Are polyphosphoinositides involved in platelet activation?

In human platelets, the amounts of triphosphoinositides (TPI) and diphosphoinositides (DPI) increase after 30 sec and level off after 120 sec of thrombin stimulation. After 180 sec of thrombin challenge, TPI and DPI increase accounts for 66 and 80%, respectively. Polyphosphoinositide changes roughly parallel the release of N-acetyl-beta-D-glucosaminidase and appear as a later event compared to aggregation and serotonin secretion. It is concluded that an increased phosphorylation of polyphosphoinositides might participate in platelets to the process of stimulus-activation coupling and might be linked to thrombin receptor occupancy. A role of DPI in platelet activation is suggested by the observation that DPI promote platelet aggregation, the mechanism of which is discussed.     

Light-mediated breakdown of phosphatidylinositol-4,5-bisphosphate in isolated rod outer segments of frog photoreceptor

When isolated frog (Rana catesbeiana) rod outer segment (ROS) fragments were incubated with [gamma-32P]ATP in the dark, only two of phospholipids, i.e., phosphatidylinositol-4-phosphate (DPI) and phosphatidic acid (PA) incorporated 32P. Upon addition of DPI (100 microM), considerable amount of 32P was incorporated into phosphatidylinositol-4,5-bisphosphate (TPI) as well as DPI and PA. Exposure of the ROS membranes to 5 sec flash of light resulted in approx. 20% decrease in the labeled TPI, while no significant effect was observed on DPI and PA. It was also observed that Ca2+ markedly accelerated the production of PA in the dark, while it reduced the 32P-incorporation into TPI. These results suggest that there is light- and/or Ca2+-dependent TPI-specific phospholipase C in ROS of vertebrate photoreceptors.     

The rapid polyphosphoinositide metabolism may be a triggering event for thrombin-mediated stimulation of human platelets

The metabolism of polyphosphoinositides was examined in human platelets activated by thrombin. The addition of thrombin to [3H]glycerol-labeled platelets induced an initial loss and a subsequent increase of the radioactivity in phosphatidylinositol-4,5-bisphosphate (TPI) without any significant change in phosphatidylinositol-4-phosphate (DPI). A marked enhancement of [32P]Pi incorporation into TPI occurred in parallel with an increase in this lipid content, which was accompanied with a conccurent decrease in phosphatidylinositol (PI). The rate of this subsequent increase in TPI was smaller than that observed in [3H]arachidonic acid-labeled platelets, suggesting that formed TPI in activated platelets may contain much greater amount of arachidonate than preexisting TPI in resting platelets. These data indicate that thrombin causes a rapid change in TPI metabolism (initial degradation of preexisting TPI and subsequent production of arachidonate-rich TPI), which might be a primary candidate to modulate thrombin-induced function in human platelets.     

Possible regulation of phospholipase C activity in human platelets by phosphatidylinositol 4',5'-bisphosphate

Phospholipase C from human platelets was found to catalyze the Ca2+-dependent degradation of phosphatidylinositol (PI), phosphatidylinositol 4'-phosphate (DPI), and phosphatidylinositol 4',5'-bisphosphate (TPI) at Ca2+ concentrations from 150 microM to 5 mM. Both DPI and TPI inhibited the hydrolysis of [2-3H]inositol-labeled PI (250 microM) in a concentration-dependent manner. The use of DPI and TPI from beef brain, both of which have fatty acid compositions different from that of soybean PI, permitted an assessment of the inhibitory effect of polyphosphoinositides on the hydrolysis of PI by phospholipase C. Fatty acid analysis of the diacylglycerols formed demonstrated that DPI and TPI, when incubated in mixture with PI, were competitive substrates for PI hydrolysis. Increasing the DPI/PI ratio from 0 to 0.3 caused a shift in the degradation of PI to DPI without greatly affecting the formation of 1,2-diacylglycerol. TPI alone, or in mixture with PI, was a poor substrate for phospholipase C. Increasing the TPI/PI ratio from 0 to 0.21, on the other hand, inhibited both PI degradation (greater than or equal to 95%) and overall formation of 1,2-diacylglycerol (greater than or equal to 82%). Kinetic analysis revealed that TPI acts as a mixed-type inhibitor with a Ki of about 10 microM. The Ka for Ca2+ in PI hydrolysis was profoundly increased from 5 to 180 microM when TPI (36 microM) was included with PI (250 microM). Optimum PI degradation under these conditions was only attained when the calcium concentration approached 4 mM. Analysis of phospholipids from unstimulated human platelets from five different donors revealed DPI/PI and TPI/PI ratios of 0.42 and 0.16, respectively. These findings, combined with the observed inhibition of PI hydrolysis by TPI at a TPI/PI ratio of 0.16, would suggest that in unstimulated platelets phospholipase C activity may be inhibited by greater than or equal to 75%. Changes in 33P-prelabeled phospholipids of intact platelets upon stimulation with thrombin indicated a transient decline in 33P label of both TPI and DPI (15 s) followed by an increase in [33P]phosphatidic acid but no change in [33P]PI. The finding that DPI is selectively degraded by phospholipase C in mixture with PI at DPI/PI ratios determined to be present in unstimulated platelets indicates that DPI may be more important than PI in the formation of 1,2-diacylglycerol which is believed to serve as precursor of arachidonic acid for thromboxane biosynthesis. Furthermore, the results suggest that in human platelets TPI may serve as modulator for the formation of 1,2-diacylglycerol from inositol phospholipids.     

Polyphosphoinositide metabolism in rat brain: effects of neuropeptides, neurotransmitters and cyclic nucleotides

This study describes effects of various peptides, neurotransmitters and cyclic nucleotides on brain polyphosphoinositide metabolism in vitro. The interconversion of the polyanionic inositol phospholipids was studied by incubation of a lysed crude mitochondrial/synaptosomal fraction with [gamma-32P]-ATP. The reference peptide ACTH1-24 stimulated the formation of radiolabelled phosphatidylinositol 4,5-diphosphate (TPI) and inhibited that of phosphatidic acid (PA). Substance P inhibited both TPI and PA labelling, whereas beta-endorphin inhibited that of PA without any effect on TPI. Morphine had no effect at any concentration tested, whereas high concentrations of naloxone inhibited the labelling of both PA and TPI. Naloxone did not counteract the effects of ACTH1-24. The other peptides tested (lysine 8-vasopressin and angiotensin II) were without any effect. Under the conditions used, adrenaline, noradrenaline and acetylcholine did not affect the labelling of the (poly)phosphoinositides. Both dopamine and serotonin, however, dose-dependently inhibited the formation of radiolabelled TPI and PA. Low concentrations of cAMP stimulated TPI, but higher concentrations had an overall inhibitory effect on the labelling of TPI, PA and especially phosphatidylinositol 4-phosphate (DPI). The cyclic nucleotide did not mediate or counteract the effects of ACTH, and cGMP was without any effect. These results are discussed in the light of current ideas on the mechanism of action of neuropeptides.     

Gonadotropin-releasing hormone rapidly alters polyphosphoinositide metabolism in rat granulosa cells

This report describes the rapid effects of GnRH and an agonist [D-Ala6, des-Gly10] GnRH ethylamide (GnRHa) on polyphosphoinositide metabolism in rat granulosa cells. As indicated by the depletion of cellular levels of 32P-prelabeled triphosphoinositide (TPI) and diphosphoinositide (DPI), GnRHa rapidly stimulated the hydrolysis of TPI and DPI. The effect of GnRHa was maximal at the earliest time point examined (30 sec) and preceded GnRHa-induced increases in labeling of phosphatidylinositol. A specific GnRH antagonist had no effect on TPI or DPI levels, but prevented the polyphosphoinositide depletion induced by GnRH. LH did not stimulate depletion of 32P-polyphosphoinositides. The rapid and specific effects of GnRH on polyphosphoinositide depletion may represent an early and possibly initiating event in the action of GnRH.     

Coupling of polyphosphoinositide breakdown with calcium efflux in formyl-methionyl-leucyl-phenylalanine-stimulated rabbit neutrophils

Exposure of rabbit neutrophils to formyl-methionyl-leucyl-phenylalanine (FMLP) induced the efflux of 45Ca2+ from pre-labeled cells which was almost complete within 30 s. On the other hand, FMLP-induced 45Ca2+ influx did not become apparent until 60 s after stimulation. When [3H]arachidonic acid-labeled neutrophils were stimulated with FMLP, the radioactivities in phosphatidylinositol 4,5-biphosphate (TPI) and phosphatidylinositol 4-phosphate (DPI) significantly decreased in parallel with the induction of 45Ca2+ efflux. In contrast, degradation of polyphosphoinositides in [3H]glycerol-labeled neutrophils was not significant until 60 s. Taken together, these results indicate that the early degradation of polyphosphoinositides, especially of those rich in arachidonic acid is closely associated with the initial efflux of calcium in FMLP-stimulated rabbit neutrophils. The study of resynthesis of polyphosphoinositides by measuring 32Pi incorporation into these lipids is also presented.     

Accessibility of glucose 6-phosphate: phosphohydrolase to antibody attack in modified microsomal vesicles

Upon subcellular fractionation of (murine) Friend erythroleukaemic cells (FELCs), purified plasma membranes were identified by their high enrichment in specific marker enzymes and typical plasma membrane lipids. When FELCs were incubated for short periods with ³²P_i before cell fractionation, the lipid-bound radioactivity was almost exclusively present in phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI), and its distribution closely matched that of the plasma membrane markers. In addition, purified plasma membranes actively incorporated ³²P from [γ-³²P]ATP into polyphosphoinositides, and the specific activities of the involved kinases were again mostly enriched in the plasma membrane fraction.     

PHOSPHOINOSITIDES AND OTHER PHOSPHOLIPIDS IN SYMPATHETIC GANGLIA AND NERVE TRUNKS OF RATS

Abstract— Paired vagus nerves, phrenic nerves or superior cervical sympathetic ganglia from adult white rats were incubated for 4 h at 37°C in a bicarbonate-buffered physiological solution containing glucose and ³²P₁. At the end of incubation triphosphoinositide (TPI) contained more ³²P than any other lipid in the vagus nerves and was second only to phosphatidylcholine (PC) in the phrenic nerves. In the sympathetic ganglia phosphatidylinositol (PI) contained more ³²P than did TPI, but both had less than PC. Conducted nerve impulses, initiated by electrical stimulation during the final 3 h of incubation, caused a highly significant increase in the [32P]-labelling of PI in ganglia (as previously reported) probably decreased the labelling of TPI in the vagus nerves, and decreased the labelling of phosphatidylethanolamine (PE) in the ganglia. Addition to the incubation medium of §- or γ-hexachlorocyclohexane (analogs of inositol) reversibly blocked transmission through the sympathetic ganglia at concentrations less than 0·1 mM. The §-isomer also blocked conduction along axons at similar concentrations; only the γ-isomer (lindane) exerted a selective effect on synaptic transmission. In the ganglia, the §-isomer increased the [³²P]-labelling of PI and diphosphoinositide (DPI) relative to that of PC. The γ-isomer did not affect the relative labelling of PI in the ganglia, whereas it decreased that of TPI, but only at relatively high concentrations. Thus, various affects of the hexachlorocyclohexanes were not explicable by assuming that they acted as analog inhibitors of inositol metabolism. In the ganglia, the hexachlorocyclohexanes reduced the effect of neuronal activity on the labelling of PI in proportion to the extent by which they blocked transmission. This metabolic effect was therefore presumed to be secondary to a ganglionic blocking action.     

Effects of Corticotropin-(1–24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract: Effects of the neuropeptide corticotropin-(1–24) -tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 ×g supernatant fraction [30–50% (NH₄)₂SO₄ precipitate; ASP_30–50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The ³²P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [γ-³²P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP_30–50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30–60 s), and it was dependent on the presence of Mg²⁺ in the incubation medium; in general it was inhibited by high concentrations (>0.2 mM) of Ca²⁺. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50–100 μ of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 μM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP_30–50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca²⁺-independent; thus the neuropeptide effects were not influenced by either 1 μM EGTA or low concentrations of Ca²⁺ (50 μ.M). We conclude that a relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.     

Calcium- and Calmodulin-Dependent Phosphorylation of Diphosphoinositide in Acetylcholine Receptor-Rich Membranes from Electroplax of Narke japonica

The phosphorylation of phosphoinositides in the acetylcholine receptor (AChR)-rich membranes from the electroplax of the electric fish Narke japonica has been examined. When the AChR-rich membranes were incubated with [gamma-32P]ATP, 32P was incorporated into only two inositol phospholipids, i.e., tri- and diphosphoinositide (TPI and DPI). Even after the alkali treatment of the membrane, AChR-rich membranes still showed a considerable DPI kinase activity upon addition of exogenous DPI. It is likely that the 32P-incorporation into these lipids was realized by the membrane-bound DPI kinase and phosphatidyl inositol (PI) kinase. Such a membrane-bound DPI kinase was activated by Ca2+ (greater than 10(-6) M), whereas the PI kinase appeared to be inhibited by Ca2+. The effect of Ca2+ on the DPI phosphorylation was further enhanced by the addition of ubiquitous Ca2+-dependent regulator protein calmodulin. Calmodulin antagonists such as chlorpromazine (CPZ), trifluoperazine (TFP), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the phosphorylation of DPI in the AChR-rich membranes. It is suggested that the small pool of TPI in the plasma membrane is replenished by such Ca2+- and calmodulin-dependent DPI kinase responding to the change in the intracellular Ca2+ level.     

The effects of cyclic nucleotides and some related agents on 32Pi labeling of renal polyphosphoinositides in vitro.

When rabbit kidney cortex slices were incubated in the presence of ³²P_i and dibutyrylcyclic AMP (dbcAMP)⁴ a significant decrease in the labeling of phosphatidyl inositol phosphate (DPI) but not phosphatidyl inositol bisphosphate (TPI) was observed. In the presence of 0.3 mm caffeine cyclic AMP (cAMP) produced a similar effect. Caffeine potentiated the inhibitory effect of dbcAMP. At high concentrations (3 mm) caffeine alone decreased the ³²P_i labeling of both DPI and TPI. These decreases in ³²P_i labeling were not mediated by decreases in the labeling of intracellular P_i or ATP as measured by 10-min acid-labile nucleotide phosphate (10′-ALNP). Addition of cyclic GMP (cGMP) to the incubation medium decreased the labeling of DPI and to a lesser extent that of TPI also. Addition of parathyroid hormone (PTH) to the incubation medium (in the absence of exogenous cyclic nucleotides) also decreased the ³²P_i labeling of DPI but not that of TPI. In contrast to the effects of cAMP, dbcAMP, cGMP, PTH, and caffeine, the addition of insulin to the incubation medium resulted in increased ³²P_i labeling of DPI with no effect on TPI labeling. DPI isolated from kidney cortex slices prelabeled with ³²P_i and subsequently incubated with cAMP or dbcAMP contained less label than DPI isolated from slices similarly prelabeled but subsequently incubated in the absence of either cAMP or dbcAMP. These data suggest an increased rate of DPI breakdown in the presence of elevated cAMP or dbcAMP concentrations. This hypothesis was supported by the fact that cAMP stimulated the hydrolysis of DPI but not of TPI by a polyphosphoinositide phosphodiesterase present in the supernatant fraction of rabbit kidney cortex.     

Effect of fusaric acid on phosphoinositide metabolism in the erythrocyte membranes of rats with spontaneous hypertension

2- and 4-month-old male spontaneously hypertensive rats (SHR) were injected fusaric acid at a dose of 50 mg/kg body weight. Fusaric acid increased diphosphoinositide (DPI) and triphosphoinositide (TPI) levels in erythrocyte membranes of 4-month-old SHR by 41% and 20%, respectively. 32P incorporation into TPI decreased by 24% in 2- and by 20% in 4-month-old SHR. Phosphatidylinositol metabolism remained unchanged. The results also suggest that fusaric acid normalized DPI and TPI metabolism in erythrocyte membranes of SHR.     

Effects of EGF and thrombin on inositol-containing phospholipids of cultured fibroblasts: stimulation of phosphatidylinositol synthesis by thrombin but not EGF

The effects of growth factors on inositol-containing phospholipids were investigated to test the hypothesis that alterations in their metabolism are involved in mitogenic stimulation. Thrombin and EGF stimulated comparable increases in the synthesis (30-50%) and degradation (20-40%) of phosphatidylinositol 4-monophosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) in a cell line which is mitogenically responsive to both growth factors. The increases in synthesis were time and dose dependent in a manner which was consistent with their involvement in mitogenesis; the increases were observed only under conditions where a mitogenic response occurred. While it has been suggested that an increased synthesis of phosphatidylinositol (PI) is coupled to the stimulation of DPI and TPI synthesis, we found that thrombin stimulated an early synthesis PI but EGF did not. To further evaluate the involvement of PI in thrombin-stimulated cell division we determined the time and dose dependence of the stimulated PI synthesis and found that it also occurred in a manner which was consistent with its involvement in thrombin-stimulated cell division. Furthermore, the stimulated PI synthesis was not observed with nonmitogenic proteases or in cell lines which were not responsive to thrombin. These results demonstrate that the metabolism of DPI and TPI appears closely related to the mitogenic response generated by EGF and thrombin. However, an early stimulation of PI synthesis is not coupled to this metabolism and is not necessary for mitogenic stimulation by EGF. Thus, a stimulation of PI synthesis is not a valid measure of alterations in inositol-containing phospholipids and what has been termed the "PI response."     

NOREPINEPHRINE-STIMULATED BREAKDOWN OF TRIPHOSPHOINOSITIDE OF RABBIT IRIS SMOOTH MUSCLE: EFFECTS OF SURGICAL SYMPATHETIC DENERVATION AND IN VIVO ELECTRICAL STIMULATION OF THE SYMPATHETIC NERVE OF THE EYE

Abstract— Paired iris smooth muscles from rabbits were prelabelled either in vitro by incubation for 30 min at 37°C in an iso-osmotic salt medium containing glucose, inositol, cytidine and ³²Pi, or in vivo by administration of the isotope intracamerally into each eye 1 h before death. One of the pair was then incubated at 37°C for 10 min in an unlabelled medium containing 10 mm of 2-deoxyglucose and the other was incubated in the presence of norepinephrine (NE) or other adrenergic agents. Triphosphoinositide (TPI) was found to contain more ³²P than any other phospholipid (almost 39% of total lipid radioactivity) in both the in vitro and in vivo experiments. NE (50 μm ) increased the loss of ³²P from TPI (the TPI effect') by 28–30% in the ³²P-labelled muscle. The TPI effect was accompanied by a significant increase in ³²P labelling of phosphatidic acid (PA) and phosphatidylinositol (PI) but not phosphatidylcoholine. In this tissue the TPI effect was found to be mediated through α-adrenergic receptors. At 14 days after surgical sympathetic denervation, incorporation of ³²P into phospholipids of the denervated muscle increased by an average of 6% over that of the normal muscle. The increase in TPI, PI and PA was 7%, 4% and 9% of that of the control respectively. There was little change in phospholipid content of the denervated muscle. The increase in sensitivity to NE (12.5 μm ) caused by denervation produced about 18% increase in the TPI effect and a 25% increase in the ³²P labelling of PA, but not PI. In view of our previous findings on the requirement of the TPI effect for Ca²⁺, this observation could suggest that an increase in Ca²⁺ influx, following the interaction between the neurotransmitter and its receptor could stimulate TPI-phosphodiesterase, thus leading to increased PA via increased diglyceride. This denervation-induced supersensitivity to NE appears to be postsynaptic in nature. ³²Pi was injected intracamerally into each eye 1 h before electrical stimulation of one of the sympathetic trunks. After stimulation for 30 min there was a significant loss of ³²P from TPI and a significant increase in the labelling of PI and PA of the stimulated muscle. It is concluded that TPI and its enzymes could play an important role in neurotransmission at the neuromuscular junction of smooth muscle.     

An early transient decrease in phosphatidylinositol 4,5-bisphosphate upon stimulation of rabbit platelets with acetylglycerylether phosphorylcholine (platelet activating factor)

Washed rabbit platelets labeled with [3H]inositol were stimulated with AGEPC (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (5 X 10(-10) M) for various time periods. Within 5 s of the mixing of these platelets with AGEPC, an approximately 25% decrease in the [3H]TPI (phosphatidylinositol 4,5-bisphosphate) was evident; immediately thereafter the radioactivity in TPI increased. These labeled platelets treated with various concentrations of AGEPC for only 5 s indicated a characteristic dose-related decrease in [3H]TPI. Radioactivity in phosphatidylinositol 4-phosphate also appeared to increase after AGEPC-induced stimulation of platelets. Interestingly, within 15 s a 15 to 20% decrease in [3H]PI (phosphatidylinositol) and an increase in [3H]lysoPI was observed. However, [3H]lysoPI could be related only to one-third of the decrease in [3H]PI. LysoGEPC (lyso-1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which is ineffective in the activation of platelets, was unable to cause any changes in the phosphoinositides. The fact that the status of TPI was influenced in a time- and dose-dependent manner and the rapidity with which these changes take place suggest that this inositol phospholipid may be associated closely with the early processes which accompany the interaction of AGEPC with platelets.     

Microdetermination of phosphoinositides in a single extract

A method that allows the quantification of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-biphosphate (TPI) on a nanomolar scale is presented. The method is based on the simultaneous separation of lipids on high-performance thin-layer chromatography plates, followed by a microassay for phosphorus of PI spots and a densitometric assay of DPI and TPI. The new procedure allows the determination of the phospholipids in small amounts (100 micrograms protein) of synaptosomes and synaptic plasma membranes, and in homogenates of microwave-fixed brain tissue (1 mg wet wt). The usefulness of the method is illustrated by showing the effect of Ca2+ on the breakdown of DPI and TPI in synaptosomal plasma membranes.     

Glucose inhibits insulin release when not promoting the entry of calcium into the beta-cells

The effect of antigen on the metabolism of polyphosphoinositides was investigated in sensitized rat peritoneal mast cells. Addition of antigen to rat peritoneal mast cells prelabelled with [3H]arachidonic acid resulted in a very rapid decrease in the level of phosphatidylinositol 4-phosphate (DPI) within 5 sec, which appeared to precede the breakdown of phosphatidylinositol (PI), while there was no significant decline of PI 4,5-bisphosphate (TPI). The reduced levels of these phosphoinositides returned almost to control or even slightly higher values by 300 sec in parallel with the antigen-stimulated [32P]phosphate incorporation into these lipids. This early and transient disappearance in DPI prior to that in PI was also observed in [3H]glycerol-prelabelled cells. These data suggest that DPI degradation upon stimulation by antigen in mast cells may be an initial step in the histamine release process.     

Effects of calcium ionophore A23187 and calcium antagonists on 32Pi incorporation into polyphosphoinositides of rat cortical synaptosomes

The role of Ca2+ on 32Pi incorporation into polyphosphoinositides (PPI) of rat cortical synaptosomes was studied. Stimulation of muscarinic receptor by carbachol (1 mM) resulted in a decrease in 32Pi incorporation into phosphatidylinositol-4,5-bisphophaphate (TPI) and phosphatidylinositol-4-phosphate (DPI), and an increase in 32Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA), whereas no significant effect on other membrane phospholipids was found. This response could be blocked by atropine (1 microM). The stimulatory effect of carbachol required Ca2+ in the medium; the presence of 0.5 mM EGTA blocked the effect of carbachol on PPI turnover completely. Calcium ionophore A23187, at 1 microM, had a similar effect on PPI turnover by carbachol (1 mM). At higher concentrations (10-100 microM) of A23187, the PPI turnover rate was much enhanced. Depolarization of the membrane by high potassium (60 mM) in the presence of calcium resulted in an enhanced PPI turnover, which was similar to the results of the carbachol (1 mM) effect but to a lesser extent. Calcium antagonists, diltiazem and trifluoperazine, at 10 microM could block the carbachol effect on 32Pi incorporation into PPI in this preparation. Our results suggest that the enhancement of PPI turnover in rat cortical synaptosomes by carbachol, calcium ionophore or high potassium requires Ca2+, and it can be blocked by compounds which interfere with the availability of this ion, such as EGTA or calcium antagonists.     

Distinctive subcellular alterations induced by hypertonic stress in sea urchin eggs

Sea urchin eggs continuously exposed to a hypertonic solution were ultrastructurally examined for osmotic-stress induced alterations. No fertilization membranes formed during the treatment and the surface-cortex complexes remained unaltered from the unfertilized state. However, the osmotic stress did induce a number of subcellular changes. During the first 30 minutes of the treatment the eggs formed many endoplasmic reticulum whorls and compacted Golgi body aggregations. Both of these new formations can be correlated with rapid changes in intracellular calcium, known to occur in hypertonic stressed eggs. Aggregations of mitochondria could be observed at later stages; these aggregations can also be related to subcellular stress and possible changes in internal calcium concentrations. The various morphological transitions within the cytoplasm, along with the lack of a cortical reaction in these eggs, not only supports the idea that calcium is released during parthenogenetic activation, but also suggests that this free calcium originates from stores other than the stores that are involved during fertilization or simple artificial activation.