Studies on the effects of acetylcholine and antiepileptic drugs on32Pi incorporation into phospholipids of rat brain synaptosomes

Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated³²P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated³²P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the³²P labeling of phospholipids nor on the specific radioactivity of [³²P]ATP. Omission of Na⁺ drastically reduced both the³²P labeling of synaptosomal phospholipids and the specific radioactivity of [³²P]ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate Na⁺ and Ca²⁺ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca²⁺ and Na⁺ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues.Abbreviations used ACh Acetylcholine - PA phosphatidic acid - PI phosphatidylinositol - poly PI polyphosphoinositides (diphosphoinositide and triphosphoinositide) - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - S.A. specific radioactivity     

Effects of insulin on phenylephrine-induced activation of phosphorylase and phosphatidylinositol turnover in isolated hepatocytes

Treatment of isolated hepatocytes with the alpha-agonist phenylephrine led to a rapid increase in the activity of phosphorylase a and an increase in the rate of 32P incorporation into phosphatidylinositol. After pretreatment of the cells with insulin, this activation of phosphorylase was reduced by about 50% but there was no alteration in either the basal or phenylephrine-stimulated rate of phosphatidylinositol turnover. This difference in the sensitivity of these two processes to insulin was observed at all times and concentrations of phenylephrine examined. Direct measurement of phosphatidylinositol breakdown and phosphatidic acid formation confirmed that the activation of the phosphatidylinositol cycle by phenylephrine was not blocked by insulin. These data suggest that insulin antagonism of alpha-adrenergic effects on glycogenolysis in liver is mediated at a step distal to hormone binding to the alpha 1-receptor and activation of inositol lipid breakdown but prior to intracellular Ca2+ mobilization.     

Calium, prostaglandins and the phosphatidylinositol effect in exocrine gland cells

The hypothesis that arachidonic acid metabolism might be involved in Ca-mobilization mechanisms in exocrine gland cells was investigated. Arachidonate (10⁻⁴M) failed to stimulate protein secretion from slices of pancreas, parotid or lacrimal glands and failed to stimulate ⁸⁶Rb efflux from parotid or lacrimal glands. The stimulation of protein secretion (all three glands) or ⁸⁶Rb efflux (parotid and lacrimal glands) by appropriate secretagogues was unaffected by 10⁻⁵M indomethacin. Eicosatetraynoic acid (2×10⁻⁵M) inhibited ⁸⁶Rb efflux due to carbachol but not that due to physalaemin or ionomycin. Nordihydroguaiaretic acid inhibited lacrimal and parotid gland responses only at high (10⁻⁴M) concentration. Collectively, these results argue against an obligatory role for arachidonate metabolites in Ca-mediated responses of these exocrine glands.In the exocrine glands activation by neurotransmitters (or analogs) of receptors that mobilize cellular Ca also stimulates the incorporation of ³²PO₄ into phosphatidylinositol (1–3). Michell (4,5) has suggested that in some manner this alteration in phospholipid metabolism may be functionally responsible for the opening of surface membrane Ca gates which presumably precedes the expression of a number of Ca-mediated responses by the exocrine cell. That this reaction probably preceeds Ca mobilization is deduced primarily from two experimental observations. First, receptor activation of phosphatidylinositol turnover is not prevented by Ca omission (6–8). Second, the effect is not mimicked by the divalent cationophore A-23187, while other effects of receptor activation are mimicked by this compound (7–9).There has also been some speculation as to the manner in which altered phosphatidylinositol metabolism might be involved in the Ca-gating mechanism (10–14). One such hypothesis suggests that receptor activation may lead to phosphatidylinositol breakdown which in turn leads to the release of free arachidonate (13, 14). As free arachidonate is generally believed to be the rate-limiting substrate for prostaglandin synthesis (15), the resulting prostaglandins might act to mobilize Ca or might act in concert with Ca (13, 14). There is evidence for this hypothesis for the mouse pancreas, where exogenous arachidonate and prostaglandins can stimulate amylase release (13). The effects of arachidonate, carbachol, caerulein and pancreozmin were all antagonized by sub-micromolar concentrations of indomethacin (13), a potent cyclooxygenase inhibitor (15). Additionally, recent reports have demonstrated stimulation by acetylcholine of prostaglandin E synthesis in mouse pancreas (16, 17).The purpose of this study was to examine the general applicability of this hypothesis by investigating the effects of arachidonate and substances that inhibit prostaglandin formation in two other exocrine tissues that show a prominent phosphatidylinositol turnover — the rat parotid and lacrimal glands.     

The stimulation of inositol lipid metabolism that accompanies calcium mobilization in stimulated cells: defined characteristics and unanswered questions

It now appears to be generally agreed that the 'phosphatidylinositol response', discovered in 1953 by Hokin & Hokin, occurs universally when cells are stimulated by ligands that cause an elevation of the ionized calcium concentration of the cytosol. The initiating reaction is almost certainly hydrolysis of an inositol lipid by a phosphodiesterase. Phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate all break down rapidly under such circumstances. However, we do not yet know which of these individual reactions is most closely coupled to receptor stimulation, nor do we know where in the cell it occurs. With many stimuli, inositol phospholipid breakdown is closely coupled to occupation of receptors and appears not to be a response to changes in cytosol [Ca2+]: this provoked the suggestion that it may be a reaction essential to the coupling between activation of receptors and the mobilization of Ca2+ within the cell. In a few situations, however, it appears probable that inositol lipid breakdown can occur as a result of the rise in cytosol [Ca2+] that follows receptor activation: such observations gave rise to the alternative opinion that inositol lipid breakdown cannot be related to stimulus-response coupling at calcium-mobilizing receptors. It now seems likely that these two views are too rigidly polarized and that some cells probably display both receptor-linked and Ca2+-controlled breakdown of inositol lipids. Both may sometimes occur simultaneously or sequentially in the same cell.     

Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[³H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [³²P]P_i, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [¹⁴C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca²⁺ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [³²P]P_i and [¹⁴C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [³²P]P_i labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A₂ and C. Whereas mobilization of cellular Ca²⁺ appears sufficient to cause activation of phospholipase A₂ and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca²⁺ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A₂ and C and their specific physiological roles in pancreatic secretion remain to be elucidated.     

Effect of Short-Term Salinity on Lipid Metabolism and Ion Accumulation in Tomato Roots

To examine the ion accumulation and membrane lipid metabolism in response to salinity we compared two tomato cvs. Pera and Hellfrucht Fruhstamm (HF), considered to be salt-tolerant and sensitive respectively. Na⁺ and K⁺ accumulation was significantly higher in roots of cv. Pera after 24 h and 72 h of 100 mM NaCl. While in cv. HF, a temporary increase in K⁺ accumulation at 24 h was accompanied by a sustained increase in Na⁺ content. Both cultivars enhanced incorporation of [³²P]orthophosphate into phosphatidylinositol 4,5-bisphosphate at 24 h and 72 h of NaCl. In parallel to the increase of phosphatidylinositol 4,5-bisphosphate a decrease in phosphorylation of phosphatidic acid and phosphatidylcholine were observed in the sensitive cv. HF. Structural and signal lipid changes in response to salinity were more evident in the sensitive cv. HF. Salt tolerant cv. Pera accumulated Na⁺ ions in the roots without considerable modifications in lipid metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inositol lipids and cell stimulation in mammalian salivary gland

The rat parotid salivary gland shows marked alterations in phospholipid metabolism when stimulated by certain agonists. These agonists are those which cause cellular Ca mobilization by activation of muscarinic, alpha-adrenergic or peptidergic (substance P) receptors. The phospholipid changes apparently reflect the activation of a phosphoinositide-phosphatidic acid cycle, the precise pathways of which are not known with certainty. The observed effects include (1) an increased labelling by 32PO4 of phosphatidylinositol and phosphatidic acid, (2) net synthesis of phosphatidic acid, (3) net breakdown of phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate. These effects apparently do not require the presence of extracellular Ca or the release of internal Ca and cannot be produced by the artificial introduction of Ca into the cytosol with Ca ionophores. These findings are consistent with the view that a receptor-mediated alteration in phosphoinositide metabolism represents an early step in the stimulus-response pathway in the parotid acinar cell. It has been suggested that phosphatidic acid synthesis might be of central importance in mediating Ca influx and that PIP2 breakdown might play a role in activation of Ca release. Evidence for these latter ideas is for the present largely circumstantial.     

Differences in Ca2+ mobilization induced by alpha-adrenergic agonist and phosphatidic acid in cultured hepatocytes

In an attempt to elucidate the relationship between phosphatidylinositol breakdown and alpha-adrenergic responses, effects of phosphatidic acid and phosphatidylinositol related metabolites on Ca²⁺ mobilization and glucose output in cultured hepatocytes were examined. Norepinephrine induced the net ⁴⁵Ca²⁺ efflux from preloaded cells and stimulated glucose output via alpha-adrenergic receptor stimulation, whereas phosphatidic acid caused ⁴⁵Ca²⁺ uptake to cells and did not stimulate glucose output. Myo-inositol-monophosphate, diglyceride and arachidonic acid, which are released by phosphatidylinositol breakdown, had no effect on ⁴⁵Ca²⁺ efflux and glucose output in cells. These results suggest that phosphatidic acid and phosphatidylinositol related metabolites can not mimic the alpha-adrenergic actions in cultured hepatocytes.     

Sodium channel activation does not alter lipid metabolism in cultured neuroblastoma cells

The interaction of voltage-sensitive Na⁺-channels and membrane lipid metabolism was examined by incubating cultured neuroblastoma cells with neurotoxins which alter the voltage-dependent relationship between the closed and open conformation of the channel protein. Guanidinium flux rate, a measure of Na⁺-channel activation, was increased 10-fold by the combined action of veratridine (100 M) and scorpion venom (28 g/ml). This response was completely blocked by tetrodotoxin (1 M). Under the same experimental conditions, the toxins did not increase the efflux of [³H]arachidonic acid from prelabeled cell membrane lipids or stimulate uptake of exogenous [³H]arachidonic acid. In addition, altering membrane fatty acid composition by incubating cells for 24 hr in a medium containing 50 M arachidonic or oleic acid did not alter guanidinium flux rates relative to that of control cultures. When cells were pulsed with³²Pi for 60 min and stimulated by veratridine plus scorpion venom for an additional 30 min, uptake of³²Pi into phosphatidylinositol as reduced; stimulating cells with bradykinin, a receptor agonist which activates the inositol cycle, promoted a 3.8 fold increase. Polyphosphoinositide turnover was not affected by Na⁺-channel activation, but was stimulated by bradykinin. These results suggest that voltage-sensitive Na⁺-channel activation in cultured neuroblastoma cells can function independent of membrane phospholipid and fatty acid metabolism.     

The relationship between the incorporation of 32P into phosphatidic acid and phosphatidylinositol in rat parotid acinar cells

Muscarinic and α-adrenergic stimulation of rat parotid acinar cells increases the turnover of phosphatidylinositol and phosphatidic acid. It is thought that this is initiated by hydrolysis of phosphatidylinositol, which would predict an increase in ³²P incorporation into phosphatidic acid before phosphatidylinositol. We have demonstrated an increase in ³²P incorporation into the former within 1 minute and into the latter by 2 minutes. The initial rapid rate of ³²P incorporation into phosphatidic acid slows, and the ³²P content reaches a steady state after 15 minutes. During the first 2 minutes after the addition of atropine to carbamylcholine stimulated cells, ³²P is lost from phosphatidic acid, and an equal amount is gained by phosphatidylinositol, after which ³²P incorporation equals that of the control. In cells prelabelled with ³²P, carbamylcholine, in the presence of oligomycin stimulated the loss of ³²P from phosphatidylinositol but had no effect on phosphatidic acid.     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

IV. Metabolism of inositides and the activation of platelets

Platelet activation is associated with the active metabolism of inositide lipids. Phosphodiesteratic cleavage of phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate is a consequence of receptor-coupled mechanisms. Degredation of phosphatidylinostiol-4,5-biphosphate is Ca²⁺ -insensitive while that of phosphatidylinositol requires Ca²⁺. The phosphodiesteratic breakdown of these inositides induces the formation of 1,2-diacylglycerol which is rapidly phosphorylated to phosphatidic acid. These biochemical changes might be related to fundamental mechanisms of amplication involved in the process of platelet activation. Phosphatidic acid constitutes an ubiquitous marker for the action of a wide variety of platelet stimuli.     

Inactivation of (Na+ + K+)-stimulated ATPase by a cytotoxic protein from cobra venom in relation to its lytic effects on cells

The mechanism of action of the cytotoxic protein P₆ isolated from cobra venom (Naja naja) which shows preferential cytotoxicity particularly to Yoshida sarcoma cells has been studied by its effects on the membrane-bound enzyme (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of a variety of cell systems. Evidence obtained with Yoshida sarcoma cells, dog and human erythrocytes and three tissue culture cell lines KB (human oral carcinoma), Hela (human cervix carcinoma) and L-132 (human lung embryonic) shows that inhibition of (Na⁺ + K⁺)-ATPase by the P₆ protein can be correlated with its lytic activity. (Na⁺ + K⁺)-ATPase of Yoshida sarcoma membrane fragments inactivated by P₆ protein could be reconstituted by the addition of phosphatidylserine and phosphatidic acid. It is conceivable that lysis of cells by the P₆ protein may be due to an imbalance of K⁺ and Na⁺ in the cell which leads to swelling and disintegration of the membrane structure. Observations indicate that the P₆ protein combines with membrane constituents of susceptible cells. The overall evidence suggests that both the specificity of its protein structure and the highly basic nature of the P₆ protein are factors which enable it to compete with the lipid moiety maintaining the (Na⁺ + K⁺)-ATPase of the susceptible cells in proper conformation for activity.     

Corticotropin-(1--24)-tetracosapeptide affects protein phosphorylation and polyphosphoinositide metabolism in rat brain.

1. Effects of corticotropin-(1--24)-tetracosapeptide on the endogenous phosphorylation of proteins and lipids were studied in a membrane/cytosol fraction prepared from a lysed crude mitochondrial/synaptosomal fraction. 2. The labelling of proteins and lipids was monitored by incubation of the subcellular fraction for 10s with [gamma-32P]ATP. 3. The phosphorylation of proteins was dose-dependently inhibited by the peptide (40% of control incubations at 100 microM-corticotropin). 4. Of the membrane phospholipids only phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidic acid became labelled. Corticotropin dose-dependently increased the formation of phosphatidylinositol bisphosphate and inhibited the production of phosphatidic acid (470% and 50% respectively of control incubations, at 100 microM of the peptide) and had no effect on phosphatidylinositol phosphate. 5. Phosphatase activity was observed to act on phosphatidylinositol bisphosphate, phosphatidylinositol phosphate and phosphoprotein but not on phosphatidic acid. 6. Corticotropin interacted with the kinases rather than with the phosphatases. 7. The formation of phosphatidylinositol bisphosphate and phosphatidic acid was maximal at 1--10mM-Mg2+ in the absence of Ca2+, and the production of phosphatidylinositol phosphate was maximal at 30mM-Mg2+. 8. The basal value of lipid phosphorylation decreased with increasing Ca2+ concentration. 9. Ca2+ abolished the effect of corticotropin on phosphatidylinositol bisphosphate formation (470%, 190% and 100% of control incubations at respectively 0, 0.1 and 1 mM-Ca2+). 10. The data provide evidence that the effects of corticotropin on protein phosphorylation and on polyphosphoinositide metabolism in brain membranes are related.     

The Role of Phospholipids in Plasma Membrane ATPase Activity in Vigna radiata L. (Mung Bean) Roots and Hypocotyls

Root and hypocotyl plasma membrane H⁺-ATPases were partially purified from deoxycholate-solubilized fractions of microsomes in mung bean (Vigna radiata L.) plants in the presence of glycerol. Certain properties of the ATPases and the manner in which phospholipids affect their activity were compared. Root ATPase was similar to hypocotyl ATPase with respect to substrate specificity, salt stimulation, pH dependence, K_m for ATP·Mg²⁺ and inhibitor sensitivity, except for inhibition by vanadate. Both purified ATPases required phospholipids for their activation. Optimum concentrations of exogenously added phospholipid mixture (asolectin) to hypocotyl and root ATPase mixture were 0.03% and 1.0%, respectively. Root ATPase activation did not decrease if more than 1.0% asolectin was added. Qualitatively, phosphatidylserine and phosphatidylcholine brought about greater ATPase activation than other phospholipids. The hypocotyl ATPase was activated by phosphatidylinositol, phosphatidylserine and phosphatidylglycerol to a greater extent than the root ATPase. Root, but not hypocotyl ATPase, was slightly inhibited by the addition of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. The hypocotyl plasma membrane contained phosphatidylinositol + phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and unsaturated fatty acids in greater abundance than the root plasma membrane. The differential activation of the plasma membrane ATPases may arise from these differences.     

Mechanisms of action of calcium-mobilizing agonists: some variations on a young theme

It is now accepted that many hormones and neurotransmitters exert their effects through G protein-mediated activation of a phospholipase C, which breaks down phosphatidylinositol bisphosphate. This releases inositol trisphosphate, which mobilizes intracellular calcium, and diacylglycerol, which, in turn, activates protein kinase C. However, recent evidence indicates that other mechanisms are involved. In some cells, the increases in cytosolic calcium elicited within 1-2 s by high concentrations of agonists or at later times by low, physiological concentrations of agonists occur without any detectable changes in inositol phosphates and calcium mobilization, and result from the opening of plasma membrane channels that are permeable to Ca2+. This response appears to be mediated more directly by G proteins. These findings question the postulated roles of inositol phosphates and calcium mobilization in the stimulation of calcium influx. Measurements of the mass and fatty acid composition of the inositol phospholipids and of the diacylglycerol and phosphatidic acid generated by agonists in several cell types indicate that phosphatidylinositol bisphosphate is probably a minor source of these lipids. On the other hand, measurements of phosphatidylcholine, choline, and phosphocholine indicate that this phospholipid is a major source, and that its breakdown involves both phospholipase C and D. These findings indicate that phosphatidylcholine breakdown may be more important than phosphoinositide hydrolysis in the regulation of protein kinase C and perhaps other cell functions.     

An Endogenous Na+, K+-ATPase Inhibitor Enhances Phosphoinositide Hydrolysis in Neonatal but Not in Adult Rat Brain Cortex

The effect of an endogenous Na⁺, K⁺-ATPase inhibitor, termed endobain E, on phosphoinositide hydrolysis was studied in rat brain cortical prisms and compared with that of ouabain. As already shown for ouabain, a transient effect was obtained with endobain E; maximal accumulation of inositol phosphates induced by endobain E was 604 ± 138% and 186 ± 48% of basal values in neonatal and adult rats, respectively. The concentration-response plot for the interaction between endobain E and phosphoinositide turnover differed from that of ouabain, thus suggesting the involvement of distinct mechanisms. In the presence of endobain E plus ouabain at saturating concentrations, no additive effect was recorded, suggesting that both substances share at least a common step in their activation mechanism of inositol phosphates metabolism or that they enhance phosphatidylinositol 4,5-biphosphate breakdown from the same membrane precursor pool, until its exhaustion. Experiments with benzamil, a potent blocker of Na⁺/Ca²⁺ exchanger, showed that it partially and dose-dependently inhibited endobain E effect. These results indicate that the endogenous Na⁺, K⁺-ATPase inhibitor endobain E, like ouabain, is able to stimulate phosphoinositide turnover transiently during postnatal brain development.     

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.     

A role of membranes in the activation of a new multifunctional protein kinase system.

A new multifunctional protein kinase, which normally exists as an inactive form in the soluble fraction in mammalian tissues, attaches to membranes to exhibit full enzymatic activity. A low concentration of Ca2+ is absolutely necessary for this activation. This process is reversible. cAMP shows no effect. The active factors in membranes are phosphatidylinositol, phosphatidylserine, phosphatidic acid, diphosphatidylglycerol, and phosphatidylethanolamine in that order. Phosphatidylcholine and sphingomyelin are far less effective. Cytoplasmic as well as other membrane fractions from various tissues are active in supporting the enzymatic activity. A possible role of this Ca2+ and phospholipid-activated protein kinase system in transmembrane control is proposed.