Increased Phosphatidylinositol Turnover Research Articles

Scorpion venom ( Leiurus quinquestriatus) elicits accumulations of inositol phosphates and cyclic AMP in guinea pig cortical synaptoneurosomes

Scorpion ( Leiurus quinquestriatus) venom (ScV) stimulated accumulations of cyclic AMP and turnover of phosphatidylinositol in guinea pig cortical synaptoneurosomes. The concentrations of ScV that were necessary to increase cyclic [ 3H]AMP accumulation were lower than those required to stimulate formation of [ 3H]inositol phosphates from phosphatidylinositol. In the presence of 10 μM 2-chloroadenosine, ScV induced a dose-dependent synergistic accumulation of cyclic AMP with an EC 50 value that was comparable to the EC 50 required for stimulation of phosphatidylinositol turnover. Tetrodotoxin partially inhibited cyclic AMP accumulations elicited by ScV indicating that at least part of such responses are due to activation of voltage-dependent sodium channel. Tetrodotoxin virtually completely blocked formation of inositol phosphate stimulated by ScV. High concentrations of Mg 2+ (30 mM) did not block responses to ScV indicating that release of neurotransmitters was not involved. Membrane potential changes could not be detected at concentrations of ScV that triggered the biochemical responses. Stimulation of phosphatidylinositol turnover by ScV appears to depend on an increase in influx of Na + in synaptoneurosomes, presumably due to slowing of the inactivation of voltage-dependent sodium channels by α-scorpion toxin, a component of ScV. At least in part, the stimulation of cyclic AMP accumulation by ScV correlates with increases in phosphatidylinositol turnover.

Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED 50 for TPA-stimulated progesterone production was 3 nM, which is close to the known K m for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.

Effects of Isoproterenol Pretreatment on Phosphatidylinositol Turnover in Rat Parotid Gland

The effects of isoproterenol pretreatment on phosphatidylinositol turnover in rat parotid slices were studied to elucidate the relationship between β- and α1-adrenoceptors. 32P-Labeling of phosphatidylinositol in parotid slices was increased by an α1- and α2-agcmist (epinephrine and norepinephrine) and α1-agonists (methoxamine and phenylephrine), but not by an α2-agonist (clonidine) and a β-agonist (isoproterenol). Prazosin inhibited the increase in phosphatidylinositol turnover elicited by epinephrine, but propranolol did not. These results indicate that the stimulation of phosphatidylinositol turnover elicited by adrenergic agonists is mediated by activation of α1-adrenoceptors in the parotid glands. Isoproterenol pretreatment of the parotid slices caused a significant increase in 32P-labeling of phosphatidylinositol and a decrease in that of phosphatidic at i The epinephrine- or phenylephrine-induced increase in 32P-labeling of phosphatidylinositol were further enhanced by the isoproterenol pretreatment of the slices. In the isoproterenol-treated membranes of the parotid glands, [3H]prazosin binding to a-i-receptors increased, but [3H]dihydroalprenolol binding to β-receptors did not. These findings indicate that the acceleration of phosphatidylinositol turnover induced by the isoproterenol pretreatment may be associated with an increase in α1-adrenoceptor binding sites which might have appeared as a result of the isoproterenol pretreatment of the parotid slices.

Calcium-independent phosphatidylinositol response in gonadotropin-releasing-hormone-stimulated pituitary cells.

This paper describes the effect of gonadotropin-releasing hormone (GnRH, gonadoliberin) on phospholipid metabolism in cultured rat pituitary cells. The cells were incubated with [32P]Pi to label endogenous phospholipids (10-60 min) and then stimulated with GnRH for up to 60 min. Cellular phospholipids were separated by two-dimensional t.l.c. and the radioactivity was determined. Phosphatidylinositol (PI), a minor constituent of cellular phospholipids (7.7%), was the major labelled phospholipid, accounting for 45% of the total radioactivity, at early periods after pulse labelling. On the other hand, phosphatidylcholine, the major cellular phospholipid (37%), was labelled only to 32% of the total radioactivity. The remaining label was distributed among phosphatidylethanolamine (4.2%), cardiolipin (3.4%), phosphatidic acid (PA, 2.5%), and phosphatidylserine (1.8%). GnRH doubled 32P labelling of PA and PI significantly at 1 and 5 min of incubation respectively in the presence or absence of extracellular Ca2+. Labelling of other phospholipids was not affected by GnRH treatment. The half-maximal stimulating dose (ED50) for PI labelling and lutropin release was 0.75 nM and 0.5 nM respectively, and the stimulatory effect was blocked by the potent GnRH antagonist [D-Glp1,pClPhe2,D-Trp3,6]GnRH. GnRH-stimulated PA and PI labelling could not be demonstrated after 1 and 45 min of incubation respectively, or when the prelabelling was conducted for 60 min rather than 10 min. These results suggest heterogeneous compartmentalization of gonadotroph PA and PI pools and that increased PI turnover might be a transducing signal for Ca2+ gating that follows gonadotroph GnRH-receptor activation.

Lipopolysaccharide and phorbol esters induce differentiation but have opposite effects on phosphatidylinositol turnover and Ca2+ mobilization in 70Z/3 pre-B lymphocytes.

We have recently shown that both lipopolysaccharide (LPS) and the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) induce differentiation in the transformed murine pre-B lymphocyte cell line 70Z/3 by enhancing Na+-H+ exchange across the plasma membrane through an amiloride-sensitive transport system (Rosoff, P.M., Stein, L.F., and Cantley, L.C. (1984) J. Biol. Chem. 259, 7056-7060). These data suggested that the activation of protein kinase C indirectly by LPS and directly by TPA was the critical step in the initiation of differentiation in these cells. We extend these observations to show that LPS rapidly stimulates an increase in phosphatidylinositol turnover, leading to a rise in the levels of diacylglycerol and inositol 1,4,5-trisphosphate and a concomitant decrease in the amount of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. There is also a rapid elevation of intracellular free [Ca2+] which is independent of the presence of extracellular Ca2+ or Na+. These results suggest that the increase in cytosolic [Ca2+] is due to release of cation from internal stores. TPA, which also causes differentiation in these cells, and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, have opposite effects from LPS on both phosphatidylinositol turnover and cellular Ca+ mobilization. These data suggest that protein kinase C inhibits the activity of phospholipase C. Thus protein kinase C plays a pivotal role in the regulation of mitogen-induced differentiation in these cells by both transducing a positive stimulus to the Na+-H+ exchange system as well as feedback regulating its own stimulatory pathway.

Inhibition of lymphocyte proliferation by detergent-solubilized mouse liver membranes.

The role of phenomena analogous to fibroblast contact inhibition in lymphocyte growth regulation is controversial, although it is clear that direct cell-cell contact is vital to immunoregulation and accessory cell function. An extract of mouse liver plasma membrane proteins, referred to as suppressive liver extract (SLE), that suppresses the growth of 3T3 fibroblasts also inhibited the mitogen-induced proliferation of murine lymphocytes. A dose of 20 micrograms/ml SLE was less than 95% suppressive of proliferation in both mouse T and mouse B cells treated with a variety of mitogens. B cell growth factor, while increasing DNA synthesis overall in mitogen-stimulated B cells, did not change the extent of SLE suppression, which suggests that the SLE does not interfere with lymphocyte-growth factor interactions. In exploring a sequence of B cell activation events, we discovered that SLE had no effect on the early activation event of increased phosphatidylinositol turnover. Blastogenesis, however, was inhibited in mitogen-stimulated, SLE-treated B cells. The maximum suppressive effect was observed if the SLE was added within 8-12 h of the mitogenic stimulus. SLE did not affect the viability of cells in culture. These results point to a possible unity of regulatory mechanisms between contact inhibition in fibroblasts and the processes of immunoregulation.

Prostaglandin synthesis linked to phosphatidylinositol turnover in isolated rat glomeruli

Prostaglandins produced by the glomerulus are important factors in controlling glomerular function. The controlling step, i.e., the release of arachidonic acid from the phospholipids by either phospholipase A 2 and/or C, remains poorly defined. The present studies were designed to determine which factors control arachidonic acid turnover and prostaglandin synthesis in glomeruli. As tools we used the calcium ionophore A23187, mepacrine, a phospholipase inhibitor, trifluoperazine, a calmodulin antagonist, and angiotensin II. A23187 (2 μM) caused a significant stimulation of both prostaglandin E 2 and prostaglandin F 2α synthesis (measured by radioimmunoassay), which was associated with increased phosphatidylinositol turnover (measured by [ 14C]arachidonic acid and [ 32P]orthophosphate incorporation). Surprisingly, trifluoperazine (10–100 μM) also progressively increased synthesis of both prostaglandins, which was accompanied by increased phosphatidic acid/phosphatidylinositol turnover and decreased phosphatidylinositol content. In contrast, phosphatidylcholine and phosphatidylethanolamine turnover were significantly inhibited by trifluoperazine and their total content remained unaffected. Mepacrine (1 mM) decreased prostaglandin synthesis and both phosphatidylcholine and phosphatidylethanolamine turnover, and had no consistent effect on phosphatidylinositol turnover in control glomeruli. Mepacrine did, however, inhibit both A23187 or trifluoperazine-induced increase in phosphatidylinositol turnover. Angiotensin II increased turnover of phosphatidylinositol and also phosphatidylcholine, as determined by incorporation of [ 14C]arachidonic acid. Thus, all agents that increased prostaglandin synthesis also enhanced phosphatidylinositol turnover. The exact pathway of arachidonic acid release remains to be determined.

GONADOTROPIN-RELEASING HORMONE STIMULATES PHOSPHATIDYLINOSITOL LABELING AND PROSTAGLANDIN E PRODUCTION IN LEYDIG CELLS

The direct effects of gonadotropin releasing hormone (GnRH) upon testicular function include a rapid (integral of 20 min) receptor-mediated increase in phosphatidylinositol (PI) turnover. Incubation of rat interstitial cells with the super-agonist [D-Ala6]desGly10-GnRH N ethylamide (GnRHa) resulted in increased incorporation of [32P]Pi into PI (2-fold at 20 min). The effect on phospholipid turnover was followed by increased prostaglandin E (PGE) and testosterone production (3 h; with ED50 values of 0.5 and 0.75 nM respectively). It is concluded that increased PI turnover and PGE production might be involved in mediating the direct testicular effects of GnRH and its agonists.

Receptor-mediated increases in phosphatidylinositol turnover in neuron-like cell lines.

Muscarinic receptors found in the N1E-115 mouse neuroblastoma cell line were tested for their ability to mediate stimulation of phosphatidylinositol (PI) turnover. This study was facilitated by the development of a new solvent system (acetone:butanol:acetic acid:water, 3:5:1:1) for the rapid and consistent separation of PI by one-dimensional thin-layer chromatography. Cholinergic stimulation caused as much as a 680% increase in the incorporation of 32P into PI. Enhanced incorporation of 32P into PI could be measured as early as 4 min after stimulation began. By 20 min, the rate of incorporation by stimulated cells had decreased to that of unstimulated cells, indicating desensitization. The magnitude of the response was dependent on the extent of receptor occupancy, and the response elicited by a saturating dose of carbamylcholine was blocked completely by 10(-7) M atropine, a specific muscarinic antagonist. Chronic stimulation, known to cause a loss of receptor binding sites, led to a 90% decrease in the maximum response even after a 40-min withdrawal period. Replacement of Na+ ions in the medium with choline or K+ severely impaired the ability of the cells to incorporate added 32P into PI (90 and 50%, respectively). Removal of the putative second messenger Ca2+ for short periods of time by the addition of excess EGTA did not alter either basal or muscarinic-stimulated PI turnover.

Control of phosphatidylinositol turnover in adrenal glomerulosa cells

The purpose of the present experiments was to compare the effects on phosphatidylinositol metabolism of agents stimulating aldosterone secretion. Glomerulosa cells, isolated from rat adrenals, were incubated in the presence of one of the following stimuli: angiotensin II, elevated potassium concentration, corticotropin, dibutyryl cyclic AMP and prostaglandin E 2. Of all these substances, only angiotensin II stimulated the incorporation of [ 32Piphosphate into phosphatidylinositol. The effect was already detected 2.5 min and was still maintained 60 min after the onset of stimulation. A slight enhancement of the incorporation into other phospholipids was observed in the first minutes of stimulation. Cycloheximide abolished the effect of angiotensin II on aldosterone production, but not on phosphatidylinositol synthesis. In cells prelabelled with [ 32P]phosphate, radioactivity in phosphatidylinositol relative to that in other phospholipids decreased in response to angiotensin II within 5 min. This indicates that angiotensin II induces a specific breakdown of phosphatidylinositol. Corticotropin failed to enhance the incorporation of [ 32P]phosphate into phosphatidylinositol and other phospholipids in isolated fasciculate-reticularis cells. The results suggests that although both angiotensin II and potassium are presumed to act through changes in calcium metabolism, angiotensin alone generates the calcium signal by increased phosphatidylinositol turnover.

Role of alpha 1 adrenoceptors in the turnover of phosphatidylinositol and of alpha 2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin⪢phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha 1 adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ⪢prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha 2 adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha 1 and alpha 2 adrenoceptors but about 90% of the binding sites were alpha 2. These data support the hypothesis that alpha 2 effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha 1 adrenoceptor activation.

Evidence against dopaminergic and further support for alpha-adrenergic receptor involvement in the pineal phosphatidylinositol effect.

Abstract: Several α‐adrenergic receptor agonists and antagonists were used to strengthen the earlier findings that the stimulation by (‐)‐norepinephrine of 32P1 incorporation into acidic phospholipids, especially phosphatidylinositol, in the rat pineal gland is mediated through α‐adrenergic receptors. Dopamine was able to induce similar stimulation, although always to a smaller extent than equimolar concentrations of norepinephrine. The dopaminergic agonists apomorphine and piribedil did not increase phosphatidylinositol labeling. A number of antagonists considered to act primarily at dopaminergic or α‐adrenergic receptors respectively completely prevented dopamine from exerting its effect. Both types of antagonists also were able to inhibit in varying degree the elevation of phospholipid labeling induced by norepinephrine. Dopamine increased phosphatidylinositol turnover without first being converted to norepinephrine, inasmuch as dopamine β‐hydroxylase inhibitors had no influence on dopamine activity. Dopamine and α‐agonists competitively activated the receptors involved in the phospholipid effect. The conclusion drawn from the several lines of evidence is that only α‐adrenergic receptors are concerned with the changes in pineal phospholipid metabolism brought about by the various agonists used and that the action of dopamine occurs through these receptors rather than through discrete dopaminergic receptors.

Secretagogues for lysosomal enzyme release as stimulants of arachidonyl phosphatidylinositol turnover in rabbit neutrophils

Summary [ 14 C]arachidonic acid incorporation into phosphatidylinositol from rabbit neutrophils pretreated with cytochalasin B was increased within 2 min by the synthetic peptide formyl-methionyl-leucyl-phenylalanine and the Ca 2+ ionophore A23187. A high concentration of the peptide elicited only a small increase in phosphatidylinositol turnover when [ 14 C]palmitic acid was employed as precursor. These data coincide with our earlier studies which identified a Ca 2+ -dependent increase in arachidonyl phosphatidylinositol turnover during adrenocorticotropin and A23187-induced activation of steroid production and release in adrenocortical cells. It is concluded that an increase in arachidonyl phosphatidylinositol turnover mediated by a Ca 2+ -dependent phospholipase A 2 may be a general mechanism for altering membrane function during secretory activity.

Colchicine inhibits phosphatidylinositol turnover induced in lymphocytes by concavalin A.

RECENTLY colchicine has been shown to affect various membrane properties such as the lateral mobility of receptors and other surface membrane markers and the agglutination of different cell types. Many of the described phenomena also involve concanavalin A (con A); for example, phagocytosis by rabbit polymorphonuclear leukocytes (PMNs) leads to a selective depletion of con A binding sites which is prevented by colchicine1, con A inhibition of lymphocyte immunoglobulin receptor capping is reversed by colchicine2–4 and con A-induced aggregation of PMNs, fibroblasts and hepatoma cells is inhibited by colchicine5–7. In addition to these effects on membrane properties, colchicine also has been shown to inhibit con A-induced lymphocyte transformation at an early stage in the sequence of events leading to thymidine incorporation and blast transformation8,9. These various considerations led us to determine if colchicine could alter phosphatidylinositol (PI) turnover in lymphocytes since it is known that increased PI turnover is an early event induced by con A and other mitogenic lectins10–12.

Investigation of the relationship between cell-surface calcium-ion gating and phosphatidylinositol turnover by comparison of the effects of elevated extracellular potassium ion concentration on ileium smooth muscle and pancreas

Incubation of fragments of guinea-pig ileum smooth muscle in the presence of an elevated extracellular K+ concentration, which causes an increase in cell-surface Ca2+ permeability and thus leads to contraction, caused a marked increase in phosphatidylinositol turnover, as assessed by incorporation of 32Pi. This response was not diminished by atropine or propylbenzilycholine mustard, two muscarinic cholinergic antagonists, and was therefore not caused by the release of endogenous acetylcholine within the tissue. In contrast, exposure of guinea-pig pancreas fragments to high extracellular [K+], which does not increase cell-surface Ca2+ permeability or evoke secretion, did not cause an increase in phosphatidylinositol turnover, even though such an increase was triggered by carbamoylcholine, which is a secretagogue. These observations are consistent with a suggested function for phosphatidylinositol breakdown in the mechanisms of cell-surface Ca2+ gates.

Effects of calcium-antagonistic drugs on the stimulation by carbamoylcholine and histamine of phosphatidylinositol turnover in longitudinal smooth muscle of guinea-pig ileum.

A number of drugs classed as calcium antagonists, spasmolytics, non-specific receptor antagonists or receptor antagonists with multiple sites of action were tested to determine whether they prevent the stimulation of phosphatidylinositol turnover caused in various tissues by the activation of receptors which increase cell-surface Ca2+ permeability. The experiments were done with fragments of longitudinal smooth muscle from guinea-pig ileum; these were incubated in vitro with 32Pi and either 100 muM-carbamoylcholine or 100 muM-histamine, in the presence of antagonistic drugs at concentrations at least sufficient to cause complete blockade of smooth-muscle contraction. The phosphatidylinositol response to carbamoylcholine was not changed by cinchocaine, papaverine, nifedipine, dibenamine, amethocaine, cinnarizine, lidoflazine, methoxyverapamil, prenylamine or two antimuscarinic alkane-bis-ammonium compounds, and the response to histamine was unaffected by the first four drugs. In contrast, phenoxybenzamine prevented the increase in phosphatidylinositol labelling caused by either carbamoylcholine or histamine. The insensitivity of the phosphatidylinositol response to most of the drugs provides further experimental support for the conclusion that the receptor-stimulated phosphatidylinositol breakdown which initiates the increase in phosphatidylinositol turnover is not caused by an increase in intracellular Ca2+. The simplest interpretation of the available information appears to be that phosphatidylinositol breakdown plays a role in the coupling between the receptor-agonist interaction and the opening of cell-surface Ca2+ gates [Michell, R. H. (1975) Biochim. Biophys. Acta 415, 81-147]. If this is correct, then phenoxybenzamine must exert its inhibitory effects on phosphatidylinositol breakdown early in this sequence of events, but the drugs must act at a stage later than phosphatidylinositol breakdown. The unexpected difference in the effects of dibenamine and phenoxybenzamine, which are chemically very similar, may provide a useful experimental tool with which to explore the way in which activated receptors provoke the opening of cell-surface Ca2+ gates.

Phosphoinositide metabolism and insulin secretion from isolated rat pancreatic islets.

Since many cell types have been shown to respond to extracellular stimulation with a rapid increase in phosphatidylinositol turnover, the present studies were undertaken to determine whether carbohydrate-stimulated insulin secretion from the isolated rat pancreatic islet is accompanied by detectable alterations in the phosphatidylinositol metabolism of this tissue. We have demonstrated that rat pancreatic islets incubated with tritiated myo-inositol rapidly incorporate radioactivity into islet phosphatidylinositol. Incubation of prelabeled islets with elevated concentrations of carbohydrates which stimulate insulin secretion (D-glucose and D-mannose) results in a decrease in the recovery of lipid-bound radioactivity, whereas incubation with carbohydrates which do not stimulate insulin secretion (D-galactose and myo-inositol) has no effect upon the recovery of lipid-bound radioactivity. Within 2 min of exposure of prelabeled islets to elevated concentrations of D-glucose, a decrease in the recovery of [2-3H]myo-inositol-derived radioactivity in islet phosphatidylinositol can be demonstrated. When islets prelabeled with [2-3H]myo-inositol are perifused with elevated concentrations of D-glucose or D-mannose (but not D-galactose or myoinositol) a rapid and transient increase in the rate of extracellular release of water-soluble radioactivity is observed. Since a significant fraction of the radioactivity released under these conditions is in the form of myo-inositol phosphate, cyclic myo-inositol-1,2-phosphate, and glycerophosphorylmyo-inositol, it is presumably derived from the cleavage of labeled islet phosphatidylinositol. It is speculated that alterations in the metabolism of myo-inositol-containing phospholipids may have a functional role in the process of insulin secretion from the pancreatic beta cell.

The relationship of calcium to receptor-controlled stimulation of phosphatidylinositol turnover. Effects of acetylcholine, adrenaline, calcium ions, cinchocaine and a bivalent cation ionophore on rat parotid-gland fragments

The possibility that Ca2+ ions are involved in the control of the increased phosphatidylinositol turnover which is provoked by alpha-adrenergic or muscarinic cholinergic stimulation of rat parotid-gland fragments has been investigated. Both types of stimulation provoked phosphatidylinositol breakdown, which was detected either chemically or radiochemically, and provoked a compensatory synthesis of the lipid, detected as an increased rate of incorporation of 32Pi into phosphatidylinositol. Acetylcholine had little effect on the incorporation of labelled glycerol, whereas adrenaline stimulated it significantly, but to a much lower extent than 32P incorporation: this suggests that the response to acetylcholine was entirely accounted for by renewal of the phosphorylinositol head-group of the lipid, but that some synthesis de novo was involved in the response to adrenaline. The responses to both types of stimulation, whether measured as phosphatidylinositol breakdown or as phosphatidylinositol labelling, occurred equally well in incubation media containing 2.5 mm-Ca2+ or 0.2 mm-EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid]. Incubation with a bivalent cation ionophore (A23187) led to a small and more variable increase in phosphatidylinositol labelling with 32Pi, which occurred whether or not Ca2+ was available in the extracellular medium: this was not accompanied by significant phosphatidylinositol breakdown. Cinchocaine, a local anaesthetic, produced parallel increases in the incorporation of Pi and glycerol into phosphatidylinositol. This is compatible with its known ability to inhibit phosphatidate phosphohydrolase (EC 3.1.3.4) and increase phosphatidylinositol synthesis de novo in other cells. These results indicate that the phosphatidylinositol turnover evoked by alpha-adrenergic or muscarinic cholinergic stimuli in rat parotid gland probably does not depend on an influx of Ca2+ into the cells in response to stimulation. This is in marked contrast with the K+ efflux from this tissue, which is controlled by the same receptors, but is strictly dependent on the presence of extracellular Ca2+. The Ca2+-independence of stimulated phosphatidylinositol metabolism may mean that it is controlled through a mode of receptor function different from that which controls other cell responses. Alternatively, it can be interpreted as indicating that stimulated phosphatidylinositol breakdown is intimately involved in the mechanisms of action of alpha-adrenergic and muscarinic cholinergic receptor systems.

Relationship between enhanced turnover of phosphatidylinositol and lymphocyte activation by mitogens

1. Various lectins [phaseolus vulgaris phytohaemagglutinin, Glycine max (soy-bean) agglutinin, Triticum vulgaris (wheat-germ) agglutinin and Axinella polyploides agglutinin] and antibodies to pig Ig (immunoglobulin) that are found by pig lymphocytes were assessed in terms of their capacities to stimulate lymphocyte transformation and to enhance phosphatidylinositol turnover. Transformation was measured after 45h of culture by incorporation of [6-(3)H]thymidine into DNA, whereas phosphatidylinositol metabolism was assessed after 1h of cultuis and G. max agglutinins and rabbit antibodies to pig Ig) increased phosphatidylinositol turnover, but non-transforming agents (T. vulgaris and A. polyploides agglutinins and Fab fragments of rabbit antibodies to pig Ig) failed to induce any significant enhancement. Subsequent cross-linkage of the bound, non-transforming Fab fragments with a goat antiserum to rabbit Ig stimulated transformation and phosphatidylinositol turnover. 3. Each transforming agent gave characteristic optimal dose responses that were similar for both phosphatidylinositol turnover and transformation. 4. The results indicate that activation of T- and B-lymphocytes is accompanied by enhanced phosphatidylinositol turnover and that in the case of B-cells this enhancement depends on the cross-linkage of surface receptors. They are consistent with the proposal that turnover represents an essential early step in the transformation process.

Role of inositol cyclic phosphate in stimulated tissues.

THYROID-STIMULATING hormone markedly stimulates phosphatidylinositol turnover in sheep thyroid gland. The enzyme catalysing the degradation of phosphatidylinositol in animal tissues can split the phospholipid, with the formation of a diglyceride and a new cyclic ester, D-myoinositol-1: 2-cyclic phosphate as well as D-myoinositol-1-phosphate2,3. Increased phosphatidylinositol turnover has been described in a variety of conditions in which cells are stimulated to secrete or into other hyperactivity. The physiological possibilities of this degradation are obvious, and it was suggested2,3 that the catabolism of phosphatidylinositol and the release of inositol cyclic phosphate might be more directly relevant to the cell stimulation than to the biosynthesis involved in the turnover. Such an hypothesis is attractive, for an accumulation of phosphatidylinositol has never been demonstrated during stimulated conditions. In addition Ca2+ is an essential requirement for the formation of inositol cyclic phosphate as well as playing an important part in secretory processes4.