Two phases of inositol polyphosphate and diacylglycerol production at fertilisation

[3H]Inositol and [3H]arachidonic acid were used to label polyphosphoinositide phospholipids in sea urchin eggs. Both [3H]inositol polyphosphate (InsP3) and [3H]diacylglycerol (DAG) increase at fertilisation. An early increase in InsP3 occurs as the sperm-induced calcium transient crosses the egg and exocytosis occurs; a later increase in InsP3 as calcium declines and the protein kinase C-dependent Na/H antiporter causes the cytoplasmic pH in increase. These results support suggestions that a calcium-induced hydrolysis of phosphatidylinositol bisphosphate occurs at fertilisation, that the production of diacylglycerol may be essential for exocytosis and that diacylglycerol production at fertilisation stimulates the Na/H antiporter. The increase in [3H]inositol polyphosphate as calcium declines indicates that this second messenger may have some function later in the cell cycle.     

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells

We have previously shown that inositol trisphosphate (IP3) releases Ca2+ from a nonmitochondrial pool of permeabilized rat pancreatic acinar cells (Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1984) Nature 306, 67-69). This pool was later identified as endoplasmic reticulum (Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Biol. 81, 241-253). As IP3 is produced by hydrolysis of phosphatidylinositol bisphosphate on activation of many "Ca2+-mobilizing receptors," our observation supported the proposal that IP3 functions as a second messenger to release Ca2+ from the endoplasmic reticulum. We have here used the same preparation of permeabilized acinar cells to study the relationship of secretagogue-induced Ca2+ release and IP3 production. We show that: 1) secretagogue-induced Ca2+ release in permeabilized cells is accompanied by a parallel production of inositol trisphosphate. 2) When the secretagogue-induced increase in intracellular free Ca2+ concentration was abolished by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering, secretagogue-induced IP3 production was unimpaired. 3) When secretagogue-induced IP3 production was reduced by inhibiting phospholipase C with neomycin, secretagogue-induced Ca2+ release was also abolished. 4) When the IP3 breakdown was reduced either by lowering the free Mg2+ concentration of the incubation medium or by adding 2.3-diphosphoglyceric acid, the rise in IP3 and the release of Ca2+ induced by secretagogues were both increased. These results further support the role of IP3 as a second messenger to induce Ca2+ mobilization.     

Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells

Bradykinin stimulation of inositol polyphosphate production was followed using [3H]inositol-labeled porcine aortic endothelial cells grown in culture. Bradykinin stimulated a significant increase in inositol trisphosphate (IP3) production within 15 s. This increase reached a maximum value of 5-fold above control at 30 s and returned toward baseline by 90 s. Production of inositol bisphosphate increased with time reaching 4-fold by 60 s. Bradykinin stimulated the production of IP3 and inositol biphosphate in a dose-dependent manner with an EC50 of 9 X 10(-9) M. Labeled pools of phosphatidylinositol-4,5-bisphosphate (PIPP) decreased by 50% within 30 s, corresponding to the rise in IP3, while labeled lysophosphatidylinositol pools increased 3-fold by 60 s. Pertussis toxin, a protein which ribosylates GTP-binding proteins, did not inhibit bradykinin-stimulated inositol polyphosphate production. Incubation of labeled cells in the absence of extracellular Ca2+ also did not affect bradykinin-stimulated inositol polyphosphate production. Further, A23187, a Ca2+ ionophore, failed to stimulate PIPP metabolism. Finally, Ca2+ influx into cell monolayers occurred with a time course which paralleled rather than preceded the increase in IP3 levels. These data suggest that bradykinin stimulates phospholipase C metabolism of PIPP to IP3 by a mechanism which does not contain a pertussis toxin sensitive GTP-binding protein. Also, this receptor-linked phospholipase C activity does not appear to be activated by extracellular Ca2+ influx. The results support the proposal that IP3 production initiates Ca2+ mobilization and suggest that the calcium-dependent step in arachidonate release is distal to IP3 production.     

A role for rat inositol polyphosphate kinases rIPK2 and rIPK1 in inositol pentakisphosphate and inositol hexakisphosphate production in rat-1 cells

Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.     

Neutrophil cathepsin G increases calcium flux and inositol polyphosphate production in cultured endothelial cells

Exposure of endothelial cells (ENDO) to human neutrophil cathepsin G (CG) increases albumin flux across the endothelial monolayer. Since calcium influences cell shape and barrier function of ENDO monolayers, the current study was designed to determine if CG acted through alterations in Ca2+ homeostasis in ENDO. The role of Ca2+ in the increased permeability of ENDO monolayers to albumin after exposure to CG was studied by using ENDO monolayers cultured on polycarbonate filters. Exposure of ENDO monolayers to CG in the presence of the Ca2+-antagonist lanthanum partially prevented the increase in albumin flux, but exposure in the presence of agents that block voltage-regulated calcium channels did not block the increase in albumin flux. To monitor the effect of CG on Ca2+-flux, ENDO were labeled with 45Ca2+ and changes in Ca2+ flux were monitored by the release of 45Ca2+. From 1 to 15 minutes after exposure of ENDO to CG, there was increased release of 45Ca2+ compared with control cells. Calcium channel blocking agents did not inhibit the increased release of 45Ca2+, but lanthanum partially blocked the increase. The increased release of Ca2+ appeared to be due, at least in part, to activation of phospholipase C because there was an increase both in inositol polyphosphate species and in diglycerides after incubation of ENDO with CG. These studies support the hypothesis that CG increases the flux of calcium in ENDO, that this increase in Ca2+ flux may result from activation of phospholipase C, and that this system may be involved in the decreased barrier properties of the ENDO after CG exposure.     

Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2+ induced by vasopressin in isolated hepatocytes

Addition of vasopressin to rat hepatocytes prelabeled with myo-[2-3H]inositol resulted in a very rapid decrease [3H]phosphatidylinositol 4,5-bisphosphate (Ptd-Ins-4,5-P2) which was paralleled by increases of up to 3-fold in the levels of [3H]inositol trisphosphate (Ins-P3) and [3H]inositol bisphosphate (Ins-P2). Increases of [3H]inositol phosphate (Ins-P) were not detected until about 5 min after hormone addition. These data indicate that the major pathway for hormone-induced lipid breakdown in liver is through a phosphodiesterase for PtdIns-4,5-P2 and that decreases of phosphatidylinositol are a secondary result of increased PtdIns-4,5-P2 resynthesis. Using the fluorescent Ca2+ indicator Quin 2, cytosolic free Ca2+ increased from 160 nM to about 400 nM after vasopressin addition to hepatocytes and preceded the conversion of phosphorylase b to a. Half-maximal and maximal increases of cytosolic free Ca2+ and phosphorylase a activity were observed at 0.2 and 1 nM vasopressin, respectively. The dose-response curve for the initial rate of cytosolic free Ca2+ increase was very similar to those obtained for the initial rates of Ins-P3 production and PtdIns-4,5-P2 breakdown. Pretreatment of hepatocytes with Li+ caused a 3--4-fold potentiation of vasopressin-induced elevations of Ins-P, Ins-P2, and Ins-P3, with half-maximal effects at 0.5, 1, and 5 mM, respectively. The calculated maximal concentrations of Ins-P3 in cells treated with 20 nM vasopressin were 10 and 30 microM, respectively, without and with Li+. Lithium did not affect the initial rate of inositol polyphosphate production or Ca2+ mobilization. The increase of Ins-P3 which correlated with peak cytosolic free Ca2+ elevation was about 0.6 microM. In a saponin-permeabilized hepatocyte preparation, Ins-P3 (1 microM) caused Ca2+ release from a vesicular, ATP-dependent Ca2+ pool. The data presented here suggest that Ins-P3 may be a second messenger for the mobilization of intracellular Ca2+ by hormones in liver.     

Dissociation of inositol trisphosphate from diacylglycerol production in Rous sarcoma virus-transformed fibroblasts

The metabolism of phosphatidylinositol (PI) and related intermediates was studied in uninfected and Rous sarcoma virus-(RSV) infected chicken embryo fibroblasts (CEFs). Cells infected with wild-type RSV exhibited twofold increases in steady-state concentrations of inositol trisphosphate (IP3) and inositol bisphosphate (IP2) as compared to uninfected CEFs. In addition, increased concentrations of IP3 and IP2 were observed in CEFs infected with the RSV temperature-sensitive transformation mutant NY72-4 when maintained at the permissive temperature (35 degrees C) for greater than 24 h. Slight increases were observed in the amounts of inositol lipids in RSV-transformed cells. Phosphoinositol metabolic changes were related to transformation and not to viral infection since CEFs infected with NY72-4, maintained at the nonpermissive temperature (41.5 degrees C), revealed amounts of phosphoinositols similar to that of uninfected cells. CEFs infected with a transformation-defective virus exhibited PI metabolic changes intermediate between those of transformed and nontransformed cells. NY72-4 CEF exhibited no increase in phosphoinositol concentrations before 8 h incubation at 35 degrees C, indicating that the transformation-specific changes in inositol metabolism were a delayed event. Furthermore, inositol turnover was not activated during this time. In contrast to the case of inositol metabolism, significant increases in diacylglycerol (DAG) concentrations were observed within 15-30 min after shift of NY72-4 CEFs to 35 degrees C. These findings suggest that (a) the major changes in inositol metabolism are specific for RSV-transformed cells; (b) transformation-specific changes in phosphoinositol content in RSV-infected CEFs are not an early effect of the expression of pp60v-src; and (c) increases in the DAG content of transformed cells occur before changes in inositol metabolism, indicating that DAG may be derived from other lipid sources.     

Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [3H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a "receptor(s)" which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively.     

Dissociation between aggregation and superoxide production in human granulocytes

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.     

The isolation and characterization of inositol polyphosphate 4-phosphatase

We previously identified an alternative pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) in calf brain. The enzyme responsible for the degradation of Ins(1,3,4)P3 was designated as inositol polyphosphate 4-phosphatase (Bansal, V. S., Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 9644-9647). We have now purified this enzyme 3390-fold from calf brain-soluble fraction. The isolated enzyme has an apparent molecular mass of 110 kDa as determined by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme migrates as a protein of 105 kDa, suggesting that it is monomeric. Among various 4-phosphate-containing inositol polyphosphates, the enzyme hydrolyzes only Ins(1,3,4)P3 and inositol 3,4-bisphosphate (Ins(3,4)P2), yielding inositol 1,3-bisphosphate and inositol 3-phosphate as products. The inositol polyphosphate 4-phosphatase has apparent Km values of 40 and 25 microM for Ins(1,3,4)P3 and Ins(3,4)P2, respectively. The maximum velocities for these two substrates are 15-20 mumol of product/min/mg protein. Ins(1,3,4)P3 is a competitive inhibitor of Ins(3,4)P2 hydrolysis with an apparent Ki of 27 microM implying that the same active site is involved in hydrolysis of both substrates. The final enzyme preparation retained a small inositol polyphosphate 3-phosphatase activity (less than 2% of rate of inositol polyphosphate 4-phosphatase activity) which most likely reflects a contaminant. The enzyme displays maximum activity between pH 6.5 and 7.5. It is not inhibited by Li+, Ca2+, or Mg2+ except at 10 mM divalent ions. Mn2+ inhibits enzyme at high concentrations IC50 = 1.5 mM.     

Effect of angiotensin II and III on inositol polyphosphate production in differentiated NG108-15 hybrid cells

Neuroblastoma x glioma hybrid cells (NG108-15), differentiated by treatment with 1.5% dimethyl sulfoxide (DMSO) and 0.5% fetal bovine serum, were used to measure the effect of angiotensin II and III (ANG II and ANG III) on the generation of inositol polyphosphates. ANG II increased the synthesis of inositol monophosphates (IP1), inositol diphosphates (IP2), and inositol trisphosphates (IP3) with maximal responses observed at 300, 120, and 30 sec, respectively. The percent increases above basal values at the maximal responses were 140% +/- 9% (IP1), 142% +/- 4% (IP2), and 132% +/- 4% (IP3). This effect was not attenuated by pretreatment of the cells with pertussis toxin. Furthermore, both ANG II and ANG III increased the production of inositol polyphosphates in a dose-dependent manner with ED50 values of 145 nM and 11 nM, respectively. We conclude that differentiated NG108-15 cells express an ANG III selective receptor that mediates phosphatidylinositol breakdown through a pertussis toxin insensitive G-protein.     

Reduced inositol polyphosphate accumulation and inositol supply induced by lithium in stimulated cerebral cortex slices.

The ability of lithium to interfere with phosphoinositide metabolism in rat cerebral cortex slices has been examined by monitoring the accumulation of CMP-phosphatidate (CMP-PtdOH) and the reduction in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels. A small accumulation of [14C]CMP-PtdOH was seen in slices prelabelled with [14C]cytidine and stimulated with carbachol (1 mM) or Li+ (1 mM). However, simultaneous addition of both agents for 30 min produced a 22-fold accumulation, with Li+ producing a half-maximal effect at a concentration of 0.61 +/- 0.19 mM. Kinetic studies revealed that the effects of carbachol and Li+ on CMP-PtdOH accumulation occurred with no initial lag apparent under these conditions and that preincubation with myo-inositol (10 or 30 mM) dramatically attenuated CMP-PtdOH accumulation. myo-Inositol could also attenuate the rate of accumulation of CMP-PtdOH when added 20 min after carbachol and Li+; these effects were not observed when equimolar concentrations of scyllo-inositol were added. Use of specific radioreceptor assays allowed the mass accumulations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 to be monitored. Following a lag of 5-10 min, Li+ resulted in a marked reduction in the accumulation of both inositol polyphosphates resulting from muscarinic-cholinergic stimulation. Preincubation of cerebral cortex slices with myo- (but not scyllo-) inositol delayed, but did not prevent, the reduction in the accumulation of Ins(1,4,5)P3 or Ins(1,3,4,5)P4. The results suggest that cerebral cortex, at least in vitro, is very sensitive to myo-inositol depletion under conditions of muscarinic receptor stimulation. The relationship of such depletion to the generation of inositol polyphosphate second messengers is discussed.     

A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster

Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6) and PP-IP5. Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster. Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators. We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4. To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies. Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6. Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either alpha or beta isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools. Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis. IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2. Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers.     

Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A.

Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.     

Bombesin stimulates inositol polyphosphate production in GH4C1 pituitary tumor cells: comparison with TRH

The hormones bombesin and thyrotropin-releasing hormone (TRH) stimulated formation of inositol- monophosphate, bisphosphate, trisphosphate and tetrakisphosphate with parallel time courses in GH4C1 cells, while a more polar inositol polyphosphate peak, consisting of inositol-pentakisphosphate and perhaps also inositol-hexakisphosphate, was unaffected by either hormone. Although bombesin and TRH had similar potencies in stimulating inositol trisphosphate production (Km = 30 nM and 40 nM, respectively), TRH was significantly more efficacious than bombesin. Maximal stimulation of inositol-1,4,5-trisphosphate formation by TRH was not further increased by addition of a maximally effective dose of bombesin, suggesting that the two hormones act through stimulation of a common pool of phospholipase C, and this enzyme pool can be fully stimulated by TRH, alone.     

Identification and characterization of a novel inositol polyphosphate 5-phosphatase

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4, 5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K(m) = 180 microM) than for inositol 1,4, 5-trisphosphate (K(m) = 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate.     

Lowering extracellular pH evokes inositol polyphosphate formation and calcium mobilization

Changing extracellular pH (pHo) from 7.4 to 6.1 increased [3H]inositol bis- and trisphosphates approximately 10- and 5-fold, respectively, in 15 s in human fibroblasts. [3H]Inositol phosphate increased less rapidly than the polyphosphates. Bradykinin similarly increased [3H]inositol phosphates. Shifting pHo from 7.4 to 6.0 evoked a large spike in cytosolic free Ca2+ [( Ca2+]i) which was primarily caused by the release of stored Ca2+. Changing pHo from 7.4 to 6.0 decreased cytoplasmic pH to approximately 7.0. Moderate decreases in intracellular pH had no effect on [Ca2+]i or 45Ca2+ efflux. Decreasing pHo strikingly increased 45Ca2+ efflux and decreased total cell Ca2+ similarly to bradykinin. Changing pHo from 7.4 to approximately 6.4 produced half-maximal effects on [Ca2+]i, 45Ca2+ efflux, and total Ca2+. Cycling pHo between 7.4 and 6.0 produced repetitive decreases and increases in total Ca2+. Bradykinin released the Ca2+ which was reaccumulated after an acid pulse indicating that Ca2+ had returned to the hormone-sensitive pool. Decreasing pHo also released stored Ca2+ from coronary endothelial, neuroblastoma, and umbilical artery muscle cells, but not from rat aortic smooth muscle or human epidermoid carcinoma (A431) cells. We suggest that lowering pHo stimulates a phosphoinositidase-coupled receptor by protonating a functional group with a pKa near 6.5.