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.     

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis

32P-Labelled washed rabbit platelets were incubated with 0.6 nM platelet activating factor (PAF-acether), giving a full aggregation and release response within 30-60 s. The major phospholipid changes observed under these conditions were: (1) An increased labelling of phosphatidic acid (PA) within 10 s and of phosphatidylinositol (MPI) at 30 s, reflecting the activation of the MPI cycle via the cytosolic phospholipase C; (2) an enhancement of phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI) labelling at later incubation times; (3) an early degradation of TPI with a counterbalancing formation of DPI. The latter changes suggest a receptor-mediated stimulation of TPI-phosphomonoesterase, the role of which in the mechanism of platelet activation is discussed.     

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.     

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.     

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.     

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.     

Calcium regulation of phosphatidyl inositol turnover in macrophage activation by formyl peptides

Stimulation by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP) of the guinea pig alveolar macrophage gives rise to transient production of superoxide anion (O2-). Components of the phosphatidyl inositol (PI) cycle (phosphatidic acid (PA), phosphatidyl inositol-4,5-bisphosphate (TPI) and phosphatidyl inositol-4-phosphate (DPI) were monitored using 32P in order to examine the possible association of this cycle with the FNLLP-stimulated production of O2-. Macrophage stimulation by FNLLP led to an increased flux of metabolites through the PI cycle. The level of 32P label in both TPI and DPI rapidly decreased upon exposure to FNLLP, followed by a 5-min period during which the 32P label in TPI and DPI approached prestimulated levels. During this period, there was a fivefold increase in 32P-PA. It is suggested that diacylglycerol (DAG) is the O2- -activating intermediate in the stimulated mechanism, as evidenced by the buildup of PA (for which DAG is the precursor) in parallel with the time course of O2- production. The importance of continued cycling of PI in the stimulated mechanism is demonstrated by the inhibition by LiCl of the extent, but not the initial rate, of both O2- production and the formation of 32P-PA upon peptide stimulation after 1-h preincubation with 10 mM LiCl. The influence of calcium on this mechanism was also examined. It has previously been demonstrated that intracellular availability of calcium can influence the rate and extent of O2- production. In cells preloaded with quin-2, which acts as a high-affinity sink for calcium in the cytosol, the initial rate of FNLLP-stimulated O2- production is inhibited in low (10 microM) extracellular calcium medium. High extracellular calcium (1 mM) completely reverses this inhibition and also significantly extends the time course of O2- production in both quin-2 and control cells (Stickle et al., 1984). In parallel with these effects on O2- production, varying calcium conditions is demonstrated to influence the rate and extent of PA formation. These same calcium conditions were found to have little or no effect on the initial unstimulated levels of TPI, DPI, and PA. These results indicate that the influence of an intracellular pool of calcium on O2- production may be via its influence on stimulated PI turnover.     

Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.     

Rapid Incorporation In Vivo of Intracerebrally Injected 32Pi into Polyphosphoinositides of Three Subfractions of Rat Brain Myelin

Abstract: At intervals ranging from 1 to 10 min after injection of ³²Pi into rat brain, myelin was prepared and separated into three subfractions: heavy, medium, and light. The radioactivity of total phospholipids and polyphospho-inositides (PPI) was then determined. There was rapid incorporation of ³²Pi into PPI, which contained 50–70% of the radioactivity among total brain lipids and more than 70% among myelin lipids. The myelin fraction had incorporated ³²Pi into total recovered PPI in the order of medium > heavy > light fraction: however, the order of relative specific radioactivities was heavy > light > medium. Labeling of the PPI precursors, phosphatidic acid (PA) and phos-phatidylinositol (PI), was considerably lower in the purified myelin than in total brain. The di- (DPI) and triphosphoinositides (TPI) in heavy myelin exchanged ³²Pi at rates 2 to 3 times faster than those in medium and light myelin. DPI of all subfractions of myelin exchanged much faster than TPI. The results show that the most active phosphate turnover of myelin PPI occurs in the heavy myelin fraction (probably largely consisting of myelin appurtenant regions). However, medium and light myelin (most probably representing the closely packed layers of myelin sheaths) also showed rapid turnover of PPI.     

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.     

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.     

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.     

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.     

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.     

AGEPC (platelet activating factor) induced stimulation of rabbit platelets: effects on phosphatidylinositol, di- and triphosphoinositides and phosphatidic acid metabolism

Stimulation of washed rabbit platelets with AGEPC (1-O-alkyl-2-acetyl-$\text{sn}$-glyceryl-3-phosphorylcholine) caused a 15–20% decrease in their phosphatidylinositol level within 15 seconds without affecting other major classes of phospholipids. In the same time frame the level of phosphatidic acid (PA) increased dramatically some four fold. LysoGEPC, which is inactive in stimulating rabbit platelets, did not cause any change in PI or PA. When [³²Pi] was present during the stimulation of platelets by AGEPC, the incorporation of radiolabel into PI-4-phosphate (DPI), PI-4,5-bis phosphate (TPI) and PA was enhanced significantly within one minute while the incorporation into PI increased only after one minute. These results clearly established that AGEPC induced stimulation of rabbit platelets was associated with the metabolism of inositol phospholipids and phosphatidic acid. The relevance of these findings to the mode of action of AGEPC and Ca²⁺ mobilization is also discussed.     

Cyclic AMP-dependent protein kinase and Ca2+-calmodulin stimulate the formation of polyphosphoinositides in a sarcoplasmic reticulum preparation of rabbit heart

A rabbit heart membrane fraction enriched in sarcoplasmic reticulum was incubated in a reaction mixture containing [gamma-32P]ATP. The catalytic subunit of cyclic AMP-dependent protein kinase enhanced the 32P-labelling of both phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Ca2 +-calmodulin also increased the 32P-incorporation into both polyphosphoinositides. Upon SDS gel-electrophoretic analysis of the membrane proteins, phospholamban was found to be concurrently phosphorylated by the exogenous catalytic subunit as well as by an endogenous Ca2+-calmodulin-dependent protein kinase.     

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.     

Phospholipid biosynthesis in mature human erythrocytes

Mature human erythrocytes were tested for their ability to synthetize membrane phospholipids from simple precursors: [32P]-orthophosphate (32Pi), [U-14C] glycerol, [U-14C] glucose, [U-14C] serine, and [U-14C] choline. The incorporation of these labels into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (lyso-PC), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) was measured. All the phospholipids tested incorporated 32Pi, glycerol, and glucose in a time dependent manner. According to the rate of 32Pi incorporation, three groups of phospholipids could be distinguished: 1) PA, PIP2, PIP, lyso-PC; 2) PI and PS; 3) PC and PE, which incorporated 5 x 10(3), 40, and 6 nmol 32Pi/mmol phospholipid per 1 h, respectively. Moreover, [U-14C] serine and [U14C] choline were found to incorporate into phospholipids, and PS-decarboxylase activity could be measured. The possibility that the observed incorporation was due to contamination with bacteria or other blood cells could be ruled out. Our results bring evidence for de novo phospholipid synthesis of human red blood cells.     

Binding of Ca2+ to phosphoinositols, phosphatidylserines and gangliosides

The effect of K+, Mg2+ and serotonin on the interaction between Ca2+ and different phospholipids as well as glycosphingolipids (gangliosides) was studied by equilibrium dialysis using 45Ca as tracer. The highly polar phosphatidylinositol-4,5-bisphosphate (TPI) was found to bind more Ca2+ per lipid molecule than all other lipids tested and Ca2+ could not be released as easily as in the other lipids by K+, Mg2+ and serotonin. Ca2+ is released from all lipid-Ca2+ complexes most effectively by Mg2+, serotonin is less effective but enhances K+ in its capacity to displace Ca2+ from the respective binding sites. A remarkable dissociating influence of serotonin on ganglioside-Ca2+ and phosphatidylserine-Ca2+ complexes is observed. This effect is less pronounced with phosphatidylinositol-Ca2+ complexes under comparable comparable conditions. The possible functional role of phospholipids and gangliosides in vivo is discussed with regard to the specific Ca2+-binding properties of these lipids.     

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.