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A Sorisky

Publications and source records attributed to A Sorisky.

At least 37 records · Page 2Linked to original sources
PubMed1998

The effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation.

We examined the effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation. Oil Red O staining of neutral lipid, cellular triglyceride mass, and glycerol phosphate dehydrogenase (GPDH) activity, were greater in 3T3-L1 cells cultured at 5 mM vs. 25 mM glucose. GPDH activity was 2- to 4-fold higher at 5 mM vs. 25 mM glucose over a range of insulin concentrations (0.1 to 100 nM). Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was 1.7-fold greater, and insulin-stimulated phosphoinositide 3-kinase association with IRS-1 was 2.3-fold higher, at 5 mM vs. 25 mM glucose. These effects of glucose were not caused by alterations in IRS-1 mass or cell-surface insulin binding. In preadipose cells at 5 mM glucose, expression of the leukocyte antigen-related (LAR) protein tyrosine phosphatase (negative regulator of insulin signaling) was 63% of the level at 25 mM glucose. Our data demonstrate that glucose concentration affects insulin-induced 3T3-L1 adipose cell differentiation as well as differentiation-directed insulin signaling pathways. Alterations in LAR expression potentially may be involved in modulating these responses.

3T3 Cells↗
PubMed1998

The effect of adipocyte differentiation on the capacity of 3T3-L1 cells to undergo apoptosis in response to growth factor deprivation.

OBJECTIVE: Little is known about the regulation of apoptosis in the adipocyte. Using the 3T3-L1 cell model of adipogenesis, we investigated whether induction of apoptosis by serum-starvation is differentiation-specific. METHODS: Apoptosis was assessed by phase-contrast microscopy, Hoechst staining and DNA fragmentation. Protein expression levels were evaluated by immunoblot analysis. RESULTS: Following serum deprivation, Hoechst staining revealed chromatin condensation and formation of apoptotic bodies in 3T3-L1 preadipocytes, but not in differentiated adipocytes. Similarly, although cell cultures of serum-starved 3T3-L1 preadipocytes displayed extensive apoptotic DNA fragmentation, this was barely detectable for cell cultures subjected to differentiation medium, in which 70-90% of the cells had assumed the mature phenotype. Consistent with the ability of the adipocytes to resist apoptosis, immunoblot analysis revealed that differentiated cell cultures expressed Bcl-2 and the endonuclease DNase I at higher and lower levels, respectively. CONCLUSION: Expression of cell survival genes is modulated during 3T3-L1 adipocyte differentiation, potentially contributing to the state of resistance to apoptosis observed for mature 3T3-L1 adipocytes upon growth factor deprivation.

3T3 Cells↗
PubMed1998

Tyrosine phosphatase inhibitors, vanadate and pervanadate, stimulate glucose transport and GLUT translocation in muscle cells by a mechanism independent of phosphatidylinositol 3-kinase and protein kinase C.

Vanadate and pervanadate (pV) are protein tyrosine phosphatase (PTP) inhibitors that mimic insulin to stimulate glucose transport. To determine whether phosphatidylinositol (PI) 3-kinase is required for vanadate and pV, as it is for insulin, cultured L6 myotubes were treated with vanadate and pV. The two compounds stimulated glucose transport to levels similar to those stimulated by insulin; however, while PI 3-kinase activity and the increase in the lipid products PI 3,4-bisphosphate and PI 3,4,5-trisphosphate were inhibited by wortmannin after stimulation by all three agents--insulin, vanadate, and pV--wortmannin blocked glucose transport stimulated by insulin but not vanadate or pV. Vanadate and pV stimulated the translocation of GLUTs from an intracellular compartment to the plasma membrane; this stimulation was not blocked by wortmannin, but insulin-induced GLUT translocation was inhibited. Similar results were obtained in cultured H9c2 cardiac muscle cells in which wortmannin did not inhibit glucose transport or the vanadate-induced translocation of GLUT4 in c-myc-GLUT4 transfected cells. The ser/thr kinase PKB (Akt/PKB/RAC-PK) is activated by insulin, lies downstream of PI 3-kinase, and has been implicated in signaling of glucose transport. Insulin and pV stimulated PKB activity, and both were inhibited by wortmannin. In contrast, vanadate, at concentrations that maximally stimulated glucose transport, did not significantly increase PKB activity. To determine the potential role of protein kinase C (PKC), L6 cells were incubated chronically with phorbol myristate acetate (PMA) or acutely with the PKC inhibitors calphostin C and bisindolylmaleimide. There was no inhibition of glucose transport stimulation by insulin, vanadate, or pV, and a combination of wortmannin and PKC inhibitors also failed to block the effect of vanadate and pV. In contrast, disassembly of the actin network with cytochalasin D blocked the stimulation of glucose transport by all three agents. In conclusion, vanadate and pV are able to stimulate glucose transport and GLUT translocation by a mechanism independent of PI 3-kinase and PKC. Similar to that by insulin, glucose transport stimulation by vanadate and pV requires the presence of an intact actin network.

Androstadienes↗
PubMed1998

Expression and regulation of neuronal apoptosis inhibitory protein during adipocyte differentiation.

We have used the 3T3-L1 and 3T3-F442A preadipocyte cell lines to examine the expression and regulation of neuronal apoptosis inhibitory protein (NAIP) during adipocyte differentiation. When 3T3-L1 preadipocytes differentiated into adipocytes, they developed resistance to apoptosis induced by growth factor deprivation, as assessed by terminal deoxynucleotide transferase (TdT)-mediated dUTP nick end labeling. Protein expression of NAIP was markedly elevated in 3T3-L1 and 3T3-F442A adipocytes compared with that in their fibroblast-like precursors. NAIP was also present in rat white adipocytes. In 3T3-L1 cells, the increase in NAIP occurred by day 4 of the 8-day differentiation protocol, which includes exposure of confluent preadipocytes to insulin, dexamethasone, and isobutylmethylxanthine. Incubation of confluent 3T3-L1 preadipocytes with any of these components alone had no effect on NAIP expression. When 3T3-C2 cells, a control cell line that does not differentiate, were subjected to the differentiation protocol, the low NAIP levels remained unaltered. Addition of rapamycin, a p70 S6 kinase inhibitor that blocks adipocyte differentiation, to the 3T3-L1 differentiation medium prevented the rise in NAIP expression. These data demonstrate for the first time that NAIP is expressed in adipocyte cell lines and primary adipocytes. The differentiation-dependent augmentation of NAIP protein levels in 3T3-L1 adipocytes is closely correlated with the development of resistance to apoptosis induced by growth factor deprivation, suggesting a potential role for NAIP in these cells.

3T3 Cells↗
PubMed1997

Efficacy and safety of a new hydroxymethylglutaryl-coenzyme A reductase inhibitor, atorvastatin, in patients with combined hyperlipidemia: comparison with fenofibrate.

This 24-week, randomized, open-label multicenter study evaluated the efficacy and safety of atorvastatin compared with fenofibrate in the treatment of patients with combined hyperlipidemia (CHL). Following a 6-week baseline period, 84 patients with CHL were randomly assigned to either atorvastatin treatment, 10 mg QD for 12 weeks increasing to 20 mg QD for 12 weeks, or fenofibrate treatment, 100 mg TID for 24 weeks. Changes from baseline in lipid parameters were evaluated at weeks 12 and 24. At both 10- and 20-mg doses, atorvastatin treatment resulted in significantly greater reductions in LDL cholesterol, apolipoprotein (apo) B, total cholesterol, LDL-apoB, and lipoprotein-B compared to 300-mg fenofibrate treatment (P < .05). While atorvastatin also resulted in clinically significant reductions in triglyceride, VLDL cholesterol, apoB in VLDL, triglyceride in VLDL, and apoC-III and significant increases in HDL cholesterol and apoA-I levels, fenofibrate was more effective than atorvastatin in altering all these parameters. However, by significantly affecting both the cholesterol-rich and triglyceride-rich particles, atorvastatin holds promise as a lipid-regulator able to adequately treat a broad range of patients that includes those with CHL.

Adult↗
PubMed1996

A peroxovanadium compound induces Xenopus oocyte maturation: inhibition by a neutralizing anti-insulin receptor antibody.

Synthetic peroxovanadium compounds are a new class of potent inhibitors of protein phosphotyrosine phosphatases. These compounds exhibit insulin-like activity both in vitro and in experimental animals. However, the molecular mechanism by which these compounds exert their biological effect is not well defined. We demonstrate here that several of these compounds induce Xenopus oocyte maturation in vitro, as indicated by germinal vesicle breakdown. Using one of these compounds for further studies, we show that the induction is dose-dependent and is accompanied by activation of maturation promoting factor as well as activation of Xenopus MAP kinase. Like insulin, bpV(pic) causes an acute accumulation of PI(3,4,5)P3 (phosphotidylinositol-3,4,5-trisphosphate), a product of PI 3-kinase. More importantly, bpV(pic)-induced oocyte maturation was abolished by microinjection of a neutralizing monoclonal anti-insulin receptor antibody (17A3) into oocytes or preincubation of oocytes with a PI 3-kinase inhibitor (wortmannin). These results suggest that bpV(pic) acts upstream of the Xenopus IGF-1 receptor in the induction of meiotic maturation, presumably by neutralizing an inhibitory protein tyrosine phosphatase(s) that may regulate the receptor. Finally, using an oocyte-follicle cell complex that responded to human chorionic gonadotropin (hCG) to undergo GVBD, we showed that injection of 17A3 anti-insulin receptor antibody into oocytes did not affect hCG-induced oocyte maturation.

Androstadienes↗
PubMed1996

The 3-phosphorylated phosphoinositide response of 3T3-L1 preadipose cells exposed to insulin, insulin-like growth factor-1, or platelet-derived growth factor.

We compared the pattern of 3-phosphorylated phosphoinositides produced by confluent 3T3-L1 preadipose cells upon exposure to growth factors that either induce differentiation (insulin, insulin-like growth factor-1) or do not (platelet-derived growth factor). Following addition of insulin or insulin-like growth factor-1, PI(3,4,5)P3 rapidly rose, on average, to levels tenfold over basal. PI(3,4)P2 either did not change (after insulin) or slightly increased (1.5 fold). Time course studies with insulin demonstrated that the rise in PI(3,4,5)P3 peaked by 1 minute, and levels then remained steady over 30 minutes. Dose-response experiments showed that insulin at a concentration of 1 nM was sufficient for the PI(3,4,5)P3 response. Insulin failed to increase PI(3,4)P2 at any of the time points or at any of the doses used. In contrast, after addition of platelet-derived growth factor, both PI(3,4)P2 and PI(3,4,5)P3 rose concurrently and to comparable extents. These data suggest that one possible mechanism contributing to insulin/insulin-like growth factor-1-induced 3T3-L1 preadipose cell differentiation is a distinct pattern of 3-phosphorylated phosphoinositide accumulation, defined by a prominent increase in PI(3,4,5)P3 with no (in the case of insulin), or a minimal (in the case of IGF-1), rise in PI(3,4)P2.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Expression of a constitutively activated form of protein kinase B (c-Akt) in 3T3-L1 preadipose cells causes spontaneous differentiation.

Insulin/IGF-1 is required for differentiation of 3T3-L1 adipose cells. Downstream targets of insulin/IGF-1 that lead to adipocyte differentiation appear to include Ras, phosphatidylinositol (PI) 3-kinase, Raf, and mitogen-activated protein kinase. We have tested whether protein kinase B (PKB), a serine/threonine kinase activated by PI 3-kinase, is sufficient for 3T3-L1 preadipose cell differentiation. A plasmid vector encoding a version of PKB that is constitutively activated (Gag-PKB) was expressed in 3T3-L1 preadipose cells (Gag-PKB cells). Spontaneous morphological changes indicative of adipocyte differentiation were observed in Gag-PKB cells. The cells assumed a spherical shape and they acquired characteristic lipid droplets that stained positively for Oil Red O. Northern blot analysis detected upregulation of LPL and aP2 mRNA, specific indicators of adipocyte differentiation. Our data demonstrate that constitutive activation of PKB is sufficient to trigger adipocyte differentiation.

3T3 Cells↗
PubMed1996

Evidence of adipocyte differentiation in human orbital fibroblasts in primary culture.

Graves' ophthalmopathy (GO), in which orbital tissues are infiltrated with activated T lymphocytes and hyaluronan, can manifest an overabundance of adipose tissue in the human orbit. Little is known about adipogenesis in this anatomic region. We have investigated whether orbital fibroblasts in culture possess the capacity to undergo adipocytic differentiation. Orbital tissue from patients with or without GO was placed in primary culture, and proliferating fibroblasts were sub-passaged. Confluent fibroblasts were subjected to a differentiation protocol, involving a serum-free defined medium supplemented with insulin, triiodothyronine, carbaprostacyclin, thyrotropin, dexamethasone, and isobutylmethylxanthine. Control cells were maintained in serum-free medium supplemented only with insulin. After approximately 14 days, light microscopy revealed characteristic morphologic changes of adipocyte differentiation, including cell rounding and lipid droplet accumulation. Oil Red O staining could be demonstrated in those cells. Scanning electron microscopy revealed that the cells undergoing adipogenesis contain multiple, discrete lipid droplets. The overall percentage of fibroblasts undergoing differentiation was somewhat variable, but no more than 5-10%. The adipocytes arise in close proximity to each other, and often at the periphery of the culture surface. In contrast, dermal fibroblasts and perimysial fibroblasts from extraocular muscle fail to differentiate. We conclude that a subpopulation of orbital fibroblasts is capable of adipocyte differentiation. These in vitro observations may represent the in vivo process that contributes to excess orbital adipose tissue volume in GO.

Adipocytes↗
PubMed1995

Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.

An insulin receptor substrate 1 (IRS-1)-like cDNA was isolated from a Xenopus ovary cDNA library by low-stringency hybridization using rat IRS-1 cDNA as a probe. The deduced amino acid sequence encoded by this cDNA (termed XIRS-L) is 67% identical (77% similar) to that of rat IRS-1. Significantly, all the insulin-induced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the Src homology domains of phosphatidylinositol (PI) 3-kinase, Syp and Grb2, are conserved in XIRS-L. Both mRNA and protein corresponding to the cloned XIRS-L can be detected in immature Xenopus oocytes. Recombinant XIRS-L protein produced in insect cells or a bacterial glutathione S-transferase fusion protein containing the putative PI 3-kinase binding site can be phosphorylated in vitro by purified insulin receptor kinase (IRK) domain, and the IRK-catalyzed phosphorylation renders both proteins capable of binding PI 3-kinase in Xenopus oocyte lysates. Another glutathione S-transferase fusion protein containing the C terminus of XIRS-L and including several putative tyrosine phosphorylation sites is also phosphorylated by IRK in vitro, but it failed to bind PI 3-kinase. Insulin stimulation of immature Xenopus oocytes activates PI 3-kinase in vivo [as indicated by an elevation of PI(3,4)P2 and PI(3,4,5)P3] as well as oocyte maturation (as indicated by germinal vesicle breakdown). Pretreatment of these oocytes with wortmannin inhibited insulin-induced activation of PI 3-kinase in vivo. The same treatment also abolished insulin-induced, but not progesterone-induced, germinal vesicle breakdown. These results (i) identify an IRS-1-like molecule in immature Xenopus oocytes, suggesting that the use of IRS-1-like Scr homology 2 domain-docking proteins in signal transduction is conserved in vertebrates, and (ii) strongly implicate PI 3-kinase as an essential effector of insulin-induced oocyte maturation.

Amino Acid Sequence↗
PubMed1994

Phosphatidylinositol 3,4,5-trisphosphate is formed from phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets.

Platelets accumulate PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to thrombin and thrombin-receptor-directed peptide in a GTP-dependent manner. These phosphoinositides are considered to be mediators of signaling events in a variety of cells. We have examined the metabolic route by which PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are synthesized by briefly (10 min) incubating platelets with high activities of [32P]Pi, followed by 20 or 60 s exposure to thrombin, and analysing the relative radioactivities of the individual phosphate groups in the resulting labelled PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The phosphate group possessing the highest specific activity under such non-equilibrium labelling conditions indicates the last one added in a metabolic sequence. The thrombin-stimulated rate of labelling of PtdIns(3,4)P2 was significantly slower than that of PtdIns(3,4,5)P3. Increased labelled PtdIns3P was not detected within 60 s. The measured relative radioactivities decreased in the order 3 > 5 > 4 >> 1 for PtdIns(3,4,5)P3 and 3 > 4 >> 1 for PtdIns(3,4)P2. On the basis of the results of both rate-of-labelling and specific radioactivity analyses we conclude that PtdIns(3,4,5)Pa is formed by 3-OH phosphorylation of PtdIns(4,5)P2, whereas PtdIns(3,4)P2, may be formed by 3-OH phosphorylation of PtdIns4P and/or dephosphorylation of PtdIns(3,4,5)P3. These findings point to the activation of phosphoinositide 3-kinase as a critical receptor-regulated step in thrombin-stimulated platelets.

Adenosine Triphosphate↗
PubMed1994

Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.

3T3 Cells↗
PubMed1992

Accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in thrombin-stimulated platelets. Different sensitivities to Ca2+ or functional integrin.

Differences in regulation of the accumulation of PtdIns(3,4)P2 versus that of PtdIns(3,4,5)P3 were noted in thrombin-stimulated human platelets. The rapid (within 20 s) response of PtdIns(3,4,5)P3 contrasted with a distinct lag in the accumulation of PtdIns(3,4)P2 that was followed by a pronounced increase by 90 s. The presence of 2.5 mM-CaCl2 further elevated PtdIns(3,4)P2 by 50-120%, but only at a late stage (after 90 s). Tetrapeptide RGDS (Arg-Gly-Asp-Ser), which blocks the interaction of ligands such as fibrinogen with platelet integrin alpha IIb beta 3 (GPIIb-IIIa), inhibited only the late-phase PtdIns(3,4)P2 accumulation that was associated with added Ca2+. Although stimulated tyrosine phosphorylation of platelet protein (total cell lysate) was altered by Ca2+ or RGDS, we could not identify any such proteins that were affected comparably to PtdIns(3,4)P2. In contrast to the PtdIns(3,4)P2 response, the accumulation of PtdIns(3,4,5)P3 was unaffected by Ca2+ or RGDS at any time.

Acylation↗
PubMed1992

Human platelets deficient in dense granules contain normal amounts of pp60c-src.

We have determined that pp60c-src, a protein tyrosine kinase abundant in normal platelets, is present at comparable levels in platelets that are deficient in dense granules (Hermansky-Pudlak syndrome). Relative quantitation of pp60c-src was performed by immunoblot analysis after protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data suggest that human platelet dense granules, unlike chromaffin cell secretory granules, are not the major intracellular site of localization of pp60c-src.

Albinism, Oculocutaneous↗
PubMed1991

"Thrombin" receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides.

Using three experimental approaches, we have addressed the questions of whether the presence of saturably bound thrombin plays a role in potentiating the activation of platelet phospholipase C (PLC) and/or accumulation of the 3-phosphorylated phosphoinositides (3-PPI), i.e. phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and whether the generation of tethered ligand (Vu, T-K.H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068) by thrombin can account fully for thrombin's proteolytic effects in activating platelets, as gauged by the above parameters. We have 1) measured PLC activation or 3-PPI after we have exposed platelets to thrombin for various periods and either blocked thrombin's proteolytic activity without interrupting its binding or blocked both binding and proteolytic activity of thrombin; 2) attempted to potentiate 3-PPI accumulation, using combinations of protein kinase C stimulation, Ca2+ elevation, and saturating but proteolytically inactive thrombins; and 3) compared the activation of platelets by thrombin with activation by the "thrombin" receptor-directed peptide, SFLLRNPNDKYEPF (SFLL; a portion of the tethered ligand created by thrombin's proteolytic activity), and examined the effect of thrombin on this latter activation. We conclude that the initial and sustained effects of thrombin in stimulating PLC and the accumulation of 3-PPI are completely attributable to thrombin's proteolytic activity. Further, thrombin's effects in promoting these responses can be accounted for by the actions of SFLL peptide, and by implication, formation of tethered ligand.

Amino Acid Sequence↗
PubMed1991

Protein kinase C regulates the stimulated accumulation of 3-phosphorylated phosphoinositides in platelets.

We have shown that platelets stimulated with thrombin or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), both of which activate phospholipase C and protein kinase C (PKC), show enhancement of 3-phosphorylated phosphoinositide accumulation (3-PPI). We now report the following. (1) Inhibition of thrombin- or GTP[S]-stimulated PKC by pseudo-substrate peptide (RFARK) added to permeabilized platelets markedly inhibits 3-PPI, whereas the serine/threonine phosphatase inhibitor, okadaic acid, promotes 3-PPI. PKC activity, insufficient in itself for fully activating 3-PPI, appears crucial to receptor and post-receptor stimulation of 3-PPI, even when tyrosine phosphorylation is unimpaired. (2) Alteration of Gi by ADP-ribosylation only slightly affects the stimulation of 3-PPI by thrombin, and activation of the G-protein Gi by adrenaline has no effect on 3-PPI. (3) Inhibition of PKC blocks activated secretion of platelet-derived growth factor (PDGF). However, PDGF cannot promote platelet 3-PPI, and thus cannot account for the inhibitory effects of RFARK on 3-PPI.

Adenosine Diphosphate Ribose↗
PubMed1990

Stimulated cholesterol-enriched platelets display increased cytosolic Ca2+ and phospholipase A activity independent of changes in inositol trisphosphates and agonist/receptor binding.

To investigate the mechanism of enhanced responsiveness of cholesterol-enriched human platelets, we compared stimulation by surface-membrane-receptor (thrombin) and post-receptor (AlF4-) G-protein-directed pathways. Platelets were labelled with [32P]Pi and [methyl-3H] choline chloride, incubated with sonicated lipid dispersions of various ratios of cholesterol and phospholipid, and loaded with the fluorescent Ca2+ indicator fura-2. We report the following. (1) Cholesterol enrichment enhances cytosolic Ca2+ accumulation and phospholipase A activation in response to both receptor-directed and post-receptor-directed agonists. No enhancement by cholesterol of phospholipase A activity at fixed Ca2+ concentrations is observed in lysed platelets, implying that no perturbation by cholesterol of phospholipase A/substrate interaction occurs in our preparations. (2) In both normal and cholesterol-enriched platelets, Ca2+ mobilization is promoted by a factor(s) apart from InsP3 that appear(s) to be modulated by cholesterol. A disproportionate increase in cytosolic Ca2+ relative to [32P]InsP3 is observed with increasing doses of thrombin in normal, and to a larger extent in cholesterol-enriched, platelets. When AlF4- is the agonist, there is no cholesterol-associated enhancement in [32P]InsP3 to account for the heightened Ca2+ rise seen with cholesterol enrichment. (3) Enhanced phospholipase A activation is not necessarily proportional to cytosolic Ca2+ increase. The magnitude of the increase in phospholipase A activity for a given rise in cytosolic Ca2+ is greater in cholesterol-enriched platelets that are stimulated by AlF4- than in those stimulated by thrombin. We conclude that increased membrane microviscosity associated with cholesterol enrichment may promote G-protein/phospholipase A interaction as well as the Ca2(+)-release mechanism, without significantly altering G-protein/phospholipase C interaction.

Blood Platelets↗