Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity

The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP₂), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301). In this paper, we show the effect of various concentrations of phosphatidyinositol, PIP, and PIP₂ on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP₂ at concentrations of 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids.     

Short-term treatment with cell wall degrading enzymes increases the activity of the inositol phospholipid kinases and the vanadate-sensitive ATPase of carrot cells

Treating carrot (Daucus carota L.) suspension culture cells with a mixture of cell wall degrading enzymes, Driselase, resulted in an increase in the percentage of [³H]phosphatidylinositol bisphosphate. Analysis of the lipid kinase activities in the isolated plasma membranes after whole cell treatment indicated that treatment with Driselase (2% weight/volume; the equivalent of 340 units per milliliter of hemicellulase and 400 units per milliliter of cellulase activity) or treatment with hemicellulase (31.7% weight/volume, 20.7 units per milliliter) resulted in an increase in the inositol phospholipid kinase activity. However, treatment with cellulase alone had no effect at 0.5% (weight/volume, 17.2 units per milliliter) or inhibited the kinase activity at 1% (weight/volume, 34.4 units per milliliter). The active stimulus in Driselase was heat sensitive. The plasma membrane vanadate-sensitive ATPase activity also increased when the cells were treated with Driselase. A time course study indicated that both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase responded to as little as 5 seconds of treatment with 2% Driselase. However, at the lowest concentration of Driselase (0.04%, weight/volume) that resulted in an increase in inositol phospholipid kinase activity, the ATPase activity was not affected. Because inositol phospholipids have been shown to activate the vanadate-sensitive ATPase from plants (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301), a stimulus-response pathway involving both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase activity is discussed.     

Phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and the phosphoinositol sphingolipids are found in the plasma membrane and stimulate the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

Several plasma membrane phospholipids have been studied for their ability to modulate the activity of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae. We show here that phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2), and/or the phosphatidylinositol and PIP kinases are localized primarily in the plasma membrane. Previous in vivo studies with S. cerevisiae have shown that large, rapid, and reversible changes occur in the levels of PIP and PIP2 congruent with changes in cellular ATP levels. We demonstrate here that isolated plasma membranes exhibit the same changes in PIP and PIP2 content when they are supplied with or washed free of ATP. Using a mixed micellar assay we systematically studied the efficacy of the plasma membrane lipids in sustaining the activity of the plasma membrane H(+)-ATPase. We demonstrate for the first time that a number of plasma membrane glycerophospholipids effectively stimulate the ATPase, including PIP, PIP2, and cardiolipin. Phosphoinositol-containing sphingolipids, major components of the plasma membrane, are also shown to stimulate the ATPase at significantly lower levels than the glycerophospholipids and must also be considered as important effectors in vivo.     

Changes in phosphatidylinositol metabolism in response to hyperosmotic stress in Daucus carota L. cells grown in suspension culture.

Carrot (Daucus carota L.) cells plasmolyzed within 30 s after adding sorbitol to increase the osmotic strength of the medium from 0.2 to 0.4 or 0.6 osmolal. However, there was no significant change in the polyphosphorylated inositol phospholipids or inositol phosphates or in inositol phospholipid metabolism within 30 s of imposing the hyperosmotic stress. Maximum changes in phosphatidylinositol 4-monophosphate (PIP) metabolism were detected at 5 min, at which time the cells appeared to adjust to the change in osmoticum. There was a 30% decrease in [3H]inositol-labeled PIP. The specific activity of enzymes involved in the metabolism of the inositol phospholipids also changed. The plasma membrane phosphatidylinositol (PI) kinase decreased 50% and PIP-phospholipase C (PIP-PLC) increased 60% compared with the control values after 5 min of hyperosmotic stress. The PIP-PLC activity recovered to control levels by 10 min; however, the PI kinase activity remained below the control value, suggesting that the cells had reached a new steady state with regard to PIP biosynthesis. If cells were pretreated with okadaic acid, the protein phosphatase 1 and 2A inhibitor, the differences in enzyme activity resulting from the hyperosmotic stress were no longer evident, suggesting that an okadaic acid-sensitive phosphatase was activated in response to hyperosmotic stress. Our work suggests that, in this system, PIP is not involved in the initial response to hyperosmotic stress but may be involved in the recovery phase.     

Turkey erythrocyte membranes as a model for regulation of phospholipase C by guanine nucleotides

Phosphoinositides of human, rabbit, rat, and turkey erythrocytes were radiolabeled by incubation of intact cells with [32P]Pi. Guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and NaF, which are known activators of guanine nucleotide regulatory proteins, caused a large increase in [32P]inositol phosphate release from plasma membranes derived from turkey erythrocytes, but had no effect on inositol phosphate formation by plasma membranes prepared from the mammalian erythrocytes. High performance liquid chromatography analysis indicated that inositol bisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate all increased by 20-30-fold during a 10-min incubation of turkey erythrocyte membranes with GTP gamma S. The increase in inositol phosphate formation was accompanied by a similar decrease in radioactivity in phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). GTP gamma S increased inositol phosphate formation with a K0.5 of 600 nM; guanosine 5'-(beta, gamma-imido)trisphosphate was 50-75% as efficacious as GTP gamma S and expressed a K0.5 of 36 microM. Although GTP alone had little effect on inositol phosphate formation, it blocked GTP gamma S-stimulated inositol phosphate formation, as did guanosine 5'-O-(2-thiodiphosphate). Turkey erythrocytes were also shown to express phosphatidylinositol synthetase activity in that incubation of cells with [3H] inositol resulted in incorporation of radiolabel into phosphatidylinositol, PIP, and PIP2. Incubation of membranes derived from [3H]inositol-labeled erythrocytes with GTP gamma S resulted in large increases in [3H] inositol phosphate formation and corresponding decreases in radiolabel in PIP and PIP2. The data suggest that, in contrast to mammalian erythrocytes, the turkey erythrocyte expresses a guanine nucleotide-binding protein that regulates phospholipase C, and as such, should provide a useful model system for furthering our understanding of hormonal regulation of this enzyme.     

Stimulation of polyphosphoinositide turnover upon activation of protein kinases in human erythrocytes

Activation of protein kinase C in erythrocytes by 4-beta-phorbol 12-myristate 13-acetate (PMA) resulted in a parallel stimulation (time course and dose response) of the phosphorylation of both membrane proteins (heterodimers of 107 kDa and 97 kDa, protein 4.1 and 4.9, respectively) and of phosphatidylinositol 4-phosphate (PIP) and, to a lesser extent, of phosphatidylinositol 4,5-bisphosphate (PIP2). Evidence that the effect on lipid was mediated by protein kinase C activation and not by a direct action of PMA was provided by (1) the lack of effect of a phorbol ester that did not activate protein kinase C or of PMA addition on isolated membranes from control erythrocytes, (2) the reversal of the effect in the presence of protein kinase C inhibitors (alpha-cobrotoxin, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) or trifluoperazine). PMA treatment did not change the specific activity of ATP or the content of PIP2, but increased the content of PIP and decreased that of PI, indicating that the phosphorylation or dephosphorylation reactions linking PI and PIP were the target for the action of PMA. PMA treatment had no effect on the Ca2+-dependent PIP/PIP2 phospholipase C activity measured in isolated membranes. Mezerein, another protein kinase activator, had similar effects on both protein and lipid phosphorylation, when added with alpha-cobrotoxin. Activation of protein kinase A by cAMP also produced increases in phosphorylation, although quantitatively different from those induced by protein kinase C, in proteins and PIP. Simultaneous addition of PMA and cAMP at maximal doses resulted in only a partially additive effect on PIP labelling. These results show that inositol lipid turnover can be modulated by a protein kinase C and protein kinase A-dependent process involving the phosphorylation of a common protein. This could be PI kinase or PIP phosphatase or another protein regulating the activity of these enzymes.     

Rapid decreases in phosphatidylinositol in isolated luteal plasma membranes after stimulation by luteinizing hormone

Phospholipid concentrations were determined in plasma membrane preparations from porcine corpora lutea after incubation for 15 to 120 s without or with 0.5 microgram/ml luteinizing hormone (LH) or 2 microM dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP). Treatment with LH caused a dramatic loss of 9 nmol in plasma membrane phosphatidylinositol (PI)/mg protein after 15 s of incubation, but no significant changes in other measurable phospholipids. Also, phospholipid concentrations were unchanged in untreated and dbcAMP-treated plasma membranes. The nature of the LH-induced decrease in PI was studied by incubating plasma membrane preparations for 15 s with [gamma 32P] adenosine 3',5'-triphosphate (ATP). 32P was incorporated only into three phospholipids: phosphatidic acid, phosphatidylinositol 4'-phosphate (PIP), and phosphatidylinositol 4',5'-bisphosphate (PIP2). Although LH generated small but significant increases in labeling of PIP and PIP2, less than 0.5 nmol of total phospholipids/mg protein were radiolabeled in 15 s. Phosphatidylinositol kinase activity, the enzyme that converts PI into PIP, was not affected by LH or dbcAMP treatment. However, incubation of luteal plasma membranes for 15 s with LH resulted in an increase of approximately 2 nmol 1,2-diacylglycerol/mg protein more than that observed in untreated or dbcAMP-treated plasma membranes. In summary, these experiments suggest that LH may stimulate hydrolysis of PI (and possibly PIP and PIP2) in isolated luteal plasma membranes.     

Metabolism and functions of inositol phosphates

Activation of Ca2+-mobilizing receptors rapidly increases the cytoplasmic Ca2+ concentration both by releasing Ca2+ stored in endoplasmic reticulum and by stimulating Ca2+ entry into the cells. The mechanism by which Ca2+ release occurs has recently been elucidated. Receptor activation of phospholipase C results in the hydrolysis of the plasma membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to yield two intracellular messengers, diacylglycerol (DAG) and (1,4,5)inositol trisphosphate [(1,4,5)IP3]. DAG remains in the plasma membrane where it stimulates protein phosphorylation via the phospholipid-dependent protein kinase C. (1,4,5)IP3 diffuses to and interacts with specific sites on the endoplasmic reticulum to release stored Ca2+. Receptor stimulation of phospholipase C appears to be mediated by one or more guanine nucleotide-dependent regulatory proteins by a mechanism analogous to hormonal activation of adenylyl cyclase. The actions of (1,4,5)IP3 on Ca2+ mobilization are terminated by two metabolic pathways, sequential dephosphorylation to inositol bisphosphate (IP2), inositol monophosphate (IP) and inositol or by phosphorylation to inositol tetrakisphosphate (IP4) and sequential dephosphorylation to different inositol phosphates. A sustained cellular response also requires Ca2+ entry into the cell from the extracellular space. The mechanism by which hormones increase Ca2+ entry is not known; a recent proposal involving movement of Ca2+ through the endoplasmic reticulum, possibly regulated by IP4, will be considered here.     

Adhesion to fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown

The aim of these experiments was to investigate whether inositol lipids might mediate some of the effects of extracellular matrix (ECM) on cellular form and functions. The lipid phosphatidylinositol bisphosphate (PIP2) plays a role in cytoskeletal regulation while its hydrolysis products, diacylglycerol and inositol triphosphate, serve as second messengers. We therefore measured the effect of adhesion to fibronectin (FN) on PIP2 and its hydrolysis products, in the presence and absence of the soluble mitogen PDGF. PDGF induced a threefold increase in release of water-soluble inositol phosphates in C3H 10T1/2 fibroblasts when cells were attached to FN, but had little effect in suspended cells. Suppression of inositol phosphate release in unattached cells was not due to dysfunction of the PDGF receptor or failure to activate phospholipase C-gamma; PDGF induced similar tyrosine phosphorylation of PLC-gamma under both conditions. By contrast, the total mass of phosphatidylinositol bisphosphate (PIP2), the substrate for PLC-gamma, was found to decrease by approximately 80% when cells were detached from their ECM attachments and placed in suspension in the absence of PDGF. PIP2 levels were restored when suspended cells were replated on FN, demonstrating that the effect was reversible. Furthermore, a dramatic increase in synthesis of PIP2 could be measured in cells within 2 min after reattachment to FN in the absence of PDGF. These results show that FN acts directly to stimulate PIP2 synthesis, and that it also enhances PIP2 hydrolysis in response to PDGF. The increase in PIP2 induced by adhesion may mediate some of the known effects of FN on cell shape and cytoskeletal organization, while regulation of inositol lipid hydrolysis may provide a means for integrating hormone- and ECM-dependent signaling pathways.     

The decrease in phosphatidylinositol 4,5-bisphosphate in ADP-stimulated washed rabbit platelets is not primarily due to phospholipase C activation.

Addition of 10 micron-ADP to washed rabbit platelets caused platelet shape change and aggregation without release of the contents of the amine-storage granules, and caused a transient decrease (8.8% at 10 s) in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2). By 20 s the decrease in PIP2 was no longer apparent, but by 60 s the amount of PIP2 was again decreased. Addition of thrombin (1 unit/ml), which causes platelet shape change, aggregation and the release of the contents of the amine-storage granules, caused a decrease in the amount of PIP2 (8.0% at 10 s); at 60 s the amount of PIP2 was not significantly different from that in controls. In platelets prelabelled with [3H]glycerol, the specific radioactivity of PIP2 was increased at 10 s in ADP-stimulated platelets, and unchanged in thrombin-stimulated platelets. In platelets prelabelled with [3H]inositol and incubated with 20 mM-Li+ to inhibit the degradation of the inositol phosphates to inositol, there was no increase in the labelling of inositol trisphosphate (IP3) upon stimulation with ADP. In contrast, stimulation with thrombin caused a significant increase in the labelling of IP3 at 10 s. These differences in the changes in polyphosphoinositide metabolism in ADP- and thrombin-stimulated platelets are consistent with the hypothesis that the decrease in PIP2 in ADP-stimulated platelets may be due not to degradation of PIP2 by phospholipase C, but rather to a shift in the equilibrium between PIP2 and phosphatidylinositol 4-phosphate (PIP). Increases in the labelling of phosphatidic acid at 10 s and of inositol bisphosphate and inositol phosphate after 20 s are consistent with phospholipase C being stimulated through some other mechanism that leads to the degradation of PIP and phosphatidylinositol; one possibility is that ADP causes an increase in cytoplasmic Ca2+.     

Light-Stimulated Inositol Phospholipid Turnover in Samanea saman Pulvini : Increased Levels of Diacylglycerol

Leaflet movement in Samanea saman is driven by an endogenous circadian clock and by light. We are investigating whether the effects of light on leaflet movement are mediated by increased inositol phospholipid turnover. We demonstrated previously that irradiation of excised pulvini with 15 to 30 seconds of white light decreases the levels of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate and increases the levels of inositol phosphates. We now report that the diacylglycerol level increases after 30 seconds of white light but returns to below the control level after 10 minutes of white light.     

Emerging cell biological functions of phosphatidylinositol 5 phosphate 4 kinase

The generation of phosphoinositides (PIs) with spatial and temporal control is a key mechanism in cellular organization and signaling. The synthesis of PIs is mediated by PI kinases, proteins that are able to phosphorylate unique substrates at specific positions on the inositol headgroup to generate signaling molecules. Phosphatidylinositol 5 phosphate 4 kinase (PIP4K) is one such lipid kinase that is able to specifically phosphorylate phosphatidylinositol 5 phosphate, the most recently discovered PI to generate the well-known and abundant PI, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P₂]. PIP4K appears to be encoded only in metazoan genomes, and several genetic studies indicate important physiological functions for these enzymes in metabolism, immune function, and growth control. PIP4K has recently been reported to localize to multiple cellular compartments, including the nucleus, plasma membrane, endosomal systems, and autophagosome. However, the biochemical activity of these enzymes that is relevant to these physiological functions remains elusive. We review recent developments in this area and highlight emerging roles for these enzymes in cellular organization.     

Protein kinase C-dependent phosphorylation of profilin is specifically stimulated by phosphatidylinositol bisphosphate (PIP2)

Calf spleen profilin is shown to be an in vitro substrate of purified human placental protein kinase C (PKC), with an apparent Km of 4 microM. Phosphatidylinositol bisphosphate (PIP2) was an effective activator of the profilin phosphorylation by PKC and caused a maximum 13-fold increase of Vmax with a half maximal effect at 40 micrograms/ml. The action of PIP2 was not mimicked by phosphatidylserine, phosphatidic acid or phosphatidylinositol, whereas phosphatidylinositol monophosphate was slightly stimulatory. By contrast, protein kinase C-dependent phosphorylation of histone type III-S, myelin basic protein or lipocortin-I was not affected by PIP. It is suggested that PIP2 modifies the nature of the profilin-PKC interactions.     

Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in isolated rat hepatocytes and plasma membranes.

Rat hepatocytes whose phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) had been labelled for 60 min with 32P were treated with glucagon for 10 min or phenylephrine for 2 min. Glucagon caused a 20% increase in PIP but no change in PIP2 whereas phenylephrine caused a similar increase in PIP but a 15% decrease in PIP2. Addition of both hormones together for 10 min produced a 40% increase in PIP. A crude liver mitochondrial fraction incubated with [32P]Pi and ADP incorporated label into PIP, PIP2 and phosphatidic acid. The PIP2 was shown to be in contaminating plasma membranes and PIP in both lysosomal and plasma-membrane contamination. A minor but definitely mitochondrial phospholipid, more polar than PIP2, was shown to be labelled with 32P both in vitro and in hepatocytes. The rate of 32P incorporation into PIP was faster in mitochondrial/plasma-membrane preparations from rats treated with glucagon or if 3 microM-Ca2+ and Ruthenium Red were present in the incubation buffer. Loss of 32P from membranes labelled in vitro was shown to be accompanied by formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate, and was faster in preparations from glucagon-treated rats or in the presence of 3 microM-Ca2+. It is concluded that glucagon stimulates both PIP2 phosphodiesterase and phosphatidylinositol kinase activities, as does the presence of 3 microM-Ca2+. The resulting formation of IP3 may be responsible for the observed release of intracellular Ca2+ stores. The roles of a guanine nucleotide regulatory protein and phosphorylation in mediating these effects are discussed.     

Interferon transiently modulates intranuclear signalling system in erythroleukemia Friend cells.

The effect of human recombinant DNA interferon alpha type A on nuclear inositol lipids, diacylglycerol (DAG) and DNA metabolism has been investigated in Friend erythroleukemia cells. A transient enhancement of phosphatidylinositol (4,5) - bisphosphate (PIP2) phosphorylation together with an increase of diacylglycerol mass were observed in nuclei isolated from cells treated with interferon alpha for 90 min. At the same time, a marked reduction of DNA polymerase alpha activity was observed, suggesting a possible involvement of nuclear inositol fraction in the response of the cell nucleus to interferon treatment.     

Ethanol stimulates shape change in human platelets by activation of phosphoinositide-specific phospholipase C

Administration of ethanol to human platelets resulted in a rapid shape change which was maximal within 30 s. Ethanol did not cause aggregation or secretion of ATP at any time and inhibited aggregation induced by collagen. In platelets that were loaded with the intracellular calcium indicator fura2, ethanol induced a rapid mobilization of calcium from internal, thrombin-sensitive pools. Cytosolic calcium increased to a maximum within 5 s and decreased slowly over the ensuing 5 min to near basal levels. The mobilization of calcium by ethanol coincided with the rapid formation of phosphatidic acid and a decrease in the level of phosphatidylinositol 4,5-bisphosphate, as measured in 32P-labeled platelets. In platelets labeled with myo-[2-3H]inositol, ethanol caused a 20-30% increase in the levels of inositol (1,4,5)-trisphosphate and inositol bisphosphate within 10 s. Ethanol also induced the transient phosphorylation of myosin light chain (20 kDa) and a 40 kDa protein, a known substrate for protein kinase C. The results indicate that ethanol activates phosphoinositide-specific phospholipase C in human platelets. The subsequent mobilization of intracellular calcium and activation of protein kinase C can account for the shape change induced by ethanol.     

脂多糖增加小鼠巨噬细胞多磷酸肌醇脂代谢转换

多磷酸肌醇脂（这里指ＰＩＰ和ＰＩＰ２）的代谢在细胞信息传递和膜运转中起着重要的作用．脂多糖（ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）于激活小鼠腹腔巨噬细胞（Ｍφ）初期（０．５～１ｈ）和后期（１６ｈ）明显增加来自［γ－３２Ｐ］ＡＴＰ的３２Ｐ参入Ｍφ的ＰＩＰ和ＰＩＰ２,３２Ｐ参入ＰＩＰ２的显著性大于ＰＩＰ．ＬＰＳ的这种作用在其激活Ｍφ的初期不受酪氨酸蛋白激酶抑制剂ｇｅｎｉｓｔｅｉｎ、蛋白激酶Ａ激动剂（ｆｏｒｓｋｏｌｉｎ）及百日咳毒素的影响;但佛波酯（ＰＭＡ）长时间预处理的Ｍφ（其ＰＫＣ活性被耗竭）再受ＬＰＳ刺激,［３２Ｐ］ＰＩＰ２水平较ＬＰＳ刺激未受ＰＭＡ预处理的Ｍφ明显降低．结果表明,ＬＰＳ于激活Ｍφ的初期和后期更显著增加ＰＩ４Ｐ－５激酶的活性,导致ＰＩＰ２合成增加,ＰＩＰ２合成的增加可能与Ｍφ激活时不同时期所表达的功能相关．     

Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells to stimulate phospholipase C-mediated hydrolysis of membrane phosphoinositides and subsequent formation of diacylglycerol and inositol phosphates. In intact cells, angiotensin II induces a dose-dependent increase in diglyceride which is detectable after 5 s and sustained for at least 20 min. Angiotensin II (100 nM)-stimulated diglyceride formation is biphasic, peaking at 15 s (227 +/- 19% control) and at 5 min (303 +/- 23% control). Simultaneous analysis of labeled inositol phospholipids shows that at 15 s phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP) decline to 52 +/- 6% control and 63 +/- 5% control, respectively, while phosphatidylinositol (PI) remains unchanged. In contrast, at 5 min, PIP2 and PIP have returned toward control levels (92 +/- 2 and 82 +/- 4% control, respectively), while PI has decreased substantially (81 +/- 2% control). The calcium ionophore ionomycin (15 microM) stimulates diglyceride accumulation but does not cause PI hydrolysis. 4 beta-Phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibits early PIP and PIP2 breakdown and diglyceride formation, without inhibiting late-phase diglyceride accumulation. Thus, angiotensin II induces rapid transient breakdown of PIP and PIP2 and delayed hydrolysis of PI. The rapid attenuation of polyphosphoinositide breakdown is likely caused by a protein kinase C-mediated inhibition of PIP and PIP2 hydrolysis. While in vascular smooth muscle stimulated with angiotensin II inositol 1,4,5-trisphosphate formation is transient, diglyceride production is biphasic, suggesting that initial and sustained diglyceride formation from the phosphoinositides results from different biochemical and/or cellular processes.     

Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol.

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.