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I Litosch

Publications and source records attributed to I Litosch.

At least 19 recordsLinked to original sources
PubMed2000

Regulation of phospholipase C-beta(1) activity by phosphatidic acid.

The role of phosphatidic acid (PA) in regulating phospholipase C-beta(1) (PLC-beta(1)) activity was determined. PA promoted the binding of PLC-beta(1) to sucrose-loaded unilamellar vesicles (SLUV) containing phosphatidylcholine. PA increased enzymatic activity over a range of Ca(2+) concentrations and reduced the Ca(2+) concentration required for half-maximal stimulation of activity. PA did not affect the apparent K(m) for phosphatidylinositol 4, 5-bisphosphate. Lysophosphatidic acid also enhanced the binding of PLC-beta(1) to SLUV but was less effective in stimulating enzymatic activity. Diacylglycerol, phosphatidylserine, and oleic acid had little effect on activity. Anionic and neutral detergents did not stimulate activity. PA stimulation was relatively independent of acyl chain length. Dipalmitoyl-PA (16:0) was comparable to PA from egg lecithin and dimyristoyl-PA (C14:0) in stimulating activity, while dilauroyl-PA (C12:0) was slightly less effective. A 100 kDa catalytic fragment of PLC-beta(1) lacking amino acid residues C-terminal to His(880) did not bind to PA and was insensitive to stimulation by 7-15 mol % PA. Stimulation of 100 kDa enzymatic activity required 30 mol % PA. PA increased receptor-G protein stimulation of PLC-beta(1) activity in membranes. These results demonstrate that PA stimulates basal and receptor-G protein-regulated PLC-beta(1) activity. PA stimulation occurs through both a C-terminal-dependent and an independent mechanism. The C-terminal-mediated mechanism for stimulation may constitute an important pathway for conferring specific regulation of PLC-beta(1) in response to increases in cellular PA levels.

Animals↗
PubMed1997

G-protein betagamma subunits antagonize protein kinase C-dependent phosphorylation and inhibition of phospholipase C-beta1.

Protein kinase C (PKC) isoforms phosphorylated phospholipase C-beta1 (PLC-beta1) in vitro as follows: PKCalpha >> PKCepsilon; not PKCzeta. PLC-beta3 was not phosphorylated by PKCalpha. G-protein betagamma subunits inhibited the PKCalpha phosphorylation of PLC-beta1 in a concentration-dependent manner. Half-maximal inhibition occurred with 500 nM betagamma. G-protein betagamma subunits also antagonized the PKCalpha-mediated inhibition of PLC-beta1 enzymic activity. PKCalpha, in turn, inhibited the stimulation of PLC-beta1 activity by betagamma. There was little effect of PKCalpha on the stimulation of PLC-beta1 by alphaq/11-guanosine 5'[gamma-thio]triphosphate (GTP[S]). These findings demonstrate that G protein betagamma subunits antagonize PKCalpha regulation of PLC-beta1. Thus betagamma subunits might have a role in modulating the negative feedback regulation of this signalling system by PKC.

Animals↗
PubMed1996

G-protein inhibition of phospholipase C-beta 1 in membranes: role of G-protein beta gamma subunits.

Rat liver plasma membranes reconstituted with bovine brain phospholipase C beta 1 (PLC- beta 1) exhibit a dual regulation of PLC- beta 1 activity by G-proteins. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]; 0.1 nM) produced a 20-25% inhibition of PLC- beta 1 activity within 7 min of incubation. The addition of vasopressin resulted in near-basal levels of activity in the presence of 0.1 nM GTP[S]. Clonidine had little effect on the net inhibition due to GTP[S]. A similar antagonism between carbachol and GTP[S] occurred in cerebral cortical membranes containing endogenous PLC- beta 1 activity. alpha 0/i-GDP (a mixture of GDP-liganded G0 alpha and Gi alpha) attenuated the GTP[S]-dependent inhibition of PLC- beta 1 whereas alpha 0/i-GTP[S] had no effect, suggesting an involvement of G-protein beta gamma subunits in the inhibition of PLC- beta 1. Low concentrations of beta gamma subunits inhibited PLC- beta 1 activity. Inhibition was followed by reversal to basal activity and onset of stimulation as the beta gamma concentration was increased. Inhibition by beta gamma was dependent on the presence of membranes. These results indicate that G-protein beta gamma subunits constitute a mechanism by which G-protein mediate a rapid and transient inhibition of PLC- beta 1.

Animals↗
PubMed1996

Protein kinase C inhibits the Ca(2+)-dependent stimulation of phospholipase C-beta 1 in vitro.

Protein kinase C (PKC) inhibited the Ca(2+)-dependent stimulation of a 600-fold purified phospholipase C beta 1 (PLC-beta 1). Inhibition by PKC was time-dependent, and required ATP and diacylglycerol. Inhibition was more pronounced when the PLC assay was conducted with a PIP2 substrate mixture containing phosphatidylserine, then with a substrate mixture containing phosphatidyle-thanolamine. Cyclic AMP-dependent protein kinase A did not inhibit PLC-beta 1 activity. PKC did not affect the rate of PLC-beta 1 activation by Ca2+ or the rate of PLC-beta 1 deactivation by EGTA. PLC-beta 1 purified 1700-fold was less sensitive to inhibition by PKC despite stoichiometric phosphorylation. These results demonstrate that PKC inhibits the Ca(2+)-dependent stimulation of a 600-fold purified PLC-beta 1 in vitro. Furthermore, purification of PLC-beta 1 to homogeneity results in a diminished sensitivity to inhibition by PKC, indicating that other components may participate in mediating the effect of PKC on the Ca(2+)-dependent stimulation of PLC-beta 1 in vitro.

Adenosine Triphosphate↗
PubMed1993

G protein-mediated inhibition of phospholipase C activity in a solubilized membrane preparation.

In solubilized bovine brain membrane preparations AlF4- (20 microM AlCl3 plus 10 mM NaF) and 50 nM guanosine 5-O-(2-thiotriphosphate) (GTP gamma S) promoted a rapid but transient inhibition of phospholipase C (PLC) activity. Maximal inhibition was evident within 7 min of incubation, followed by reversal of inhibition. In contrast, 10 microM GTP gamma S did not induce inhibition of PLC activity but rather produced a time-dependent stimulation of PLC activity. GTP gamma S-dependent inhibition of PLC activity was concentration-dependent with half-maximal inhibition at 1 nM. Inhibition was antagonized by guanosine 5-O-(2-thiodiphosphate (GDP beta S). Pertussis toxin delayed the onset of inhibition by GTP gamma S but did not prevent the inhibitory effect. alpha o-GTP gamma S or alpha o-GDP had little effect on PLC activity. alpha i-GTP gamma S and alpha i-GDP produced a 15% inhibition of PLC activity. Beta gamma subunits did not inhibit basal PLC activity but did attenuate the net degree of inhibition due to GTP gamma S. Inhibition was associated with a decrease in the Ca2+ sensitivity of PLC. Preincubation of membranes with anti-PLC-beta 1 antibody, but not anti-PLC-gamma 1 or anti-PLC-delta 1, prevented the GTP gamma S-mediated inhibition of PLC. These studies implicate PLC-beta 1 as an effector system that is under negative modulation by a G protein-dependent mechanism.

Animals↗
PubMed1993

Rapid stimulation of Ins (1,4,5)P3 production in rat aorta by NE: correlation with contractile state.

Rapid stimulation of Ins(1,4,5)P3 production in rat aorta by NE: correlation with contractile state. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H126-H132, 1993.--The isomeric composition of inositol phosphates generated in response to norepinephrine (NE) stimulation and the relationship of inositol phosphate production to release of intracellular Ca2+ as measured by contraction were characterized in rat aorta prelabeled with [3H]inositol. NE stimulated a rapid and transient increase in labeled D-myo-inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] levels. A maximal increase in labeled Ins(1,4,5)P3 occurred within 15 s of stimulation followed by a decline to control levels at 5 min. D-Myo-inositol 1,3,4-trisphosphate [Ins-(1,3,4)P3] and D-myo-inositol 1-monophosphate [Ins(1)P] levels also increased rapidly in response to NE. In contrast to the transient production of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1)P production was maintained in the presence of NE. Half-maximal stimulation of Ins(1,4,5)P3 production and Ca2+ release occurred at 0.3 microM NE, and maximal effects were obtained with 10 microM NE. The concentration-response curve and time course for production of Ins(1,4,5)P3 correlated with the neurotransmitter-induced Ca2+ release from intracellular stores, indicating that the level of Ins(1,4,5)P3 regulated the Ca(2+)-release mechanism. In the continued presence of NE, the intracellular pools did not completely refill with Ca2+ despite the return of Ins-(1,4,5)P3 levels to basal at 5 min. These results demonstrate that NE stimulates a rapid increase in Ins(1,4,5)P3 that correlates with contraction in Ca(2+)-free buffer. The reuptake of Ca2+ into intracellular stores is regulated by a mechanism that may not involve Ins(1,4,5)P3.

Animals↗
PubMed1991

G protein regulation of phospholipase C activity in a membrane-solubilized system occurs through a Mg2(+)- and time-dependent mechanism.

GTP-binding proteins have been implicated to function as key transducing elements in the mechanism underlying receptor activation of a membrane-associated phospholipase C activity. In the present study, the regulation of phospholipase C activity by GTP-binding proteins has been characterized in a detergent-solubilized system derived from bovine brain membranes. Guanosine-5'-(3-O-thio)triphosphate (GTP-gamma-S) and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) stimulated a dose-dependent increase in phospholipase C activity with half-maximal activation at 0.6 microM and 10 microM, respectively. The maximal degree of stimulation due to Gpp(NH)p or GTP-gamma-S was comparable. 100 microM GTP had only a slight stimulatory effect on phospholipase C activity. Adenine nucleotides, 100 microM adenylyl-imidodiphosphate and ATP, did not stimulate phospholipase C activity, indicating that specific guanine nucleotide-dependent regulation of phospholipase C activity was preserved in the solubilized state. Gpp(NH)p or GTP-gamma-S stimulation of phospholipase C activity was time-dependent and required Mg2+.Mg2+ regulated the time course for activation of phospholipase C by guanine nucleotides and the ability of guanine nucleotides to promote an increase in the Ca2+ sensitivity of phospholipase C. 200 microM GDP-beta-S or 5 mM EDTA rapidly reversed the activation due to GTP-gamma-S or Gpp(NH)p. These findings demonstrate that G protein regulation of phospholipase C activity in a bovine brain membrane- solubilized system occurs through a Mg2+ and time-dependent mechanism. Activation is readily reversible upon addition of excess GDP-beta-S or removal of Mg2+.

Animals↗
PubMed1989

Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity.

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5'[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.

Cell Membrane↗
PubMed1989

Guanine nucleotides mediate stimulatory and inhibitory effects on cerebral-cortical membrane phospholipase C activity.

In cerebral-cortical membranes, hydrolysis-resistant guanine nucleotides exert a dual regulatory effect on phospholipase C activity. Nanomolar concentrations of guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) inhibited basal phospholipase C activity, with a maximum inhibition of 30% at 10 nM. Increasing the concentration of p[NH]ppG or GTP[S] to over 10 nM resulted in a reversal of the inhibitory effect and onset of stimulation of phospholipase C activity. These inhibitory effects were blocked by 100 microM-guanosine 5'-[beta-thio]diphosphate. GTP was relatively ineffective in producing either stimulation or inhibition of phospholipase C activity. Similarly, ATP, adenosine 5'-[beta gamma-imido]triphosphate and GDP were also ineffective. Expression of the dual effects of guanine nucleotides was affected by the Mg2+ concentration. At 0.3 mM-Mg2+, both the inhibitory and the stimulatory components of p[NH]ppG action were evident. At 2.5 mM-Mg2+, only p[NH]ppG stimulation was observed. Pertussis-toxin treatment blocked the p[NH]ppG-mediated inhibition of phospholipase C activity. These results demonstrate that non-hydrolysable guanine nucleotides exert both a stimulatory and an inhibitory effect on membrane phospholipase C activity. These effects may be mediated through distinct GTP-binding proteins.

Animals↗
PubMed1988

Norepinephrine stimulates the production of inositol trisphosphate and inositol tetrakisphosphate in rat aorta.

Norepinephrine stimulated the rapid hydrolysis of [3H]phosphatidylinositol-4,5-bisphosphate in rat aorta with a maximal decrease of 30% within 60 sec of stimulation. Levels of [3H]phosphatidylinositol-4,5-bisphosphate returned to control by 5 min despite the continued presence of agonist. Hydrolysis of [3H]phosphatidylinositol-4,5-bisphosphate occurred concurrently with the formation of inositol phosphates. Inositol-tris and tetrakisphosphate levels were increased within 30 sec of agonist stimulation. Increases in inositol phosphate levels due to agonist were dose-dependent with half-maximal activation at 1 microM norepinephrine.

Animals↗
PubMed1987

Regulatory GTP-binding proteins: emerging concepts on their role in cell function.

The last few years have evidenced a tremendous expansion in our appreciation of the role of regulatory GTP-binding proteins in cellular activation. The availability of cholera and pertussis toxins to detect G proteins as well as methodological advances in the study of cellular function has afforded the opportunity to examine G protein participation in many cellular events. Regulation of adenylyl cyclase and cyclic GMP phosphodiesterase by G proteins has been demonstrated. Phosphatidylinositol-4,5-biphosphate specific phospholipase C activity appears to be subject to G protein control. G proteins regulate inward K+ and Ca2+ channels through a mechanism which may be independent of effects on the above mentioned enzymes. Certainly, the number of G proteins which have been identified from sequencing of complementary DNA affords the potential for G protein involvement in many cellular events. Only three G proteins have however been isolated and functionally characterized, Gs, Gi and transducin. Whether all the functions of these proteins have been identified remains to be seen.

Animals↗
PubMed1987

Guanine nucleotide and NaF stimulation of phospholipase C activity in rat cerebral-cortical membranes. Studies on substrate specificity.

Guanyl-5'-yl imidodiphosphate (p[NH]ppG) stimulated a rapid phospholipase C-mediated breakdown of exogenously added phosphatidylinositol 4,5-bisphosphate (PIP2) in rat cerebral-cortical membranes, with half-maximal activation at approx. 33 microM. NaF stimulated phospholipase C activity, with half-maximal activation at 0.5 mM. Stimulation of phospholipase C activity by NaF exhibited pH optima at approx. 5.5 and 7.0, with the stimulatory activity at pH 7.0 greater than that at pH 5.5. With p[NH]ppG, only stimulation at pH 7.0 was observed. Neither p[NH]ppG nor NaF stimulated hydrolysis of added phosphatidylinositol (PI) or phosphatidylinositol 4-phosphate (PIP). Mg2+ (0.5 mM) potentiated p[NH]ppG-stimulated breakdown of PIP2. Ca2+ increased basal and p[NH]ppG-stimulated breakdown of PIP2. PI breakdown was stimulated only by high Ca2+ concentrations and was unaffected by p[NH]ppG at any Ca2+ concentration examined. These results indicate that, in cerebral-cortical membranes, activation of phospholipase C by guanine nucleotides or fluoride directly increases a phospholipase C activity which specifically hydrolyses PIP2.

Animals↗
PubMed1986

Regulation of phosphoinositide breakdown by guanine nucleotides.

Phosphoinositide hydrolysis is coupled to receptor systems involved in the elevation of cytosolic Ca2+ and activation of protein kinase C. In cell-free systems, guanine nucleotides are required to transduce the effects of receptor activation to phosphoinositide breakdown. Non-hydrolyzable guanine nucleotides stimulate phosphoinositide breakdown in permeabilized cells as well as membranes prepared from salivary glands, GH3 cells, neutrophils, hepatocytes and cerebral cortical tissue. In blowfly salivary gland membranes, 5-hydroxytryptamine stimulates a guanine-nucleotide dependent breakdown of both endogenous and exogenous phosphoinositide substrate through activation of phospholipase C. These data suggest that a GTP-binding protein modulates phospholipase C activity. The identity of this GTP-binding protein has not been established but may resemble other regulatory GTP-binding proteins which have been identified as transducing proteins in a variety of receptor systems.

Adenylyl Cyclases↗
PubMed1986

5-Methyltryptamine decreases net accumulation of 32P into the polyphosphoinositides from [gamma-32P]ATP in a cell-free system from blowfly salivary glands. Activation of breakdown of the newly synthesized [32P]polyphosphoinositides.

Incubation of blowfly salivary gland homogenates with 30 microM [gamma-32P]ATP resulted in a rapid, Mg2+-dependent, synthesis of [32P]polyphosphoinositides and [32P]phosphatidic acid. 5-Methyltryptamine, in the presence of 10 microM guanosine 5'-(3-O-thio)trisphosphate, reduced the net accumulation of 32P label into phosphatidylinositol-4,5-P2 and phosphatidylinositol-4-P by 35 and 20%, respectively. 5-Methyltryptamine did not affect synthesis of [32P]phosphatidic acid. Phosphorylation of polyphosphoinositides was not affected by 5-methyltryptamine. In membranes labeled in vitro with [gamma-32P]ATP, 5-methyltryptamine stimulated a rapid breakdown of the [32P]polyphosphoinositides. These results indicate that in blowfly salivary gland homogenates, hormone stimulates breakdown of the newly synthesized polyphosphoinositides. In the presence of hormone, the rate of polyphosphoinositide synthesis does not compensate for the rate of polyphosphoinositide degradation.

Adenosine Triphosphate↗
PubMed1985

5-Methyltryptamine stimulates phospholipase C-mediated breakdown of exogenous phosphoinositides by blowfly salivary gland membranes.

5-Methyltryptamine, through a GTP-dependent mechanism, stimulated breakdown of endogenous [3H]inositol-labeled phosphoinositides in membranes prepared from blowfly salivary gland homogenates through a phospholipase C exhibiting a pH optimum of approximately 7.0. Unlabeled membranes, prepared from salivary gland homogenates, hydrolyzed exogenous [3H]phosphatidylinositol 4,5-bisphosphate substrate with generation of labeled inositol phosphates. Inositol trisphosphate formation was increased approximately 200% by 10 microM guanosine 5'-(O-thio)-trisphosphate (GTP gamma S) within 30 s. 5-Methyltryptamine, in the presence of 10 microM GTP gamma S, increased the rate of inositol trisphosphate formation by approximately 500% within 30 s. Half-maximal activation of hormone-stimulated breakdown of exogenous substrate required approximately 0.05 microM GTP gamma S. [3H]Phosphatidylinositol was also hydrolyzed during incubation with membranes, resulting in the generation of inositol, glycerol phosphoinositol, and inositol monophosphate. Formation of inositol monophosphate was stimulated approximately 30% by 10 microM GTP gamma S and 10 microM 5-methyltryptamine. Neither inositol nor glycerol phosphoinositol formation was affected by hormone. These results indicate that in a cell-free system from blowfly salivary glands, 5-methyltryptamine, through a GTP-dependent mechanism, directly activates a phospholipase C which mediates phosphoinositide hydrolysis.

Animals↗
PubMed1985

5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown.

Phosphoinositide breakdown has been linked to the receptor mechanism involved in the elevation of cytosolic Ca2+. In a cell-free system prepared from [3H] inositol-labeled blowfly salivary glands, 5-hydroxytryptamine stimulated the rapid production of inositol phosphates. Within 30 s of hormone addition, there was a 100% increase in inositol trisphosphate formation, a 70% increase in inositol bisphosphate formation, and a 90% increase in inositol monophosphate formation as compared to control homogenates incubated for the same length of time. 5-Hydroxytryptamine did not stimulate inositol or glycerol phosphoinositol formation. Half-maximal activation of inositol phosphate production was obtained with 0.33 microM 5-hydroxytryptamine. Ethylene glycol bis(beta-aminoethyl ether)-N',N',N',N'-tetraacetic acid, (EGTA) (0.3 mM) inhibited the basal formation of inositol phosphates and decreased the net accumulation of inositol bisphosphate and inositol trisphosphate due to hormone as compared to homogenates incubated in the absence of added Ca2+. EGTA, however, had little effect on the per cent stimulation of inositol phosphate production due to hormone. In homogenates, ATP, GTP or guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was required for a hormone effect. Gpp(NH)p, unlike ATP or GTP, increased the basal formation of inositol phosphates. In membranes, GTP, Gpp(NH)p, or guanosine 5'-(3-O-thio)trisphosphate (GTP gamma S) sustained a hormone effect whereas ATP was ineffective. GTP did not affect production while Gpp(NH)p and GTP gamma S increased inositol phosphate production. Half-maximal effects of Gpp(NH)p and GTP gamma S on hormone-stimulated inositol phosphate formation occurred at 10 microM and 100 nM, respectively. In the presence of 1 microM GTP gamma S, 5-methyltryptamine stimulated inositol phosphate formation within 2 s in membranes. These results indicate that in a cell-free system, GTP is involved in mediating the effects of Ca2+-mobilizing hormones on phosphoinositide breakdown.

Animals↗
PubMed1984

Phosphoinositide synthesis and Ca2+ gating in blowfly salivary glands exposed to 5-hydroxytryptamine.

Blowfly salivary glands, previously exposed to 10 microM-5-hydroxytryptamine for 30 min, demonstrated a rapid compensatory resynthesis of [3H]inositol-labelled phosphatidylinositol 4,5-bisphosphate when allowed to recover in medium containing 3-5 microM-inositol. Phosphatidylinositol 4,5-bisphosphate comprised 70% of the total [3H]-phosphoinositide, and there was a corresponding decrease in the formation of [3H]-phosphatidylinositol. Subsequent addition of 5-hydroxytryptamine produced an equivalent breakdown of the newly synthesized phosphoinositides but little 45Ca2+ gating. Increasing the inositol concentration in the medium to 300 microM produced a 14-fold stimulation of phosphatidylinositol synthesis but only a 5-fold increase in phosphatidylinositol 4,5-bisphosphate synthesis. Increasing the inositol concentration in the medium from 3 microM to 300 microM resulted in a progressively greater recovery of the 45Ca2+-gating response. At 300 microM-inositol there was an 85% recovery of 45Ca2+-gating response. These results indicate that conversion of phosphatidylinositol into phosphatidylinositol 4,5-bisphosphate occurs in blowfly salivary glands and is secondary to an initial breakdown of the phosphoinositides. Recovery of Ca2+ gating is dependent on the restoration of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate to appropriate concentrations.

Animals↗