42 results on '"Litosch I"'
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2. Relationship Between Vasopressin Activation of Rat Hepatocyte Glycogen Phosphorylase, Inhibition of $$C{{a}^{{{{2}^{ + }}}}} - M{{g}^{{{{2}^{ + }}}}}$$ and Phosphoinositide Breakdown
- Author
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Fain, J. N., primary, Lin, S.-H., additional, Litosch, I., additional, and Wallace, M., additional
- Published
- 1985
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3. G-protein inhibition of phospholipase C-beta 1 in membranes: role of G-protein beta gamma subunits
- Author
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Litosch, I
- Subjects
Cerebral Cortex ,Membranes ,Dose-Response Relationship, Drug ,Protein Conformation ,Vasopressins ,Cell Membrane ,Phospholipase C beta ,Thionucleotides ,Guanosine Diphosphate ,Clonidine ,Rats ,Isoenzymes ,Phenylephrine ,Liver ,GTP-Binding Proteins ,Guanosine 5'-O-(3-Thiotriphosphate) ,Type C Phospholipases ,Animals ,Carbachol ,Cattle ,Research Article - Abstract
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.
- Published
- 1996
4. G protein-mediated inhibition of phospholipase C activity in a solubilized membrane preparation.
- Author
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Litosch, I., primary, Sulkholutskaya, I., additional, and Weng, C., additional
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- 1993
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5. Rapid stimulation of Ins (1,4,5)P3 production in rat aorta by NE: correlation with contractile state
- Author
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Pijuan, V., primary, Sukholutskaya, I., additional, Kerrick, W. G., additional, Lam, M., additional, van Breemen, C., additional, and Litosch, I., additional
- Published
- 1993
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6. G protein regulation of phospholipase C activity in a membrane-solubilized system occurs through a Mg2(+)- and time-dependent mechanism.
- Author
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Litosch, I, primary
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- 1991
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7. Phosphatidic acid modulates G protein regulation of phospholipase C-b1 activity in membranes
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Litosch, I.
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- 2002
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8. Guanine nucleotide and NaF stimulation of phospholipase C activity in rat cerebral-cortical membranes. Studies on substrate specificity
- Author
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Litosch, I
- Abstract
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.
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- 1987
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9. Forskolin as an activator of cyclic AMP accumulation and lipolysis in rat adipocytes.
- Author
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Litosch, I, Hudson, T H, Mills, I, Li, S Y, and Fain, J N
- Abstract
Forskolin increased cyclic AMP accumulation in isolated adipocytes and markedly potentiated the elevation of cyclic AMP due to isoproterenol. In adipocyte membranes, forskolin stimulated adenylate cyclase activity at concentrations of 0.1 microM or greater. Forskolin did not affect the EC50 for activation of adenylate cyclase but did increase the maximal effect of isoproterenol. Neither the soluble nor particulate low-Km cyclic AMP phosphodiesterase activity was affected by forskolin. Low concentrations of forskolin (0.1-1.0 microM), which significantly elevated cyclic AMP levels, did not increase lipolysis, whereas similar increases in cyclic AMP levels due to isoproterenol elevated lipolysis. Forskolin did not inhibit the activation of triacylglycerol lipase by cyclic AMP-dependent protein kinase or the subsequent hydrolysis of triacylglycerol. Higher concentrations of forskolin (10-100 microM) did increase lipolysis. Both the increased cyclic AMP production and lipolysis due to forskolin were inhibited by the antilipolytic agents insulin and N6-(phenylisopropyl)adenosine. Hypothyroidism reduced the ability of forskolin to stimulate cyclic AMP production and lipolysis. These results indicate that forskolin increases cyclic AMP production in adipocytes through an activation of adenylate cyclase. Lipolysis is activated by forskolin but at higher concentrations of total cyclic AMP than for catecholamines.
- Published
- 1982
10. 5-HT-stimulated arachidonic acid release from labeled phosphatidylinositol in blowfly salivary glands
- Author
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Litosch, I., Saito, Y., and Fain, J. N.
- Abstract
In blowfly salivary glands, breakdown of phosphatidylinositol has been linked to the activation of hormone-sensitive Ca2+ channels. Addition of 5-hydroxytryptamine to blowfly salivary glands stimulated the breakdown of phosphatidylinositol prelabeled with 32P or [3H]arachidonic acid. This was associated with a transient accumulation of [3H]arachidonic-labeled diglyceride. There was no appreciable effect of 5-hydroxytryptamine on breakdown of phosphatidylethanolamine or phosphatidylcholine labeled with 32P or [3H]arachidonic acid, indicating that phosphatidylinositol was the immediate source of diglyceride. Extracellular Ca2+ was necessary for [3H]arachidonic acid but not 32P loss from phosphatidylinositol. Addition of arachidonic acid to salivary glands did not stimulate salivary gland secretion or 45Ca flux. In contrast, 5-hydroxytryptamine stimulated both salivary gland secretion and 45Ca flux. These results indicate that, although [3H]arachidonic acid is incorporated into phosphatidylinositol and its release from this phospholipid is increased by 5-hydroxytryptamine, the liberated arachidonic acid does not stimulate salivary gland secretion or 45Ca flux.
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- 1982
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11. Phosphoinositide breakdown in blowfly salivary glands
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Litosch, I., Lee, H. S., and Fain, J. N.
- Abstract
In blowfly salivary glands, 5-hydroxytryptamine stimulated a rapid and sustained loss of [3H]inositol, [32P]phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. There was a corresponding increase in labeled inositol phosphates. In the absence of Ca2+, 5-hydroxytryptamine stimulated a rapid but transient loss of labeled phosphatidylinositol 4,5-bisphosphate. By 5 min, the amount of labeled phosphatidylinositol 4,5-bisphosphate recovered to control values. The divalent ionophore A23187 stimulated loss of labeled phosphatidylinositol 4,5-bisphosphate and increased the amount of labeled phosphatidylinositol. In homogenates, Ca2+ stimulated phosphatidylinositol 4,5-bisphosphate breakdown but not phosphatidylinositol breakdown. These results suggest that hormone-stimulated breakdown of labeled phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate occurs through a phospholipase C and is relatively independent of extracellular Ca2+. There is also a Ca2+-activated conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol.
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- 1984
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12. Phosphoinositide synthesis and Ca2+ gating in blowfly salivary glands exposed to 5-hydroxytryptamine
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Sadler, K, Litosch, I, and Fain, J N
- Abstract
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.
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- 1984
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13. Forskolin as an activator of cyclic AMP accumulation and secretion in blowfly salivary glands
- Author
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Litosch, I, Saito, Y, and Fain, J N
- Abstract
Forskolin is a diterpene that activates adenylate cyclase in a variety of mammalian cells. In addition of forskolin to blowfly salivary glands increased cyclic AMP accumulation and salivary secretion. There was a small increase in transepithelial movement of labelled Ca2+. Forskolin did not induce breakdown of labelled phosphatidylinositol or inhibit the stimulation of phosphatidylinositol breakdown caused by 5-hydroxytryptamine. These data indicate that forskolin can mimic all the effects of 5-hydroxytryptamine on salivary-gland secretion that have been attributed to cyclic AMP.
- Published
- 1982
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14. Regulation of adenylate cyclase and cyclic AMP phosphodiesterase by 5-hydroxytryptamine and calcium ions in blowfly salivary-gland homogenates
- Author
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Litosch, I, Fradin, M, Kasaian, M, Lee, H S, and Fain, J N
- Abstract
Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5′-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.
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- 1982
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15. 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.
- Author
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Litosch, I, Wallis, C, and Fain, J N
- Abstract
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.
- Published
- 1985
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16. 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.
- Author
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Litosch, I, Calista, C, Wallis, C, and Fain, J N
- Abstract
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.
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- 1986
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17. Rapid changes in hepatocyte phosphoinositides induced by vasopressin.
- Author
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Litosch, I, Lin, S H, and Fain, J N
- Abstract
Vasopressin stimulated a 40% decrease in [32P]phosphatidylinositol 4,5-bisphosphate and a 15% decrease in [32P]phosphatidylinositol within 30 s of addition to hepatocytes prelabeled for 60 min with 32P. In hepatocytes prelabeled with [3H]inositol for 60 min, vasopressin produced 20% breakdown of phosphatidylinositol and 33% breakdown of phosphatidylinositol 4,5-bisphosphate within 30 s. There was a 40% increase in total phosphatidylinositol 4,5-bisphosphate within 30 s of vasopressin addition. Breakdown of phosphatidylinositol accounted for disappearance of 95% of the inositol lipid label. In hepatocytes from rats labeled in vivo with [3H]inositol, vasopressin stimulated 10% loss of labeled phosphatidylinositol. Loss of [32P]phosphatidylinositol due to vasopressin was followed by reincorporation of label to levels greater than control while 32P reuptake into phosphatidylinositol 4,5-bisphosphate did not exceed control values. With in vitro [3H]inositol-labeled hepatocytes, loss of label from the phosphoinositides was followed by reuptake of tritium label to control levels. In hepatocytes labeled in vivo with [3H]inositol, reuptake of [3H]inositol label did not occur. These data indicate that the hormone-sensitive pool of hepatocyte phosphoinositides can be labeled by both in vitro and in vivo procedures. Vasopressin induces a rapid decrease of labeled phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate within 30 s.
- Published
- 1983
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18. Guanine nucleotides mediate stimulatory and inhibitory effects on cerebral-cortical membrane phospholipase C activity
- Author
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Litosch, I
- Abstract
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.
- Published
- 1989
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19. 5-Methyltryptamine stimulates phospholipase C-mediated breakdown of exogenous phosphoinositides by blowfly salivary gland membranes.
- Author
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Litosch, I and Fain, J N
- Abstract
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.
- Published
- 1985
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20. Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity
- Author
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Litosch, I
- Abstract
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.
- Published
- 1989
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21. Decoding Gαq signaling.
- Author
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Litosch I
- Subjects
- Animals, GTP-Binding Protein alpha Subunits, Gq-G11 drug effects, Humans, Phospholipase C beta metabolism, Receptors, G-Protein-Coupled agonists, Signal Transduction drug effects, GTP-Binding Protein alpha Subunits, Gq-G11 physiology, Signal Transduction physiology
- Abstract
Gαq signals with phospholipase C-β (PLC-β) to modify behavior in response to an agonist-bound GPCR. While the fundamental steps which prime Gαq to interact with PLC-β have been identified, questions remain concerning signal strength with PLC-β and other effectors. Gαq is generally viewed to function as a simple ON and OFF switch for its effector, dependent on the binding of GTP or GDP. However, Gαq does not have a single effector, Gαq has many different effectors. Furthermore, select effectors also regulate Gαq activity. PLC-β is a lipase and a GTPase activating protein (GAP) selective for Gαq. The contribution of G protein regulating activity to signal amplitude remains unclear. The unique PLC-β coiled-coil domain is essential for maximum Gαq response, both lipase and GAP. Nonetheless, coiled-coil domain associations necessary to maximum response have not been revealed by the structural approach. This review discusses progress towards understanding the basis for signal strength with PLC-β and other effectors. Shared and effector-specific interactions have been identified. Finally, the evidence for allosteric regulation of lipase stimulation by protein kinase C, the membrane, phosphatidic acid, phosphatidylinositol-4, 5-bisphosphate and GPCR is explored. Endogenous allosteric regulators can suppress or enhance maximum lipase stimulation dependent on the PLC-β coiled-coil domain. A better understanding of allosteric modulation may therefore identify a wealth of new targets to regulate signal strength and behavior., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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22. Regulating G protein activity by lipase-independent functions of phospholipase C.
- Author
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Litosch I
- Subjects
- Animals, Humans, GTPase-Activating Proteins metabolism, Guanine Nucleotide Exchange Factors metabolism, Phosphatidic Acids metabolism, Signal Transduction, Type C Phospholipases metabolism
- Abstract
The phosphatidylinositol-specific phospholipase C (PLC) family is known to regulate physiological response through an increase in the levels of cytosolic Ca(2+). PLC hydrolyzes phosphatidylinositol-4, 5-bisphosphate (PIP2) to inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 releases the stored pool of Ca(2+). DAG stimulates protein kinase C (PKC) activity. An intriguing story is that some PLCs are also GTPase activating proteins (GAPs) or guanine nucleotide exchange factors (GEFs) to regulate the activity of their G protein. GEF and GAPs modulate the G protein GTPase cycle. GEFs catalyze the replacement of GDP with GTP to activate the G protein. GAPs accelerate the GTPase cycle to limit signaling upon removal of the activating ligand. It is not known whether GAP/GEF activity is coupled to the lipase activity of PLC, nor is it clear whether or how lipid factors may contribute to this synergistic interaction. While lipase activity is subject to allosteric regulation by mechanisms that include the signaling phospholipid, phosphatidic acid, regulation of the lipase associated GAP/GEF activity remains unexplored. This review explores the possibilities and evidence that support synergistic lipase-G protein regulatory activity in the PLC-β, PLC-δ, PLC-ε and PLC-γ subfamilies that may be mediated, in part, through phosphatidic acid. Understanding the full spectrum of PLC activities, and their regulation, is necessary to drive innovation in medicine by identifying novel targets., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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23. Regulation of phospholipase C-β(1) GTPase-activating protein (GAP) function and relationship to G(q) efficacy.
- Author
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Litosch I
- Subjects
- GTP-Binding Protein alpha Subunits, G12-G13 physiology, GTP-Binding Proteins metabolism, Guanine Nucleotide Exchange Factors physiology, Guanosine Triphosphate metabolism, Models, Biological, Phosphatidic Acids pharmacology, Signal Transduction, GTP-Binding Protein alpha Subunits, Gq-G11 physiology, GTPase-Activating Proteins metabolism, Phospholipase C beta metabolism
- Abstract
How cells regulate Gq efficacy (initiation and termination of Gq signaling) to effect response remains a central question in pharmacology and drug discovery. Phospholipase C-β1 (PLC-β1) is an effector and a GTPase activating protein (GAP) specific to Gαq. The physiological function of PLC-β1 GAP remains unclear and controversial. GAPs are generally thought to function in deactivation of Gq signaling. However, PLC-β1 GAP has also been shown to increase signaling efficiency through kinetic coupling with the ligand-activated GPCR. GPCRs function as guanine nucleotide exchange factors (GEF) on the G protein activation cycle. This article sets forth a new hypothesis that could unify these conflicting paradigms as it pertains to physiological signaling and native levels of protein. It is proposed that the physiological function of PLC-β1 GAP is context-dependent and regulated by phosphatidic acid (PA). PA stimulates PLC-β1 GAP activity. In the absence of ligand, PLC-β1 GAP does indeed deactivate Gq signaling, limiting leaky activation to set the threshold for stimulation to sharpen signal kinetics. However in the presence of activating ligand, the increase in levels of PA would stimulate PLC-β1 GAP to kinetically couple with GPCR GEF to increase signaling efficiency. We found that PA-increased Gq efficiency is dependent on signaling via the unique PLC-β1 PA binding domain.
- Published
- 2013
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24. Negative feedback regulation of Gq signaling by protein kinase C is disrupted by diacylglycerol kinase ζ in COS-7 cells.
- Author
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Litosch I
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Diacylglycerol Kinase genetics, Diglycerides metabolism, GTP-Binding Protein alpha Subunits, Gq-G11 genetics, Humans, Phospholipase D metabolism, Protein Kinase C antagonists & inhibitors, Protein Kinase C beta, Receptor, Muscarinic M1 genetics, Transfection, Diacylglycerol Kinase metabolism, Feedback, Physiological, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, Protein Kinase C metabolism, Receptor, Muscarinic M1 metabolism
- Abstract
Cellular response to G(q)-linked agonists is shaped by regulatory inputs which determine signal strength and duration. Stimulation of phospholipase C-β (PLC-β) lipase activity results in an increase in the levels of diacylglycerol (DAG) and activation of protein kinase C (PKC) activity. PKC has been implicated in the feedback regulation of G(q) signaling through actions on PLC-β and phospholipase D (PLD) lipase activity. As PKC activity is modulated by multiple layers of regulation, the physiological impact of PKC on G(q) signaling is unclear. PKC signaling can be terminated by diacylglycerol kinases (DGKs) which are regulated in a cell-specific manner. The present studies investigated the contribution of the ubiquitously expressed DGKζ isoform in the regulation of PKC signaling and G(q) response in transfected COS-7 cells. Genetic depletion of DGKζ protein with antisense oligonucleotides dramatically reduced DAG metabolism. The sustained increase in PKC signaling was associated with a pronounced inhibition of carbachol-stimulated lipase activity in cells co-transfected with m1 muscarinic receptor, Gα(q) and either with or without PLC-β(1). The data also reveal that sustained activation of PKC alone does not increase cellular PLD1 activity. Therefore, G(12)-activated RhoA is physiologically important for adequate stimulation of PLD1 activity. These data show that the impact of PKC on G(q) signal transduction is determined by the background of cell-specific processes., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2012
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25. RhoA co-ordinates with heterotrimeric G proteins to regulate efficacy.
- Author
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Litosch I
- Subjects
- Phosphatidic Acids biosynthesis, GTP-Binding Protein alpha Subunits metabolism, Phosphatidic Acids metabolism, Receptors, G-Protein-Coupled metabolism, rhoA GTP-Binding Protein metabolism
- Abstract
Heterotrimeric G proteins have a critical role in mediating signal transduction by ligand-stimulated GPCRs. While activation of heterotrimeric G proteins is known to proceed via the G protein guanine nucleotide cycle, there is much uncertainty regarding the process that determines efficacy, the extent of response across signaling pathways. Gα(GTP) can interact with multiple binding partners, including several effectors and regulators. Cross-talk by other receptor-signaling pathways can alter the response. It remains unclear whether G protein efficacy is regulated. This lack of clarity impairs our ability to predict and manipulate the pharmacological behavior of activated G proteins. This review will discuss emerging evidence that implicates monomeric RhoA in the process that regulates G(q) efficacy., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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26. Phosphatidic acid potentiates G(alpha)q stimulation of phospholipase C-beta1 signaling.
- Author
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Litosch I
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Enzyme Activation, Phospholipase D metabolism, Receptor, Muscarinic M1 genetics, Signal Transduction drug effects, Tetradecanoylphorbol Acetate pharmacology, Transfection, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, Phosphatidic Acids metabolism, Phospholipase C beta biosynthesis
- Abstract
Phosphatidic acid (PA) is interactive with G(alpha)q-linked agonists to stimulate GPCR signaling via phospholipase C-beta(1) (PLC-beta(1)). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-beta(1) activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-beta(1) and G(alpha)q. Activated G(alpha)q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-beta(1) activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-beta(1) implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-beta(1) to both PMA and carbachol. PA alone thus has little effect on PLC-beta(1) activity, but PA and PLD1 synergize with activated G(alpha)q to stimulate PLC-beta(1) signaling. Coordinate interaction with activated G(alpha)q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-beta signaling by PA in cells.
- Published
- 2009
- Full Text
- View/download PDF
27. Phosphatidic acid regulates signal output by G protein coupled receptors through direct interaction with phospholipase C-beta(1).
- Author
-
Litosch I, Pujari R, and Lee SJ
- Subjects
- Animals, COS Cells, Cell Line, Chlorocebus aethiops, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, Mutant Proteins metabolism, Phosphatidic Acids pharmacology, Phospholipase C beta metabolism, Receptors, G-Protein-Coupled metabolism, Signal Transduction
- Abstract
Phosphatidic acid (PA), generated downstream of monomeric Rho GTPases via phospholipase D (PLD) and additionally by diacylglycerol kinases (DGK), both stimulates phospholipase C-beta(1) (PLC-beta(1)) and potentiates stimulation of PLC-beta(1) activity by Galpha(q) in vitro. PA is a potential candidate for integrating signaling by monomeric and heterotrimeric G proteins to regulate signal output by G protein coupled receptors (GPCR), and we have sought to understand the mechanisms involved. We previously identified the region spanning residues 944-957, lying within the PLC-beta(1) C-terminus alphaA helix and flexible loop of the Galpha(q) binding domain, as required for stimulation of lipase activity by PA in vitro. Regulation by PA does not require residues essential for stimulation by Galpha(q) or GTPase activating activity. The present studies evaluated shorter alanine/glycine replacement mutants and finally point mutations to identify Tyr(952) and Ile(955) as key determinants for regulation by PA, assessed by both in vitro enzymatic and cell-based co-transfection assays. Replacement of Tyr(952) and Ile(955), PLC-beta(1) (Y952G/I955G), results in an 85% loss in stimulation by PA relative to WT-PLC-beta(1) in vitro. COS 7 cells co-transfected with PLC-beta(1) (Y952G/I955G) demonstrate a 10-fold increase in the EC(50) for stimulation and a 60% decrease in maximum stimulation by carbachol via Galpha(q) linked m1 muscarinic receptors, relative to cells co-transfected with WT-PLC-beta(1) but otherwise similar conditions. Residues required for regulation by PA are not essential for stimulation by G protein subunits. WT-PLC-beta(1) and PLC-beta(1) (Y952G/I955G) activity is increased comparably by co-transfection with Galpha(q) and neither is markedly affected by co-transfection with Gbeta(1)gamma(2). Inhibiting PLD-generated PA production by 1-butanol has little effect on maximum stimulation, but shifts the EC(50) for agonist stimulation of WT-PLC-beta(1) by 10-fold, producing a phenotype similar to PLC-beta(1) (Y952G/I955G) with respect to agonist potency. 1-Butanol is without effect on carbachol stimulated PLC activity in cells co-transfected with either PLC-beta(1)(Y952G/I955G) or on endogenous PLC activity, indicating that regulation by PA requires direct interaction with the PLC-beta(1) PA-binding region. These data show that endogenous PA regulates signal output by Galpha(q)-linked GPCRs in transfected cells directly through PLC-beta(1). Galpha(q) and PA may co-ordinate to regulate signaling. Regulation by PA may constitute part of a mechanism that routes receptor signaling to specific PLC isoforms.
- Published
- 2009
- Full Text
- View/download PDF
28. Structural determinants for phosphatidic acid regulation of phospholipase C-beta1.
- Author
-
Ross EM, Mateu D, Gomes AV, Arana C, Tran T, and Litosch I
- Subjects
- Binding Sites, Circular Dichroism, Enzyme Activation, Isoenzymes genetics, Mutation genetics, Phenotype, Phospholipase C beta, Type C Phospholipases genetics, Isoenzymes chemistry, Isoenzymes metabolism, Phosphatidic Acids chemistry, Phosphatidic Acids metabolism, Type C Phospholipases chemistry, Type C Phospholipases metabolism
- Abstract
Signaling from G protein-coupled receptors to phospholipase C-beta (PLC-beta) is regulated by coordinate interactions among multiple intracellular signaling molecules. Phosphatidic acid (PA), a signaling phospholipid, binds to and stimulates PLC-beta(1) through a mechanism that requires the PLC-beta(1) C-terminal domain. PA also modulates Galpha(q) stimulation of PLC-beta(1). These data suggest that PA may have a key role in the regulation of PLC-beta(1) signaling in cells. The present studies addressed the structural requirements and the mechanism for PA regulation of PLC-beta(1). We used a combination of enzymatic assays, PA-binding assays, and circular dichroism spectroscopy to evaluate the interaction of PA with wild-type and mutant PLC-beta(1) proteins and with fragments of the Galpha(q) binding domain. The results identify a region that includes the alphaA helix and flexible loop of the Galpha(q)-binding domain as necessary for PA regulation. A mutant PLC-beta(1) with multiple alanine/glycine replacements for residues (944)LIKEHTTKYNEIQN(957) was markedly impaired in PA regulation. The high affinity and low affinity component of PA stimulation was reduced 70% and PA binding was reduced 45% in this mutant. Relative PLC stimulation by PA increased with PLC-beta(1) concentration in a manner suggesting cooperative binding to PA. Similar concentration dependence was observed in the PLC-beta(1) mutant. These data are consistent with a model for PA regulation of PLC-beta(1) that involves cooperative interactions, probably PLC homodimerization, that require the flexible loop region, as is consistent with the dimeric structure of the Galpha(q)-binding domain. PA regulation of PLC-beta(1) requires unique residues that are not required for Galpha(q) stimulation or GTPase-activating protein activity.
- Published
- 2006
- Full Text
- View/download PDF
29. Regulation of phospholipase C-beta activity by phosphatidic acid: isoform dependence, role of protein kinase C, and G protein subunits.
- Author
-
Litosch I
- Subjects
- Animals, Cattle, Dose-Response Relationship, Drug, Enzyme Activation drug effects, GTP-Binding Protein alpha Subunits, Gq-G11, Heterotrimeric GTP-Binding Proteins metabolism, Humans, Isoenzymes chemistry, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Phospholipase C beta, Phosphorylation, Protein Binding, Protein Kinase C beta, Protein Kinase C-alpha, Type C Phospholipases chemistry, Heterotrimeric GTP-Binding Proteins chemistry, Isoenzymes metabolism, Phosphatidic Acids pharmacology, Protein Kinase C chemistry, Protein Subunits chemistry, Type C Phospholipases metabolism
- Abstract
Phosphatidic acid (PA) stimulates phospholipase C-beta(1) (PLC-beta(1)) activity and promotes G protein stimulation of PLC-beta(1) activity. The isoform dependence for PA regulation of PLC-beta activity as well as the role of PA in modulating regulation of PLC-beta activity by protein kinase C (PKC) and G protein subunits was determined. As compared to PLC-beta(1), the phospholipase C-beta(3) (PLC-beta(3)) isoform was less sensitive to PA, requiring greater than 15 mol % PA for stimulation. PLC-beta(3) bound weakly to PA. PKC had little effect on PA stimulation of PLC-beta(3) activity. PKC, however, inhibited PA stimulation of PLC-beta(1) activity through a mechanism dependent on the mol % PA. Stimulation by 7.5 mol % PA was completely inhibited by PKC. Increasing the PA and Ca(2+) concentration attenuated PKC inhibition. The binding of PLC-beta(1) to PA containing phospholipid vesicles was also reduced by PKC, in a manner dependent on the mol % PA. PA increased the stimulation of PLC-beta(1) activity by G alpha q but had little effect on the stimulation by beta gamma subunits. These results demonstrate that PA stimulation of PLC-beta activity is tightly regulated, suggesting the existence of a distinct PA binding region in PLC-beta(1). PA may be an important component of a receptor mediated signaling mechanism that determines PLC-beta(1) activation.
- Published
- 2003
- Full Text
- View/download PDF
30. Novel mechanisms for feedback regulation of phospholipase C-beta activity.
- Author
-
Litosch I
- Subjects
- Animals, Cell Membrane enzymology, Diglycerides metabolism, Enzyme Activation, Humans, Inositol 1,4,5-Trisphosphate metabolism, Isoenzymes metabolism, Models, Biological, Models, Genetic, Phosphatidylinositol 4,5-Diphosphate metabolism, Phospholipase C beta, Protein Kinase C metabolism, Protein Structure, Tertiary, Receptors, Cell Surface metabolism, Type C Phospholipases metabolism, Gene Expression Regulation, Isoenzymes physiology, Type C Phospholipases physiology
- Abstract
The receptor-regulated phospholipase C-beta (PLC-beta) signaling pathway is an important component in a network of signaling cascades that regulate cell function. PLC-beta signaling has been implicated in the regulation of cardiovascular function and neuronal plasticity. The Gq family of G proteins mediate receptor stimulation of PLC-beta activity at the plasma membrane. Mitogens stimulate the activity of a nuclear pool of PLC-beta. Stimulation of PLC-beta activity results in the rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate, with production of inositol-1,4,5-trisphosphate and diacylglycerol, intracellular mediators that increase intracellular Ca2+ levels and activate protein kinase C activity, respectively. Diacylglycerol kinase converts diacylglycerol to phosphatidic acid, a newly emerging intracellular mediator of hormone action that targets a number of signaling proteins. Activation of the Gq linked PLC-beta signaling pathway can also generate additional signaling lipids, including phosphatidylinositol-3-phosphate and phosphatidylinositol-3,4,5-trisphosphate, which regulate the activity and/or localization of a number of proteins. Novel feedback mechanisms, directed at the level of Gq and PLC-beta, have been identified. PLC-beta and regulators of G protein signaling (RGS) function as GTPase-activating proteins on Gq to control the amplitude and duration of stimulation. Protein kinases phosphorylate and regulate the activation of specific PLC-beta isoforms. Phosphatidic acid regulates PLC-beta1 activity and stimulation of PLC-beta1 activity by G proteins. These feedback mechanisms coordinate receptor signaling and cell activation. Feedback mechanisms constitute possible targets for pharmacological intervention in the treatment of disease.
- Published
- 2002
- Full Text
- View/download PDF
31. Regulation of phospholipase C-beta(1) activity by phosphatidic acid.
- Author
-
Litosch I
- Subjects
- Animals, Binding Sites, Brain enzymology, Cattle, Coated Vesicles metabolism, Enzyme Activation, GTP-Binding Proteins metabolism, Isoenzymes isolation & purification, Phospholipase C beta, Rats, Type C Phospholipases isolation & purification, Isoenzymes metabolism, Phosphatidic Acids metabolism, Type C Phospholipases metabolism
- Abstract
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.
- Published
- 2000
- Full Text
- View/download PDF
32. G-protein betagamma subunits antagonize protein kinase C-dependent phosphorylation and inhibition of phospholipase C-beta1.
- Author
-
Litosch I
- Subjects
- Animals, Cattle, Phospholipase C beta, Phosphorylation, GTP-Binding Proteins metabolism, Isoenzymes metabolism, Protein Kinase C metabolism, Type C Phospholipases metabolism
- Abstract
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.
- Published
- 1997
- Full Text
- View/download PDF
33. G-protein inhibition of phospholipase C-beta 1 in membranes: role of G-protein beta gamma subunits.
- Author
-
Litosch I
- Subjects
- Animals, Carbachol pharmacology, Cattle, Cell Membrane enzymology, Cerebral Cortex enzymology, Clonidine pharmacology, Dose-Response Relationship, Drug, Guanosine 5'-O-(3-Thiotriphosphate) administration & dosage, Guanosine 5'-O-(3-Thiotriphosphate) pharmacology, Guanosine Diphosphate analogs & derivatives, Guanosine Diphosphate pharmacology, Liver enzymology, Membranes enzymology, Phenylephrine pharmacology, Phospholipase C beta, Protein Conformation, Rats, Thionucleotides pharmacology, Vasopressins pharmacology, GTP-Binding Proteins physiology, Isoenzymes antagonists & inhibitors, Type C Phospholipases antagonists & inhibitors
- Abstract
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.
- Published
- 1996
- Full Text
- View/download PDF
34. Protein kinase C inhibits the Ca(2+)-dependent stimulation of phospholipase C-beta 1 in vitro.
- Author
-
Litosch I
- Subjects
- Adenosine Triphosphate metabolism, Animals, Brain enzymology, Cattle, Cyclic AMP-Dependent Protein Kinases pharmacology, Diglycerides, Isoenzymes antagonists & inhibitors, Phospholipase C beta, Phosphorylation, Type C Phospholipases antagonists & inhibitors, Calcium pharmacology, Enzyme Inhibitors pharmacology, Isoenzymes metabolism, Protein Kinase C pharmacology, Type C Phospholipases metabolism
- Abstract
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.
- Published
- 1996
35. Regulatory GTP-binding proteins: emerging concepts on their role in cell function.
- Author
-
Litosch I
- Subjects
- Animals, Ion Channels metabolism, Proto-Oncogene Proteins physiology, Proto-Oncogene Proteins p21(ras), Type C Phospholipases metabolism, GTP-Binding Proteins physiology
- Abstract
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.
- Published
- 1987
- Full Text
- View/download PDF
36. Sickle red cell calcium metabolism: studies on Ca2+-Mg2+ATPase and Ca-binding properties of sickle red cell membranes.
- Author
-
Litosch I and Lee KS
- Subjects
- Adolescent, Adult, Biological Transport, Active, Calcium pharmacology, Calmodulin pharmacology, Child, Child, Preschool, Humans, Magnesium pharmacology, Anemia, Sickle Cell metabolism, Calcium metabolism, Calcium-Transporting ATPases metabolism, Erythrocyte Membrane metabolism, Erythrocytes metabolism
- Abstract
Sickle (Hb SS) red cells, preloaded with 45Ca by reversal of hemolysis, exhibit an incomplete 45Ca extrusion, retaining approximately four times more 45Ca than normal cells. Studies indicated that neither the reduction in Hb SS cell Ca2+-Mg2+ ATPase activity (84% of normal) nor the activation of Ca2+-Mg2+ ATPase by calmodulin was sufficiently different from normal cells to attribute a major role to the calcium pump in 45Ca retention. These results suggested that 45Ca retention may reflect an alteration in the calcium-binding properties of Hb SS cell membranes. Low-affinity calcium-binding (freely dissociable) was similar in normal and Hb SS cell membranes. However, the total calcium bound with high-affinity (tightly bound) was four-to-five times greater in Hb SS cell membranes than in normal membranes. These results are compatible with the hypothesis that Hb SS cell 45Ca retention reflects an exchange of a fraction of the total 45Ca with a tightly bound calcium pool, larger in Hb SS cell membranes than in normal membranes. A comparable degree of red cell 45Ca retention, which did not correlate with the reticulocyte population, was observed in other chronic anemic states. These findings suggest that the increased high-affinity calcium binding by the membrane may be a consequence of cellular changes induced by the anemic condition.
- Published
- 1980
- Full Text
- View/download PDF
37. Synergistic activation of rat hepatocyte glycogen phosphorylase by A23187 and phorbol ester.
- Author
-
Fain JN, Li SY, Litosch I, and Wallace M
- Subjects
- Animals, Drug Synergism, Enzyme Activation drug effects, Female, Kinetics, Rats, Vasopressins pharmacology, Calcimycin pharmacology, Liver enzymology, Phorbols pharmacology, Phosphorylases metabolism, Tetradecanoylphorbol Acetate pharmacology
- Abstract
The combination of 1.6 microM 4 beta phorbol, 12 beta myristate, 13 alpha acetate (PMA) and 1 microM A23187 produced a five-fold greater stimulation of rat hepatocyte glycogen phosphorylase activity than was seen with PMA alone. Vasopressin activation of glycogen phosphorylase was comparable to that seen with PMA plus A23187. Glycogen phosphorylase activity due to PMA plus A23187 was increased significantly after 30 sec, maximal at 120 and sustained at elevated levels for 240 sec. In contrast, activation due to vasopressin was maximal at 30 sec followed by a decrease. The addition of PMA 5 min prior to the A23187 abolished the synergism between these two agents. These data are compatible with the hypothesis that diacylglycerol and Ca2+ synergistically increase glycogen phosphorylase activity in rat hepatocytes.
- Published
- 1984
- Full Text
- View/download PDF
38. Hormonal regulation of phosphatidylinositol breakdown.
- Author
-
Fain JN, Lin SH, Litosch I, and Wallace M
- Subjects
- Animals, Calcium metabolism, Cell Membrane metabolism, Deoxycholic Acid pharmacology, Diptera, Kinetics, Membrane Lipids metabolism, Salivary Glands drug effects, Salivary Glands metabolism, Serotonin pharmacology, Cyclic AMP metabolism, Phosphatidylinositols metabolism
- Abstract
Cyclic AMP and Ca2+ are intracellular mediators of hormone action. Catecholamines interact with beta adrenoceptors to activate adenylate cyclase or with alpha 2 adrenoceptors to inhibit adenylate cyclase. Alpha 1 adrenoceptor activation results in elevation of cytosol Ca2+ and an increased breakdown of phosphatidylinositol. In blowfly salivary glands, 5-hydroxytryptamine (5-HT) interacts with beta type receptors resulting in adenylate cyclase activation while alpha type receptors are involved in phosphatidylinositol breakdown and elevation of cytosol Ca2+. The link between Ca2+ mobilization and phosphatidylinositol breakdown remains to be established but breakdown of the receptor-regulated pool of phosphatidylinositol is not secondary to the rise in Ca2+. Direct addition of 5-HT to cell-free homogenates of blowfly salivary glands results in activation of phosphatidylinositol breakdown in the absence of Ca2+. In rat liver plasma membrane preparations, vasopressin increases phosphatidylinositol breakdown in the absence of Ca2+ or cytosol if deoxycholate is present. The data do not indicate whether hormone activation increases the availability of substrate to enzymatic hydrolysis or activates phospholipase C. However, they demonstrate that hormones directly accelerate phosphatidylinositol breakdown.
- Published
- 1983
- Full Text
- View/download PDF
39. Effect of thyroid status on alpha- and beta-catecholamine responsiveness of hamster adipocytes.
- Author
-
Garćia-Sáinz JA, Litosch I, Hoffman BB, Lefkowitz RJ, and Fain JN
- Subjects
- Animals, Cyclic AMP pharmacology, Epididymis, Hyperthyroidism etiology, Hypothyroidism chemically induced, Iodine deficiency, Male, Thyroid Gland physiology, Triiodothyronine, Adipose Tissue metabolism, Catecholamines metabolism, Cricetinae metabolism, Hyperthyroidism metabolism, Hypothyroidism metabolism, Mesocricetus metabolism
- Abstract
It has been suggested that part of the increased beta-catecholamine responsiveness in hyperthyroid animals is due to a decrease in alpha-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the alpha 2-adrenergic inhibition of adenylate cyclase or the alpha 1-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamsters. No effect of hyperthyroidism was found on the Kd for binding of [3H]dihydroergocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to beta-catecholamines was enhanced in adipocytes from hyperthyroid hamsters, as was lipolysis. However, in adipocytes from hyperthyroid hamsters the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on alpha-catecholamine action by enhances the activation of lipolysis by beta-catecholamine agonists.
- Published
- 1981
- Full Text
- View/download PDF
40. Norepinephrine stimulates the production of inositol trisphosphate and inositol tetrakisphosphate in rat aorta.
- Author
-
Pijuan V and Litosch I
- Subjects
- Animals, Chlorides pharmacology, In Vitro Techniques, Inositol metabolism, Inositol 1,4,5-Trisphosphate, Kinetics, Lithium pharmacology, Lithium Chloride, Male, Rats, Rats, Inbred Strains, Reference Values, Aorta, Thoracic metabolism, Inositol Phosphates biosynthesis, Muscle, Smooth, Vascular metabolism, Norepinephrine pharmacology, Sugar Phosphates biosynthesis
- Abstract
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.
- Published
- 1988
- Full Text
- View/download PDF
41. Regulation of phosphoinositide breakdown by guanine nucleotides.
- Author
-
Litosch I and Fain JN
- Subjects
- Adenylyl Cyclases metabolism, Animals, Calcium physiology, Cholera Toxin pharmacology, Cyclic AMP physiology, Enzyme Activation, Guanosine Triphosphate physiology, Humans, Membrane Proteins physiology, Transducin, Type C Phospholipases metabolism, Virulence Factors, Bordetella pharmacology, GTP-Binding Proteins physiology, Guanine Nucleotides physiology, Phosphatidylinositols metabolism
- Abstract
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.
- Published
- 1986
- Full Text
- View/download PDF
42. Hormonal activation of phosphoinositide breakdown in the presence of guanine nucleotides using a cell-free system from blowfly salivary glands.
- Author
-
Fain JN and Litosch I
- Subjects
- Animals, Cell Membrane metabolism, Cell-Free System, Female, Guanine Nucleotides pharmacology, Inositol metabolism, Male, Membrane Lipids metabolism, Radioisotope Dilution Technique, Salivary Glands metabolism, Diptera metabolism, Phosphatidylinositols metabolism
- Published
- 1987
- Full Text
- View/download PDF
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