Characterization of a phosphatidylinositol 4-phosphate-specific phosphomonoesterase in rat liver nuclear envelopes

Incubation of rat liver nuclear envelopes with [gamma-32P]ATP resulted in the synthesis of phosphatidylinositol-[4-32P]phosphate (PIP). Degradation of endogenously labeled PIP was observed upon the dilution of the labeled ATP with an excess of unlabeled ATP. This degradation was most rapid in the presence of EDTA, and was inhibited by MgCl2 and CaCl2. To further characterize the degradative activity, phosphatidylinositol[4-32P]phosphate and phosphatidylinositol [4,5-32P]bisphosphate (PIP2) were synthesized and isolated from erythrocyte plasma membranes. The 32P-labeled phospholipids were then resuspended in 0.4% Tween 80, a detergent that did not inhibit degradation of endogenously labeled PIP, and mixed with nuclear envelopes. [32P]PIP and [32P]PIP2 were degraded at rates of 2.25 and 0.04 nmol min-1 mg nuclear envelope protein-1, respectively. Only 32P was released from phosphatidyl[2-3H]inositol-[4-32P]phosphate, indicating that hydrolysis of PIP was due to a phosphomonoesterase activity (EC 3.1.3.36) in nuclear envelopes. Similarly, anion-exchange chromatographic analysis of the water-soluble products released from [32P]PIP indicated that inorganic phosphate was the sole 32P-labeled product. Hydrolysis of PIP was most rapid at neutral pH, and was not affected by inhibitors of acid phosphatase or alkaline phosphatase. Hydrolysis of PIP was also not inhibited by nonspecific phosphatase substrates, such as glycerophosphate, p-nitrophenylphosphate, AMP, or glucose 6-phosphate. Hydrolysis was stimulated by putrescine, and was inhibited by inositol 2-phosphate, spermidine, spermine, and neomycin.     

Phorbol ester-stimulated hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase D in HeLa cells. Evidence that the basal turnover of phosphoglycerides does not involve phospholipase D

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [3H]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.     

Subcellular site and mechanism of vasopressin-stimulated hydrolysis of phosphoinositides in rat hepatocytes

The intracellular site of vasopressin-induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 s of vasopressin treatment of hepatocytes prelabeled with 32Pi, the levels of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4,5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%, then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P] PI-P and [32P]PI-P2 were observed in the other subcellular fractions, and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol in the plasma membrane decreased by 15% after 45 s of vasopressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes, it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphosphoinositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (200 nM) of free calcium when assayed at low ionic strength, but the calcium requirement shifted to micromolar concentrations under isosmotic, intracellular-like, ionic conditions. Addition of vasopressin (200 nM) to the 32P-labeled plasma membranes stimulated the breakdown of 20% of the [32P]PI-P2 present in the plasma membranes in 1 min when assayed under isosmotic conditions in the presence of 2 nM MgCl2 and approximately 200 nM free calcium. This suggests that the phosphoinositide-specific phosphodiesterase is not active under normal cellular conditions, but is activated upon the addition of vasopressin to the intact cell.     

Plasma membrane-associated phosphatase activities hydrolyzing [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate

We describe a procedure of preparing [32P]phosphotyrosyl histones with minimal contamination by 32P-labeled lipids; the latter was usually found to be mixed with the phosphoproteins when the cell membrane-enriched fraction of A-431 cells was used as a source of tyrosine kinase. The phosphatase activities previously found to be associated with the plasma membranes of a human astrocytoma were resolved using purified [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate. In comparison with the phosphotyrosyl protein phosphatase, the phosphatidylinositol phosphate phosphatase activity is more active over a broad range of pH values, and its activity is inhibited by fluoride, zinc chloride, and lower concentrations of vanadate.     

Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603-7608) that serotonin induces: a very rapid formation of PI; and alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin.

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.     

Phosphodiesteratic breakdown of endogenous polyphosphoinositides in nerve ending membranes

When a membrane preparation, obtained by freezing and thawing nerve endings labeled by preincubation with 32pi, is incubated in the presence of millimolar Ca2+, there is a rapid and selective loss of label from the polyphosphoinositides and a concomitant increase in labeled inositol di- and triphosphates recovered. When the membranes are not prelabeled and are exposed to [gamma-32P]ATP under similar conditions, phosphatidate labeling is enhanced, indicating increased availability of diacylglycerol. These observations provide evidence for the presence of membrane-bound, Ca2+-stimulated phosphodiesterase activity (phospholipase C) acting on endogenous polyphosphoinositides. The implications of these findings are discussed in respect to the "phosphatidylinositol" cycle.     

Calcium promotes the accumulation of polyphosphoinositides in intact and permeabilized bovine adrenal chromaffin cells

1. Because cellular pools of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate turn over rapidly during phospholipase C stimulation, the continuing production of inositol phosphates requires continuing synthesis from phosphatidylinositol of the polyphosphoinositides. In the present study in adrenal chromaffin cells, we examined the effects of nicotinic stimulation and depolarization in intact cells and micromolar Ca2+ in permeabilized cells on the levels of labeled polyphosphoinositides. We compared the effects to muscarinic stimulation in intact cells and GTP gamma S in permeabilized cells. 2. Nicotinic stimulation, elevated K+, and muscarinic stimulation cause similar production of inositol phosphates (D. A. Eberhard and R. W. Holz, J. Neurochem. 49:1634-1643, 1987). Nicotinic stimulation and elevated K+ but not muscarinic stimulation increased the levels of [3H]inositol-labeled phosphatidylinositol phosphate by 30-60% and [3H]phosphatidylinositol bisphosphate by 25-30%. The increase required Ca2+ in the medium, was maximal by 1-2 min, and was not preceded by an initial decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. 3. In digitonin-permeabilized cells, Ca2+ caused as much as a twofold increase in [3H]phosphatidylinositol phosphate and [3H]phosphatidylinositol bisphosphate. Similarly, Ca2+ enhanced the production of [32P]phosphatidylinositol phosphate and [32P]phosphatidylinositol bisphosphate in the presence of [gamma-32P]ATP. In contrast, GTP gamma S in permeabilized cells decreased polyphosphoinositides in the presence or absence of Ca2+. 4. The ability of Ca2+ to increase the levels of the polyphosphoinositides decayed with time after permeabilization. The effect of Ca2+ was increased when phosphoesterase and phospholipase C activities were inhibited by neomycin. 5. These observations suggest that Ca2+ specifically enhances polyphosphoinositide synthesis at the same time that it activates phospholipase C.     

Elevated cytosolic Ca2+ activates phospholipase D in human platelets

We have examined the activation of phospholipase D in human platelets treated with alpha-thrombin. When incubated with 1-O-[9,10-3H2]hexadecyl-2-lysophosphatidylcholine (PtdCho) and 1-alkyl-[32P]lysoPtdCho for 2 h, platelets formed 3H/32P-labeled PtdCho in a ratio of 11:1. After incubation of such labeled platelets with alpha-thrombin for 5 min, increased accumulation of 3H/32P-labeled phosphatidic acid (PtdOH) was detected in the same ratio, indicating the action of phospholipase D. The Ca2+ ionophore A23187 and alpha-thrombin each stimulated the formation of labeled PtdOH as above in a time- and concentration-dependent manner, with only minor changes in labeled diglyceride. A23187 was able to cause increases in labeled PtdOH comparable to those observed with alpha-thrombin. beta-Phorbol 12,13-dibutyrate, an activator of protein kinase C, only slightly stimulated the accumulation of labeled PtOH. The protein kinase C inhibitor, staurosporine, totally blocked these changes but only slightly inhibited the increases in labeled PtdOH promoted by alpha-thrombin. These results suggest that an increase in intracellular Ca2+, rather than protein kinase C activity, is a major factor regulating phospholipase D in platelets exposed to alpha-thrombin. We have also examined the relative contributions of phospholipase D and diglyceride kinase (following phospholipase C action) to PtdOH accumulation in [32P]Pi-labeled platelets by comparing the 32P-specific radioactivities of PtdOH, PtdCho, and metabolic gamma-ATP in control and alpha-thrombin-exposed platelets. Based on these determinations, we conclude that 13 and 87% of incremental PtdOH in human platelets exposed to alpha-thrombin arises via phospholipase D acting on PtdCho and phospholipase C/diglyceride kinase, respectively.     

Increased phosphatidylinositol synthesis in rat embryo fibroblasts after growth stimulation and its inhibition by delta-hexachlorocyclohexane

When resting rat embryo fibroblasts are stimulated to grow, a substantial increase in phosphatidylinositol synthesis can be observed. This increase cannot be explained by increased glucose uptake or glycolysis. delta-Hexachlorocyclohexane having the same configuration as myo-inositol, inhibits phosphatidyl inositol synthesis as well as DNA synthesis and mitosis, but has no effect on phosphatidyl choline synthesis. When delta-hexachlorocyclohexane is added to fibroblast cultures during the first hours after stimulation, a delay of DNA synthesis and mitosis compared to uninhibited cultures can be observed. Since delta-hexachlorocyclohexane also inhibits the uptake of nucleotides, hexoses and amino acids, it is suggested that phosphatidylinositol is necessary for the proper functioning of those receptors and carriers which are an essential part of the early cellular processes after growth stimulation, and this role of phosphatidyl-inositol may explain its increased turnover in growing cells. The increased phosphatidylinositol synthesis could not be associated to one of the subcellular fractions. When cells were labeled with [32P]orthophosphate during the first 10 min after growth stimulation and were subsequently separated into cellular fractions such as nuclei, mitochondria, plasma membranes and microsomes, no significant differences in radioactivity of phosphatidylinositol among those fractions could be observed.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases.

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.     

Evidence that phospholipid turnover is the signal transducing system coupled to serotonin-S2 receptor sites

Upon stimulation with serotonin of washed human platelets prelabeled with [32P]orthophosphate, we found an approximately 250% increase in [32P]phosphatidic acid (PA) formation, a small decrease in [32P]phosphatidylinositol 4,5-bisphosphate, and a concomitant increase in [32P]phosphatidylinositol 4-phosphate. Using [3H]arachidonate for prelabeling, [3H]diacylglycerol accumulated transiently at 10 s after addition of the agonist, [3H]PA increased but to a lower extent compared to 32P-labeled lipid, and the formation of both [3H]polyphosphoinositides increased. The serotonin-induced dose-dependent changes in [32P]PA correlate with its effect on the changes in slope of aggregation of platelets. The potency of 13 drugs to antagonize the serotonin-induced PA formation closely corresponds to both their potency to inhibit platelet aggregation and their binding affinity for serotonin-S2 receptor sites. It is suggested that at least part of the signal transducing system following activation of the serotonin-S2 receptors involves phospholipase C catalyzed inositol lipid breakdown yielding diacylglycerol which is subsequently phosphorylated to PA.     

Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase.

Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.     

Chemoattractant receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in human polymorphonuclear leukocyte membranes. Requirement for a guanine nucleotide regulatory protein

Incubation of plasma membranes from human polymorphonuclear leukocytes (PMNs) with [gamma-32P]ATP in the presence of MgCl2 resulted in the formation of 32P-labeled phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Membranes from PMN specific and azurophil granules synthesized only PIP, suggesting that PIP2 metabolism is confined to the plasma membrane in PMNs. Further incubations of the labeled plasma membranes for 60 s in the presence of 1 mM CaCl2 resulted in the hydrolysis of approximately 40 and 50% of the labeled PIP and PIP2, respectively. In the presence of 2 microM added CaCl2, PIP and PIP2 levels were unchanged by incubation with either the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) at 0.1 microM or by 10 microM GTP; however, addition of fMet-Leu-Phe plus GTP together resulted in a 11 and 28% decrease in PIP and PIP2, respectively. These treatments had no effect on PA levels. No additional radiolabeled organic-soluble products were detected after treatment with fMet-Leu-Phe plus GTP. Incubation of intact PMNs, with the Bordetella pertussis toxin (islet-activating protein) eliminated the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in the isolated plasma membranes, but did not inhibit PIP2 degradation in the presence of 1 mM CaCl2. These results provide the first direct evidence that the fMet-Leu-Phe receptor in PMN membranes is coupled to polyphosphoinositide hydrolysis through an islet-activating protein-sensitive guanine nucleotide regulatory protein.     

Rapid increase in inositol pentakisphosphate accumulation by nicotine in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with nicotine (10 microM), a large and transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s, then declined to the basal level at 2 min. Nicotine also induced [3H]inositol tetrakisphosphate (InsP4) and [3H]inositol hexakisphosphate (InsP6) accumulation with a slower time course and a lesser magnitude than [3H]InsP5. The peaks of [3H]InsP4, [3H]InsP5 and [3H]InsP6 coincided with those of 32P radioactivity, when cells were doubly labeled with [3H]inositol and inorganic 32P. These results suggest that inositol pentakisphosphate is rapidly increased by nicotine, a cholinergic agonist, in cultured adrenal chromaffin cells.     

Stimulation by glucose and carbamylcholine of phospholipase C in pancreatic islets

Phosphoinositide hydrolysis in intact pancreatic islet cells was investigated in an indirect but dynamic manner by monitoring the efflux of radioactivity from islets prelabelled with [3H]inositol. A rise in glucose concentration provoked a rapid, modest but sustained increase in effluent radioactivity, this phenomenon being abolished in the absence of extracellular Ca2+ or presence of verapamil. The release of [3H]inositol was also stimulated at high extracellular K+ concentration, but not by gliclazide. Whether in the presence or absence of glucose, carbamylcholine provoked a marked increase in effluent radioactivity. The response to the cholinergic agent was decreased in the presence of verapamil or absence of extracellular Ca2+ and abolished in the presence of atropine or LiCl. These results suggest that an increase in cytosolic Ca activity, as caused by glucose or membrane depolarization, may cause activation of phospholipase C. In response to cholinergic agents, however, the enzymic activation, although modulated by Ca2+ availability, may result directly from the occupation of muscarinic receptors.     

Vasopressin-stimulated phosphorylation of rat liver phospholipid methyltransferase in isolated hepatocytes

Addition of vasopressin (1 microM) to isolated rat hepatocytes prelabeled with [32P]phosphate was accompanied by a 250% increase in the phosphorylation of phospholipid methyltransferase. Vasopressin-stimulated phospholipid methyltransferase phosphorylation was time- and dose-dependent. 32P-labeled phospholipid methyltransferase was recovered by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. After electrophoresis, phospholipid methyltransferase was electroeluted from the polyacrylamide gel and subjected to tryptic digestion or HCl hydrolysis. Analysis of 32P-labeled peptides reveals only one site of phosphorylation and the analysis of [32P]phosphoamino acids indicates that phosphoserine is the only labeled amino acid.     

Metabolic regulation during early frog development: flow of glycolytic carbon into phospholipids in Xenopus oocytes and fertilized eggs

32P-labeled glucose 6-phosphate, [32P]phosphoenolpyruvate, and [gamma-32P]ATP were injected into oocytes and fertilized eggs of Xenopus laevis, and the incorporation of the 32P label was followed into phospholipids. Several classes of phospholipids incorporated 32P label from the injected glycolytic intermediates, including lysophosphatidic acid, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol phosphates, inferring de novo synthesis of these lipids from dihydroxyacetone phosphate or glycerol 3-phosphate. Injection of [gamma-32P]ATP into oocytes and fertilized eggs led to labeling of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, indicating an active phosphatidylinositol cycle in resting oocytes and fertilized eggs. Maturation and fertilization of the oocyte led to a qualitative change in phosphatidylinositol metabolism, increased labeling of phosphatidylinositol phosphate compared to phosphatidylinositol bisphosphate (either from glycerol 3-phosphate or from ATP). This change occurs late in the maturation process, and the new pattern of phosphatidylinositol metabolism is maintained during the rapid cleavage stages of early embryogenesis.     

Plasma membrane lipid metabolism of petunia petals during senescence

The specific activities of 6 enzymes, which are involved in the synthesis and catabolism of membrane lipids, were monitored in plasma membranes isolated from petunia petals during senescence. These included phosphatidylinositol (PI) kinase (EC 2.7.1.67), phosphatidylinositol monophosphate (PIP) kinase (EC 2.7.1.68). diacylglycerol (DAG) kinase (EC 2.7.1.107), phospholipase A (EC 3.1.1.4) and PIP- and PIP₂-phospholipase C˙(EC 3.1.4.3). Using endogenous substrate, the [³²P]PA and [³²P]PIP₂ formation increased to 140 and 200%, respectively, of the day 1 value by 4 days after harvest. There was no significant change in [³²P]PIP formation during the same time period. On the fifth day the petals wilted and the [³²P]PA and [³²P]PIP formation declined significantly. In contrast, the [³²P]PIP₂ formation remained high in the day 5 petals. When the lipid kinase activities were assayed in the membranes in the presence of exogenous substrate the specific activity of all of the enzymes increased. and the changes in [³²P]PA production over the 5-day period were similar to those observed with endogenous substrate. When exogenous PI and PIP were added, however, there was no longer an increase in [³²P]PIP₂ formation by plasma membranes of day 4 petals and [³²P]PIP formation significantly decreased. The relative decrease in PIP and PIP₂ formation by day 4 membranes when exogenous substrate was added may have resulted from differences in the lipase activities in the day 1 and day 4 membranes. The plasma membrane A-type phospholipase activity increased throughout the 5 day period, and phospholipase C activity increased two-fold between day 1 and day 4. Such changes in the metabolism of the plasma membrane lipids during flower senescence would affect the ability of the petals to use inositol phospholipid-based signal transduction pathways.