Inositol Bisphosphate Research Articles

SHIP2: an emerging target for the treatment of type 2 diabetes mellitus.

With the rapid increase in the number of patients developing type 2 diabetes mellitus and the lack of optimal therapies, much focus has been placed on the insulin-signaling pathway in the discovery of novel drug targets. Phosphatidyl Inositol 3-Kinase (PI3K) is central to mediating insulin-s metabolic effects. PI3K catalyzes the generation of phosphatidyl inositol (3,4,5) triphosphate (PIP(3)). Inhibition of PI3K activity results in a blockade of insulin signaling including glucose uptake and glyocogen synthesis. Thus, PIP(3) is a critical mediator of insulin action. A family of phosphatidyl inositol phosphatases have been identified that counter-regulate PI3K activity by hydrolyzing PIP(3) to phosphatidyl inositol bisphosphate at either the 3' or 5' position of the inositol ring. Mice lacking one of these enzymes, Src-Homology Inositol Phosphatase-2 (SHIP2), demonstrate increased insulin sensitivity, suggesting that pharmacological inhibition of SHIP2 could alleviate insulin resistance. Recent studies demonstrate elevated SHIP2 expression is associated with insulin resistance in human patients. Comparing the studies on SHIP2 and other phosphatases suggests how inhibition of SHIP2 leads to increased insulin sensitivity without deleterious effects. This review focuses on the emergence of SHIP2 as a target in the insulin-signaling pathway for the treatment of type 2 diabetes.

Phospholipase Cδ 1 does not mediate Ca 2+ responses in neonatal rat cardiomyocytes

Phospholipase C (PLC) activation in neonatal rat ventricular cardiomyocytes (NRVM) generates inositol(1,4,5)trisphosphate (Ins(1,4,5)P 3) in response to elevations in Ca 2+ or inositol(1,4)bisphosphate in response to G protein stimulation. Overexpression of PLCδ 1 increased total [ 3H]inositol phosphate (InsP) content and elevated [ 3H]Ins(1,4,5)P 3, but failed to increase [ 3H]InsP responses to the Ca 2+ ionophore A23187. Antisense PLCδ 1 expression reduced endogenous PLCδ 1 content but did not decrease the A23187 response. In permeabilized NRVM, [ 3H]InsP responses to elevated Ca 2+ were not inhibited by Ins(1,4,5)P 3, even at concentrations 1000-fold greater than required for selective inhibition of PLCδ 1. Taken together these data provide evidence that PLCδ 1 does not mediate the InsP response to elevated Ca 2+ in NRVM.

An Autocrine Switch Governing Pulsatile Secretion

Episodic secretion of gonadotropin-releasing hormone (GnRH) from hypothalamic neurons is required for normal gonadal function. GnRH release depends on the intracellular calcium concentration [Ca 2+ ] i and is sensitive to adenosine 3′,5′-monophosphate (cAMP). The mechanisms governing pulsatile secretion, however, have remained unclear, although an autocrine contribution from GnRH itself has been postulated. Krsmanovic et al. investigated coupling of the GnRH receptor (GnRHR), which stimulates several heterotrimeric guanosine triphosphate (GTP)-binding protein (G protein)-mediated pathways, to various G protein α subunits and implicated a GnRH-induced switch in coupling in the pulsatile secretion of GnRH. The authors used Western analysis of rat primary hypothalamic neurons or immortalized GnRH neurons to monitor membrane association of different G protein α subunits. GnRH decreased membrane-associated Gα q/11 subunits (indicative of Gα q/11 activation) and stimulated a dose-dependent increase in inositol bisphosphate and inositol trisphosphate production and in [Ca 2+ ] i , whereas GnRH antagonists, which increased membrane-associated Gα q/11 , abolished pulsatile secretion. GnRH had a biphasic effect on cAMP production, stimulating production at nanomolar concentrations but inhibiting it at micromolar concentrations. Pharmacological analysis combined with selective G protein subunit overexpression indicated that both Gα s and Gα i were stimulated at low concentrations of GnRH but only Gα i was stimulated at high concentrations. The authors developed a model in which low concentrations of GnRH, acting through Gα s and Gα q/11 , increase [Ca 2+ ] i , stimulate production of cAMP, and stimulate GnRH secretion, until extracellular GnRH concentrations become high enough to trigger the switch to Gα i coupling. Elevated GnRH leads to decreased cAMP production and consequently less GnRH secretion, thereby reactivating the stimulatory cycle. L. Z. Krsmanovic, N. Mores, C. E. Navarro, K. K. Arora, K. J. Catt, An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion. Proc. Natl. Acad. Sci U.S.A . 111 , 2969-2974 (2003). [Abstract] [Full Text]

Squamous cell carcinomas fail to respond to the prodifferentiating actions of 1,25(OH)2D: why?

1,25(OH)2D regulates a number of cellular events which contribute to its ability to stimulate differentiation of the keratinocyte. 1,25(OH)2D raises the intracellular calcium (Cai) level in part by increasing the expression of the calcium receptor (CaR). This sensitizes the cell to extracellular calcium, triggering the signaling pathway coupled to the CaR, which results in a rise in Cai. 1,25(OH)2D induces the family of phospholipases C (PLC). These enzymes mediate the hydrolysis of phosphatidyl inositol bisphosphate (PIP2) to form inositol tris phosphate (IP3) and diacylglycerol (DG), which stimulate calcium release from intracellular stores and activate protein kinases C (PKC), respectively. The CaR and other G protein coupled receptors signal through PLC-beta, whereas tyrosine kinase growth factor receptors such as the EGF receptor signal through PLC-gamma. Calcium and PKC regulate the expression of genes in part by controlling the levels and activity of AP-1 transcription factors. 1,25(OH)2D also directly induces structural genes such as involucrin, a substrate for transglutaminase, which crosslinks it to other substrates to form the cornified envelope. 1,25(OH)2D regulates gene expression by activating the vitamin D receptor (VDR), a transcription factor, which, in combination with the retinoid X receptor (RXR) or retinoid A receptor (RAR), binds to its vitamin D response elements (VDRE) in the promoters of genes whose expression it regulates. The VDR also binds to one of two coactivator complexes, Mediator/DRIP (VDR interacting proteins) or p160/SRC (steroid hormone receptor complex), complexes which link the VDR to the RNA polymerase complex. We have recently discovered that the binding of VDR to these complexes is sequential. Binding to Mediator/DRIP occurs in the undifferentiated keratinocyte, but as the cell differentiates, DRIP(205) (the key protein of the DRIP complex binding to the VDR) levels fall, and p160/SRC binding takes over. We hypothesize that this sequential replacement of Mediator/DRIP by p160/SRC is critical for differentiation. Squamous cell carcinomas (SCC) fail to respond to the prodifferentiating actions of 1,25(OH)2D. These cells have normal levels of VDR and normal binding of VDR to VDREs. However, they fail to down-regulate DRIP(205) such that the p160/SRC complex fails to bind to VDR. This lack of sequential binding of these coactivator complexes to the VDR, we believe, maintains the cell in a state of continued proliferation and blocks the ability of 1,25(OH)2D to induce the expression of genes required for the differentiation process.

HERG K(+) channel activity is regulated by changes in phosphatidyl inositol 4,5-bisphosphate.

Autonomic stimulation controls heart rate and myocardial excitability and may underlie the precipitation of both acquired and hereditary arrhythmias. Changes in phosphatidyl inositol bisphosphate (PIP2) concentration results from activation of several muscarinic and adrenergic receptors. We sought to investigate whether PIP2 changes could alter HERG K(+) channel activity in a manner similar to that seen with inward rectifier channels. PIP2 (10 micromol/L) internally dialyzed increased the K(+) current amplitude and shifted the voltage-dependence of activation in a hyperpolarizing direction. Elevated PIP2 accelerated activation and slowed inactivation kinetics. When 10 micromol/L PIP2 was applied to excised patches, no significant change in single channel conductance occurred, indicating that PIP2-dependent effects were primarily due to altered channel gating. PIP2 significantly attenuated the run-down of HERG channel activity that we normally observe after patch excision, suggesting that channel run-down is due, in part, to membrane depletion of PIP2. Inclusion of a neutralizing anti-PIP2 monoclonal antibody in whole cell pipette solution produced the opposite effects of PIP2. The physiological relevance of PIP2-HERG interactions is supported by our finding that phenylephrine reduced the K(+) current density in cells coexpressing alpha1A-receptor and HERG. The effects of alpha-adrenergic stimulation, however, were prevented by excess PIP2 in internal solutions but not by internal Ca(2+) buffering nor PKC inhibition, suggesting that the mechanism is due to G-protein-coupled receptor stimulation of PLC resulting in the consumption of endogenous PIP2. Thus, dynamic regulation of HERG K(+) channels may be achieved via receptor-mediated changes in PIP2 concentrations.

Abscisic acid-induced changes in inositol metabolism in Spirodela polyrrhiza.

In response to abscisic acid (ABA), the duckweed Spirodela polyrrhiza (L.) activates a developmental pathway that culminates in the formation of dormant structures known as turions. Levels of the mRNA encoding D-myo-inositol-3-phosphate synthase (EC.5.5.1.4) which converts glucose-6-phosphate to inositol-3-phosphate, increase early in response to ABA. In order to understand the role of this enzyme in turion formation, we have investigated changes in inositol metabolism in ABA-treated plants. Here, we show that ABA-treatment leads to a 3-fold increase in free inositol, which peaks 2 d after treatment. This increase is followed by sequential increases in inositol phosphates and in accumulation of inositol hexakis-phosphate (InsP6), in particular. In addition, we observed an early increase in a novel inositol bisphosphate which is not directly on the pathway to InsP6. In control plants, we observed synthesis and turnover of both inositol pentakisphosphate and InsP6. Two compounds more polar than InsP6 (diphosphoinositol polyphosphates) were present in both ABA-treated and control plants. Together, this suggests that the role of InsP6 in plants may be more complex than simply that of a storage compound during dormancy.

Enhanced phospholipase C activity in the cultured skin fibroblast obtained from patients with coronary spastic angina: possible role for enhanced vasoconstrictor response

OBJECTIVESWe measured phospholipase C (PLC) activity in the cultured skin fibroblasts obtained from patients with and without coronary spasm and examined its correlation with coronary artery vasomotility.BACKGROUNDCoronary artery vasomotility is enhanced in coronary spastic angina (CSA), but no information is available for the intracellular signaling. In spontaneously hypertensive rats, PLC activity in the skin fibroblasts has been shown to be enhanced.METHODSSkin fibroblasts obtained from 24 patients with CSA—14 with organic coronary artery disease (CAD) and 12 control subjects—were cultured by the explant method. Activity of PLC was determined by incubating the membrane fraction with 3H-phosphatidyl inositol bisphosphate and by quantifying 3H-inositol trisphosphate. In patients with CSA and control subjects, the relations between PLC activity and coronary artery basal tone and constrictor response to intracoronary acetylcholine (ACh) were examined.RESULTSActivity of PLC (pmol/protein [mg] per min) was 1.74 ± 0.19 in patients with CSA; 0.90 ± 0.12 in patients with CAD; and 0.65 ± 0.07 in control subjects (p < 0.001, patients with CSA vs. patients with CAD and control subjects; p = NS, patients with CAD vs. control subjects). According to the Lineweaver-Burk plot, Michaelis constant (μmol/liter) of PLC was 28 ± 4 in patients with CSA; 49 ± 14 in patients with CAD; and 56 ± 10 in control subjects (p < 0.05, patients with CSA vs. control subjects), whereas the maximal velocity was not different between the three groups. There were significant positive correlations between PLC activity and both basal tone (p = 0.0108) and response to ACh (p = 0.0053). Western blot analysis using membrane fraction demonstrated that 89% of PLC isoenzymes detected was of the δ1 isoform.CONCLUSIONSBecause the PLC activity measured was genetically defined and was positively correlated with coronary artery vasomotility, enhanced PLC activity may be involved in the pathogenesis of coronary spasm.

Analysis of Inositol Phospholipid Turnover During Lymphocyte Activation

Receptor-mediated activation of phospholipase C (PLC) leads to the hydrolysis of membrane inositol phospholipids, generating diacylglycerol (DAG) and water-soluble inositol phosphates. This signaling mechanism is used by antigen receptors on T and B cells that have been implicated as mediators of receptor-induced influx of extracellular Ca(2+). This unit provides protocols that describe the resolution of InsP by Dowex anion-exchange chromatography. This technique provides a reliable means of separating inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, but does not resolve isomers of these. An Alternate Protocol describes the separation of inositol phosphates by anion-exchange HPLC. A protocol for resolution of inositol phospholipids by thin-layer chromatography (TLC) is also provided.

Interaction of vimentin with actin and phospholipids.

Vimentin intermediate filaments are a major cytoskeletal constituent of cells of mesenchymal origin. They have been colocalized with a variety of intracellular structures such as actin filaments and the plasma membrane. Labeled actin filaments, observed in vitro by fluorescence microscopy, break in the presence of polymerizing vimentin; the time course is consistent with stopped-flow measurements of vimentin polymerization. This breakage phenomenon appears to be specific for vimentin. Inhibition of vimentin network formation was observed with phosphatidyl inositol phosphate (PI(4)P) and phosphatidyl inositol bisphosphate (PI(4,5)P2), but not phosphatidyl choline (PC), phosphatidyl serine (PS), or phosphatidyl inositol (PI). Taken together, these results indicate a specific interaction of vimentin with F-actin and polyphosphoinositide lipids.

Effects of tumor necrosis factor alpha (TNF alpha) on the phospholipid metabolism of Tetrahymena pyriformis.

The effect of (0.05 ng ml-1 and 0.1 ng ml(-1)) TNF alpha on the phospholipid metabolism of Tetrahymena pyriformis was studied. The amount of phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), diacylglycerol (DAG), arachidonic acid (AA) and ceramide was higher, but the phosphatidyl inositol 4 phosphate (PIP) and phosphatidyl inositol bis-phosphate (PIP2) as well, as sphingomyelin (SM) content was lower in TNF alpha-treated cells than in the controls. In the culture medium (secreted forms) this situation was reversed. There were differences in the results gained by incorporation of [3H]-palmitic acid or 32P into the phospholipids. To control the functional effects of TNF alpha in Tetrahymena, the rate of cell division, the condensation of chromatin, the viability of cells and morphometrical values have been studied. The cytokine reduced cell growth, altered morphometric indices and increased chromatin condensation, however cell viability was not influenced. The results demonstrate the effects of TNF alpha at a low level of evolution, what is realized by changes in the phospholipid metabolism participating in signalling pathways.

Lithium chloride does not inhibit the proliferation of L6 myoblasts by decreasing intracellular free inositol.

We conducted a series of experiments to determine whether lithium chloride (LiCl) inhibited the proliferation of L6 myoblasts by reducing the availability of intracellular free inositol. After the myoblasts were plated in DMEM + 10% fetal bovine serum (FBS) for 24 h, medium was replaced with DMEM + 10% FBS containing 0 (control), 5, 10, or 20 mM LiCl. Cell number, protein content, and [3H]thymidine incorporation into DNA were determined at 24-h intervals. Control cells exhibited a 3.8-fold increase in cell number by 96 h in culture. Although 5 mM LiCl did not affect the rate or extent of proliferation, 10 and 20 mM LiCl caused 36 and 86% decreases, respectively (P < .05), in cell number by 96 h in culture. The effects of LiCl could not be overcome by the addition of free inositol (up to 20 mM) to the medium. Lithium chloride caused 4.6- and 7.3-fold increases (P < .05) in lactate dehydrogenase activity in culture media after 96 h of exposure to 10 and 20 mM LiCl, respectively, indicating loss of viability after chronic treatment. However, the acute effects of LiCl after 24 h of treatment were reversible, as indicated by a rapid resumption of proliferation following removal of LiCl. Concentrations of 5, 10, and 20 mM LiCl caused 4.7-, 8.2-, and 9.1-fold increases (P < .05), respectively, in the accumulation of [3H]inositol within the inositol monophosphate pool. Treatment of cells with 10 and 20 mM LiCl also increased (P < .05) label recovered as inositol bisphosphate. Rather than depress phosphoinositide synthesis, the addition of 10 and 20 mM LiCl dose-dependently increased (P < . 05) the incorporation of [3H]inositol into phosphatidylinositol and phosphatidylinositol-4-phosphate. These results indicate that LiCl does not decrease proliferation of L6 myoblasts via a depletion in the intracellular free inositol pool. Instead, LiCl may block the hydrolysis of phosphatidyl inositides.

The pharmacokinetics and metabolism of α-trinositol ( d-myo-inositol 1,2,6-trisphosphate) : Studies involving intravenous infusion in rats, metabolism in human platelets and leukocytes, and dephosphorylation by bovine alkaline phosphatases

In the present study the disposition of α-trinositol in rats, with special emphasis on its metabolism, was investigated. The drug was administered as an intravenous bolus dose (18.2 μmol/kg) immediately followed by a short term intravenous infusion (48.8 μmol/kg/h) of [ 3H]α-trinositol over 1.5 h. In the plasma samples, only unchanged α-trinositol, InsP 2, InsP 1, inositol and water were detected which indicates that during metabolism of α-trinositol in the rat only these metabolites are formed. The plasma concentration–time profile of α-trinositol showed a very fast decline following the cessation of the intravenous infusion, which probably is due to rapid metabolism and therefore a relative high total clearance of the drug, 23±4.3 ml/min/kg. The volume of distribution at steady state is 3.5±1.5 l/kg in the rat. To gain further insight into the metabolism of α-trinositol, we have also examined the possible metabolism by blood cells using intact human platelets, platelet homogenates, intact human neutrophils and also α-trinositol incorporated into human platelets using reversible electropermeabilization. Intact platelets and neutrophils showed little ability to metabolize α-trinositol when measured over a one hour incubation period. Platelet homogenates also showed little ability to metabolize α-trinositol under conditions where inositol 1,4,5-trisphosphate was rapidly metabolized to both inositol bisphosphate and inositol tetrakisphosphate. When α-trinositol was encapsulated into human platelets during reversible electropermeabilization significant metabolism was observed, with the main metabolite being InsP 1. We have also examined α-trinositol metabolism with alkaline phosphatases from bovine intestinal mucosa, kidney and liver. The intestinal enzyme showed extensive dephosphorylation of α-trinositol to Ins(1,2)P 2 and Ins(2)P 1 The kidney and liver enzyme showed slower dephosphorylation of α-trinositol with the end product being Ins(1,2)P 2.

Studies of inositol phosphate export from neuronal tissue in vitro.

Recent in vivo microdialysis studies have demonstrated the presence of extracellular levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] that can be increased in a concentration-dependent manner by muscarinic receptor activation. The aim of the present study was to determine whether extracellular levels of Ins(1,4,5)P3 could be measured in vitro. Despite rapid increases in internal Ins(1,4,5)P3 levels after stimulation with 1 mM carbachol, there was no change in external levels in both rat brain cortical slices and human neuroblastoma SH-SY5Y cells. Suprafusion of myo-[3H]inositol-prelabelled hippocampal slices with 1 mM carbachol caused an increase in 3H-inositol phosphates over basal levels in the perfusate after 10 min, reaching a peak (223 +/- 56% of basal) 20 min after suprafusion with carbachol was started. This response to carbachol was potentiated in the presence of 30 mM K+. Analysis of the individual 3H-inositol phosphates in the perfusate revealed that levels of [3H]inositol monophosphate, [3H]inositol bisphosphate, [3H]inositol trisphosphate, and [3H]inositol tetrakisphosphate were all significantly increased. A similar increase in extracellular 3H-inositol phosphates was demonstrated in SH-SY5Y cells incubated with 1 mM carbachol for 30 min. This response was again enhanced by 30 mM K+, although the intracellular response was not potentiated. Possible roles for extracellular inositol phosphates are discussed.

Effect of Chronic Ethanol Exposure on Myocardial Phosphoinositide Turnover

The effect of chronic ethanol consumption (2 months) on atrial contractility and the myocardial phosphoinositide signaling system was examined in rat heart. Two months of ethanol consumption was not associated with changes in heart weight-to-body weight ratios; however, developed twitch tension was significantly lower in the ethanol atria compared with the control atria. Cytosolic and membrane-associated phospholipase C activity in atrial and ventricular tissue was measured and ethanol consumption was only associated with changes in ventricular cytosolic phospholipase C activity. When examining alpha(1)-adrenergic stimulated phosphoinositide turnover in [3H]inositol radiolabeled left atria, no differences in phenylephrine (10 microM)-stimulated inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate were found between groups before or at various times after the addition of phenylephrine. It is concluded that short-term ethanol consumption is associated with depressed contractile function, but not the development of hypertrophy or changes in alpha(1)-adrenoreceptor-stimulated phosphoinositide turnover.

5-Hydroxytryptamine stimulates phosphoinositide hydrolysis and contraction in the ovine umbilical artery

5-Hydroxytryptamine-stimulated contraction and generation of inositol phosphates (InsPs) were investigated in isolated ovine umbilical artery from near term pregnant sheep. 5-Hydroxytryptamine produced a concentration-dependent contraction in the presence or absence of external Ca 2+, however, the contractile response in Ca 2+-free medium was depressed by 67% ( P<0.05). The initial increase of either inositol monophosphate (InsP 1) or inositol bisphosphate (InsP 2) was not apparent, although a significant rise in both InsP 1 and InsP 2 was observed at 30 min (107 and 100% over basal level, P<0.05). The generation of inositol trisphosphate (InsP 3) was rapid and reached its peak at 60–90 s (35% over basal level, P<0.05), followed by a second rise at 30 min (96% over basal level, P<0.05). The generation of InsPs stimulated by 5-hydroxytryptamine was also concentration-dependent. In agreement with previous contraction studies, the generation of InsPs stimulated by 5-hydroxytryptamine was blocked by 10 nM ketanserin, a specific 5-HT 2A receptor antagonist. A temporal study revealed that the generation of InsP 3 coincided with the phasic component of the contractile response induced by 5-hydroxytryptamine. Our results suggest that through activation of 5-HT 2A receptors, the generation of InsP 3 plays a critical role in the contraction induced by 5-hydroxytryptamine in the ovine umbilical artery.

Inositol Phosphate Metabolism During Myocardial Ischemia

Inositol phosphate release in intact heart in response to norepinephrine involves primarily release of inositol(1,4)bisphosphate (Ins(1,4)P2) rather than inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) but Ins(1,4,5)P3release predominates under conditions of post-ischemic reperfusion. In the current study, effects of myocardial ischemia on inositol phosphate responses were examined. Global myocardial ischemia in rat ventricle caused a reduction in the content of [3H]Ins(1,4)P2(70–90%) and [3H]Ins(1,4,5)P3(46%) and altered the pattern of norepinephrine stimulation such that increases in [3H]Ins(1,4,5)P3were observed. Simulated ischemia in isolated right atria or isolated ventricular myocytes (PO216–20 mmHg, pH 6.7, KCl 10 mm) produced similar changes. Reduction in O2in the absence of other changes reduced the content of [3H]Ins(1,4)P2(79%) in right atria whereas hypoxia and reduced pH were required to alter the [3H]Ins(1,4,5)P3response. Progressive reduction in atrial ATP content using metabolic inhibitors caused a parallel decrease in [3H]Ins(1,4,5)P3content (r=0.96) without affecting [3H]Ins(1,4)P2or the isomers of InsP1, showing that levels of Ins(1,4)P2and Ins(1,4,5)P3are regulated differently in the heart. These findings show that effects of ischemia on inositol phosphates in heart are complex and multifactorial, with Ins(1,4)P2being affected under more moderately ischemic conditions than required for alterations in Ins(1,4,5)P3. These studies also demonstrate that ischemia produces similar effects on the release and metabolism of inositol phosphates in heart regardless of the ischemic model or the myocardial preparation used.

Metabolism of inositol 1,4,5-trisphosphate in Candida albicans: significance as a precursor of inositol polyphosphates and in signal transduction during the dimorphic transition from yeast cells to germ tubes.

The metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was examined in yeast cells and germ tubes of Candida albicans. Methods have been developed for analysis of the two key metabolic enzymes, Ins(1,4,5)P3, kinase and phosphatase. ATP-dependent Ins(1,4,5)P3 kinase activity was detected predominantly in the soluble fraction of cell extracts and exhibited a Km of approximately 9 microM. The apparent Km of Ins(1,4,5)P3 phosphatase for Ins(1,4,5)P3 was approximately 480 microM. The slow rate of dephosphorylation of Ins(1,4,5_P3 to inositol bisphosphate suggests a lower importance of the phosphatase within cells compared to the kinase. Since both yeast cells and germ tubes of C. albicans rapidly phosphorylated Ins(1,4,5)P3 to inositol tetrakisphosphate and inositol penta/hexakisphosphate, it is suggested that Ins(1,4,5)P3 has an important role as a precursor for production of these compounds. A sustained increase in cellular Ins(1,4,5)P3 levels was observed during germ tube formation and, prior to the onset of germination between 1 and 2 incubation, the Ins(1,4,5)P3 content increased up to eightfold. Transient increases in the level of Ins(1,4,5)P3 were also observed during yeast-like growth of C. albicans. The possible role and relative importance of Ins(1,4,5)P3 as a precursor for inositol polyphosphates and in signal transduction involving Ca2+ release from internal stores is discussed.

Protein Kinase C Regulation of ATP-Induced Phosphoinositide Hydrolysis in Bovine Aorta Endothelial Cells

This study investigated the mechanism of protein kinase C-mediated inhibition of ATP-induced phospholipase C activation in cultured bovine aorta endothelial cells (BAEC). In BAEC labeled with 3H-inositol, phorbol myristate acetate (PMA) prevented ATP-induced inositol bisphosphate and inositol trisphosphate formation. In membranes prepared from these PMA-treated cells, Ca2+-, sodium fluoride,- GTPγS-, and ATP plus GTPγS-stimulated inositol bisphosphate, but not inositol trisphosphate, formation was inhibited. Inositol trisphosphate phosphatase activity was not altered in membranes from PMA-treated BAEC. These results suggest that 1) protein kinase C inhibits ATP-induced phospholipase C activation in BAEC through interference with the coupling of phospholipase C with a G-protein and through an effect on phospholipase C itself, and 2) different mechanisms are responsible for the inhibition by protein kinase C of the phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate and phosphatidyl-inositol phosphate.

Thromboxane A2 mediates the stimulation of inositol 1,4,5-trisphosphate production and intracellular calcium mobilization by bradykinin in neonatal rat ventricular cardiomyocytes.

Bradykinin is a mediator of the protection of myocardium by angiotensin I-converting enzyme/kininase II inhibitors. We reported that the activation of B2 bradykinin receptors in neonatal rat cardiac myocytes in primary culture was followed by hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (IP3). Here we examine the regulation of IP3 formation stimulated by bradykinin. Activation of myocytes with 1 mu/L bradykinin increased IP3 production from 117 +/- 8.3 to 1011 +/- 48.6 pmol/mg protein. Treatment of the cells with 10 mu/L indomethacin or 1 mu/L dexamethasone partially blocked this bradykinin-induced response. Moreover, either U73122, a phospholipase C inhibitor, or (p-amylcinnamoyl) anthranilic acid, a phospholipase A2 inhibitor, blunted the IP3 response to bradykinin. Because thromboxane A2 stimulates inositol bisphosphate metabolism in guinea pig atria, we also investigated the effect of the thromboxane A2 receptor antagonist BM 13177 (1 mu/L), which strongly attenuated the stimulated IP3 production. Since thromboxane A2 appears to partly mediate the IP3 response to bradykinin, we examined the effect of the stable thromboxane A2 mimetic U46619. Control cultures were stimulated more by U46619 than by bradykinin (1629 +/- 14.5 versus 1011 +/- 48.6 pmol IP3/mg protein). This property of U46619 was selectively antagonized by BM 13177. Inhibition of either phospholipase C or phospholipase A2 blunted the IP3 response to U46619. Short-term (30 minutes) activation of protein kinase C with phorbol 12-myristate 13-acetate (10 pmol/L to 1 mu/L) attenuated the IP3 accumulation in response to bradykinin; the effect of phorbol 12-myristate 13-acetate was reversed with 1 mu/L staurosporine, a protein kinase C inhibitor. Treatment with 1 microgram/mL cholera toxin or pertussis toxin for 4 hours amplified the IP3 response to 10 nmol/L bradykinin from 570 +/- 20.0 to 1150 +/- 51.3 and to 1016.7 +/- 21.9 pmol/mg protein. Bradykinin mobilized 9.4% of intracellular calcium stores in cardiomyocytes as assessed by chlortetracycline-based fluorometry, and this effect of bradykinin was blocked by BM 13177 or the B2 bradykinin receptor blocker Hoe 140 by more than 70%. In functional studies, bradykinin (1 mu/L) increased by 12% the twitch contractile force of neonatal rat ventricular strips paced at threshold intensity, but this was unaffected by BM 13177. In conclusion, in cardiomyocytes, bradykinin enhances IP3 production mostly via phospholipase A2 stimulation and thromboxane A2 formation. This prostanoid in turn stimulates its receptor and activates phospholipase C, which then splits phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol. The effect of bradykinin on phospholipase C, via thromboxane A2, is negatively regulated by protein kinase C activation.

The Luteinizing Hormone/Chorionic Gonadotropin Receptor Has Distinct Transmembrane Conductors for cAMP and Inositol Phosphate Signals

The luteinizing hormone/chorionic gonadotropin receptor is a member of the seven-transmembrane receptor family. It is coupled, presumably via Gs and Gq, to two signal pathways involving adenylyl cyclase/cAMP and phospholipase C/inositol phosphate (IP). Little is known about the events prior to G-protein coupling: for example, whether these signals are generated from a single or multiple independent origins and mechanisms, when and where they diverge, and how they are transduced. We report novel observations that the cAMP signal and the IP signal originate and diverge upstream of G-protein coupling. The generation of these two signals independently involves Lys583 in exoloop 3 of the rat receptor. For this study, Lys583 of the receptor was substituted with a panel of amino acids, and mutant receptors were assayed for hormone binding and induction of cAMP, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. No substitutions for Lys583 were permissible for cAMP induction, despite successful surface expression and hormone binding. In contrast, several substitutions were permissible for IP induction. Our results suggest two distinct transmembrane signal conductors for cAMP and inositol phosphate signals and imply particular models of receptor activation not previously suggested.