The role of calcium in agonist-stimulated hydrolysis of phosphatidylinositol in mouse pancreas

The effects of Ca²⁺ on agonist-stimulated hydrolysis of myo-[2-³H]inostol-labelled phosphatidylinositol in mouse pancreas in vitro, were studied. The increase in cytosol Ca²⁺ concentration produced by the ionophore A23187 did not stimulate the breakdown of phosphatidylinositol. Cholecystokinin-octapeptide (CCK-8) stimulated the hydrolysis of phosphatidylinositol under conditions in which intracellular calcium stores were depleted. The breakdown of phosphatidylinositol was stimulated by bethanechol and CCK-8 in Ca²⁺-free Krebs solution, and the addition of Ca²⁺ to the medium potentiated the effects of these agonists. Lanthanum significantly reduced bethanechol and CCK-8 stimulated hydrolysis of phosphatidylinositol in Krebs solution, but was without effect in Ca²⁺-free Krebs solution. The results of this study support the proposal that PI hydrolysis does not occur as a result of Ca²⁺ mobilization and may be involved in Ca²⁺ gating in the pancreas.     

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.     

Phosphatidylinositol hydrolysis and phosphatidylinositol 4′,5′-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca²⁺-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca²⁺ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of ³²PO₄ into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca²⁺-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This ³²PO₄ turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4′,5′-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca²⁺ deficiency. This phosphatidylinositol 4′,5′-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca²⁺ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4′,5′-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4′,5′-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca²⁺ mobilization.     

Structural preferences of phosphatidylinositol and phosphatidylinositol-phosphatidylethanolamine model membranes influence of Ca2+ and Mg2+

The structural preferences of soya phosphatidylinositol in isolation and in mixtures with soya phosphatidylethanolamine, and the influence of Ca²⁺ and Mg²⁺ on these preferences, have been examined employing ³¹P-NMR and freeze-fracture techniques. It is shown that phosphatidylinositol assumes the bilayer organization on hydration both in the presence and absence of Ca²⁺ and Mg²⁺. In mixed systems with H_II phase) phosphatidylethanolamine, phosphatidylinositol induces lipidic particle structure at low (<10 mol%) concentrations and bilayer structure at higher levels. In systems containing 15 or 20 mol% phosphatidylinositol, Ca²⁺ (but not Mg²⁺) can induce H_II phase structure. The results indicate that phosphatidylinositol is a more effective agent than other acidic phospholipids for stabilizing bilayer structure, particularly when high levels of divalent cations are present. These findings are discussed in terms of functional roles of phosphatidylinositol and mechanisms whereby Ca²⁺ induces structural reorganization in mixed systems containing acidic phospholipids and phosphatidylethanolamine.     

PCaPs,possible regulators of PtdInsP signals on plasma membrane

In plants, Ca²⁺, phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates are major components of intracellular signaling. Several kinds of proteins and enzymes, such as calmodulin (CaM), protein kinase, protein phosphatase, and the Ca²⁺ channel, mediate the signaling. Two new Ca²⁺-binding proteins were identified from Arabidopsis thaliana and named PCaP1 and PCaP2 [plasma membrane (PM)-associated Ca²⁺(cation)-binding protein 1 and 2]. PCaP1 has an intrinsically disordered region in the central and C-terminal parts. The PCaP1 gene is expressed in most tissues and the PCaP2 gene is expressed predominantly in root hairs and pollen tubes. We recently demonstrated that these proteins are N-myristoylated, stably anchored in the PM, and are bound with phosphatidylinositol phosphates, especially PtdInsP₂s. Here we propose a model for the switching mechanism of Ca²⁺-signaling mediated by PtdInsPs. Ca²⁺ forms a complex with CaM (Ca²⁺-CaM) when there is an increase in the cytosol free Ca²⁺. The binding of PCaPs with Ca²⁺-CaM causes PCaPs to release PtdInsPs. Until the release of PtdInsPs, the signaling is kept in the resting state.Key words: calcium signal, calmodulin, inositol phosphate, intrinsically disordered protein, myristoylation, phosphatidylinositol phosphate, plasma membrane     

Role of phosphatidylinositol turnover in alpha1 and of adenylate cyclase inhibition in alpha2 effects of catecholamines

Ligand binding and pharmacological studies have indicated that alpha-adrenergic receptors can be divided into alpha₁ and alpha₂. We suggest that alpha₁ receptors mediate those metabolic effects of alpha catecholamines which involve phosphatidylinositol turnover and the release of bound intracellular Ca²⁺ as well as the entry of extracellular Ca²⁺. In contrast, alpha effects of catecholamines are due to non-specific inhibition of adenylate cyclase through a mechanism independent of Ca²⁺. A similar classification for the effects of both histamine and serotonin suggests that they have separate type 1 or alpha receptors for Ca²⁺ dynamics which are different from type 2 or beta receptors which regulate adenylate cyclase.There is a significant correlation between hormone effects on phosphatidylinositol turnover and elevation of intracellular Ca²⁺. The available data suggest that the turnover of membrane-bound phosphatidylinositol is involved in Ca²⁺ gating in rat hepatocytes, rat and hamster adipocytes and blowfly salivary glands. In hamster adipocytes adenylate cyclase activity is also inhibited by alpha₂ catecholamines through a Ca²⁺ independent mechanism.     

Phosphatidylinositol turnover in mitogen-activated lymphocytes. Suppression by low-density lipoproteins

Low-density (LD) lipoproteins inhibit phytohaemagglutinin-enhanced turnover of phosphatidylinositol in human peripheral lymphocytes. Turnover was assessed by ³²P incorporation into phospholipids and by loss of ³²P from [³²P]phosphatidylinositol. Inhibition of lipid turnover by LD lipoproteins is not the result of a change in the amount of phytohaemagglutinin required for maximum cellular response. Neither phytohaemagglutinin nor LD lipoproteins influence ³²P incorporation into phosphatidylethanolamine and phosphatidylcholine during the first 60min after mitogenic challenge. The extent of inhibition of phosphatidylinositol turnover by LD lipoproteins depends on the concentration of LD lipoproteins present in the incubation medium: 50% of maximum inhibition occurs at a low-density-lipoprotein protein concentration of 33μg/ml and maximum inhibition occurs at low-density-lipoprotein protein concentrations above 100μg/ml. Phytohaemagglutinin stimulates ³²P incorporation into phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. However, LD lipoproteins abolish ³²P incorporation into phosphatidylinositol without affecting incorporation into phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. The ability of LD lipoproteins to inhibit phytohaemagglutinin-induced phosphatidylinositol turnover is mimicked by EGTA. Furthermore, inhibition of LD lipoproteins by phytohaemagglutinin-induced ³²P incorporation into phosphatidylinositol correlates directly with inhibition by LD lipoproteins of Ca²⁺ accumulation. These results suggest that Ca²⁺ accumulation and turnover of phosphatidylinositol are coupled responses in lymphocytes challenged by mitogens. The step in phosphatidylinositol metabolism that is sensitive to LD lipoproteins and, by inference, that is coupled to Ca²⁺ accumulation is release of [³²P]phosphoinositol from phosphatidylinositol.     

A calcium-activated polyphosphoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes

Haemoglobin-free human erythrocyte ghosts that were prepared in the presence of EDTA and were then exposed to Ca²⁺ showed a substantial loss of phosphatidylinositol phosphate and phosphatidylinositol diphosphate, measured either chemically or by loss of ³²P from the lipids of prelabelled membranes. At the same time there was, as reported previously (Allan, D. and Michell, R.H., (1976) Biochim. Biophys. Acta 455, 824–830), an approximately equivalent rise in the diacylglycerol content of the membranes. Analysis of the ³²P-labelled water-soluble material released during this process showed that the major products were inositol diphosphate and inositol triphosphate. No change was seen in the phosphatidylinositol or phosphatidate content of the membranes, and there was no Ca²⁺-activated loss of ³²P from the phosphatidate of prelabelled membranes: this suggests that Ca²⁺ did not activate phosphoinositide phosphomonoesterases or phosphatidate phosphomonoesterase in human erythrocyte membranes. It is concluded that human erythrocyte membranes contain at their cytoplasmic surface a Ca²⁺-activated phosphodiesterase that is active against both phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Rabbit erythrocytes also contained this enzyme, but in these cells there was also evidence for the presence of a Ca²⁺-activated phosphatidate phosphomonoesterase.     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.     

A Novel Phosphorylated Lipid Counteracts Activation of the Renal Plasma Membrane (Ca2+ + Mg2+)ATPase by Endogenous Phosphatidylinositol-4-Phosphate

The plasma membrane (Ca²⁺ + Mg²⁺)ATPase is activated by acidic phospholipids in reconstituted systems. In this report it is shown that reversible phosphorylation of endogenous phosphatidylinositol regulates the renal plasma membrane (Ca²⁺ + Mg²⁺)ATPase, and that a novel phosphorylated lipid that can be isolated from the same membrane strongly counteracts the stimulatory effect of phosphatidylinositol-4-phosphate.     

Phospholipid metabolism in calcium-regulated differentiation in cultured murine keratinocytes

The present findings associate phospholipid alteration, particularly the turnover of phosphatidylinositol, in Ca²⁺ induced differentiation of keratinocytes. These conclusions are based on the hydrolysis of ¹⁴C-AA from prelabeled PI and the accumulation ¹⁴C-DG and ¹⁴C-PA after cells are switched from low to normal concentrations of extracellular Ca²⁺. This novel finding implies that the biological changes which accompany keratinocyte differentiation after switch from low to normal extracellular medium may be due at least in part to increased accumulation of PA and DG which are major deacylation and reacylation products of phosphatidylinositol. A second interesting finding in these studies is the marked transformation of ¹⁴C-AA into lipoxygenase products by proliferating keratinocytes cultured in low Ca²⁺ medium when compared to differentiating cells cultured in normal Ca²⁺. The significance of decreased generation of lipoxygenase products in epidermal differentiation deserve further exploration.     

Control of membrane fusion by phospholid head groups I. Phosphatidate/phosphatidylinositol specificity

We have studied the characteristics of fusion of large unilamellar vesicles composed of phosphatidate and phosphatidylinositol alone and in mixtures with other naturally occurring phospholipids. Fusion was induced by the addition of Ca²⁺ or Mg²⁺ and was monitored by detecting the mixing of aqueous vesicle contents. Release of vesicle contents was measured by dequenching of carboxyfluorescein fluorescence. Aggregation was monitored by 90° light scattering. The results indicated striking differences with respect to the fusion capacity of the different vesicles. Phosphatidate vesicles fuse in the presence of both Ca²⁺ and Mg²⁺ at threshold concentration ranges of 0.03–0.1 mM (Ca²⁺) and 0.07–0.15 mM (Mg²⁺) depending on the pH of the medium, 8.5-6.0, respectively. In contrast, phosphatidylinositol vesicles do not fuse with either Ca²⁺ or Mg²⁺ even at 50 mM concentrations, in spite of aggregation induced by both cations in the range of 5–10 mM. A large difference in terms of fusion capacity is retained even when these two phospholipids are mixed with phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in 2 : 2 : 4 : 2 molar ratios. The results are discussed in terms of the molecular mechanism of membrane fusion and the possible role of the metabolic interconversion of phosphatidylinositol to phosphatidate as an on-off control system for membrane fusion phenomena involved in secretion.     

Mechanism of desensitization of neutrophil response to N-formylmethionylleucylphenylalanine by slow rate of receptor occupancy. Studies on changes in Ca2+ concentration and phosphatidylinositol turnover

Previous studies on the regulation of responses of neutrophils to fMet-Leu-Phe have demonstrated the relevance of the role of the rate of occupation of the receptors by the stimulant. When this rate is decreased by presenting the peptide to neutrophils over a period of time by means of an infusion pump, the activation of the respiratory burst and of the secretion is greatly depressed or is absent. This paper deals with further investigations on the mechanisms of this desensitization, which previous results have shown to consist of an uncoupling between the ligand-receptor complexes and the target for cell responses, caused by the deceleration of the initial rate of occupation of the receptors. The data presented here demonstrate that this desensitization is not linked to the formation of a negative intermediate such as cAMP, but is associated with: (i) a depression of the rate and magnitude of the phosphatidylinositol response (activation of phospahtidylinositol turnover measured as modification of incorporation of [³²P]P_i and [³H]glycerol into phosphatidylinositol and phosphatidic acid); (ii) a deceleration of the rate of the release of bound Ca²⁺, without a decrease in the total quantity of Ca²⁺ liberated (measured as fluorescence changes of chlorotetracycline treated neutrophils); (iii) a slower rise of cytosolic free Ca²⁺ concentration [Ca²⁺]_i, without a decrease in the magnitude of the final increase of [Ca²⁺]_i (monitored with Quin 2). These findings, which are discussed in relation to the recent hypotheses on the transduction reactions of receptor-mediated stimuli for neutrophil responses, are consistent with a mechanism of desensitization involving decreased production of diacylglycerol by the hydrolysis of phosphatidylinositol and deficient activation of Ca²⁺-phospholipid-dependent protein kinase C.     

Protein-mediated intermembrane contact specifically enhances Ca2+-induced fusion of phosphatidate-containing membranes

Ca²⁺-induced fusion of glycolipid-phospholipid vesicles containing several different anionic phospholipids was investigated, with and without lectin-mediated intervesicle contact. In vesicles containing phosphatidylserine, phosphatidylinositol or its mono- or diphosphate as the anionic phospholipid fusion was induced only at 1–10 mM Ca²⁺ both in the absence and presence of lectin. In contrast, the Ca²⁺-threshold for fusion of phosphatidate-containing vesicles was reduced to ?0.1 mM Ca²⁺ by lectin-mediated intermembrane contact.     

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca²⁺ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca²⁺ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca²⁺ permeability. The effect was not specific for Ca²⁺. Other divalent cations were translocated in the order Mn²⁺ > Mg²⁺  Ca²⁺ ? Sr²⁺  Ba²⁺. The permeability of liposomes to the monovalent cation, Na⁺, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.     

Effects of Ca ionophore A23187 and Ca deficiency on pancreatic phospholipids and amylase release in vitro

The Ca²⁺ ionophore A23187 elicits a transient increase in pancreatic amylase release in vitro, and this is accompanied by a transient decrease in phosphatidyl inositol concentration. Effects of ionophore A23187 and carbachol on amylase release and phosphatidylinositol breakdown are dependent on medium Ca²⁺. These results suggest that major secretagogue-induced, pancreatic phospholipid changes follow, rather than precede, changes in Ca²⁺ in the pancreas.     

Control of membrane fusion by phospholipid head groups II. The role of phosphatidylethanolamine in mixtures with phosphatidate and phosphatidylinositol

Membrane fusion induced by Ca²⁺ and Mg²⁺ in large unilamellar vesicles composed of mixtures of phosphatidylethanolamine with phosphatidate and phosphatidylinositol was studied by means of a fluorescence assay for the intermixing of internal aqueous contents of the vesicles. The threshold concentrations of Ca²⁺ or Mg²⁺ required for fusion increased only moderately when up to 80 mol% phosphatidylethanolamine was included with phosphatidate at pH 7.4, but no fusion could be detected in vesicles containing 70 mol% phosphatidylcholine even at high concentrations of Ca²⁺ or Mg²⁺. Phosphatidate-phosphatidylethanolamine (1 : 4) vesicles could be induced to fuse by 0.1 mM Ca²⁺ in the presence of a Mg²⁺ concentration which alone was insufficient for fusion. When equimolar amounts of phosphatidylethanolamine was included with phosphatidylinositol, the vesicles were susceptible to fusion by Ca²⁺, although pure phosphatidylinositol vesicles themselves merely aggregate and do not fuse (Sundler, R. and Papahadjopoulos, D. (1981) Biochim. Biophys. Acta 649, 743–750, accompanying paper). The role of phosphatidylethanolamine acyl chains, and hence the possible involvement of the bilayer-hexagonal (H_II) transition in membrane fusion, was examined by the temperature dependence of Ca²⁺-induced fusion in phosphatidylinositol-dimyristoylphosphatidylethanolamine (1 : 1) vesicles. Fusion was strictly dependent on the gel-liquid crystalline transition of the mixture and not on the phase behavior of the phosphatidylethanolamines. Comparable fusion rates were obtained for both egg yolk phosphatidylethanolamine and dimyristoylphosphatidylethanolamine at 50°C. As the dimyristoylphosphatidylethanolamine does not convert to a non-bilayer phase in this temperature range, we conclude that the bilayer-hexagonal transition is not necessary for membrane fusion. We propose that the dehydration characteristics of the phospholipids and their metal ion complexes are the critical factors determining fusion suceptibility of phospholipid membranes.     

Protein-mediated intermembrane contact specifically enhances Ca-induced fusion of phosphatidate-containing membranes

Ca²⁺-induced fusion of glycolipid-phospholipid vesicles containing several different anionic phospholipids was investigated, with and without lectin-mediated intervesicle contact. In vesicles containing phosphatidylserine, phosphatidylinositol or its mono- or diphosphate as the anionic phospholipid fusion was induced only at 1–10 mM Ca²⁺ both in the absence and presence of lectin. In contrast, the Ca²⁺-threshold for fusion of phosphatidate-containing vesicles was reduced to 0.1 mM Ca²⁺ by lectin-mediated intermembrane contact.     

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.     

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.