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PubMed1992

Hippocampal phosphoinositide turnover is altered by hippocampal sympathetic ingrowth and cholinergic denervation.

Cholinergic denervation of the hippocampus, by medial septal (MS) lesions, results in an unusual neuronal rearrangement in which peripheral sympathetic nerves, which originate from the superior cervical ganglia, grow into the hippocampal formation. To assess the functional significance of hippocampal sympathetic ingrowth (HSI), hydrolysis of phosphoinositides was examined in three groups: control, MS lesions + sham ganglionectomy (HSI group); and MS lesions + ganglionectomy (MSGx; no ingrowth). Four months after surgery, both norepinephrine (NE) and carbachol were found to produce a dose-dependent increase in the hydrolysis of hippocampal phosphoinositides in all groups. However, the presence of HSI, when compared to control and MSGx groups, significantly enhanced the turnover of phosphoinositides when stimulated by carbachol, but not NE. In further studies, the time course of this effect was studied. One week after surgery, carbachol-stimulated phosphoinositide turnover was equivalent among all groups; by 2 weeks, phosphoinositide turnover was enhanced in the HSI and MSGx group; by 4 weeks, PI turnover was markedly diminished in the MSGx group when compared to both the HSI and control groups, which were equivalent to each other. To ensure that the ganglionectomy alone did not alter phosphoinositide turnover, a ganglionectomy-alone group was studied at the 4-week time point. In this group, phosphoinositide turnover was equivalent to controls, suggesting no influence of the superior cervical ganglia on this response. In all groups, atropine inhibited carbachol-stimulated phosphoinositide turnover. These results suggest that both cholinergic denervation (i.e., MSGx group) and HSI produce marked functional alterations in hippocampal metabolic activity.

Animals↗
PubMed1989

Regulation of oxytocin-induced phosphoinositide breakdown in adipocytes by adenosine, isoproterenol and insulin.

In rat adipocytes, the breakdown of phosphoinositides labelled by a 3 h incubation with [3H]inositol resulted in the accumulation of labelled inositol mono-, bis- and trisphosphates in the presence of oxytocin, vasotocin or vasopressin. Oxytocin at a concentration of 1 nM markedly increased phosphoinositide breakdown. Incubation of adipocytes both during the 3 h labelling and the 10 min breakdown period in a low adenosine medium (presence of adenosine deaminase) or high adenosine medium (presence of 0.1 microM N6-(phenylisopropyl)adenosine) (PIA) did not affect basal or ligand-stimulated phosphoinositide breakdown. The addition of 1 microM PIA only during the measurement of phosphoinositide breakdown variably stimulated basal breakdown but significantly potentiated that due to oxytocin. Isoproterenol similarly had little effect on basal but inhibited oxytocin stimulation of phosphoinositide breakdown. Insulin did not affect basal or ligand-stimulated phosphoinositide breakdown in the low or high adenosine medium. However, in adipocytes incubated in the absence of added adenosine deaminase or PIA, insulin stimulated basal accumulation of inositol phosphates by about 20% and inhibited that due to oxytocin by about 20%. There was no significant effect of insulin on the stimulation by vasopressin or vasotocin of phosphoinositide breakdown. These results indicate that, in adipocytes, phosphoinositide breakdown stimulated by oxytocin is enhanced by adenosine, inhibited by isoproterenol and, under some conditions is inhibited by insulin.

Adenosine↗
PubMed1994

Interaction of protein kinase C and phosphoinositides: regulation by polyamines.

Phosphatidylinositol 4,5-bisphosphate (PIP2) activates protein kinase C (PKC) in the presence of phosphatidylserine and calcium. Recently it has been demonstrated that direct interaction of PKC with PIP2 in the absence of divalent cation inactivates this kinase. In the present study, the interaction of natural aliphatic polyamines with phosphoinositides was investigated for its possible relevance to PKC-mediated protein phosphorylation. PKC/phosphoinositide interaction was studied by monitoring the changes in (a) intrinsic fluorescence of the enzyme, and (b) PKC activity (protamine sulphate or histone III-S as substrate). All the phosphoinositides: PIP2, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol (PI) inactivated PKC with an IC50 of 0.4 microM for PIP2, 5 microM for PIP and 10 microM for PI. Hydrogenated PIP2 behaved similarly to that of natural PIP2. Time-dependent studies showed very rapid inactivation of PKC by PIP2. The polyamines spermine and spermidine at physiological concentrations protected PKC from phosphoinositides-mediated inactivation when added prior to PKC interaction with phosphoinositides. Putrescine was least effective. Addition of spermine or spermidine to PKC/phosphoinositides incubation mixture did not reverse PKC activity indicating that the inactivation of PKC by phosphoinositides is irreversible. Fluorescence quenching experiments showed that phosphoinositides inactivate PKC by inducing conformational changes of the enzyme that are prevented by spermine. We propose that polyamines protect PKC and possibly other protein kinase from phosphoinositides-mediated inactivation, and that inactivation of protein kinases by phosphoinositides may not have physiological relevance.

Animals↗
PubMed1991

Effects of mastoparan and related peptides on phosphoinositide breakdown in HL-60 cells and cell-free preparations.

In differentiated HL-60 cells the amphiphilic peptide mastoparan induces a dose-dependent stimulation of phosphoinositide breakdown with an EC50 value of 9 microM. Such stimulation can be markedly reduced by pretreatment of the cells with pertussis toxin (100 ng/ml, 2 h). In membranes obtained from differentiated HL-60 cells, guanine nucleotides stimulate the formation of IP2 and IP3. Calcium ions also induce phosphoinositide breakdown in this preparation independent of the presence of guanine nucleotides. In HL-60 cell membranes, mastoparan inhibited GTP gamma S-stimulation of phosphoinositide breakdown with an IC50 value of 3 microM. Such inhibitory activity of mastoparan also was present in membranes from cells pretreated with pertussis toxin. Calcium-induced stimulation of phosphoinositide breakdown was not significantly inhibited by mastoparan. The analogs mastoparan-X and polistes mastoparan had similar inhibitory activity, whereas the analog des-Ile1-Asn2-mastoparan was inactive. In permeabilized HL-60 cells mastoparan also inhibited phosphoinositide breakdown. Another amphiphilic peptide, melittin, was inactive in HL-60 intact cells, but similar to mastoparan, inhibited guanine nucleotide-induced phosphoinositide breakdown in HL-60 cell membranes and permeabilized cells. Thus, mastoparan peptides can stimulate phosphoinositide breakdown in intact HL-60 cells, probably through the interaction with a guanine nucleotide binding protein. In permeabilized cells and in cell membranes, mastoparan induces inhibition of guanine nucleotide-mediated phosphoinositide breakdown presumably through an interaction with an intracellular site. The inhibitory action of mastoparan and melittin is probably related to the amphiphilic character of these peptides.

Cell Membrane↗
PubMed1995

Effects of kappa-opioid receptor agonists on stimulated phosphoinositide hydrolysis in rat kidney.

To determine the effects of kappa-opioid receptor agonists on phosphoinositide metabolism in rat renal cortex, tissue slices labelled with [3H]inositol were stimulated with norepinephrine or carbachol alone or in combination with the kappa-opioid receptor agonists, ethylketocyclazocine, trans-3,4-dichloro-N-methyl-N-[2-(pyrrolindinyl)-cyclohexyl)- benzeneacetamide (U50,488) and nalorphine. Both norepinephrine and carbachol stimulated phosphoinositide hydrolysis (measured in a LiCl buffer) concentration- and time-dependently. The EC50 and maximal stimulation of phosphoinositide hydrolysis for norepinephrine and carbachol were approximately 3 microM and 0.15 dpm released/dpm incorporated, respectively. Concentrations up to 1 mM of ethylketocyclazocine, U50,488 or nalorphine alone did not affect phosphoinositide hydrolysis. However, ethylketocyclazocine and U50,488 decreased 10 microM norepinephrine-stimulated phosphonositide hydrolysis concentration-dependently, each with an approximate IC50 of 30 microM. In contrast, nalorphine had no effect on norepinephrine-stimulated phosphoinositide hydrolysis. In addition, concentrations of up to 1 mM ethylketocyclazocine or U50,488 did not alter carbachol-stimulated phosphoinositide hydrolysis. The inhibitory effect of U50,488 and ethylketocyclazocine on norepinephrine-stimulated phosphoinositide hydrolysis was blocked by the selective kappa-opioid receptor antagonist, nor-binaltorphimine. These results indicate that kappa 1-opioid receptor stimulation may affect phosphoinositide metabolism in rat renal cortex by modulating the subcellular effects of renal alpha 1-adrenoceptor activation.

Animals↗
PubMed1994

Regulation of the phosphoinositide cascade by polyamines in brain.

The endogenous polyamines spermidine and spermine enhanced guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S)-stimulated phosphoinositide turnover with EC50 values of 100 +/- 30 and 50 +/- 15 microM, respectively, whereas the synthetic polyamines N,N'-bis(3-aminopropyl)-1,3-propanediamine and -ethylenediamine inhibited GTP-gamma-S-stimulated phosphoinositide turnover, with maximal inhibition at 1 mM. Kinetic analysis of GTP-gamma-S-stimulated phosphoinositide turnover in the absence and presence of spermidine showed that the Km for GTP-gamma-S was not changed (1,303 +/- 270 and 1,069 +/- 214 nM, respectively), whereas the Vmax was increased by 206% (1,566 +/- 141 and 4,792 +/- 84 cpm, respectively), indicating that spermidine and GTP-gamma-S acted at different sites. Spermidine also enhanced Ca(2+)-stimulated phosphoinositide turnover in the absence of GTP-gamma-S by decreasing the Ca2+ requirement of the phosphoinositide-specific phospholipase C. Arcaine and agmatine, polyamine antagonists at the NMDA receptor complex, did not block the effects of spermidine on GTP-gamma-S- and Ca(2+)-induced phosphoinositide turnover, suggesting that the spermidine effects are not mediated through these specific polyamine sites. Furthermore, spermidine increased the level of [3H]phosphatidylinositol 4-phosphate (EC50 = 120 +/- 10 microM), without affecting significantly the levels of [3H]-phosphatidylinositol and [3H]phosphatidylinositol 4,5-bis-phosphate. Collectively these data indicate that the enhanced phosphoinositide turnover induced by spermidine in the presence of GTP-gamma-S or Ca2+ is mediated through multiple levels of the phosphoinositide turnover cascade.

Agmatine↗
PubMed1999

Structural and biochemical evaluation of the interaction of the phosphatidylinositol 3-kinase p85alpha Src homology 2 domains with phosphoinositides and inositol polyphosphates.

Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85alpha subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85alpha SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85alpha SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85alpha SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85alpha. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85alpha SH2 domains.

3T3 Cells↗
PubMed1999

Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.

Phosphoinositides are both substrates for second messenger-generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking. Phosphatidylcholine-specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD-dependent cellular processes is not known. To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function. This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides. Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are inactive against cellular lipid substrates. Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides. The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo. Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.

Amino Acid Sequence↗
PubMed1992

The epithelial phenotype of human neuroblastoma cells express bradykinin, endothelin, and angiotensin II receptors that stimulate phosphoinositide hydrolysis.

The neuroblastoma line SK-N-SH consists of distinct and interconverting cell types, which include a neuroblast phenotype (SH-SY5Y), an epithelial phenotype (SH-EP), and an intermediate cell type (SH-IN). In SH-SY5Y cells, only muscarinic receptor activation produced stimulation of phosphoinositide turnover, whereas in SH-EP cells, where muscarinic receptors are not present, the peptides bradykinin, endothelin, and angiotensin II stimulated phosphoinositide hydrolysis with EC50 values of 16, 6, and 0.7 nM, respectively, and a rank order of maximal effects of bradykinin greater than endothelin greater than angiotensin II. Fetal calf serum at concentrations between 1 and 10% was also a potent stimulator of phosphoinositide hydrolysis in SH-EP cells but not in SH-SY5Y cells. In the intermediate cell clone, SH-IN, phosphoinositide hydrolysis was stimulated not only by muscarinic receptors, but also by endothelin, bradykinin, and serum, an indication that this cell type harbors all the kinds of receptors that are differentially expressed in the other two cell types. The effects of the three peptides--bradykinin, endothelin, and angiotensin II--on phosphoinositide hydrolysis in SH-EP cells were additive, a result suggesting that the three kinds of receptors may activate distinct transducer proteins and/or phospholipase C subtypes. Pretreatment of intact SH-EP cells with pertussis toxin under conditions sufficient to ADP-ribosylate 90-95% of the endogenous guanine nucleotide regulatory protein substrates did not impair the ability of any of the receptors to stimulate phosphoinositide hydrolysis in any of the cell types. In contrast, short-term exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (1 microM) abolished the stimulation of phosphoinositide hydrolysis mediated by peptide receptors in SH-EP cells and partially inhibited that by muscarinic receptors in SH-SY5Y cells. Prolonged incubation of SH-EP cells with phorbol ester resulted in a recovery of receptor responsiveness, the extent and rate of which were different for each receptor type. In contrast, there was no recovery of responsiveness for muscarinic receptors in SH-SY5Y cells. The pattern of phorbol ester-mediated effects depended on the cell rather than on the receptor type. In fact, muscarinic receptor responsiveness in SH-IN, the intermediate cell type, was desensitized by and recovered from treatment with phorbol esters in a manner more similar to peptide receptors in SH-EP than to muscarinic receptors in SH-SY5Y. These data suggest that the transduction mechanisms by which distinct receptor types are coupled to phosphoinositide hydrolysis in the three cell phenotypes differ in sensitivity to feedback regulation by protein kinase C.

Angiotensin II↗
PubMed1987

Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides.

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in 32Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of 32Pi into this pool is slow. Results are quite different when [3H]inositol is the precursor utilized. Incorporation of [3H]inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of [3H]phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the [3H]inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of [3H]inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing [3H]inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of [3H]inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.

Animals↗
PubMed1985

Serotonin-stimulated phosphoinositide turnover: mediation by the S2 binding site in rat cerebral cortex but not in subcortical regions.

In rat cerebral cortex, serotonin (5-HT) stimulates phosphoinositide turnover with an EC50 of 1 microM in the presence of pargyline. The EC50 is 16-fold higher in the absence of pargyline. Selective S2 antagonists inhibit 5-HT-stimulated phosphoinositide turnover. Schild analysis of the blockade by ketanserin of the 5-HT effect gives an estimated Kd of ketanserin for the phosphoinositide-linked receptor of 11.7 nM, which agrees with the Kd (3.5 nM) of [3H]ketanserin for the S2 site. Furthermore, MK-212, 5-HT and 5-fluorotryptamine stimulate phosphoinositide turnover with potencies that resemble their potencies at the S2 but not the S1 binding site. Of 11 agonists tested, the tryptamine derivatives tend to be more efficacious than the piperazine derivatives. The selective S1 agonist 8-hydroxy-2-(di-N-propylamino)tetralin is inactive at stimulating phosphoinositide turnover. No significant relationship exists between the regional distributions of 5-HT-stimulated phosphoinositide turnover and S2 binding sites. Furthermore, the S2 antagonist ketanserin is less potent and less efficacious in hippocampus and limbic forebrain than in cerebral cortex. These data suggest that 5-HT-stimulated phosphoinositide turnover is linked to the S2 binding site in rat cerebral cortex. However, 5-HT increases phosphoinositide turnover in subcortical regions by mechanisms other than stimulation of the S2 receptor.

Animals↗
PubMed1998

Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase.

Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17-35. Use of a synthetic peptide (hep I), containing amino acids 17-35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of alpha-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2-3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In 32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.

Amino Acid Sequence↗
PubMed2001

Phosphoinositide-specific phospholipase Cbeta1 expression is not linked to nerve growth factor-induced differentiation, cell survival or cell cycle control in PC12 rat pheocromocytoma cells.

Recent reports have highlighted that phosphoinositide-specific phospholipase Cbeta1 expression is linked to neuronal differentiation in different experimental models. We sought to determine whether or not this is also true for nerve growth factor (NGF)-induced neuronal differentiation of rat PC12 cells. However, we did not find differences in the expression of both the forms of phosphoinositide-specific phospholipase Cbeta1 (a and b) during sympathetic differentiation of these cells. Also, PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 were not more susceptible to the differentiating effect of NGF. Furthermore, since it is well established that phosphoinositide-specific phospholipase Cbeta1 affects cell proliferation, we investigated whether or not PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 showed differences in survival to serum deprivation and cell cycle, when compared to wild type cells. Nevertheless, we did not find any differences in these parameters between wild type cells and the overexpressing clones. Interestingly, in PC12 cells the overexpressed phosphoinositide-specific phospholipase Cbeta1 did not localize to the nucleus, but by immunofluorescence analysis, was detected in the cytoplasm. Therefore, our findings may represent another important clue to the fact that only when it is located within the nucleus phosphoinositide-specific phospholipase Cbeta1 is able to influence cell proliferation.

Animals↗
PubMed2000

Effects of pertussis toxin and galpha-protein-specific antibodies on phosphoinositide hydrolysis in rat brain membranes after cholinergic denervation and hippocampal sympathetic ingrowth.

Cholinergic denervation of the hippocampal formation, via medial septal lesions, induces peripheral noradrenergic fibers, originating from the superior cervical ganglion, to grow into the hippocampus. We have previously reported that cholinergic denervation and hippocampal sympathetic ingrowth differentially affect guanosine-5'-O-(3-thiotriphosphate)- as well as guanosine-5'-O-(3-thiotriphosphate) + carbachol-stimulated polyphosphoinositide hydrolysis, suggesting an alteration in G proteins and/or the entire receptor complex. To examine the type of G protein which may be involved in these effects, rat dorsal hippocampal membranes were preincubated with pertussis toxin in the presence of guanosine-5'-O-(3-thiotriphosphate) and guanosine-5'-O-(3-thiotriphosphate) + carbachol. Pertussis toxin reduced guanosine-5'-O-(3-thiotriphosphate) in all groups, while guanosine-5'-O-(3-thiotriphosphate) + carbachol-stimulated phosphoinositide hydrolysis was reduced in controls and animals without sympathetic ingrowth but not in animals with hippocampal sympathetic ingrowth. This suggests that pertussis toxin-sensitive G proteins may be involved in the mediation of phosphoinositide hydrolysis. To confirm this hypothesis, membranes were preincubated with antibodies to Galphao and Gq/11. The Go antibody significantly decreased guanosine-5'-O-(3-thiotriphosphate) in all groups, while guanosine-5'-O-(3-thiotriphosphate) +carbachol-stimulated phosphoinositide hydrolysis was reduced only in hippocampal sympathetic ingrowth. Impairment of guanosine-5'-O-(3-thiotriphosphate) and carbachol-stimulated phosphoinositide hydrolysis was also decreased in all groups when preincubated with Gq/11 antibody. To determine whether hippocampal sympathetic ingrowth or cholinergic denervation altered the concentration of various G proteins, immunoblotting methodology was utilized. Gq/11 concentrations were found to be equivalent among groups. The density of Go1, Go2, and Go3 isoforms was significantly increased in the cholinergic denervation, while in the hippocampal sympathetic ingrowth only group Go3 was significantly increased. When assessed as total Go protein, density was increased significantly only in the cholinergic denervation group. Overall, these results suggest that hippocampal sympathetic ingrowth and cholinergic denervation induce alterations in phosphoinositide hydrolysis through both the Gq/11 and the Go proteins and that the coupling between muscarinic receptor and G protein is the possible site which affects changes in phosphoinositide turnover. Our results also suggest that cholinergic denervation and hippocampal sympathetic ingrowth may mediate phosphoinositide hydrolysis through an effect on different isoforms of the same G protein.

Animals↗
PubMed2000

Analysis of phosphoinositides in protein trafficking.

Phosphoinositides are key regulators of vesicle-mediated protein trafficking. Their roles include recruiting vesicle coat and effector proteins to the site of budding and promoting vesicle fusion. The intracellular levels of phosphoinositides and their localization to intracellular membranes are critical to their functions. An analytical procedure was developed that optimizes the recovery of radiolabeled cellular phosphoinositides. Quantitative analyses of yeast cellular phosphoinositides indicated that this approach is useful for examining the intracellular membrane phosphoinositide compositions related to trafficking phenomena. The approach will also enable investigators to determine whole-plant phosphoinositide compositions that have been difficult to achieve in the past. These analytical advances should be generally applicable to studies of phosphoinositide dynamics related to membrane trafficking in yeast, plant, and animal cells.

Arabidopsis↗
PubMed1996

Chronic dexamethasone administration decreases noradrenaline-stimulated, but not serotonin-stimulated, phosphoinositide metabolism in the rat brain.

The present study was undertaken to investigate the effects of chronic administration of dexamethasone on the noradrenaline- and serotonin-stimulated (5-HT-stimulated) phosphoinositide metabolism in hippocampus and frontal cortex of the rat brain. For determination of phosphoinositide metabolism, slices from selected regions of the rat brain (hippocampus or frontal cortex) were loaded with myo- [3H] inositol and stimulated with the agonists (noradrenaline or 5-HT) in the presence of LiCl (7.5 mM). Administration of dexamethasone (1 mg/kg/day) every 2nd day for 14 days markedly reduced the noradrenaline-stimulated phosphoinositide metabolism in the rat hippocampus (IP1: 60% of the control value). In the rat frontal cortex, the noradrenaline-stimulated phosphoinositide metabolism was less depressed by the chronic administration of dexamethasone (IP1: 84% of the control value). However, the chronic administration of dexamethasone did not affect the 5-HT-stimulated phosphoinositide metabolism in the rat brain. The binding characteristics of alpha 1 -adrenoceptors and 5-HT2A receptors were unaffected by the chronic treatment with dexamethasone. These results indicate that chronic administration of dexamethasone induces regional and neurotransmitter-specific changes of phosphoinositide metabolism in rat brain. The results suggest that the reduction of noradrenaline-stimulated phosphoinositide metabolism is due to modification of the intracellular signal transduction system.

Animals↗
PubMed1992

Regulation of phosphoinositide hydrolysis in cultured astrocytes by sphingosine and psychosine.

The effects of sphingosine and psychosine on phosphoinositide hydrolysis in primary cultured astrocytes were determined. Exposure to sphingosine produced a dose-dependent stimulation of phosphoinositide hydrolysis requiring the presence of external Ca++ for optimal activity. The addition of 10 microM norepinephrine resulted in a stimulation additional to that with sphingosine. The alpha 1-antagonist prazosin completely inhibited norepinephrine-induced phosphoinositide hydrolysis but had no effect on that produced by sphingosine. Psychosine (108 microM), when co-incubated with sphingosine, produced complete inhibition of sphingosine-induced phosphoinositide hydrolysis at all doses of sphingosine tested (33-668 microM). Likewise, psychosine totally inhibited norepinephrine-induced phosphoinositide hydrolysis. The protein kinase C inhibitor staurosporine (1 microM) had no effect on sphingosine-induced phosphoinositide hydrolysis. These findings suggest that lysosphingolipids such as sphingosine and psychosine may play an important role in the regulation of phosphoinositide turnover in astrocytes by a mechanism dependent on extracellular Ca++ and independent of the alpha 1-adrenergic receptor and protein kinase C.

Alkaloids↗
PubMed1988

Selective inhibition of excitatory amino acid-stimulated phosphoinositide hydrolysis in the rat hippocampus by activation of protein kinase C.

The relative roles of protein kinase C in regulating excitatory amino acid-, cholinoceptor-, and adrenoceptor-stimulated phosphoinositide hydrolysis were studied. Slices of rat hippocampus were prelabeled with [3H]-myo-inositol, and agonist-induced [3H]-phosphoinositide hydrolysis was measured by the formation of [3H]-inositol monophosphate ([3H]-IP) in the presence of lithium ion. Activation of protein kinase C with phorbol 12,13-dibutyrate (PDB) (10(-6) M) completely inhibited ibotenate (IBO) (10(-3) M)-induced [3H]-phosphoinositide hydrolysis. Half-maximal inhibition was observed at about 10(-7) M PDB. Higher concentrations of PDB were required to inhibit stimulation of [3H]-IP by either carbachol (CARB) (10(-3) M) or norepinephrine (NE) (10(-4) M, and only partial inhibition could be attained. Preincubation with staurosporine (STAURO) (10(-5) M) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) (10(-4) M), inhibitors of protein kinase C, potentiated IBO- but not CARB- or NE-induced stimulation of [3H]-IP. PDB inhibition of IBO- or NE-stimulated [3H]-phosphoinositide hydrolysis was reversed by co-addition of STAURO or H-7. In the case of IBO + STAURO, this reversal was to the potentiated level observed with STAURO alone. Enhanced agonist stimulation and reversal of PDB inhibition were also produced by STAURO when [3H]-phosphoinositide hydrolysis was stimulated by either L-glutamate or quisqualate. These experiments show that direct activation of protein kinase C by PDB leads to inhibition of phosphoinositide hydrolysis mediated by excitatory amino acid receptors, cholinoceptors, or adrenoceptors. However, the enhanced agonist-stimulated phosphoinositide hydrolysis elicited by inhibitors of protein kinase C suggests that, when protein kinase C is indirectly activated, only excitatory amino acids rapidly inhibit further receptor-coupling.

Alkaloids↗