Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock

The inositol phospholipids phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) comprise 14.8, 1.2, and 0.3 mol %, respectively, of Dunaliella salina phospholipids. In isolated plasma membrane fractions, PIP and PIP2 are highly concentrated, together comprising 9.5 mol % of plasmalemma phospholipids. The metabolism of these inositol phospholipids and phosphatidic acid (PA) is very rapid under normal growth conditions. Within 5 min after introduction of 32Pi into the growth medium, over 75% of lipid-bound label was found in these quantitatively minor phospholipids. Within 2 min after a sudden hypoosmotic shock, the levels of PIP2 and PIP dropped to 65 and 79%, respectively, of controls. Within the same time frame, PA rose to 141% of control values. These data suggest that a rapid breakdown of the polyphosphoinositides may mediate the profound morphological and physiological changes which allow this organism to survive drastic hypoosmotic stress. In contrast to hypoosmotic shock, hyperosmotic shock induced a rise in PIP2 levels to 131% of control values, whereas the level of PA dropped to 56% of controls after 4 min. These two different types of osmotic stress affect inositol phospholipid metabolism in a fundamentally opposite manner, with only hypoosmotic shock inducing a net decrease in polyphosphoinositides.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: II. Calcium Requirement

The calcium requirement for agonist-dependent breakdown of phosphatidylinositol and polyphosphoinositides has been examined in rat cerebral cortex. The omission of added Ca2+ from the incubation medium abolished [3H]inositol phosphate accumulation from prelabelled phospholipid induced by histamine, reduced that due to noradrenaline and 5-hydroxytryptamine, but did not affect carbachol-stimulated breakdown. EC50 values for agonists were unaltered in the absence of Ca2+. Removal of Ca2+ by preincubation with EGTA (0.5 mM) abolished all responses, but complete restoration was achieved by replacement of Ca2+. The EC50 for Ca2+ for histamine-stimulated [3H]inositol phosphate accumulation was 80 microM. Noradrenaline-stimulated breakdown was antagonised by manganese (IC50 1.7 mM), but not by the calcium channel blockers nitrendipine or nimodipine (30 microM). The calcium ionophore A23187 stimulated phosphatidylinositol/polyphosphoinositide hydrolysis with an EC50 of 2 microM, and this response was blocked by EGTA. Omission of Ca2+ or preincubation with EGTA or Mn2+ (EC50 = 230 microM) greatly enhanced the incorporation of [3H]inositol into phospholipids. The IC50 for Ca2+ in inhibiting incorporation was 25 microM. The results show that different receptors mediating phosphatidylinositol/polyphosphoinositide breakdown in rat cortex have quantitatively different Ca2+ requirements, and it is suggested that rigid opinions regarding phosphatidylinositol/polyphosphoinositide breakdown as either cause or effect of calcium mobilisation in rat cortex are inappropriate.     

Differential activation of phosphatidylinositol deacylation and a pathway via diphosphoinositide in macrophages responding to zymosan and ionophore A23187

The inositol phospholipids of peritoneal macrophages were prelabeled with [3H]inositol to enable studies on the enzymatic mechanisms of stimulus-induced phosphatidylinositol breakdown. Ionophore A23187 induced a rapid breakdown of phosphatidylinositol in the presence of Ca2+ with 25% loss occurring within 5 min. The main water-soluble product of this breakdown was identified as inositol diphosphate. Since the accumulation of inositol diphosphate far exceeded the concomitant decrease in polyphosphoinositides, an increased phosphorylation of phosphatidylinositol must have preceded, or accompanied, the degradation of diphosphoinositide. The degradation of phosphatidylinositol induced by A23187 was shown to be strictly dependent on Ca2+. The monovalent cation ionophore monensin and platelet-activating factor increased the level of diphosphoinositide but caused no net degradation of inositol phospholipids. The same effect was seen with ionophore A23187 in the absence of Ca2+. Zymosan particles also induced extensive degradation of phosphatidylinositol. Products of phosphodiesterase-catalyzed cleavage of inositol lipids were observed, but the pathway of deacylation dominated as evidenced by the accumulation of lysophosphatidylinositol and glycerophosphoinositol. Deacylation was also enhanced in response to concanavalin A. Thus, in mouse peritoneal macrophages phosphatidylinositol breakdown occurred primarily by deacylation or via diphosphoinositide, depending on the stimulus, rather than through a phosphatidylinositol phosphodiesterase reaction.     

Enhancement by Potassium of Carbachol-Stimulated Inositol Phospholipid Breakdown in Rat Cerebral Cortical Miniprisms: Comparison with Other Depolarising Agents

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.     

Thyrotropin-releasing hormone rapidly activates the phosphodiester hydrolysis of polyphosphoinositides in GH3 pituitary cells. Evidence for the role of a polyphosphoinositide-specific phospholipase C in hormone action

The early actions of thyrotropin-releasing hormone (TRH) have been studied in hormone-responsive clonal GH3 rat pituitary cells. Previous studies had demonstrated that TRH promotes a "phosphatidylinositol response" in which increased incorporation of [32P]orthophosphate into phosphatidylinositol and phosphatidic acid was observed within minutes of hormone addition. The studies described here were designed to establish whether increased labeling of phosphatidylinositol and phosphatidic acid resulted from prior hormone-induced breakdown of an inositol phosphatide. GH3 cells were prelabeled with [32P]orthophosphate or myo-[3H]inositol. Addition of TRH resulted in the rapid disappearance of labeled polyphosphoinositides, whereas levels of phosphatidylinositol and other phospholipids remained unchanged. TRH-promoted polyphosphoinositide breakdown was evident by 5 S and maximal by 15 s of hormone treatment. Concomitant appearance of inositol polyphosphates in [3H]inositol-labeled cells was observed. In addition, TRH rapidly stimulated diacylglycerol accumulation in either [3H]arachidonic- or [3H]oleic acid-labeled cultures. These results indicate that TRH rapidly causes activation of a polyphosphoinositide-hydrolyzing phospholipase C-type enzyme. The short latency of this hormone effect suggests a proximal role for polyphosphoinositide breakdown in the sequence of events by which TRH alters pituitary cell function.     

The polyphosphoinositide phosphodiesterase of erythrocyte membranes

1. A new assay procedure has been devised for measurement of the Ca(2+)-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [(32)P]P(i). They are incubated under appropriate conditions for activation of the phosphodiesterase and the released (32)P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca(2+). For both esters, optimum rates of release were seen at pH6.8-6.9. Mg(2+) did not provoke release of either ester. 3. Ca(2+) provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca(2+). 4. In the presence of Mg(2+), the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca(2+) and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg(2+)-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.     

Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid

The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.     

Evidence for differential activation of arachidonic acid metabolism in formylpeptide- and macrophage-activation-factor-stimulated guinea-pig macrophages.

Alterations of phospholipid and arachidonic acid metabolism were studied by treatment of guinea-pig peritoneal-exudate macrophages with chemotactic peptide, formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) and macrophage activation factor (MAF). The chemotactic peptide caused a rapid rearrangement in inositol phospholipids, including a breakdown of polyphosphoinositides within 30s, followed by a resultant formation of phosphatidylinositol (PI), diacylglycerol, phosphatidic acid and non-esterified arachidonic acid within 5 min. In addition to these sequential alterations, arachidonic acid was released mainly from PI. On the other hand, MAF induced a slow liberation of arachidonic acid, mainly from phosphatidylethanolamine (PE) and phosphatidylcholine (PC) by phospholipase A2 after the incubation period of 30 min, but not any rapid changes in phospholipids. Treatment of macrophages for 15 min with fMet-Leu-Phe produced the leukotrienes (LTs) B4, C4 and D4, prostaglandins (PG) E2 and F2 alpha and thromboxane (TX) B2. In contrast, MAF could not stimulate the production of arachidonic acid metabolites during the incubation period of 15 min, but could enhance that of PGE2, PGF2 alpha, TXB2 and hydroxyeicosatetraenoic acids at 6 h. However, the stimulated formation of LTs was not detected at any time. These results indicate that the effects of fMet-Leu-Phe on both phospholipid and arachidonic acid metabolism are very different from those mediated by MAF.     

Polyphosphoinositide breakdown as the initiating reaction in receptor-stimulated inositol phospholipid metabolism

All cell-surface receptors that bring about a rise in cytosol Ca2+ concentration upon stimulation appear also to provoke enhanced metabolism of inositol phospholipids. For many years, it has been thought that the initiating reaction in this response is phosphodiesterase-catalysed breakdown of phosphatidylinositol (PtdIns). However, recent experiments with hepatocytes, parotid gland and blowfly salivary gland have demonstrated very rapid breakdown of phosphatidylinositol-4, 5-bisphosphate (PtdIns4,5P2), and maybe also of PtdIns4P, in cells stimulated by Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, muscarinic cholinergic, substance P and 5-hydroxytryptamine). As with the disappearance of PtdIns that had been studied previously, this response is not Ca2+-mediated and shows a receptor occupation dose-response curve. The PtdIns 'breakdown' studied previously was probably utilization of PtdIns for resynthesis of polyphosphoinositides to replace the degraded PtdIns4,5P2. We suggest that the primary event in receptor-stimulated inositol phospholipid metabolism is phosphodiesterase attack upon PtdIns4,5P2 to yield 1,2-diacylglycerol and inositol-1,4, 5-trisphosphate, and that this is an essential coupling event in a general mechanism by which receptors mobilize Ca2+ in the cytosol of stimulated cells.     

Metabolism of inositol phosphates in parotid cells: implications for the pathway of the phosphoinositide effect and for the possible messenger role of inositol trisphosphate

Rat parotid acinar cells were used to investigate the time course of formation and breakdown of inositol phosphates in response to receptor-active agents. In cells preincubated with [3H]inositol and in the presence of 10 mM LiCl (which blocks hydrolysis of inositol phosphate), methacholine (10(-4)M) caused a substantial increase in cellular content of [3H]inositol phosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Subsequent addition of atropine (10(-4) M) caused breakdown of [3H]inositol trisphosphate and [3H]inositol bisphosphate and little change in accumulated [3H]inositol phosphate. The data could be fit to a model whereby inositol trisphosphate and inositol bisphosphate are formed from phosphodiesteratic breakdown of phosphatidylinositol bisphosphate and phosphatidylinositol phosphate respectively, and inositol phosphate is formed from hydrolysis of inositol bisphosphate rather than from phosphatidyl-inositol. Consistent with this model was the finding that [3H]inositol trisphosphate and [3H]inositol bisphosphate levels were substantially increased in 5 sec while an increase in [3H]inositol phosphate was barely detectable at 60 sec. These results indicate that in the parotid gland the phosphoinositide cycle is activated primarily by phosphodiesteratic breakdown of the polyphosphoinositides rather than phosphatidyl-inositol. Also, the results show that formation of inositol trisphosphate is probably sufficiently rapid for it to act as a second messenger signalling internal Ca2+ release in this tissue.     

Effects of luteinizing hormone on phosphoinositide metabolism in rat granulosa cells

Rat granulosa cells isolated from mature Graafian follicles were incubated with luteinizing hormone under various conditions in order to follow the synthesis and degradation of phospholipids. During acute incubations, luteinizing hormone provoked rapid and concentration-dependent increases in the incorporation of 32PO4 into phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides. Similarly, luteinizing hormone provoked increases in labeling of phosphatidylinositol and the polyphosphoinositides when granulosa cells were incubated with myo-[2-3H]inositol. When granulosa cells were prelabeled with 32PO4 in order to label phosphatidylinositol to constant specific radioactivity (4 h), luteinizing hormone treatment significantly increased 32P-phosphatidylinositol levels (23%). Comparable increases (27%) in the cellular concentrations of phosphatidylinositol were observed in response to luteinizing hormone. In pulse-chase experiments employing 32PO4 - or [3H]inositol-prelabeled cells, luteinizing hormone did not alter phospholipid degradation. In addition, luteinizing hormone did not stimulate degradation of polyphosphoinositides. These results demonstrate that: (a) luteinizing hormone has selective effects on phospholipid metabolism in rat granulosa cells which involve phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides, (b) luteinizing hormone increases net levels of phosphatidylinositol and presumably phosphatidic acid and the polyphosphoinositides, and (c) luteinizing hormone does not increase phospholipid degradation. Our findings suggest that luteinizing hormone provokes increases in de novo synthesis of phosphatidylinositol in rat granulosa cells. These changes in phospholipid metabolism may be important for steroidogenesis and other enzymatic processes during treatment with luteinizing hormone.     

Sympathetic denervation impairs agonist-stimulated phosphatidylinositol metabolism in rat parotid glands.

Substance P, muscarinic and alpha-adrenoceptor agonists stimulated the incorporation of [3H]inositol into phosphatidylinositol in rat parotid gland slices. Surgical denervation of the sympathetic input to the rat parotid gland by superior cervical ganglionectomy produced marked reductions in these responses. The stimulated incorporation of radiolabelled precursors into phosphatidylinositol is a measure of its resynthesis after receptor-mediated breakdown of inositol phospholipids. We therefore examined the enzymic site of the lesion induced by sympathetic denervation using parotid gland slices labelled with either [3H]inositol or [32P]phosphate and stimulated with substance P. Receptor-activated phospholipase C attack upon [3H]inositol phospholipids was assayed by measuring the formation of [3H]inositol 1-phosphate in the presence of 10 mM-Li+ to inhibit further breakdown. It was not affected by denervation. Substance P elicited a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and this response was reduced in the denervated gland. The second step in stimulated phosphatidylinositol turnover, phosphorylation of diacylglycerol to phosphatidate was not affected by denervation. Sympathetic denervation appears to induce a specific enzymic lesion in the parotid gland that impairs receptor-stimulated resynthesis of phosphatidylinositol from phosphatidate. This change in membrane lipid metabolism may be related to a number of the effects of sympathetic denervation, such as agonist supersensitivity, reduced gland cell proliferation and induction of new surface receptors.     

Vasopressin does not hydrolyze polyphosphoinositides in rabbit papillary collecting tubule cells

Changes in phosphatidylinositol metabolism are suggested to be involved in the mechanism of action of many membrane active hormones. We studied the effect of vasopressin on polyphosphoinositide metabolism in rabbit papillary collecting tubule cells to assess if the hydrolysis of these phospholipids is involved in transmembrane signaling. Rabbit papillary collecting tubule cells grown in monolayers for 5 days were labeled to constant specific activity with [3H]inositol. The temporal changes in [3H]inositol-labeled phospholipids were assessed in response to vasopressin. Similarly, water-soluble inositides were monitored after separation by ion exchange chromatography. Intracellular Ca2+ was monitored by use of the fluorescent indicator dye, quin2. Vasopressin (10(-7) M) did not increase the hydrolysis of phosphoinositides over a 5 min period when compared with controls. Similarly, there was no increase in water-soluble phosphoinositols during the same interval. Pretreating the cells with LiCl (10 mM) did not produce any increase in inositol 1-phosphate when stimulated with vasopressin but did in response to bradykinin. Finally, vasopressin did not increase cytosolic Ca2+ and did not increase the release of prostaglandin E2 into the media under our experimental conditions. We conclude that vasopressin does not stimulate prostaglandin E2 in rabbit papillary collecting tubule cells, does not initiate hydrolysis of polyphosphoinositides and does not increase cytosolic Ca2+. Thus these cells lack V1 receptor coupling mechanisms.     

Is Inositol Bisphosphate the Product of A23187 and Carbachol-Mediated Polyphosphoinositide Breakdown in Synaptosomes?

Synaptosomes have been isolated from rat cerebral cortex and labelled in vitro with [32P]orthophosphate and myo-[2-3H]inositol. Subsequent addition of the Ca2+ ionophore A23187 in the presence of 2 mM extrasynaptosomal Ca2+ raised intrasynaptosomal free [Ca2+] to greater than 2 microM from a resting level of 200 nM and led to rapid breakdown of polyphosphoinositides. This was accompanied by a small increase in the level of inositol monophosphate, greatly enhanced accumulation in inositol bisphosphate, but no detectable increase in inositol trisphosphate. Depolarising (25 mM) extrasynaptosomal K+ produced a smaller increase in intrasynaptosomal free [Ca2+] (to around 400 nM) and a proportional increase in inositol bisphosphate radioactivity. Carbachol (1 mM) alone elicited only limited polyphosphoinositide breakdown and inositol mono- and bisphosphate formation, but this was greatly increased in the presence of 25 mM K+. The effect of carbachol in the presence of depolarising K+ was time- and dose-dependent and was antagonised by atropine (10 microM). There was no detectable accumulation of inositol trisphosphate in the presence of carbachol, K+, or carbachol plus K+, even after short (30 s.) incubations. The lack of inositol trisphosphate accumulation does not appear to result from rapid formation of inositol tetrakisphosphate or from enhanced breakdown of the trisphosphate in synaptosomes.     

Insulin and oxytocin effects on phosphoinositide metabolism in adipocytes

The effects of hormones on phosphoinositide metabolism were examined in rat adipocytes prelabeled with 32Pi or [3H]inositol. Oxytocin and vasopressin produced large decreases in labeled polyphosphoinositides and increases in phosphatidic acid and inositol phosphates, whereas insulin was without effect, although it stimulated lipogenesis from glucose. Likewise, insulin did not elevate 1,2-diacylglycerol measured chemically by high pressure liquid or thin-layer chromatography in fat cells or pads. It also did not increase the radioactivity in 1,2-diacylglycerol in ghosts prepared from fat cells previously labeled with [3H]arachidonic acid, although oxytocin and vasopressin increased this. It is therefore concluded that insulin does not stimulate the breakdown of polyphosphoinositides to yield 1,2-diacylglycerol and inositol phosphates in adipocytes and that the insulin-like actions of oxytocin must be due to other changes. Insulin induced small, but significant and equal increases (40% at 30 min) in the incorporation of [3H] inositol into phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in adipocytes. The effects were not dependent upon glucose and were not evident before 15 min. Oxytocin also produced large increases in the labeling of the three phosphoinositides. Insulin stimulated the incorporation of [3H]glycerol into the three phosphoinositides and also phosphatidic acid, phosphatidylserine, and phosphatidylethanolamine by 50-100% in cells incubated without glucose. No changes in the labeling of glycerol 3-phosphate, lysophosphatidic acid, phosphatidylcholine, and triacylglycerol were detected, and there was a small increase (30%) in 1,2-diacylglycerol labeling. It is concluded that insulin increases the synthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, and phosphatidylserine in fat cells partly by stimulating a reaction(s) located between glycerol 3-phosphate and phosphatidic acid in the biosynthetic pathway.     

Activation of polyphosphoinositide metabolism at artificial maturation of Patella vulgata oocytes.

The metabolism of polyphosphoinositides (PPI) has been investigated during the meiosis reinitiation of the oocytes of a prosobranch mollusk, the limpet Patella vulgata. Meiosis reinitiation which leads to germinal vesicle breakdown (GVBD) and metaphase-1 spindle formation was artificially induced by treating the prophase-blocked oocytes with 10 mM NH4Cl, pH 8.2. This treatment, which results in a rise in intracellular pH, triggered a general increase in polyphosphoinositide synthesis. Determinations of phosphorus content showed that maturation induced a 30 to 50% increase in both phosphatidylinositol (PI) and phosphatidylinositol-1 monophosphate (PIP) concentrations. Incorporations of 32PO4 and [3H]inositol have been measured in three classes of polyphosphoinositides: PI, PIP, and phosphatidylinositol 4,5-bisphosphate (PIP2). By comparing incorporation rates of the radiolabeled precursors into PPI before and after meiosis reinitiation, we found that artificial maturation by ammonia induced a 50-fold increase in the turnover of these lipids. No significant burst of inositol 1,4,5-trisphosphate (IP3) was observed after maturation. We suggest that modifications in PPI metabolism occurring at maturation of Patella oocytes might ensure the formation of an important stock of PPI that would be available for the profuse production of IP3, the messenger responsible for the Ca2+ signal at fertilization.     

Inositol lipids in Friend erythroleukemia cells: evidence for changes in nuclear metabolism after differentiation.

The incorporation of 32Pi into phospholipids was studied in Friend erythroleukemia cells either induced or not to erythroid differentiation with 4 mM hexamethylenebisacetamide (HMBA). The effect of the differentiating agent on the recovery of radiolabelled phospholipids was compared in whole cells, isolated nuclei and nuclear matrix after in vivo labelling for 1 hr. The procedure employed for the isolation of nuclei was demonstrated to allow only negligible lipid redistribution caused by cell manipulations. Among the lipids extractable from nuclei, acidic phospholipids, and particularly polyphosphoinositides, were more represented than in whole cells, while small differences were found in the other phospholipid classes examined. The comparison between the uninduced and induced condition showed that the relative amounts of nuclear inositol lipids were modified by HMBA treatment of the cells, with a decreased recovery of phosphatidylinositol 4,5 bisphosphate. These results indicate that phosphatidylinositol and its phosphorylation products synthesized in vivo show a different metabolism in nuclei and whole cells. They appear to be tightly bound nuclear components, also present in membrane-deprived nuclei and nuclear matrix, and are probably related to the nuclear events involved in erythroid differentiation.     

Evidence for glucose-responsive and -unresponsive pools of phospholipid in pancreatic islets

The effect of glucose on the metabolism of phospholipids in pancreatic islets was studied with three radioactive phospholipid precursors, [32P]orthophosphate, [3H]myoinositol, and [3H]arachidonic acid, to determine the conditions necessary for studying the breakdown of prelabeled phospholipids. Islets were incubated in the presence of a radioactive precursor for 60 or 90 min and in the presence of either 3.3 or 16.7 mM glucose to prelabel phospholipids. To study the breakdown of prelabeled phospholipid, the unincorporated precursor was removed and the islets were reincubated for 15 or 20 min under conditions that either did or did not stimulate insulin release. Prelabeling in the presence of a noninsulinotropic concentration of glucose (3.3 mM) supported the incorporation of precursors into almost all islet phospholipids studied. Prelabeling in an insulinotropic concentration of glucose (16.7 mM) increased the incorporation of precursors into a number of phospholipids even more; and reincubation in 16.7 mM glucose caused a rapid loss of radioactivity from specific phospholipids (phosphatidylinositol and/or phosphatidylcholine, depending on the precursor). This breakdown was observed only when islets had been prelabeled in 16.7 mM glucose. The amount of radioactivity lost from phospholipid corresponded roughly to the additional amount incorporated during the prelabeling in the high concentration of glucose. Radioactivity in phospholipids in islets prelabeled in 3.3 mM glucose or in nonsecretagogue metabolic fuels, such as malate plus pyruvate, did not decrease when the islets were subsequently exposed to 16.7 mM glucose, nor did it decrease in 3.3 mM glucose when these islets had been prelabeled in 16.7 mM glucose. Glyceraldehyde, an insulin secretagogue, but not galactose or L-glucose which are not insulin secretagogues, stimulated phospholipid breakdown in islets that had been prelabeled in 16.7 mM glucose. Depriving islets of extracellular calcium, a condition that inhibits insulin release, inhibited phospholipid breakdown. The results suggest that pancreatic islets contain a glucose-responsive and a glucose-unresponsive phospholipid pool. The glucose-responsive pool becomes labeled and undergoes rapid turnover only under stimulatory conditions and may play a role in the stimulus-secretion coupling of insulin release.     

Kinetic Analysis of A23187-Mediated Polyphosphoinositide Breakdown in Rat Cortical Synaptosomes Suggests that Inositol Bisphosphate Does Not Arise Primarily by Degradation of Inositol Trisphosphate

The kinetics of polyphosphoinositide breakdown and inositol phosphate formation have been studied in rat cortical synaptosomes labelled in vitro with myo-[2-3H]inositol. Intrasynaptosomal Ca2+ concentrations have been varied by the use of Ca-EGTA buffers or by adding the ionophore A23187 in the presence and absence of 1 mM Ca2+. The former studies have revealed that, at very low (20 nM) intrasynaptosomal free Ca2+ levels, inositol bisphosphate, but not inositol monophosphate levels are reduced. Addition of A23187 in the absence of added Ca2+ gives rise to greatly enhanced inositol bisphosphate accumulation, which is further enhanced if 1 mM Ca2+ is present in the extrasynaptosomal medium. At all time points examined (down to 2 s after adding ionophore), the ratio of inositol trisphosphate/inositol bisphosphate accumulation does not exceed 0.2, and calculations based on inositol bis- and trisphosphate breakdown rates in synaptosomal lysates suggest that only a minority of the inositol bisphosphate arises from degradation of inositol trisphosphate. Addition of ionophore in the presence (but not in the absence) of 1 mM Ca2+ leads to rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and ATP and slower breakdown of phosphatidylinositol 4-phosphate (PtdInsP). The rates of loss of PtdinsP2 and ATP are very highly correlated, suggesting that polyphosphoinositide resynthesis may be limited by ATP availability at high Ca2+ levels. Analysis of 32P-labelled synaptosomes also reveals that A23187 produces Ca2+-dependent losses of PtdInsP2, PtdInsP, ATP, and GTP radioactivity and a marked increase in the radioactivity of a compound distinct from nucleotides or any of the lipid breakdown products tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic phosphatidylinositol kinase activity in continuously endotoxemic rats.

The activity of phosphatidylinositol (PI) kinase and the content and fatty acid composition of inositol phospholipids (IPLs) were analyzed in the livers of rats that had been continuously infused with Escherichia coli endotoxin (ET) or saline for 30 h. Maximal enzymatic activity in total liver membrane fractions was observed in the presence of 1 mM ATP, 20 mM MgCl2, exogenously added 0.3 mM PI and Triton X-100 (0.25%). The activity of PI kinase for endogenous and exogenous PI was 43 and 79% higher respectively, in ET- as compared with saline-infused rats. The Km of the enzyme for ATP was not altered (0.175 mM), while the apparent Vmax was higher for ET- as compared with saline-infused rats (0.48 and 0.38 nmol of phosphatidylinositol 4-phosphate formed/mg protein per min, respectively). The ET-induced higher activity of PI kinase was paralleled by a 68-78% increase in the content of polyphosphoinositides (PPI), while PI content was unchanged. All IPLs from livers of endotoxemic rats had a lower content of arachidonic acid. We demonstrate for the first time that ET can directly and/or indirectly stimulate the net synthesis of PPI in liver cells. This effect could serve to modulate the PPI derived signals by increasing the availability of the substrate phosphatidylinositol 4,5-bisphosphate.