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C P Downes

Publications and source records attributed to C P Downes.

At least 19 recordsLinked to original sources
PubMed1995

Kinetic analysis of phospholipase C beta isoforms using phospholipid-detergent mixed micelles. Evidence for interfacial catalysis involving distinct micelle binding and catalytic steps.

Phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)-P2) hydrolysis by three different beta-isoforms of phospholipase C (PLC) was examined to investigate the catalytic action of these extracellular signal-regulated enzymes. Depletion of phospholipase C from solution by incubation with sucrose-loaded vesicles of differing compositions followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles from which an equilibrium association constant of PLC with PtdIns (4,5)P2 could be determined. A mixed micellar system was established to assay PLC activity using dodecyl maltoside, which behaved as an essentially inert diluent of PtdIns (4,5)P2 with respect to PLC beta activity. Kinetic analyses were performed to test whether PLC beta activity was dependent on both bulk PtdIns (4,5)P2 concentration and surface concentration in the micelles as has been shown for other lipid metabolising enzymes. Each of the PLC beta isoforms behaved similarly in these analyses, which indicated the involvement of at least two binding events. Interfacial Michaelis constants were calculated to be between 0.1-0.2 mol fraction for all three enzymes, and Ks (the equilibrium dissociation constant of PLC for lipid) ranged between 100-200 microM. The apparent multiple interfacial binding events did not appear to result from lipid-induced PLC beta oligomerization implying that PLC beta monomers possess more than one lipid-binding site. Surface dilution of PLC-catalyzed PtdIns (4,5)P2 hydrolysis was assessed in the presence of increasing concentrations of various nonsubstrate phospholipids, which profoundly reduced PLC activity, suggesting that these lipids may inhibit enzyme action. The data indicate that G protein-regulated isoforms of PLC operate with separate lipid binding and catalytic steps and imply that under physiological conditions, PLC beta isoforms operate under first-order conditions. These findings may have implications for the mechanisms of regulation of PLC beta s by G protein subunits.

Animals↗
PubMed1995

Cooperation of Src homology domains in the regulated binding of phosphatidylinositol 3-kinase. A role for the Src homology 2 domain.

Fibroblasts transformed by the v-Src oncoprotein exhibit elevated activity of the enzyme phosphatidylinositol 3'-kinase (PI 3-kinase), which binds to, and is activated by, a wide range of receptor tyrosine kinases as well as v-Src and transforming polyoma middle T/c-Src complexes. Here we consider the role of the v-Src homology (SH) domains, SH3 and SH2, and the tyrosine kinase catalytic domain, in the stimulation of v-Src-associated PI 3-kinase activity in response to rapid activation of the oncoprotein. As shown by others, we find that the v-Src SH3 domain tightly binds the PI 3-kinase p85 regulatory subunit in normal growing chicken embryo fibroblasts. However, we also find that in transformed cells there is additional efficient binding of PI 3-kinase to the v-Src SH2 domain in a catalytically active form. Furthermore, the binding of p85 to the SH2 domain, which is almost undetectable in quiescent cells, is rapidly stimulated upon activation of temperature-sensitive v-Src and consequent cell cycle entry, demonstrating that binding is a target for regulation. We also show that v-Src-associated PI 3-kinase differs considerably from PDGF receptor-associated enzyme by a different mode of binding, a lack of substantial allosteric activation, and a dependence on the tyrosine kinase activity of v-Src. The rapidly induced binding and activation of PI 3-kinase thus provides sensitive regulation of recruitment of PI 3-kinase to its substrates and into other signaling complexes at the cell membrane, which involves all the Src homology domains.

Amino Acid Sequence↗
PubMed1995

The mechanism of muscarinic receptor-stimulated phosphatidylinositol resynthesis in 1321N1 astrocytoma cells and its inhibition by Li+.

The coupling of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C to resynthesis of phosphatidylinositol (PtdIns) and the ability of Li+ to inhibit this after cellular inositol depletion were studied in 1321N1 astrocytoma cells cultured in medium +/- inositol (40 microM). In inositol-replete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0-30 min) approximately > or = 20-fold activation of phospholipase C, whereas prolonged (> 60 min) stimulation turned over PtdIns equal to the cellular total mass, involving approximately 80% of the cellular PtdIns pool without reducing PtdIns concentrations significantly. PtdIns resynthesis was achieved by a similar, initial agonist activation of PtdIns synthase. The dose dependency for carbachol stimulation of PtdIns synthase and phospholipase C was similar (EC50 approximately 20 microM) as was the relative intrinsic activity of muscarinic receptor partial agonists. This demonstrates the tight coupling of phosphoinositide hydrolysis to resynthesis and suggests this is achieved by a direct mechanism. In inositol-replete or depleted cells basal concentrations of inositol and CMP-phosphatidate were respectively approximately 20 mM or < or = 100-500 microM and approximately 0.1 or approximately > or = 1-10 pmol/mg of protein. Comparison of the effects of agonist +/- Li+ on the concentrations of these cosubstrates for PtdIns synthase suggest that accelerated activity of this enzyme is differentially driven by stimulated increases in the amounts of CMP-phosphatidate or inositol in inositol-replete or depleted cells, respectively. Thus, the preferential capacity of Li+ to impair stimulated phosphoinositide turnover in systems expressing low cellular inositol can be attributed to its ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated PtdIns resynthesis.

Astrocytoma↗
PubMed1995

Multiple roles of phosphatidylinositol 3-kinase in regulation of glucose transport, amino acid transport, and glucose transporters in L6 skeletal muscle cells.

Phosphatidylinositol 3-kinase (PI3k) activity is required for the insulin stimulation of glucose transport in adipocytes and Chinese hamster ovary cells. Wortmannin (WM), an inhibitor of PI3k, inhibits the stimulation of glucose transport by insulin and the gain of glucose transporters at the cell surface. However, the effect of inhibition of PI3k on the maintenance of the basal and the insulin-stimulated glucose transport and on the intracellular donor pool of glucose transporters has not been clarified. Here we show that in L6 skeletal muscle cells in culture WM significantly inhibits the basal PI3k activity (by 40%), decreases the levels of phosphatidylinositol 3,4-phosphate and 3,4,5-phosphate (by about 50%) and abolishes the activation of the enzyme by insulin. WM inhibited the basal rate of transport of glucose (by 45%) and of amino acids through system A (by 25%) and abolished their stimulation by insulin. Insulin caused a transient increase in PI3k activity and PI3k products that returned to basal levels within 40 min, whereas glucose and amino acid transport remained elevated. Under these conditions, WM reduced the rate of glucose and amino acid transport back to basal levels. In unstimulated cells, WM decreased significantly the GLUT4 glucose transporter content at the plasma membrane and prevented the ability of insulin to recruit transporters to this membrane. Interestingly, the intracellular pools of the GLUT3 and GLUT4 glucose transporters were significantly reduced in response to WM treatment alone. We conclude that in muscle cells PI3k activity is required to maintain basal and insulin-stimulated glucose and amino acid transport, as well as to develop the stimulation of the two transport processes in response to the hormone. We hypothesize that PI3k, likely through production of phosphatidylinositol 3,4-phosphate and 3,4,5-phosphate, regulates the basal plasma membrane glucose transporter recycling and the organization of the transporter intracellular pool, in addition to being an insulin signal.

Amino Acids↗
PubMed1994

The turkey erythrocyte beta-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein alpha subunits.

By contrast with mammalian beta-adrenergic receptors, the avian isoform elicits two distinct effector responses, activation of adenylate cyclase and polyphosphoinositide-specific phospholipase C (PLC) leading to the accumulation of both cyclic adenosine monophosphate (cyclic AMP) and inositol phosphates. We have investigated the mechanisms of beta-adrenergic receptor signalling in turkey erythrocytes. Stimulation of adenylate cyclase by the beta-adrenergic-receptor agonist isoprenaline exhibits a 30-fold lower EC50 than that for PLC activation, which may indicate a marked receptor reserve for the former effector. Similar Ki values were obtained for the inhibition of both responses by four beta-adrenergic antagonists, arguing that a single receptor population is responsible for both effects. Antibodies raised against G-protein peptide sequences were used to show that the identity of the G-protein mediating the PLC response was an avian homologue of G11, the level of expression of which was very similar to that of the stimulatory G-protein of adenylate cyclase, Gs. Thus a single population of beta-adrenergic receptors apparently interacts with distinct G-proteins to activate different effectors. The stoichiometries of the receptor-G-protein-effector interactions are therefore similar for both second-messenger responses and the data are discussed in terms of the different efficacies observed for each response.

Adenosine Diphosphate↗
PubMed1994

Inositol trisphosphate metabolism in Saccharomyces cerevisiae: identification, purification and properties of inositol 1,4,5-trisphosphate 6-kinase.

Ins(1,4,5)P3 metabolism was examined in Saccharomyces cerevisiae extracts. S. cerevisiae contains readily detectable Ins(1,4,5)P3 kinase activity that is predominantly soluble, but phosphomonoesterase activity acting on Ins(1,4,5)P3 was not detected in either soluble or particulate preparations from this organism. We have purified the kinase activity approximately 685-fold in a rapid four-step process, and obtained a stable preparation. The enzyme has an apparent native molecular mass of approximately 40 kDa, and displays Michaelis-Menten kinetics with respect to its two substrates, ATP and Ins(1,4,5)P3. The Km for ATP was 2.1 mM, and that for Ins(1,4,5)P3 was 7.1 microM. The enzyme appeared to be the first step in the conversion of Ins(1,4,5)P3 into an InsP5, and the partially purified preparation contained another activity that converted the InsP4 product into an InsP5. The InsP4 product of the partially purified kinase was not metabolized by human erythrocyte ghosts and co-chromatographed with an Ins(3,4,5,6)P4 [L-Ins(1,4,5,6)P4] standard, identifying it as D-Ins(1,4,5,6)P4. The yeast enzyme is thus an Ins(1,4,5)P3 6-kinase. This activity may be an important step in the production of inositol polyphosphates such as InsP5 and InsP6 in S. cerevisiae.

Adenosine Triphosphate↗
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

A G-protein beta gamma-subunit-responsive phosphoinositide 3-kinase activity in human platelet cytosol.

Thrombin activates phosphoinositide 3-kinase (PI 3-kinase) in platelets via a mechanism involving G-proteins, possibly of both the heterotrimeric and the low molecular weight families. We have investigated the regulation of PI 3-kinase present in platelet cytosol, and we show that this activity can be stimulated by a mixed preparation of G-protein beta gamma-subunits. This stimulation is reversed by preincubation of the beta gamma-subunits with GDP-liganded alpha-subunits. The beta gamma-stimulated activity is inhibited by wortmannin, a recently identified inhibitor of PI 3-kinase in other systems. In addition, the activity associates with PDGF receptor phosphotyrosyl peptide and monoclonal antibody designed to interact with the p85 subunit of PI 3-kinase. We suggest that this beta gamma-sensitive activity is related to previously identified forms of PI 3-kinase.

Androstadienes↗
PubMed1994

Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C.

The activity of a beta-isoform of phospholipase C (PLC) partially purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.

Animals↗
PubMed1994

The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.

Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP2 availability. The potential for complex regulation of PLC and PtdIns synthase is discussed.

Culture Media↗
PubMed1994

Visualization of agonist-stimulated inositol phospholipid turnover in individual neurons of the rat cerebral cortex and hippocampus.

A novel autoradiographic method to identify individual neurons responding to neurotransmitter stimulation with increased phosphoinositide turnover is described. When phosphoinositide-coupled receptors are activated, phosphatidylinositol 4,5-bisphosphate is hydrolysed by phospholipase C generating the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. During prolonged receptor stimulation, both second messengers are actively recycled to maintain the effective intracellular levels of agonist-sensitive phosphoinositides. Lithium ions inhibit this recycling pathway by blocking the recovery of free inositol from inositol 1,4,5-trisphosphate thus leading to the accumulation of phosphatidyl cytidine monophosphate, a membrane bound molecule which is the activated precursor of the synthesis of phosphoinositides. Therefore, addition of excess myo-inositol reverts the effects of lithium inhibition. Thus, taking advantage of this fact and using [3H]cytidine as precursor, phosphatidyl [3H]cytidine monophosphate accumulation was induced in rat neocortical and hippocampal slices after muscarinic or metabotropic glutamate receptor stimulation. The labelled slices were then fixed, dehydrated and embedded in Durcupan resin. Semithin sections (1 micron thick) were cut and exposed to autoradiographic emulsion for several weeks. Biochemical analysis of the incorporation of [3H]cytidine into the chloroform extracted (containing lipids) and the alkali-solubilized (containing nucleic acids and proteins) fractions were carried out in parallel with morphological studies. The stimulation of both receptor types induced labelling of neurons in neocortex and hippocampus. In labelled cells silver grains were characteristically observed over the cytoplasm surrounding the nucleus and main dendritic processes. The anatomical location and distribution of labelled cells as well as the levels of response obtained in both brain regions studied, was found to be receptor specific. Inclusion of 30 mM myo-inositol in the incubation media reversed completely both the accumulation of phosphatidyl [3H]cytidine monophosphate and the labelling of cells, thus demonstrating that the label detected autoradiographically corresponds to phosphatidyl [3H]cytidine monophosphate. It is concluded that the method is sensitive and specific, allowing identification of individual neurons in both neocortical and hippocampal slices and after stimulation of both muscarinic and metabotropic glutamate receptor subtypes. The method may open a new means to study the phosphoinositide second messenger signalling pathway and the cells in which it takes place.

Animals↗
PubMed1994

Involvement of calcium influx in muscarinic cholinergic regulation of phospholipase C in cerebellar granule cells.

Inositol phosphate accumulation on carbachol stimulation of rat cerebellar granule cells shows a marked dependence on factors affecting cytosolic Ca2+ concentration ([Ca2+]c). After 5 min, potassium depolarisation caused a modest accumulation of inositol phosphates but augmented the response to carbachol by a factor of 2-3. These effects of potassium were dependent on an extracellular source of calcium and could be partially blocked by specific (nifedipine) and nonspecific (verapamil) calcium channel blockers. Measurements of [Ca2+]c under a range of stimulatory conditions demonstrated a close correlation between the elevation of [Ca2+]c and agonist-stimulated phospholipase C (PLC) activity. The maximal potentiation of carbachol-stimulated inositol phosphate accumulation was achieved using 20 mM KCl, which increased [Ca2+]c from approximately 20 to approximately 75 nM, indicating the involvement of relatively low threshold Ca2+ channels and the high sensitivity of the relevant PLC to small changes in [Ca2+]c. By contrast, increases in [Ca2+]c induced by the Ca2+ ionophore ionomycin were associated with more modest and less potent effects on agonist-stimulated PLC. These results demonstrate a cooperative interaction between a receptor/G protein-regulated PLC and voltage-stimulated elevations of [Ca2+]c, which may function to integrate ionotropic and metabotropic signalling mechanisms in cerebellar granule cells.

Animals↗
PubMed1993

Direct labelling of hormone-sensitive phosphoinositides by a plasma-membrane-associated PtdIns synthase in turkey erythrocytes.

We have previously characterized phosphatidylinositol (PtdIns) synthase and PtdIns/myo-inositol-exchange enzyme activities in ghost membranes prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Vaziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that PtdIns synthase activity is relatively enriched in plasma-membrane preparations of turkey erythrocytes and that inositol phospholipids labelled by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated phospholipase C (PLC), which is present also in ghost preparations. Specific-radioactivity measurements of [3H]PtdIns from ghosts labelled to equilibrium under conditions favouring [3H]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly access approx. 14% of the total erythrocyte ghost PtdIns. Approx. 16% of the [3H]PtdIns labelled by the PtdIns synthase reaction can be phosphorylated to polyphosphoinositides, which are then hydrolysed by the receptor- and G-protein-stimulated PLC. Since the mass of PtdIns declines to a similar extent as [3H]PtdIns during stimulation in the presence of guanine nucleotides and ATP, it is evident that both the labelled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma membranes were also labelled by [3H]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities respectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-22981], but the labelled lipids were sensitive to purinergic-receptor-stimulated hydrolysis in reconstitution assays using partially purified turkey erythrocyte PLC. The results strongly suggest that at least a portion of the PtdIns synthase in turkey erythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids.

Animals↗
PubMed1993

The characteristics, capacity and receptor regulation of inositol uptake in 1321N1 astrocytoma cells.

The uptake of inositol into 1321N1 astrocytoma cells was studied by measurement of the accumulation of free [3H]inositol within the intracellular pool. Uptake occurs via a saturable transporter with apparent Km for inositol approximately 40 microM and Vmax approximately 180 pmol/min per mg of protein, which permits intracellular inositol concentrations to exceed those of the medium by a factor of approximately 500. At extracellular concentrations up to 500 microM, inositol uptake is highly dependent (> or = 85%) on the presence of Na+ in the medium, and at physiological extracellular inositol concentrations, allows inositol to achieve an intracellular concentration of approximately 20 mM, indicating an active process driven by the Na+ gradient. Despite this, uptake was only minimally impaired or was unaffected by ouabain (1 mM) or dinitrophenol (1 mM). Consistent with a carrier-mediated mechanism, uptake was competitively blocked by phlorhizin (K1 approximately 125 microM). Uptake was also inhibited by carbachol and histamine, which act respectively via muscarinic and H1 receptors in these cells to stimulate phospholipase C. Inhibition by carbachol was dose-dependent (EC50 approximately 3-30 microM) and blocked by atropine. Inhibition by carbachol (1 mM) was non-competitive, resulting from approximately 50% decrease in the Vmax for uptake without affecting the Km and was persistent over 30-90 min. Inhibition by carbachol and histamine was independent of extracellular Ca2+ and was reproduced by phorbol ester, but not by Ca2+ ionophore or stimulation of adenylate cyclase. These results imply that receptors which couple to phospholipase C may mediate inhibition of inositol uptake via protein kinase C. The data are discussed in relation to inositol homoeostasis in resting and stimulated cells.

Astrocytoma↗
PubMed1993

Subcellular distribution of agonist-stimulated phosphatidylinositol synthesis in 1321 N1 astrocytoma cells.

In an inositol-depleted 1321 N1 astrocytoma cell line, propranolol at 0.5 mM concentration and carbachol in the presence of Li+ induce a large increase (30-60-fold) in the amount of CMP-phosphatidate, the lipid substrate of PtdIns synthase. The actions of both agents on CMP-phosphatidate accumulation were reversed by co-incubation with 1 mM inositol. In cells grown in the presence of 40 microM inositol the propranolol- and carbachol-mediated CMP-phosphatidate accumulation was much smaller (2-4-fold). Propranolol- and carbachol-mediated increases in CMP-phosphatidate accumulation were at least additive in both inositol-replete and -depleted cells. The subcellular distribution of accumulated CMP-phosphatidate was investigated by sucrose-density-gradient centrifugation of a lysate of inositol-depleted cells. There were two coincident peaks of carbachol-stimulated [3H]CMP-phosphatidate and PtdIns synthase activity, respectively. The first peak of accumulated [3H]CMP-phosphatidate and PtdIns synthase activity is characteristic of a 'light vesicle' fraction, since it sediments at sucrose densities similar to that of endocytosed 125I-transferrin. The later peak, containing both carbachol-stimulated [3H]CMP-phosphatidate and PtdIns synthase activity, has a distribution in the gradient that is similar to NADPH-cytochrome c reductase activity, an endoplasmic-reticulum marker. By contrast, propranolol-stimulated [3H]CMP-phosphatidate accumulates in membranes which sediment as a single peak corresponding to the endoplasmic-reticulum marker. These observations suggest that agonist-stimulated PtdIns synthesis occurs in the endoplasmic reticulum and in at least one additional membrane compartment which is insensitive to propranolol, an inhibitor of endoplasmic-reticulum phosphatidate phosphohydrolase.

Astrocytoma↗