5HT2-serotonergic and H2-histaminergic receptors. VIP-elicited accumulations of cyclic AMP are also augmented through stimulation of alpha 1-adrenergic, 5HT2-serotonergic and H1-histaminergic receptors. Activation of these amine receptors also increases the turnover of phosphatidylinositols in [3H]inositol-labeled guinea pig cerebral cortical slices. Norepinephrine causes a 5-fold, serotonin a 1.2-fold, and histamine a 2.5-fold increase in accumulations of [3H]inositol phosphates. 2-Chloroadenosine, vasoactive intestinal peptide, baclofen, and somatostatin have no effect on phosphatidylinositol turnover, nor do the last two agents augment accumulations of cyclic AMP elicited by 2-chloroadenosine. The data suggest a possible relationship between turnover of phosphatidylinositol and the augmentations of the cyclic AMP accumulations elicited by biogenic amines in brain slices.

2-Chloroadenosine↗
PubMed1992

The possible involvement of protein kinase C(s) and inositol phosphate metabolism in the basal but not in the prolactin stimulated casein release by the lactating rabbit mammary epithelial cell.

The secretagogue effect of prolactin (PRL) on casein release by epithelial mammary cells has been previously related to stimulation of the phospholipase A2-arachidonic acid cascade. In order to determine whether other intracellular pathways are implicated in this secretagogue effect, different agents acting on protein kinase C (PKC) and phospholipase C (PLC) activity have been assessed in vitro in lactating rabbit mammary gland fragments. Phorbol ester (20 nm TPA and 1-oleoyl-2-acetyl-sn-glycerol (10 microM (OAG) stimulated newly synthesized casein secretion and potentiated the PRL secretatogue effect. However, 100 microM quercetin, 100 microM H-7 and 5 and 20 nM staurosporine did not inhibit the latter effect. Exogenous PLC did not stimulate casein secretion. PRL did not affect production of inositol phosphates (IPs) during 10 or 60 min exposure. These results show that PKC activation may increase basal levels of casein secretion, and demonstrate that PRL does not act primarily via PKC activation or by PLC activation to stimulate casein secretion.

Alkaloids↗
PubMed1989

Angiotensin II stimulates both inositol phosphate production and human placental lactogen release from human trophoblastic cells.

We studied the functional significance of the binding of angiotensin-II (AII) to human placentas. Human trophoblastic cell suspensions were prepared by trypsin digestion of minced tissue. Cell incubations with increasing doses of [125I](SAR1)AII, ranging from 0.01-2.5 nmol/L, were carried out for 20 min at 37 C. The results indicated the presence of specific low capacity [4300 +/- 1300 (+/- SE) sites/cell], high affinity (Kd = 0.38 +/- 0.06 nmol/L) binding sites for [125I](Sar1)AII. This binding was specific for AII analogs. When placental cells were preloaded with 40 microCi/mL [3H]myoinositol for 2 h at 37 C, AII stimulation resulted in a dose-dependent increase in inositol phosphate (InsP) production [EC50 = 1.4 +/- 0.4 (+/- SE) nmol/L], as measured by ion exchange chromatography. (Sar1)AII also stimulated InsP production, with an EC50 of 0.3 +/- 0.2 nmol/L. AII-stimulated production of InsP was completely blocked by the antagonist (Sar1,Ala8)AII. AII also stimulated human placental lactogen release from trophoblastic cells in a dose-dependent fashion. The EC50 was 18 +/- 9 pmol/L, and the stimulation was blocked by (Sar1,Ala8)AII, as found for AII-stimulated InsP production. These results suggest that stimulation of human placental lactogen release by AII may be mediated by activation of phospholipase-C, which, in turn, produces phosphoinositide breakdown. The results, therefore, provide evidence of a physiological role for the renin-angiotensin system within the human placenta.

Angiotensin II↗
PubMed1987

Transmembrane signalling via the T11-dependent pathway of human T cell activation. Evidence for the involvement of 1,2-diacylglycerol and inositol phosphates.

It has previously been shown that some anti-T11 monoclonal antibodies, when used in combination, can activate the human T cell line Jurkat to produce interleukin 2. In this study, we investigate the mechanism by which perturbation of different epitopes of T11 molecules induces activation in Jurkat cells. We show that this activation is initiated by a T11-mediated increase in the concentration of free cytoplasmic calcium ions ([Ca2+]i). The initial increment in [Ca2+]i can occur when extracellular Ca2+ is depleted by EGTA, indicating that Ca2+ from intracellular stores is mobilized. As an early response to extracellular signals provokes a rapid breakdown of a class of lipid known collectively as the phosphoinositides, we measured the levels of phosphatidylinositol bisphosphate (PIP2) which is hydrolyzed to generate inositol triphosphates (IP3), the putative mobilizer of Ca2+ from internal stores and 1,2-diacylglycerol (DAG), the physiological activator of protein kinase C. Monoclonal antibodies directed either against different epitopes of T11 molecules or the T3-Ti antigen receptor complex provoke a rapid breakdown of PIP2, the parental product from which IP3 and DAG derive. In addition antibodies to either the T11 molecules or T3-Ti antigen receptor complex induce marked elevations in IP3, other inositol phosphate compounds and DAG. Taken together, these data indicate that, during T cell activation, due to the perturbation of T11 molecules or T3-Ti antigen receptor complex, membrane phosphoinositides are specifically hydrolyzed. This hydrolysis of phosphoinositides generates two putative second messengers such as IP3 and DAG, which mobilizes Ca2+ from intracellular stores and stimulates protein phosphorylation, respectively.

Antibodies, Monoclonal↗
PubMed1987

Effects of PDGF on inositol phosphates, Ca2+, and contraction of mesangial cells.

Platelet-derived growth factor (PDGF) is a potent mitogen and vasoactive polypeptide for aortic smooth muscle. Because contractile glomerular mesangial cells synthesize a PDGF-like molecule and may respond to PDGF released by infiltrating cells at the site of glomerular inflammation, we studied the effects of exogenous, highly purified PDGF on 1) contraction of cultured rat mesangial cells and 2) membrane phosphoinositide turnover and cytosolic free calcium ([Ca2+]i), as putative mechanisms of membrane signal transduction. PDGF, 10(-11) and 10(-10) M, contracted 56.1 +/- 5.2 and 72.9 +/- 6.4% of the cells, respectively, with an average decrease of cross-sectional area of 22.0 +/- 2.6 and 28.1 +/- 2.7% of basal, as assessed by image-analysis microscopy. PDGF also rapidly increased total water-soluble inositol phosphates, measured after anion-exchange chromatography on perchloric acid-extracted cells, and simultaneously raised [Ca2+]i, measured by the fluorescent intracellular probe fura-2, from basal levels of 83.1 +/- 6.8 to a peak of 229.4 +/- 20.0 nM. We conclude that PDGF stimulates contraction of rat mesangial cells via a phospholipase C-dependent pathway, with potential relevance to the control of glomerular hemodynamics and mesangial proliferation in immune-mediated glomerular disease.

Angiotensin II↗
PubMedDate not supplied

A novel calcium sensor stimulating inositol phosphate formation and [Ca2+]i signaling expressed by GCT23 osteoclast-like cells.

Osteoclast activity is inhibited by elevated [Ca2+]o; however, the underlying molecular mechanism is unknown. We used the human osteoclast-like cells GCT23 to elucidate their cation-sensing properties. Cells responded to elevated [Ca2+]o with rapid concentration-dependent [Ca2+]i transients (EC50 = 7.8 mm, time to peak 44 +/- 4 sec) that were due to release from intracellular stores, followed by Ca2+ influx across the plasma membrane. Ca2+ store depletion by thapsigargin, endothelin-1, or bradykinin activated calcium entry pathways. Cells responded similarly to Ni2+ and Cd2+ with albeit slower kinetics (EC50 <10 microm and <100 microm, times to peak 140 +/- 25 sec and 150 +/- 24 sec, respectively). The three cations stimulated inositol phosphate production (two-fold, p <.02) similar to bradykinin (2.5-fold, p <. 002), which activates a phospholipase C (PLC)-coupled receptor in GCT23 cells. The cells did not respond to 0.1-1 mM Gd3+ or neomycin B, indicating that the parathyroid calcium receptor (PCaR) is not functionally expressed. In confirmation, PCaR could not be detected by reverse transcriptase polymerase chain reaction in GCT23 cells and in mouse osteoclasts, and the calcimimetic compound NPS R-568 failed to produce the left shift of the concentration-response curve characteristic for PCaR. Our data demonstrate for the first time that cation sensing by osteoclast-like GCT23 cells is mediated by a PLC-coupled receptor that is not identical to PCaR.

Animals↗
PubMed1999

Muscarinic cholinoceptor subtypes mediating tracheal smooth muscle contraction and inositol phosphate generation in guinea pig and rat.

The effects of the muscarinic cholinoceptor antagonists atropine (non-selective), pirenzepine (M1-selective), methoctramine (M2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; M3-selective) were examined on the responsiveness of guinea pig and rat tracheal tissue to acetylcholine and carbachol. Results indicate that smooth muscle contraction in isolated tracheal tissue from both species was mediated primarily by muscarinic M3 cholinoceptors. The effects of atropine, pirenzepine and 4-DAMP were similar against the contractile actions of acetylcholine and carbachol in both species and in epithelium-intact and epithelium-denuded tissue. In contrast, differences in the effects of methoctramine in antagonising contractile responses to acetylcholine and carbachol were observed between the two species and following epithelium removal in the guinea pig. Thus, whilst this study has found that tracheal smooth muscle contraction in the guinea pig and rat is mediated primarily by muscarinic M3 cholinoceptors, anomalies in the functional inositol phosphate generation results obtained with the muscarinic cholinoceptor antagonists highlight species differences in the actions of acetylcholine and carbachol in eliciting smooth muscle contraction suggesting the possible existence of functional non-M3 muscarinic cholinoceptors.

Animals↗
PubMed1990

Cell surface markers, inositol phosphate levels and membrane potential of lymphocytes from young and old human patients.

It is well known that most physiological functions change with aging, including the immune response. Data concerning the aging of lymphocyte subpopulations are conflicting. The antigen density of peripheral blood lymphocytes has been determined by fluorescently tagged OKT-3, OKT-4, OKT-8, OKT-11 and OKM1 monoclonal antibodies in a carefully selected aged (over 87 years) population, and compared to that of young subjects. A substantial difference was found in the percentage distribution of OKT8 and OKM1 subsets. The volume of lymphocytes of the elderly population was significantly less than that of the young. The effect of various monoclonal antibodies on phosphatidylinositol breakdown has also been studied. It was found that only OKT3, acting through the CD3 antigen receptor, was able to induce inositol phosphate formation in both young and elderly, although in the latter population this occurred at a lower level. Because the plasma membrane plays a regulatory role in this process, an important and sensitive functional parameter, the membrane potential, was also monitored and influenced by changing the extracellular K+ concentration. The lymphocytes of the elderly population responded less sensitively to changes in extracellular potassium concentration.

Adult↗
PubMed1993

Roles of cyclic AMP and inositol phosphates in the luteolytic action of cloprostenol, a prostaglandin F2 alpha analogue, in marmoset monkeys (Callithrix jacchus).

The luteolytic response to a prostaglandin F2 alpha analogue, cloprostenol, was investigated in vivo and in vitro at defined stages of the luteal phase. In vivo administration of cloprostenol to female marmoset monkeys on day 3 after ovulation had no effect on plasma progesterone concentrations, whereas administration on day 14 after ovulation reduced plasma progesterone to preovulatory concentrations within 4 h. To identify the cellular basis for this luteolytic action, marmoset luteal tissue obtained on days 3, 6 and 14 after ovulation was incubated in vitro and progesterone production, cAMP accumulation and phosphoinositide (PI) turnover measured in response to cloprostenol, human chorionic gonadotrophin (hCG) with or without cloprostenol, or dibutyryl-cAMP with or without cloprostenol. Progesterone production was stimulated by both hCG and dbcAMP at all stages of the luteal phase. Although neither hCG nor dbcAMP had any significant effects on PI turnover, hCG also increased cAMP accumulation. In marmoset luteal tissue obtained on day 3 after ovulation, cloprostenol had no significant effect on basal or hCG/dbcAMP-stimulated progesterone production but significantly stimulated PI turnover. In contrast, on days 6 and 14 after ovulation, cloprostenol significantly inhibited hCG- and dbcAMP-stimulated progesterone production and the cAMP response to hCG, but had no significant effect on PI turnover. Since progesterone production by the marmoset corpus luteum depends on the luteotrophic support of luteinizing hormone (LH), these observations suggest that the luteolytic action of cloprostenol in vivo involves the inhibition of LH/hCG action at sites both prior and subsequent to cAMP accumulation. However, such luteolytic effects do not appear to require the generation of inositol phosphates by increased PI turnover.

Animals↗
PubMed1990

Intact antigen receptor-mediated generation of inositol phosphates and increased intracellular calcium in CD4 CD8 T lymphocytes from MRL lpr mice.

The predominant T lymphocytes that accumulate in the peripheral lymphoid tissues of mice homozygous for the lpr gene bear the phenotype CD3+CD4-CD8-. By certain functional criteria these cells would appear to have impaired CD3-mediated signal transduction, in that they do not respond to alloantigen and produce little if any detectable IL-2 or other lymphokines. However, the signal pathway appears adequate for achieving other T cell functions, including induction of high affinity IL-2R, and thymic deletion. To clarify the basis of this seeming discrepancy, we examined transmembrane signal transduction in T cell subsets of lpr/lpr (lpr) and +/+ mice, as defined by increased [Ca2+]i and the generation of inositol phosphates (InsPs). Stimulation of lpr CD4-CD8- cells with anti-CD3 antibody produced prompt and sustained increases in the concentration of [C2+]i and in InsPs. Similar responses occurred in mature T cells from lpr and +/+ mice, except for the somewhat slower kinetics of their increased [Ca2+]i. In marked distinction to the anti-CD2-mediated response, Con A, even in high doses, could not stimulate any increase of [Ca2+]i in lpr CD4-CD8- cells, and only modest increases in InsPs. Mature T cells, whether of lpr or +/+ origin, yielded normal increased [Ca2+]i with Con A. The reason for the differences in signal transduction between anti-CD3 and Con A stimulation of lpr CD4-CD8- cells may relate to the absence of surface structures on these immature T cells that are required for activation by Con A but not by anti-CD3. The data demonstrate that the CD3 complex in lpr CD4-CD8- T cells can couple to phospholipase C to hydrolyze phosphoinositides. These activation properties of lpr CD4-CD8- T cells have interesting functional parallels to thymocytes at the time of thymic selection, as well as tolerance induction of mature T lymphocytes.

Animals↗
PubMed2003

Thiomersal enhances the binding of histamine to the H1 receptor, but not histamine-stimulated inositol phosphate formation.

Thiomersal (thimerosal) was a weak inhibitor of the binding of [(3)H]mepyramine to histamine H(1) receptors in guinea-pig cerebellar membranes (11 +/- 1% inhibition at 10 microM, 32 +/- 3% inhibition at 300 microM). However, in the concentration range 3-30 microM, thiomersal enhanced the binding of histamine to the H(1) receptor, as reflected by the displacement of curves of histamine inhibition of [(3)H]mepyramine binding to lower concentrations, without any change in the Hill coefficient. The ratio of the IC50 values (the concentration giving 50% inhibition) in the absence and presence of thiomersal increased from 1.8 with 3 microM to 3.6 with 30 microM thiomersal. There was no consistent effect of thiomersal at concentrations of 30 microM and below on curves of mepyramine inhibition of [(3)H]mepyramine binding. In the presence of 10 microM thiomersal histamine-induced accumulation of inositol phosphates in U373 MG astrocytoma cells was partially inhibited (37 +/- 8% inhibition of the maximum response), without any significant change in the EC50 (the concentration giving the half maximal response) for histamine. Thus although histamine binding was potentiated by thiomersal, there was no potentiation of an H(1) receptor-mediated functional response.

Animals↗
PubMed2000

Single amino acid of g(alpha16) (Ala(228)) is responsible for the ability of chemoattractant C5a receptor to induce G(alpha16)-mediated inositol phosphate release.

Our previous study suggested that the region encompassing residues 220-240 on G(alpha16) is important in coupling with C5a receptor (Lee et al. (1995) Mol. Pharmacol. 47, 218-223). When aligned sequences are compared in the residue 220-240 segment of G(alpha16), there is a block of eight amino acids extending from residue 227 to residue 234 (227-Ile-Ala-Leu-Ile-Tyr-Leu-Ala-Ser-234) in G(alpha16) that is replaced by a heterologous block extending from amino acid residue 224 to residue 231 (224-Thr-Ser-Ile-Met-Phe-Leu-Val-Ala-231) in G(alpha11). In order to identify the specific amino acid residue necessary for coupling to C5a receptor within the extension of eight amino acids in G(alpha16), a series of chimeric G(alpha11)/G(alpha16) cDNA constructs and mutant G(alpha16) cDNAs were expressed. Then the ability of chimeras and mutant proteins to mediate C5a-induced release of inositol phosphate in transfected Cos-7 cells was tested. The results show that single amino acid Ala(228) is responsible for conferring about 40-50% of the activity of G(alpha16) induced by C5a receptor stimulation.

Alanine↗
PubMed1988

Effect of exogenous ATP upon inositol phosphate production, cationic fluxes and insulin release in pancreatic islet cells.

Endogenous ATP is thought to play a key regulatory role in nutrient-stimulated insulin release. The present study deals with the effect of exogenous ATP and its stable analog alpha, beta-methylene ATP upon pancreatic islet function. Both alpha, beta-methylene ATP (5.0 microM to 0.2 mM) and ATP (0.3-3.0 mM) caused a rapid and concentration-related increase in insulin output by rat islets incubated or perfused at an intermediate concentration of D-glucose (8.3 mM). The effect of the ATP analog faded out at both lower and higher D-glucose concentrations. In the presence of 8.3 mM D-glucose, ATP also increased both 86Rb and 45Ca outflow from prelabelled islets. The cationic response to ATP persisted in the absence of extracellular Ca2+ and, hence, was reminiscent of that evoked by cholinergic agents. Like carbamylcholine, ATP caused a dose-related increase in the production of [3H]inositol phosphates from prelabelled islets or tumoral islet cells (RINm5F line). The latter effect was duplicated by alpha, beta-methylene ATP and unaffected by atropine. It is speculated that ATP, liberated together with insulin at the exocytotic site, might participate in a positive feedback control of insulin release.

Adenosine Triphosphate↗
PubMed1989

Muscarinic cholinergic induced secretin subsensitivity in rat isolated pancreatic acini. Effects on amylase release, cyclic adenosine monophosphate and inositol phosphate formation.

In this study, dispersed rat pancreatic acini exhibited secretin subsensitivity in their capacity to release amylase after preexposure to increasing concentrations of the muscarinic cholinergic agonist carbamylcholine. The present study also explores the potential mechanisms involved in this cellular desensitization phenomenon. Secretin subsensitivity of pancreatic acini pre-exposed to 10(-4) M carbamylcholine was observed only at secretin concentrations above 10(-8) M. The desensitized cells had not recovered 3 h after the cholinergic agonist exposure. In these acini, the adenylate cyclase pathway remained unaltered because cholera toxin, forskolin, and 8-Br-cAMP still induced weak, but normal, amylase release when compared with control acini. In vivo administration of pertussis toxin failed to protect the dispersed pancreatic acini against carbamylcholine-induced secretin subsensitivity. Moreover, cAMP production by these acini in response to secretin, cholera toxin, and forskolin was similar to that observed in control acini. Secretin stimulation of inositol phosphate (InsP1, InsP2, InsP3) production after carbamylcholine pre-exposure remained equivalent to that observed in acini that had never been exposed to the cholinergic agonist. Thus, after muscarinic cholinergic agonist exposure, pancreatic acini showed secretin subsensitivity in their capacity to release enzyme. This phenomenon appears to result from modifications at post-second messenger loci.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Overexpression of G11alpha and isoforms of phospholipase C in islet beta-cells reveals a lack of correlation between inositol phosphate accumulation and insulin secretion.

It has been suggested that insulin secretion from pancreatic islets may be mediated in part by activation of phospholipases C (PLCs) and phosphoinositide hydrolysis. The purpose of this study was to determine whether the relatively modest fuel-stimulated insulin secretion responses of rodent beta-cell lines might be explained by inadequate expression or activation of PLC isoforms. We have found that two insulinoma cell lines, INS-1 and betaG 40/110, completely lack PLC-delta1 expression but have levels of expression of PLC-beta1, -beta2, -beta3, -delta2, and -gamma1 that are similar to or slightly reduced from those found in fresh rat islets. Adenovirus-mediated overexpression of PLC-delta1, -beta1, or -beta3 in INS-1 or betaG 40/110 cells results in little or no enhancement in inositol phosphate (IP) accumulation and no improvement in insulin secretion when the cells are stimulated with glucose or carbachol, despite the fact that the overexpressed proteins are fully active in cell extracts. Overexpression of PLC-beta1 or -beta3 in normal rat islets elicits a larger increase in IP accumulation but, again, has no effect on insulin secretion. Because the effect of carbachol on insulin secretion is thought to be mediated through muscarinic receptors that link to the Gq/11 class of heterotrimeric G proteins, we also overexpressed G11alpha in INS-1 cells, either alone or in concert with overexpression of PLC-beta1 or -beta3. Overexpression of G11alpha enhances IP accumulation, an effect slightly potentiated by co-overexpression of PLC-beta1 or -beta3, but these maneuvers do not affect glucose or carbachol-stimulated insulin secretion. In sum, our studies show a lack of correlation between IP accumulation and insulin secretion in INS-1 cells, betaG 40/110 cells, or cultured rat islets. We conclude that overexpression of PLC isoforms and/or G11alpha is not an effective means of enhancing fuel responsiveness in the insulinoma cell lines studied.

Adenoviridae↗
PubMed1993

The biological relevance of the binding of calcium ions by inositol phosphates.

The binding of Ca2+ (chelation) by myo-inositol polyphosphates at pH 7.0 was studied using a Ca(2+)-sensitive electrode. Glucose 6-phosphate (used as a model for a monophosphate) bound Ca2+ with an affinity of 152 +/- 31 liters/mol and a molar ratio of 0.94 +/- 0.02. Inositol 3,4-bisphosphate, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol hexakisphosphate showed affinities of 9.0 +/- 2.1 x 10(3), 6.3 +/- 1.5 x 10(3), 6.2 x 10(4), and 1.92 +/- 0.47 x 10(5) liters/mol, respectively, and molar ratios of 0.92 +/- 0.49, 0.95 +/- 0.10, 0.75, and 2.5 +/- 0.5. In general, the affinity increased with the number of phosphate substituents on the inositol ring, although the stereochemistry is also expected to be important. This suggests that for the physiologically relevant inositol phosphates (tris-, tetrakis-, pentakis-, and hexakis-) half-maximal Ca2+ binding will occur in the Ca2+ concentration range of approximately 5 x 10(-6) to 2 x 10(-4) M. This range lies between the basal intracellular and the fee extracellular Ca2+ levels (10(-7) and 10(-3) M), respectively, and may therefore be of physiological importance. Chelation provides a possible simple explanation for the inhibition by Ca2+ of inositol 1,4,5-trisphosphate binding to its receptor in rat cerebellum and other tissues. It may also have a role in limiting inositol phosphate-mediated increases in intracellular Ca2+.

Binding Sites↗