Stimulation of inorganic-phosphate incorporation into phosphatidylinositol in rat thoracic aorta mediated through V1-vasopressin receptors.

Vasopressin stimulates the incorporation of [32P]Pi into phosphatidylinositol but not into other phospholipids in rat thoracic aorta strips. The relative abilities of three vasopressin analogues to stimulate phosphatidylinositol labelling in rat aorta are similar to their relative pressor potencies in vivo and to their relative potencies in stimulating the metabolism of rat hepatocytes, but very different from their relative antidiuretic potencies. The vasopressor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin competitively inhibits [Arg8]vasopressin-stimulated phosphatidylinositol labelling in rat aorta with a pA2 of 8.1. It is concluded that the Ca2+-mobilizing vasopressin receptors (V1-receptors) of the rat aorta stimulate phosphatidylinositol metabolism, probably by enhancing phosphatidylinositol breakdown.     

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.     

Involvement of phosphoinositide metabolism in potentiation by adrenaline of ADP-induced aggregation of rabbit platelets.

Changes in phosphoinositide metabolism were examined in washed rabbit platelets stimulated with 0.5 microM-ADP, 50 microM-adrenaline, or ADP and adrenaline in combination. Adrenaline does not stimulate platelet aggregation when used alone, but does potentiate aggregation stimulated by ADP. In platelets prelabelled with [32P]Pi and [3H]glycerol, adrenaline was found to potentiate the ADP-induced changes in platelet phospholipids, causing larger increases in the amount and labelling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid than was observed with ADP alone. The combination of ADP and adrenaline did not produce a greater decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) than was produced by ADP alone. In platelets prelabelled with [3H]inositol, adrenaline potentiated the increases in labelling of inositol phosphate and inositol bisphosphate stimulated by ADP; no increase in inositol trisphosphate labelling was detected with ADP alone or with the combination of ADP and adrenaline. Phentolamine, an alpha-adrenergic-receptor antagonist, blocked potentiation by adrenaline of ADP-induced changes in phosphoinositide metabolism. Propranolol and sotalol, beta-adrenergic-receptor antagonists, augmented the potentiation; this is consistent with the concept that the effect of adrenaline is mediated by beta-adrenergic receptors. The effect of adrenaline on phosphoinositide metabolism appears to be to potentiate the mechanisms by which ADP causes turnover of PIP and possibly degradation of PI, rather than the mechanism by which PIP2 is decreased.     

The phosphoinositides exist in multiple metabolic pools in rabbit platelets.

The labelling of the phosphoinositides and phosphatidic acid in washed rabbit platelets incubated with [32P]phosphate or [3H]glycerol was studied in the presence of isotope and after unincorporated isotope had been removed. With both isotopes the increase in the specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2) lagged behind that of phosphatidylinositol 4-phosphate (PIP) but the specific radioactivity remained higher after unincorporated isotope had been removed. This result was consistent with the presence of a second pool of PIP2, which interconverted slowly with the pool of PIP2 which was in direct equilibrium with PIP, proposed to explain the increase in specific radioactivity of PIP2 which accompanies the decrease in amount of PIP2 at 10 s in ADP-stimulated platelets. In platelets labelled with [3H]glycerol, the specific radioactivity of PIP2 became higher than that of PIP and the specific radioactivity of PIP became higher than that of phosphatidylinositol (PI). These results were interpreted to indicate that there were two pools of PIP; of these the pool with the higher specific radioactivity was the precursor of PIP2. Similarly, two pools of PI were proposed. The presence of pools of the phosphoinositides with different specific radioactivities necessitates the measurement of chemical amount of these compounds when studying the effect of stimulation of the platelets, since changes in labelling may not accurately reflect changes in the amount of the phosphoinositides.     

Sodium ion and the neutrotransmitter-stimulated 32P labelling of phosphoinositides and other phospholipids in the iris muscle

The effects of Na+, other cations and the neurotransmitters, acetylcholine and norepinephrine on 32Pi incorporation into phospholipids of the rabbit iris smooth muscle were investigated [1]. The basal 32P-labelling of phospholipids including phosphatidic acid, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine and the polyphosphoinositides increased with Na+ concentration [2]. The neurotransmitter-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine is dependent on the presence of extracellular Na+ [3]. The monovalent cation requirement for Na+ specific. Of the monovalent cations Li+, NH+4, K+, Choline+ and Tris, only Li+ partially substituted for Na+ [4]. A significant decrease in 32P labelling of phospholipids in response to acetylcholine was observed when Ca2+ and/or K+ were added to an isoosmotic medium deficient of Na+ [5]. Ouabain, which blocks the Na+-pump, inhibited the basal 32Pi incorporation into phosphatidylcholine and the acetylcholine-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine [6]. It was suggested that phosphoinositide breakdown is associated with Ca2+ influx as we have previously reported (Akhtar, R.A. and Abdel-Latif, A.A. (1978) J. Pharmacol. Exp. Ther. 204, 655-668) and that the enhanced 32P-labelling of phosphoinositides could be associated with Na+ outflux, via the Na+-pump mechanism.     

Characterization of a phosphatidylinositol 4-phosphate-specific phosphomonoesterase in rat liver nuclear envelopes

Incubation of rat liver nuclear envelopes with [gamma-32P]ATP resulted in the synthesis of phosphatidylinositol-[4-32P]phosphate (PIP). Degradation of endogenously labeled PIP was observed upon the dilution of the labeled ATP with an excess of unlabeled ATP. This degradation was most rapid in the presence of EDTA, and was inhibited by MgCl2 and CaCl2. To further characterize the degradative activity, phosphatidylinositol[4-32P]phosphate and phosphatidylinositol [4,5-32P]bisphosphate (PIP2) were synthesized and isolated from erythrocyte plasma membranes. The 32P-labeled phospholipids were then resuspended in 0.4% Tween 80, a detergent that did not inhibit degradation of endogenously labeled PIP, and mixed with nuclear envelopes. [32P]PIP and [32P]PIP2 were degraded at rates of 2.25 and 0.04 nmol min-1 mg nuclear envelope protein-1, respectively. Only 32P was released from phosphatidyl[2-3H]inositol-[4-32P]phosphate, indicating that hydrolysis of PIP was due to a phosphomonoesterase activity (EC 3.1.3.36) in nuclear envelopes. Similarly, anion-exchange chromatographic analysis of the water-soluble products released from [32P]PIP indicated that inorganic phosphate was the sole 32P-labeled product. Hydrolysis of PIP was most rapid at neutral pH, and was not affected by inhibitors of acid phosphatase or alkaline phosphatase. Hydrolysis of PIP was also not inhibited by nonspecific phosphatase substrates, such as glycerophosphate, p-nitrophenylphosphate, AMP, or glucose 6-phosphate. Hydrolysis was stimulated by putrescine, and was inhibited by inositol 2-phosphate, spermidine, spermine, and neomycin.     

脂多糖增加小鼠巨噬细胞多磷酸肌醇脂代谢转换

多磷酸肌醇脂（这里指ＰＩＰ和ＰＩＰ２）的代谢在细胞信息传递和膜运转中起着重要的作用．脂多糖（ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）于激活小鼠腹腔巨噬细胞（Ｍφ）初期（０．５～１ｈ）和后期（１６ｈ）明显增加来自［γ－３２Ｐ］ＡＴＰ的３２Ｐ参入Ｍφ的ＰＩＰ和ＰＩＰ２,３２Ｐ参入ＰＩＰ２的显著性大于ＰＩＰ．ＬＰＳ的这种作用在其激活Ｍφ的初期不受酪氨酸蛋白激酶抑制剂ｇｅｎｉｓｔｅｉｎ、蛋白激酶Ａ激动剂（ｆｏｒｓｋｏｌｉｎ）及百日咳毒素的影响;但佛波酯（ＰＭＡ）长时间预处理的Ｍφ（其ＰＫＣ活性被耗竭）再受ＬＰＳ刺激,［３２Ｐ］ＰＩＰ２水平较ＬＰＳ刺激未受ＰＭＡ预处理的Ｍφ明显降低．结果表明,ＬＰＳ于激活Ｍφ的初期和后期更显著增加ＰＩ４Ｐ－５激酶的活性,导致ＰＩＰ２合成增加,ＰＩＰ２合成的增加可能与Ｍφ激活时不同时期所表达的功能相关．     

Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin.

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.     

Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603-7608) that serotonin induces: a very rapid formation of PI; and alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.     

Comparison of muscarinic and alpha-adrenergic receptors in rat atria based on phosphoinositide turnover

The effects of muscarinic and alpha-adrenergic receptor stimulation on phosphoinositide turnover in rat atria have been compared. Despite the similar densities of muscarinic receptors in rat left and right atria, 0.1 mM carbachol increased [32P]phosphate incorporation into phosphatidylinositol (PI) by 35% (p less than 0.05) in left atria but had no effect in right atria. By contrast to the small muscarinic receptor effect, stimulation of alpha 1-adrenergic receptors by 0.1 mM methoxamine produced a more than two fold increase in [32P]phosphate incorporation into PI in both left and right atria, despite the reported smaller density of alpha-adrenergic receptors in rat atria compared to muscarinic receptors. Enhanced phosphate labelling by methoxamine did not occur in phospholipids other than PI, and was blocked by the alpha-adrenergic antagonist, phentolamine (20 microM). The results indicate that the majority of the muscarinic receptors in rat atria are not coupled to phosphoinositide turnover. If indeed the observed enhancement in [32P]-phosphate labelling by carbachol reflects phosphoinositide turnover, and assuming equal coupling efficiencies of muscarinic and adrenergic receptors, it is calculated that not more than 2% of the muscarinic receptors in rat left atria are coupled to this response.     

Phosphoinositide turnover enhanced by angiotensin II in isolated rat glomeruli

To clarify the signal transduction mechanism of angiotensin II in renal glomeruli, we studied the effect of the hormone on phospholipid metabolism using isolated rat glomeruli. Stimulation of the glomeruli pulse-chase labeled with [3H]glycerol by angiotensin II caused a rapid (within 15 s) breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) with a concurrent production of 1,2-diacylglycerol. This effect of angiotensin II was in a dose-dependent manner within the range from 10(-12) M to 10(-6) M, and was inhibited by saralasin. Angiotensin II also decreased the 3H radioactivity of PIP slightly only at 15 s and increased that of phosphatidic acid after 15 s, with no significant effect upon the labelings of phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) within 1 min. The change in phospholipid metabolism by angiotensin II was similar when the glomeruli were labeled with [32P]orthophosphate: the decrease in the labeling of PIP2 and the increase in the labeling of phosphatidic acid after 15 s. In addition, 32P labeling of PI increased after 2 min. These results suggest that angiotensin II, after binding to glomerular receptors, induces initial PIP2 hydrolysis to diacylglycerol and subsequent resynthesis of PIP2 through phosphoinositide turnover.     

Surgical Sympathetic Denervation Increases α1Adrenoceptor-Mediated Accumulation of myo-Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle

Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: the effects of NE on PIP2 breakdown, measured as myo-inositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: The EC50 values (microM) for NE-induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1 microM). The EC50 values (microM) for NE-induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE-induced muscle contraction was blocked by prazosin (1 microM). The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. Denervation increased significantly (15-18%) the basal labelling of phosphoinositides from myo-[3H]inositol, but not from 32P or [14C]arachidonic acid. Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+-ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between alpha1 receptors and PIP2 breakdown into IP3 and 1,2-diacylglycerol (DG) more efficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inositol Lipid Metabolism in Rat Hippocampal Formation Slices: Basal Metabolism and Effects of Cholinergic Agonists

Incubation of rat hippocampal formation slices under steady-state conditions with [3H]inositol leads to only three phospholipids becoming labelled: phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. All three lipids incorporate [32P]Pi into their phosphodiester phosphate group with the polyphosphoinositides also incorporating this tracer into their monoester phosphate groups. As the concentrations of these lipids remain constant during these labelling processes we conclude that the phosphodiester phosphate, the inositol moiety, and the monoester phosphate groups undergo metabolic turnover in hippocampal formation slices incubated in vitro. The rate of incorporation of [3H]inositol into all three inositol phospholipids was stimulated by the addition of methacholine to the medium. Moreover, following steady-state labelling of the inositol lipids with [3H]inositol, methacholine in the presence of 10 mM LiCl caused a transient fall of 13% in the radiochemical concentration of phosphatidylinositol 4,5-bisphosphate after only 30 s stimulation and a fall of 15% in the radiochemical concentration of phosphatidylinositol after 30 min. Concomitantly, there was an approximately stoichiometric rise in the radiochemical concentration of inositol phosphates. Thus, we suggest that methacholine stimulates an inositol phospholipid phosphoinositidase C in rat hippocampal formation slices.     

Phospholipids of Trypanosoma cruzi: increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation.

We have studied the effect of carbamoylcholine in Trypanosoma cruzi epimastigote forms prelabelled with [32P]-Pi. Suspensions of cells were incubated at 28 degrees C to measure changes in the levels of [32P]-labelled phospholipids after stimulation. The presence of this cholinergic agonist induced changes in the phosphoinositide metabolism; a shift in the levels of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid (PA) was observed, whereas the levels of the other glycerophospholipids were not changed. This study shows that carbamoylcholine either directly or indirectly influences changes in phosphoinositide metabolism.     

Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.

We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.     

Rapid decreases in phosphatidylinositol in isolated luteal plasma membranes after stimulation by luteinizing hormone

Phospholipid concentrations were determined in plasma membrane preparations from porcine corpora lutea after incubation for 15 to 120 s without or with 0.5 microgram/ml luteinizing hormone (LH) or 2 microM dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP). Treatment with LH caused a dramatic loss of 9 nmol in plasma membrane phosphatidylinositol (PI)/mg protein after 15 s of incubation, but no significant changes in other measurable phospholipids. Also, phospholipid concentrations were unchanged in untreated and dbcAMP-treated plasma membranes. The nature of the LH-induced decrease in PI was studied by incubating plasma membrane preparations for 15 s with [gamma 32P] adenosine 3',5'-triphosphate (ATP). 32P was incorporated only into three phospholipids: phosphatidic acid, phosphatidylinositol 4'-phosphate (PIP), and phosphatidylinositol 4',5'-bisphosphate (PIP2). Although LH generated small but significant increases in labeling of PIP and PIP2, less than 0.5 nmol of total phospholipids/mg protein were radiolabeled in 15 s. Phosphatidylinositol kinase activity, the enzyme that converts PI into PIP, was not affected by LH or dbcAMP treatment. However, incubation of luteal plasma membranes for 15 s with LH resulted in an increase of approximately 2 nmol 1,2-diacylglycerol/mg protein more than that observed in untreated or dbcAMP-treated plasma membranes. In summary, these experiments suggest that LH may stimulate hydrolysis of PI (and possibly PIP and PIP2) in isolated luteal plasma membranes.     

Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.     

3H]Inositol incorporation into phosphoinositides of pig reticulocytes

Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) of pig reticulocytes were extensively labelled when these cells were incubated with [3H]inositol. In marked contrast, a total lack of [3H]inositol labelling of phosphoinositides was observed in mature erythrocytes. Phosphoinositides of both reticulocytes and mature erythrocytes were labelled with 32P but the labelling in reticulocytes was several-fold higher than in mature erythrocytes. Inclusion of Ca2+ (2 mM)+ ionophore A23187 (2 micrograms/ml) during the labelling experiments substantially reduced the radioactivity incorporation into phosphoinositides of reticulocytes. When [3H]inositol-prelabelled reticulocytes were treated with Ca2+ + A23187 the levels of radioactive PI and PIP2 did not change significantly. However, the PIP pool exhibited a remarkable sensitivity to Ca2+ as shown by a 75% increase in its radioactivity over the control. The ability to incorporate [3H]inositol into phosphoinositides remains transitorily intact in the reticulocyte stage. Thus, pig reticulocytes offer a suitable model in which to explore the physiological role of phosphoinositides in relation to cellular maturation process.     

Activation of (arachidonyl) phosphatidylinositol turnover in rabbit neutrophils by the calcium ionophore A23187.

The addition of the Ca2+ ionophore A23187 to rabbit neutrophils stimulated [14C]arachidonic acid incorporation into phosphatidylinositol and lysosomal enzyme secretion. A significant increase in phosphatidylinositol labelling was observed after a 2 min exposure to 0.1 microM-ionophore A23187. Maximum increases in rate of labelling were obtained with 1 microM-ionophore A23187 within 1 min, declining to basal rates after 15 min. Similarly, maximum rate of enzyme release occurred during the first 2 min of exposure to ionophore and release was essentially complete by 15 min. Threshold and peak ionophore A23187 concentrations for stimulating both processes were identical. In contrast with the specificity of phosphatidylinositol labelling induced by 1 microM-ionophore A23187 in the absence of cytochalasin B, ionophore also significantly stimulated labelling of phosphatidylserine and phosphatidylethanolamine in the presence of cytochalasin B. With a threshold ionophore concentration (0.1 microM), the enhanced incorporation of arachidonate was relatively specific for phosphatidylinositol in cytochalasin-treated cells. Ionophore A23187 did not accelerate labelling of phosphatidylinositol by [14C]acetate or [14C]glycerol, indicating that ionophore A23187 does not stimulate phosphatidylinositol synthesis de novo, although it did promote [14C]palmitate and [32P]Pi incorporation into neutrophil phosphatidylinositol. However, the increase in phosphatidylinositol labelling with these latter precursors was generally slower in onset and much more modest in magnitude than that observed with arachidonic acid. These results support the hypothesis that a Ca2+-dependent phospholipase, which acts on the arachidonate moiety of phosphatidylinositol, is responsible for initiating at least certain of the membrane events coupled to the release of secretory product from the neutrophil.