Early effects of luteinizing hormone on mitochondrial phosphoinositides in ovarian follicles

It is not clear if luteinizing hormone (LH) stimulates breakdown as well as synthesis of phosphoinositides in ovarian tissue. Possibly, LH stimulation results in hydrolysis of ovarian phosphoinositides in discrete subcellular compartments while increasing their synthesis at other sites. To investigate this hypothesis, we determined the effects of LH on phosphoinositide metabolism in whole homogenates and mitochondria of ovarian follicles. Medium (3-7 mm) follicles from porcine ovaries were preincubated for 2 h in phosphate (PO4)-free medium with 32PO4, and incubated without or with LH (1 microgram/ml). Phosphatidylinositol (PI) and related compounds, phosphatidic acid (PA), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2), accounted for 40% of the radiolabeled phospholipids in whole homogenates and over 60% in mitochondria from preincubated follicles. After 5 min, LH caused a significant decrease in radiolabeling of PIP2 and PIP in mitochondria, but not in whole homogenates. Luteinizing hormone increased radiolabeling of PIP2, PIP, PI and PA within 10 min in whole homogenates, and within 20 to 30 min in mitochondria. This delayed increase in radiolabeling of mitochondrial phosphoinositides after LH treatment was accompanied by decreases in PIP2, PIP and PI radiolabeling in whole homogenates. Follicles also were preincubated for 4 h with [3H]inositol, then for 15 min with 10 mM LiCl (an inhibitor of inositol phosphate hydrolysis). Inositol phosphate accumulation in 30 min was 2.7 times higher in homogenates of LH-treated follicles then in untreated follicles. Also, LH significantly decreased inositol bisphosphate, but did not change inositol trisphosphate accumulation. Accumulation of inositol phosphates in mitochondria was not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.     

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.     

Changes in the concentration and fatty acid composition of phosphoinositides induced by hormones in hepatocytes

The hormonal regulation of phosphoinositide levels in isolated hepatocytes was studied using chemical means. Extracted inositol phospholipids were adsorbed to neomycin-coated glass beads and then eluted and quantitated by charring after separation by thin layer chromatography on silica gel. The amounts (in nanograms/mg wet weight) of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) were 20 +/- 1, 16 +/- 1, and 1790 +/- 140, respectively). Incubation of the cells with 100 nM vasopressin decreased the value for PIP2 to 10 +/- 0.2 at 15 s, 12 +/- 1.5 at 1 min, and 14 +/- 2.1 at 5 and 30 min. In contrast, the hormone increased 1,2-diacylglycerol plus phosphatidate by over 200 ng/mg wet weight at 5 min under similar conditions (Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chem. 262, 15309-15315). PIP2 was also significantly decreased at 15 s by angiotensin II (100 nM), ATP (100 microM), and epinephrine (1 microM). In contrast, PIP was not significantly changed, and PI was significantly decreased (by approximately 15%) at later times (15 and 30 min). The changes in phosphoinositide mass were well correlated with changes in labeled phosphoinositides in hepatocytes previously incubated with [3H]inositol for 90 min. The amounts of inositol phospholipids in liver plasma membranes (in micrograms/mg protein) were 2.1 +/- 0.2 for PIP2, 0.24 +/- 0.03 for PIP, and 23 +/- 4 for PI. Comparison of these values with those for whole cells suggests that PIP2 is enriched in the plasma membrane, whereas PIP is present elsewhere in the cell. The fatty acid composition of whole cell PIP2 showed significant differences from that of PI. The percentages of palmitic, stearic, linoleic, and arachidonic acids were, respectively, 14, 41, 10, and 25 for PIP2 and 10, 34, 7, and 37 for PI. Vasopressin treatment for 15 s did not alter the fatty acid composition of PIP2. The corresponding fatty acid percentages for liver plasma membranes were 13, 41, 11, and 21 for PIP2 and 8, 34, 0, and 40 for PI. The fatty acid composition of PIP in whole cells and plasma membranes resembled that of PIP2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thrombin-induced breakdown of phosphoinositides in platelets from patients with NIDDM.

We have examined thrombin-induced metabolism of phosphoinositides in the platelets from fifteen NIDDM (non-insulin-dependent diabetes mellitus) patients and fifteen healthy subjects (control). The diabetic patients were divided into two groups. One group (group I) had diabetic retinopathy (microangiopathy) and the other group (group II) had atherosclerosis of great vessels (macroangiopathy). In platelets incubated with [32P] orthophosphate for 80 min, the incorporation of 32P radioactivity into phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was significantly lower in the group II than in the control. The addition of thrombin induced a marked decrease in PIP2 radioactivity at 10 sec in platelets from group I compared with that from the control. These results suggest that the breakdown of polyphosphoinositides is increased in platelets from diabetic subjects with retinopathy, and also that the formation of polyphosphoinositides is decreased in the platelets from diabetic subjects with macroangiopathy.     

Correlation of receptor sequestration with sustained diacylglycerol accumulation in angiotensin II-stimulated cultured vascular smooth muscle cells

Angiotensin II stimulates sequential phospholipase C-mediated hydrolysis of initially the polyphosphoinositides and subsequently phosphatidylinositol (PI) in cultured rat aortic smooth muscle cells resulting in biphasic, sustained formation of diacylglycerol (DG). The mechanisms underlying this delayed induction of sustained DG accumulation are unknown but may be related to cellular events including processing of the angiotensin II receptor-ligand complex. In the present study, we characterized the kinetics of angiotensin II receptor sequestration and studied the effects of interventions which interfere with receptor processing on the pattern of angiotensin II-induced DG formation and phosphoinositide hydrolysis. Conversion of the angiotensin II receptor to an acid-resistant form was temperature-dependent, with half-times of 1.5 min at 37 degrees C and 7 min at 19 degrees C. Reducing the temperature to 25 or 19 degrees C caused a marked temporal separation between the two phases of DG accumulation. There was a close temporal correlation between the effect of temperature on receptor sequestration and on sustained DG accumulation. Furthermore, phenylarsine oxide (5 min, 10 microM), which inhibited angiotensin II receptor internalization, also selectively inhibited the sustained phase of DG accumulation (81 +/- 6% inhibition). Monensin and chloroquine, which interfere with receptor processing through the lysosomal-degradative pathway, had no effect on angiotensin II-induced DG formation in these cells, suggesting that the processing event important to hormonally induced sustained DG accumulation occurs early in the internalization pathway, probably at the level of the plasma membrane. Moreover, the acid-resistant state of the angiotensin II receptor-ligand complex retained its ability to signal, since removal of the surface signal by competitive antagonism with Sar1-Ile8-angiotensin II or acid-wash only slowly reversed accumulation of DG and depression of total cell calcium. These experiments support our previous observation that the initial and sustained phases of angiotensin II-induced diacylglycerol formation in vascular smooth muscle are differentially controlled and suggest that an early event in the cellular processing of the angiotensin II-receptor complex is essential to maintenance of DG accumulation.     

Potassium depletion selectively inhibits sustained diacylglycerol formation from phosphatidylinositol in angiotensin II-stimulated, cultured vascular smooth muscle cells

Potassium depletion decreases blood pressure in vivo and blunts the pressor response to angiotensin II (ang II) without down-regulating the receptor. In cultured rat aortic smooth muscle cells, the ang II-induced signaling sequence is biphasic with rapid hydrolysis of the polyphosphoinositides producing an early (15 s) diacylglycerol (DG) peak and a transient rise in inositol trisphosphate (IP3) and more delayed phosphatidylinositol (PI) hydrolysis resulting in sustained DG formation (peak at 5 min). Exposure of intact vascular smooth muscle cells to low potassium growth medium for 24 h or acutely potassium-depleting cells with nigericin causes selective, marked inhibition of late DG formation (5-min peak inhibited by 60 +/- 8% and 84 +/- 7%, respectively). The early cell response, namely polyphosphoinositide hydrolysis, inositol bis- and trisphosphate production and the 15-s DG peak, is not affected. Analysis of 125I-ang II-binding data reveals no significant differences in either receptor number or binding affinity (Kd) in potassium-depleted cells. Together with its marked inhibitory effect on sustained ang II-induced DG formation, acute potassium depletion effectively blocks internalization of 125I-ang II: there is no significant internalization of the ligand after 5 min at 37 degrees C versus 64 +/- 7% internalization in control cells. Thus, potassium depletion does not alter ang II binding or initial membrane signaling in rat aortic smooth muscle but blocks ligand internalization and selectively and markedly inhibits the development of direct PI hydrolysis and sustained diacylglycerol formation. These findings suggest a role for ligand-receptor processing in generating the sustained cell response and potentially explain the lower blood pressure and decreased pressor response to ang II seen in hypokalemic states in vivo. Furthermore, the ability of K+ depletion to alter secondary signal generation may provide insight into the mechanisms underlying the K+ dependence of a variety of cell functions.     

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.     

Kinetic analysis of the formation of inositol 1:2-cyclic phosphate in carbachol-stimulated pancreatic minilobules. Half is formed by direct phosphodiesteratic cleavage of phosphatidylinositol

The issue as to whether there is direct phosphodiesteratic cleavage of phosphatidylinositol (PI), in addition to that of phosphatidylinositol 4,5-bisphosphate (PIP2), on agonist stimulation of cells has been controversial. In an attempt to resolve this issue, we have studied the kinetics of the formation and breakdown of the cyclic inositol phosphates. This approach is fairly straightforward, since the turnover of the cyclic inositol phosphates is very slow as compared to that of the noncyclic inositol phosphates and proceeds from inositol 1:2-cyclic 4,5-trisphosphate to inositol 1:2-cyclic phosphate (I(c1:2)P) directly by dephosphorylation without any branching pathways, in contrast to the multiple branchpoints of the noncyclic inositol phosphate pathway. Mouse pancreatic minilobules were prelabeled with [3H]inositol for 30 min, followed by washing to remove free inositol. They were then stimulated with carbachol for 30 min. The inositol cyclic polyphosphates reached steady state at 10-15 min, and I(c1:2)P reached steady state at 25 min. We blocked the action of carbachol by addition of an excess of atropine at 30 min, and the rate of disappearance of the three cyclic inositol phosphates was measured. From these data, the contribution of the inositol cyclic polyphosphate pathway to I(c1:2)P was calculated, which was 40-50% of total I(c1:2)P formation. Thus, 40-50% of the I(c1:2)P formed must have been derived from direct phosphodiesteratic cleavage of PI. This approach should prove useful in measuring the relative contributions of PI hydrolysis and PI phosphorylation (phosphatidylinositol 4,5-bisphosphate hydrolysis) in the overall PI cascade.     

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t_1/2=14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5–10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.Abbreviations I(1,4,5)P₃ inositol 1,4,5-trisphosphate - IP total inositol phosphate fraction - IPL total inositol lipid fraction - mAChR muscarinic acetylcholine receptor - NMS N-methylscopolamine - Oxo-M oxotremorine-M - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PPI phosphoinositide - QNB quinuclidinyl benzilate Special issue dedicated to Dr. Bernard W. Agranoff     

C5a reduces formyl peptide-induced actin polymerization and phosphatidylinositol(3,4,5)trisphosphate formation, but not phosphatidylinositol (4,5) bisphosphate hydrolysis and superoxide production, in human neutrophils.

We investigated phospholipid signal transduction, calcium flux, O2- anion production and actin polymerization after stimulation with the C fragment and chemoattractant, C5a, and then determined how C5a pretreatment affected subsequent responses to formyl peptide in human neutrophils. We have previously demonstrated that the novel lipids, phosphatidylinositol trisphosphate (PIP3) and phosphatidylinositol(3,4)P2 (PI(3,4)P2), rise transiently in neutrophils after activation with formyl peptide. Furthermore, the rise in PIP3 parallels actin polymerization. In this study, neutrophils activated with C5a exhibited two distinct G protein-dependent signal pathways involving different phosphoinositides: 1) [32P]PI(4,5)P2 hydrolysis and [32P]PA production, and 2) the transient formation of D-3-phosphorylated phosphoinositides, [32P]PIP3 and [32P]PI(3,4)P2. When neutrophils were preincubated with C5a for 5 min before stimulation with formyl peptide, [32P]PI(4,5)P2 hydrolysis was unchanged, and [32P]PA production and O2- formation were slightly enhanced compared with controls stimulated with formyl peptide in the absence of C5a. In contrast, [32P]PIP3 production, right angle light scatter, and actin polymerization were all reduced 35 to 40%. Therefore, these data support the hypothesis that PIP3 plays a role in chemotaxis but not superoxide formation.     

Platelet-derived growth factor stimulates rapid polyphosphoinositide breakdown in fetal human fibroblasts

The addition of human platelet-derived growth factor (PDGF) to confluent, quiescent cultures of human diploid fibroblasts induced the rapid breakdown of cellular polyphosphoinositides. The levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) decreased by 30 to 40% within 1 min after exposure of the cells to PDGF. The levels of PIP and PIP2 returned to their initial values within 3 and 10 min, respectively, after PDGF addition. The level of PI continued to increase after it had returned to control values and was up threefold within 30 min after PDGF addition. In cells prelabeled with myo-[3H]inositol PDGF caused an eightfold increase in the levels of inositol trisphosphate (IP3) within 2 min. Lesser increases, twofold and 1.3-fold, respectively, were seen in levels of inositol bisphosphate (IP2) and inositol monophosphate (IP). Within 10 min after PDGF addition the levels of all three inositol phosphates had decreased to control values. The levels of IP3 measured 2 min after PDGF addition depended on the PDGF concentration and were maximal at 5-10 ng/ml of PDGF. Similar concentrations of PDGF stimulate maximal cell growth and DNA synthesis in these cells.     

Kinetic analysis of guanosine 5'-O-(3-thiotriphosphate) effects on phosphatidylinositol turnover in NRK cell homogenates

Addition of the guanine nucleotide analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to [3H]inositol-labeled NRK cell homogenates resulted in rapid breakdown of cellular polyphosphoinositides. GTP gamma S stimulated phospholipase C, resulting in a more than 4-fold increase in the hydrolysis rates of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis(phosphate) (PIP2). No significant effect of GTP gamma S on direct phosphatidylinositol (PI) hydrolysis was detected. There was an increase in water-soluble inositols, with inositol tris(phosphate) (IP3) levels increasing at least 10 times over the decrease seen in PIP2, indicating that PIP kinase activity was also accelerated following GTP gamma S addition. Inositol 1,4,5-tris(phosphate) peaked rapidly after GTP gamma S addition (less than 2 min) while inositol 1,3,4-tris-(phosphate) was produced more slowly and leveled off after approximately 10 min. The differential equations describing conversion between intermediates in the PI turnover pathway were solved and fitted to data obtained from both [3H]inositol and [32P]phosphate fluxes by nonlinear least-squares analysis. GTP gamma S effects on the pseudo-first-order rate constants for the lipase, kinase, and phosphatase steps were determined from the analysis. From these measurements it can be estimated that, in the presence of GTP gamma S and calcium buffered to 130 nM, hydrolysis of PIP2 accounts for at least 10 times as much diacylglycerol as direct PI breakdown despite the 100-fold excess of PI over PIP2. From the kinetic model it is predicted that small changes in the activities of PI and PIP kinases can have large but different effects on the level of IP3 and diacylglycerol following GTP gamma S addition. These results argue that regulation of PI and PIP kinases may be important for determining both cellular IP3 and diacylglycerol levels.     

Bradykinin induces the bi-phasic production of lysophosphatidyl inositol and diacylglycerol in a dorsal root ganglion X neurotumor hybrid cell line, F-11

Bradykinin acts on the dorsal root ganglion X neuroblastoma hybrid cell line F-11 to stimulate the rapid elevation of inositol trisphosphate (IP3) and intracellular calcium. We now show an equally rapid release of arachidonyl labeled diacylglycerol (DAG), (243 +/- 32% of control). This first peak of diacylglycerol production was inhibitable by either pretreatment with 200 ng/ml of pertussis toxin overnight or by 10 nM tetradecanoylphorbol acetate (TPA). In addition, a second, more sustained release occurred, plateauing at approximately five minutes (304 +/- 16%). The second peak of DAG was unaffected by these TPA or pertussis pre-incubations. Simultaneous analysis of inositol-labeled phospholipids showed that the initial IP3 and DAG peaks corresponded to initial decreases in phosphoinositides PIP2 and PIP whereas PI increased slightly over this same time period. In contrast, at 5-30 minutes, PIP2 and PIP returned to normal levels, but PI gradually decreased to 75% of control values. Likewise, TPA blocked this early PIP and PIP2 breakdown, but had no effect on the delayed breakdown of monophosphatidylinositol (PI). Bradykinin also induced an equally rapid increase in lysophosphatidyl inositol (lyso-PI) with a peak around 10-30 seconds, and a second more sustained peak after 10 minutes. This production of lyso-PI was not affected by prior treatment with TPA or pertussis toxin. The initial and the sustained phases of diacylglycerol production probably result from different biochemical mechanisms and/or substrates.     

Differences in the metabolism of inositol and phosphoinositides by cultured cells of neuronal and glial origin

Phosphoinositide and inositol metabolism was compared in glioma (C6), neuroblastoma (N1E-115) and neuroblastoma X glioma hybrid (NG 108-15) cells. All cell lines had similar proportions of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Neuroblastoma and hybrid cells had almost identical phospholipid and phosphoinositide compositions and similar activities for the enzymes metabolizing polyphosphoinositides (PI kinase, PIP phosphatase, PIP kinase, PIP2 phosphatase, PIP2 phosphodiesterase). Glioma cells differed by having greater proportions of ethanolamine plasmalogen and sphingomyelin, lower PIP kinase, 3-5-fold higher PIP phosphatase activity and 10-15-fold greater PIP2 phosphodiesterase activity. Higher PIP phosphatase and PIP2 diesterase activities appear to be characteristic of cells of glial origin, since similar activities were found in primary cultures of astroglia. Glioma cells also metabolize inositol differently. In pulse and pulse-chase experiments, glioma cells transported inositol into a much larger water-soluble intracellular pool and maintained a concentration gradient 30-times greater than neuroblastoma cells. Label in intracellular inositol was less than in phosphoinositides in neuroblastoma and exchanged rapidly with extracellular inositol. In glioma, labeling of intracellular inositol greatly exceeded that of phosphoinositides. As a consequence, radioactivity in prelabeled phosphoinositides could not be effectively chased from glioma cells by excess unlabeled inositol. Such differences between cells of neuronal and glial origin suggest different and possibly supportive roles for these two cell types in maintaining functions regulated through phosphoinositide-linked signalling systems in the central nervous system.     

Diacylglycerol formation from phosphatidylcholine in angiotensin II-stimulated vascular smooth muscle cells.

In cultured vascular smooth muscle cells (VSMC), angiotensin II (Ang II) induces a biphasic diacylglycerol (DAG) formation peaking at 15 sec and 5 min. Although it has been well established that the first peak is produced by the hydrolysis of inositol 4,5-bisphosphate (PIP2), the origin of the second DAG peak has never been examined in detail. In the present paper, we provide evidence that the second peak of DAG formation in Ang II-stimulated VSMC originates mainly from PC.     

Alpha-thrombin-induced inositol phosphate formation in G0-arrested and cycling hamster lung fibroblasts: evidence for a protein kinase C-mediated desensitization response

In resting Chinese hamster fibroblasts (CCL39) alpha-thrombin rapidly induces the breakdown of phosphoinositides. Accumulation of inositol phosphates (IP), measured in the presence of Li+, is detectable within 5s (seconds) of thrombin stimulation. Formation of inositol tris- and bisphosphates slightly precedes that of inositol monophosphate, indicating that thrombin activates primarily the phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate. Initial rates of IP production increase with thrombin concentration, with no apparent saturability over the range 10(-4)-10 U/ml. Thrombin-induced phosphoinositide hydrolysis rapidly desensitizes (t1/2 less than 5 min), but a residual activity, corresponding to about 10% of the initial stimulation is sustained for at least 9 h, in contrast with the undetectable activity of G0-arrested cells. This apparent desensitization may be due to a feedback regulation by protein kinase C, since pretreatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly inhibits (by up to 70%) subsequent thrombin-induced inositol phosphate formation. Conversely, growth factor deprivation of CCL39 cells results in a progressive increase of thrombin-induced phosphoinositide hydrolysis, from the very low level of exponentially growing cells to the maximal level of G0-arrested cells. This "up regulation" was found maximal in A51, a very well growth-arrested CCL39 derivative, and reduced or virtually abolished in two tumoral and growth factor-relaxed derivatives of CCL39. Although preliminary, this observation suggests that a persistent activation of phosphatidyl inositol breakdown might operate in variants selected for autonomous growth.     

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.     

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.     

Molecular Species of Diacylglycerols and Phosphoglycerides and the Postmortem Changes in the Molecular Species of Diacylglycerols in Rat Brains

The molecular species of 1,2-diacyl-sn-glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse-phase HPLC. The total amount and the concentration of each species were quantified by using 1,2-distearoyl-sn-glycerol (18:0-18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn-1 and sn-2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0-20:4 (35%), 16:0-18:1 (15%), 16:0-16:0 (9%), and 16:0-20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse-phase HPLC. PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0-20:4 species (50% in PI and approximately 65% in PIP and PIP2). PS contained mainly the 18:0-22:6 (42%) and 18:0-18:1 (24%) species. PE was mainly composed of the 18:0-20:4 (22%), 18:0-22:6 (18%), 16:0-18:1 (15%), and 18:0-18:1 (15%) species. In PC the main molecular species were 16:0-18:1 (36%), 16:0-16:0 (19%), and 18:0-18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40-50 nmol/g in 0 min to 210-290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven- to 10-fold) were found for the 18:0-20:4 and 16:0-20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.