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PubMed1998

Specific binding of phosphatidylinositol 4,5-bisphosphate to calcium-dependent activator protein for secretion (CAPS), a potential phosphoinositide effector protein for regulated exocytosis.

The calcium-dependent activator protein for secretion (CAPS) is a novel neural/endocrine-specific cytosolic and peripheral membrane protein required for the Ca2+-regulated exocytosis of secretory vesicles. CAPS acts at a stage in exocytosis that follows ATP-dependent priming, which involves the essential synthesis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In the present studies, CAPS is shown to bind liposomes that contain acidic phospholipids and binding was markedly enhanced by inclusion of PtdIns(4,5)P2 but not other phosphoinositides in the absence of Ca2+. PtdIns(4,5)P2, but not other phosphoinositides including PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, altered the susceptibility of CAPS to proteolysis by trypsin and proteinase K, suggesting that phosphoinositide binding promoted a conformational change. Photoaffinity labeling studies with a photoactivatable benzoylcinnimidyl acyl chain derivative of PtdIns(4,5)P2 confirmed the phosphoinositide-binding properties of CAPS and suggested a hydrophobic aspect of the interaction. CAPS, as one of very few characterized proteins with a binding specificity for 4-, 5-phosphorylated inositides over 3-phosphorylated inositides, may function in regulated exocytosis as an effector of PtdIns(4,5)P2.

Animals↗

PubMed2000

SAC1 encodes a regulated lipid phosphoinositide phosphatase, defects in which can be suppressed by the homologous Inp52p and Inp53p phosphatases.

The yeast protein Sac1p is involved in a range of cellular functions, including inositol metabolism, actin cytoskeletal organization, endoplasmic reticulum ATP transport, phosphatidylinositol-phosphatidylcholine transfer protein function, and multiple-drug sensitivity. The activity of Sac1p and its relationship to these phenotypes are unresolved. We show here that the regulation of lipid phosphoinositides in sac1 mutants is defective, resulting in altered levels of all lipid phos- phoinositides, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. We have identified two proteins with homology to Sac1p that can suppress drug sensitivity and also restore the levels of the phosphoinositides in sac1 mutants. Overexpression of truncated forms of these suppressor genes confirmed that suppression was due to phosphoinositide phosphatase activity within these proteins. We have now demonstrated this activity for Sac1p and have characterized its specificity. The in vitro phosphatase activity and specificity of Sac1p were not altered by some mutations. Indeed, in vivo mutant Sac1p phosphatase activity also appeared unchanged under conditions in which cells were drug-resistant. However, under different growth conditions, both drug sensitivity and the phosphatase defect were manifest. It is concluded that SAC1 encodes a novel lipid phosphoinositide phosphatase in which specific mutations can cause the sac1 phenotypes by altering the in vivo regulation of the protein rather than by destroying phosphatase activity.

Amino Acid Sequence↗

PubMed2001

Phosphoinositide-specific inositol polyphosphate 5-phosphatase IV inhibits Akt/protein kinase B phosphorylation and leads to apoptotic cell death.

Phosphoinositide-specific inositol polyphosphate 5- phosphatase IV has the affinity for PI(3,4,5)P(3) (K(m) = 0.65 microM) that is approximately 10-fold greater than the other inositol polyphosphate 5-phosphatases, which use this substrate including SHIP, OCRL, and 5ptase II, suggesting that it may be important in controlling intracellular levels of this metabolite. We created cell lines stably expressing the enzyme to study its effect on cell function. We found that overexpression of 5ptase IV in 293 cells caused the rapid depletion of both PI(4,5)P(2) and PI(3,4,5)P(3) in cells with corresponding increases in the products, PI(4)P and PI(3,4)P(2), changing the balance of two phosphoinositol products of phosphoinositide 3-kinase, PI(3,4)P(2) and PI(3,4,5)P(3), in the cell. One of the targets of these phosphoinositides is the serine/threonine kinase Akt, which plays an important role in the control of apoptosis. We were able to address the relative roles of PI(3,4)P(2) and PI(3,4,5)P(3) in the activation of Akt by selective depletion of these phosphoinositides in cells stably transfected with 5ptase IV and inositol polyphosphate 4-phosphatase (4ptase I). In cells transfected with 4ptase I, the level of PI(3,4)P(2) was reduced, and PI(3,4,5)P(3) was increased. Expression of the two enzymes had the opposite effect on the phosphorylation of Akt in response to stimulation with growth factors or heat shock. Akt phosphorylation was inhibited in cells expressing 5ptase IV but increased in 4ptase I cells and correlated with the intracellular level of PI(3,4,5)P(3) and not that of PI(3,4)P(2). The inhibition of Akt phosphorylation in cells expressing 5ptase IV makes them highly susceptible to FAS-induced apoptosis, whereas overexpressing of the 4ptase I protects cells from apoptosis. Our results place 5ptase IV as a relevant biological regulator of PI3K/Akt pathway in cells.

Apoptosis↗

PubMed2002

The phox homology (PX) domain-dependent, 3-phosphoinositide-mediated association of sorting nexin-1 with an early sorting endosomal compartment is required for its ability to regulate epidermal growth factor receptor degradation.

Recent studies have shown that phox homology (PX) domains act as phosphoinositide-binding motifs. The majority of PX domains studied show binding to phosphatidylinositol 3-monophosphate (PtdIns(3)P), an association that allows the host protein to localize to membranes of the endocytic pathway. One issue, however, is whether PX domains may have alternative phosphoinositide binding specificities that could target their host protein to distinct subcellular compartments or allow their allosteric regulation by phosphoinositides other than PtdIns(3)P. It has been reported that the PX domain of sorting nexin 1 (SNX1) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) (Zhong, Q., Lazar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S. D., and Gill, G. N. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6767-6772). In the present study, we have shown that whereas SNX1 binds PtdIns(3,4,5)P(3) in protein:lipid overlay assays, in liposomes-based assays, binding is observed to PtdIns(3)P and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) but not to PtdIns(3,4,5)P(3). To address the significance of PtdIns(3,4,5)P(3) binding, we examined the subcellular localization of SNX1 under conditions in which plasma membrane PtdIns(3,4,5)P(3) levels were significantly elevated. Under these conditions, we failed to observe association of SNX1 with this membrane. However, consistent with the binding to PtdIns(3)P and PtdIns(3,5)P(2) being of more physiological significance was the observation that the association of SNX1 with an early endosomal compartment was dependent on a 3-phosphoinositide-binding PX domain and the presence of PtdIns(3)P on this compartment. Finally, we have shown that the PX domain-dependent/early endosomal association of SNX1 is important for its ability to regulate the targeting of internalized epidermal growth factor receptor for lysosomal degradation.

Animals↗

PubMed2005

The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.

14-3-3 Proteins↗

PubMed2006

The polybasic region that follows the plant homeodomain zinc finger 1 of Pf1 is necessary and sufficient for specific phosphoinositide binding.

The plant homeodomain (PHD) zinc finger is one of 14 known zinc-binding domains. PHD domains have been found in more than 400 eukaryotic proteins and are characterized by a Cys(4)-His-Cys(3) zinc-binding motif that spans 50-80 residues. The precise function of PHD domains is currently unknown; however, the PHD domains of the ING1 and ING2 tumor suppressors have been shown recently to bind phosphoinositides (PIs). We have recently identified a novel PHD-containing protein, Pf1, as a binding partner for the abundant and ubiquitous transcriptional corepressor mSin3A. Pf1 contains two PHD zinc fingers, PHD1 and PHD2, and functions to bridge mSin3A to the TLE1 corepressor. Here, we show that PHD1, but not PHD2, binds several monophosporylated PIs but most strongly to PI(3)P. Surprisingly, a polybasic region that follows the PHD1 is necessary for PI(3)P binding. Furthermore, this polybasic region binds specifically to PI(3)P when fused to maltose-binding protein, PHD2, or as an isolated peptide, demonstrating that it is sufficient for specific PI binding. By exchanging the polybasic regions between different PHD fingers we show that this region is a strong determinant of PI binding specificity. These findings establish the Pf1 polybasic region as a phosphoinositide-binding module and suggest that the PHD domains function down-stream of phosphoinositide signaling triggered by the interaction between polybasic regions and phosphoinositides.

Amino Acid Motifs↗

PubMed1995

Diet and disease alter phosphoinositide composition and metabolism in murine polycystic kidneys.

Because diet can affect the progression of polycystic kidney disease (PKD) and because renal phosphoinositide metabolism is altered in mice with PKD, the effects of diet and disease on phosphoinositide composition and metabolism were examined in kidneys of mice with PKD. The phosphatidylinositol-phosphate (PIP) to phosphatidylinositol (PI) molar ratio was higher (0.034 +/- 0.003 vs. 0.023 +/- 0.001, P < 0.01) and the PI-bisphosphate (PIP2) to PIP molar ratio was lower (0.70 +/- 0.08 vs. 1.19 +/- 0.10, P < 0.05) in kidneys of mice with PKD [DBA/2FG-pcy (pcy)] compared with normal controls (DBA/2J). When initial incorporation (reflecting synthesis) of [3H]inositol into renal phosphoinositides in mice injected with [3H]inositol was measured, the [3H]PIP to [3H]PI ratio was higher in the diseased kidneys compared with normal kidneys (0.016 +/- 0.001 vs. 0.013 +/- 0.001, P < 0.05), whereas the [3H]PIP2 to [3H]PIP ratio was not significantly different. In a study using dietary manipulations that alter the progression of PKD in pcy mice (6 vs. 25% casein and sunflower seed oil vs. fish oil in a 2 x 2 design), animals were injected intraperitoneally with [3H]inositol 5 h before killing. In these animals, the [3H]PIP2 to [3H]PIP ratio seemed to be the best indicator of disease progression. In addition, kidney weight (as altered by diet) was positively correlated (r = 0.62, P = 0.02) with the level of the [3H]PI-3-P isomer relative to total [3H]PIP in the kidney. These results demonstrate that alterations in dietary protein level and lipid composition can modulate renal phosphoinositide signal transduction in mice with PKD.

Animals↗

PubMed1987

Early effects of luteinizing hormone on mitochondrial phosphoinositides in ovarian follicles.

It is not clear if luteinizing hormone (LH) stimulates breakdown as well as synthesis of phosphoinositides in ovarian tissue. Possibly, LH stimulation results in hydrolysis of ovarian phosphoinositides in discrete subcellular compartments while increasing their synthesis at other sites. To investigate this hypothesis, we determined the effects of LH on phosphoinositide metabolism in whole homogenates and mitochondria of ovarian follicles. Medium (3-7 mm) follicles from porcine ovaries were preincubated for 2 h in phosphate (PO4)-free medium with 32PO4, and incubated without or with LH (1 microgram/ml). Phosphatidylinositol (PI) and related compounds, phosphatidic acid (PA), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2), accounted for 40% of the radiolabeled phospholipids in whole homogenates and over 60% in mitochondria from preincubated follicles. After 5 min, LH caused a significant decrease in radiolabeling of PIP2 and PIP in mitochondria, but not in whole homogenates. Luteinizing hormone increased radiolabeling of PIP2, PIP, PI and PA within 10 min in whole homogenates, and within 20 to 30 min in mitochondria. This delayed increase in radiolabeling of mitochondrial phosphoinositides after LH treatment was accompanied by decreases in PIP2, PIP and PI radiolabeling in whole homogenates. Follicles also were preincubated for 4 h with [3H]inositol, then for 15 min with 10 mM LiCl (an inhibitor of inositol phosphate hydrolysis). Inositol phosphate accumulation in 30 min was 2.7 times higher in homogenates of LH-treated follicles then in untreated follicles. Also, LH significantly decreased inositol bisphosphate, but did not change inositol trisphosphate accumulation. Accumulation of inositol phosphates in mitochondria was not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2006

A novel pathway of cell growth regulation mediated by a PLA2alpha-derived phosphoinositide metabolite.

The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A2 action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl3 rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and alpha-adrenergic receptors, respectively. The enzyme involved here is phospholipase A2 IValpha (PLA2 IValpha), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA2 IValpha leads to stimulation of PCCl3 cell growth. The involvement of a PLA2 IValpha-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA2 IValpha(1-522). Modulation of PCCl3 cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl3 cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.

Animals↗

PubMed1989

Potentiated endothelin-1-induced phosphoinositide hydrolysis in atria and mesenteric artery of DOCA-salt hypertensive rats.

The effect of endothelin-1 on the phosphoinositide pathway was studied in slices of atria and mesenteric artery from normotensive and hypertensive (DOCA-salt) rats. Endothelin-1 induced a dose-dependent increase in inositol monophosphate production in both groups, but the reactivity of the phosphoinositide pathway was enhanced in DOCA-salt hypertensive rats. Endothelin-1 was more potent than noradrenaline and activation by these two agonists combined was additive. The phosphoinositide activation induced by endothelin-1 was independent of extracellular calcium, and was not prevented by nifedipine treatment or by removing calcium from the incubation medium. Endothelin-1 is a potent direct activator of the phosphoinositide pathway in cardiovascular tissues, and this effect is potentiated in DOCA-salt hypertension.

Animals↗

PubMed1992

Angiotensin II increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells via the angiotensin II AT1 receptor.

OBJECTIVES: The aim of this study was to determine which angiotensin II receptor (AT receptor) mediates proto-oncogene expression and phosphoinositide metabolism in vascular smooth muscle cells in vitro. DESIGN: The AT receptor antagonists DuP753 (losartan), an AT1 antagonist, and PD 123319, an AT2 antagonist, were used to characterize AT receptors on cultured vascular smooth muscle cells derived from the rat mesenteric artery and to identify which receptor subtype mediates the angiotensin II-induced increase in proto-oncogene expression and phosphoinositide metabolism. METHODS: Rat mesenteric artery vascular smooth muscle cells were grown using standard cell culture methods. Proto-oncogene induction was measured using Northern blotting. Phosphoinositide breakdown was assessed by measuring [3H]-inositol phosphates released from prelabelled cells. RESULTS: Receptor-binding studies revealed that the AT1 receptor predominated on vascular smooth muscle cells. Incubation of quiescent cells with 0.1 mumol/l angiotensin II resulted in a 65% increase in total [3H]-inositol phosphates released compared with unstimulated cells and in a rapid accumulation of c-fos messenger RNA (mRNA). Pre-incubation of the cells with 10(-5) mol/l PD 123319 had no effect on either total inositol phosphates release or c-fos mRNA induction. Both responses, however, were totally abolished by pre-incubation of the cells with 10(-5) mol/l losartan or saralasin. CONCLUSIONS: Angiotensin II acts through the AT1 receptor to increase c-fos expression and phosphoinositide turnover in vascular smooth muscle cells. These mechanisms may be important in angiotensin II-induced smooth muscle hypertrophy.

Angiotensin II↗

PubMed1988

Excitatory amino acid agonist-antagonist interactions at 2-amino-4-phosphonobutyric acid-sensitive quisqualate receptors coupled to phosphoinositide hydrolysis in slices of rat hippocampus.

Studies were carried out to define the relative affinities and intrinsic activities of excitatory amino acid agonists that activate receptor sites coupled to phosphoinositide hydrolysis in brain. Slices of rat hippocampus were prelabeled with myo-[3H]inositol, and agonist stimulation was indexed by measuring the accumulation of [3H]inositol monophosphate [( 3H]IP) in the presence of Li+. It was observed that ibotenic (IBO) and quisqualic (QUIS) acids both elicit highly significant, concentration-dependent stimulation of phosphoinositide hydrolysis. Whereas maximal stimulation by IBO (10(-3) M) was four- to fivefold over basal values, the maximal effect of QUIS (10(-4) M) was less (about twofold). Based on the relative concentrations required for 50% maximal stimulation, QUIS was 20 times more potent than IBO. Stimulation of phosphoinositide hydrolysis by either IBO or QUIS was additive to the effects of nonexcitatory amino acid agonists (carbachol and norepinephrine) in this tissue. However, the stimulatory effects of IBO plus QUIS were not additive. At greater than or equal to 10(-4) M, QUIS significantly inhibited phosphoinositide hydrolysis by a maximal stimulatory concentration of IBO (10(-3) M) to a level observed with QUIS alone. Other excitatory amino acid agonists, including kainate, N-methyl-D-aspartate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), had no stimulatory effects at concentrations as high as 10(-3) M. The D,L or L forms of 2-amino-4-phosphonobutyric acid (AP4), but not D-AP4, significantly enhanced [3H]IP levels to approximately 135% of basal values.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1989

In vivo determination of 5-hydroxytryptamine receptor-stimulated phosphoinositide turnover in rat brain.

Phosphoinositide turnover stimulated by 5-hydroxytryptamine (5-HT) receptors in the intact rat brain was studied using an in vivo method. Phosphoinositides in the rat brain were prelabeled with [3H]inositol injected into the lateral cerebral ventricles. The rats were killed by microwave irradiation after 48 h and the contents in the frontal cortex of 3H-inositol phosphates, [3H]inositol-1-monophosphate [( 3H]IP1), [3H]inositol-1,4-bisphosphate [( 3H]IP2), and a mixture of [3H]inositol-1,4,5-trisphosphate and [3H]inositol-1,3,4-trisphosphate [( 3H]IP3) were assayed by HPLC. Lithium treatment (10 mEq/kg, i.p., 2 h before) increased the content of [3H]IP1 and [3H]IP2. 5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) and quipazine, 5-HT agonists, significantly increased the amount of 3H-inositol phosphates under lithium pretreatment. The response to 5-MeODMT was inhibited by ritanserin, a 5-HT2 antagonist, but not by (-)-propranolol, a 5-HT1 antagonist. These results suggest that phosphoinositide turnover in the rat frontal cortex in vivo is stimulated by 5-HT2 receptor activation. It is considered that this method will be useful for measurement of 5-HT2 receptor-stimulated phosphoinositide turnover in vivo to examine the in vivo effects of various psychotropic drugs such as antidepressants.

Animals↗

PubMed1989

Inhibition of excitatory amino acid-stimulated phosphoinositide hydrolysis in the neonatal rat hippocampus by 2-amino-3-phosphonopropionate.

The effects of excitatory amino acid agonists and alpha-amino-omega-phosphonocarboxylic acid antagonists on phosphoinositide hydrolysis in hippocampal slices of the 7-day neonatal rat were examined. Significant stimulation of [3H]inositol monophosphate formation was observed with ibotenate, quisqualate, L-glutamate, L-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, L-homocysteate, and kainate. N-Methyl-D-aspartate had no effect. Of these agonists, ibotenate and quisqualate were the most potent and efficacious. Stimulations by ibotenate and quisqualate were partially inhibited by L-2-amino-4-phosphonobutyrate (10(-3) M), but this antagonist had no effect on L-glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate. At 10(-3) M, D,L-2-amino-3-phosphonopropionate completely inhibited ibotenate and quisqualate stimulations, partially inhibited L-glutamate stimulation, and had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-, kainate-, or carbachol-induced [3H]inositol monophosphate formation. Concentration-effect experiments showed D,L-2-amino-3-phosphonopropionate to be five times more potent as an antagonist of ibotenate-stimulated phosphoinositide hydrolysis than L-2-amino-4-phosphonobutyrate. Thus in the neonatal rat hippocampus, like in the adult rat brain, D,L-2-amino-3-phosphonopropionate is a selective and relatively potent inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis. Because this glutamate receptor is uniquely sensitive to D,L-2-amino-3-phosphonopropionate, these studies provide further pharmacological evidence for the existence of a novel excitatory amino acid receptor subtype that is coupled to phosphoinositide hydrolysis in brain.

Alanine↗

PubMed1993

Lithium modulation of phosphoinositide signaling system in rat cortex: selective effect on phorbol ester binding.

Recent work indicates that the therapeutic action of lithium may be mediated through perturbation of postreceptor second messenger systems. To elucidate further the postreceptor cellular sites of action(s) of lithium, the effect of chronic lithium treatment on various components of the receptor-activated phosphoinositide pathway was investigated. We found that chronic administration of lithium (0.2% LiCl, 21 days) to adult male rats did not significantly affect phosphoinositide hydrolysis in cerebral cortical slices induced by carbachol (1 mM) or NaF (10 mM). Nor did the same treatment alter the carbachol (1 mM) potentiation of guanosine 5'-(gamma-thio)triphosphate (30 microM) stimulation of phosphoinositide hydrolysis (an index of receptor/G protein coupling) in cortical membranes. Immunoblotting studies revealed no changes in the levels of G alpha q/11 immunoreactivity in the cortex after chronic lithium treatment. The levels of protein kinase C, as revealed by specific binding of [3H]phorbol dibutyrate ([3H]PDBu), were significantly reduced in the cytosolic fraction and increased in the particulate fraction of rat cortex after chronic lithium, whereas the KD of [3H]PDBu binding remained relatively constant. A small and insignificant decrease in the density of [3H]inositol 1,4,5-trisphosphate binding was also found in the cortex. The above data suggest that chronic lithium treatment affects neither the muscarinic cholinergic-linked phosphoinositide turnover nor the putative G protein alpha subunit (G alpha q/11) responsible for phospholipase C activation. However, a possible translocation and activation of protein kinase C activity may be significant in the therapeutic effect of this mood-stabilizing agent.

Animals↗

PubMed1992

Bradykinin stimulation of phosphoinositide hydrolysis in guinea-pig ileum longitudinal muscle.

1. Bradykinin (BK)-induced contraction of ileal smooth muscle is assumed to be due to phosphoinositide hydrolysis but this has never been reported. We have investigated whether BK receptors are linked to this transduction mechanism in guinea-pig ileum longitudinal muscle and determined whether these receptors are equivalent to those labelled in [3H]-BK binding assays. 2. In membranes prepared from longitudinal muscle, [3H]-BK bound to a single class of sites with high affinity. Characterization of the binding with BK analogues indicated that the radioligand selectivity labelled a B2 type receptor. 3. BK significantly elevated tissue levels of [3H]-inositol phosphates in longitudinal muscle slices preincubated with [3H]-myo-inositol. The agonists potencies of BK, Lys-BK, Met-Lys-BK, Tyr5-BK and Tyr8-BK were in agreement with their relative potencies in the binding assay. The B1 receptor agonist des-Arg9-BK, did not stimulate inositol phosphate production. The response to BK was blocked by known B2 receptor antagonists but not by the B1 antagonist des-Arg9, Leu8-BK. 4. BK-induced phosphoinositide hydrolysis was unaffected by exposure of muscle slices to either atropine or indomethacin. 5. The results indicate that the B2 receptors linked to phosphoinositide turnover in ileal longitudinal muscle exhibit properties similar to those involved in contractile responses. Also, the receptor mediating the phosphoinositide response is likely to be that labelled in the [3H]-BK binding studies.

Animals↗

PubMed1994

Modulatory effects of NMDA on phosphoinositide responses evoked by the metabotropic glutamate receptor agonist 1S,3R-ACPD in neonatal rat cerebral cortex.

1. The effect of NMDA-receptor stimulation on phosphoinositide signalling in response to the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) has been examined in neonatal rat cerebral cortex slices. 2. Total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 5 mM LiCl, in [3H]-inositol pre-labelled slices was concentration-dependently increased by 1S,3R-ACPD (EC50 16.6 microM) and, at a maximally effective concentration, 1S,3R-ACPD (300 microM) increased [3H]-InsPx accumulation by 12.8 fold over basal values. 3. [3H]-InsPx accumulation stimulated by 1S,1R-ACPD was enhanced by low concentrations of NMDA (3-30 microM), but not by higher concentrations (> 30 microM). [3H]-InsPx accumulations stimulated by 1S,3R-ACPD in the absence or presence of 10 microM NMDA were linear with time, at least over the 15 min period examined; however, in the presence of 100 microM NMDA the initial enhancement of 1S,3R-ACPD-stimulated phosphoinositide hydrolysis progressively decreased with time. 4. In the presence of a maximal enhancing concentration of NMDA (10 microM), the response to 1S,3R-ACPD (300 microM) was increased 1.9 fold and the EC50 for agonist-stimulated [3H]-InsPx accumulation decreased about 4 fold. The enhanced response to the metabotropic agonist was concentration-dependently inhibited by competitive and uncompetitive antagonists of NMDA-receptor activation. 5. 1S,3R-ACPD also stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation with an initial peak response (5-6 fold over basal) at 15 s decaying to a smaller (2 fold), but persistent elevated accumulation (1-10 min). 6. Co-addition of 10 or 100 MicroM NMDA enhanced the initial peak Ins(1,4,5)P3 response to 1S,3RACPD.However, the enhancing effect was only maintained over 10 min in the presence of 1O Micro MNMDA, whilst in contrast, 100 MicroM NMDA ceased to cause a significant enhancement of the metabotropic response by 5 min and completely suppressed lS,3R-ACPD-stimulated Ins(1,4,5)P3 accumulation at 10 min.7. Both basal and 1S,3R-ACPD-stimulated Ins(1,4,5)P3 accumulations were reduced when slices were incubated in nominally Ca2"-free medium. Under these conditions only a concentration-dependent enhancement of the response was observed (EC50 for NMDA facilitation of lS,3R-ACPD-stimulated Ins(1,4,5)P3 accumulation of 32 MicroM).8. These experiments have revealed that at low concentrations, NMDA can dramatically potentiate1S,3R-ACPD-stimulated phosphoinositide hydrolysis, probably by a Ca2"-dependent facilitation of agonist-stimulated phosphoinositide-specific phospholipase C activity. Higher concentrations of NMDA result in time-dependent inhibition of the metabotropic agonist-stimulated response. We believe the former effect could be fundamental in glutamate receptor 'cross-talk', whereas the latter may reflect a Ca2+-dependent neurotoxic effect of NMDA on the neonatal cerebral cortex slices.

2-Amino-5-phosphonovalerate↗

PubMed1996

Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor-stimulated phosphoinositide turnover.

1. The abilities of the four diastereoisomers of 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) to stimulate, and the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methylcarboxyphenylglycine (MCPG) to inhibit, phosphoinositide turnover in neonatal rat cerebral cortex have been studied. Two indices of phosphoinositide cycle activity were assessed; inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation, and total inositol phosphate [3H]-InsPx accumulation (in the presence of Li+) in myo-[3H]-inositol prelabelled slices. 2. The diastereoisomers of ACPD stimulated each response with a rank order of potency of 1S, 3R > 1R, 3R > 1S, 3S >> 1R, 3S. The response to 1R, 3R-ACPD was largely prevented by pre-addition of the NMDA-receptor antagonist, MK-801, or omission of extracellular Ca2+, suggesting that this isomer acts indirectly on phosphoinositide responses through activation of NMDA-type ionotropic glutamate receptors. In contrast, the responses to 1S, 3R- and 1S, 3S-ACPD were unaffected by prior addition of MK-801, but were blocked by MCPG. 3. The concentration of 1S, 3R-ACPD required to half-maximally stimulate the Ins(1,4,5)P3 response (-log EC50 (M), -4.09 +/- 0.10) was significantly higher than that required to exert a similar effect on [3H]-InsPx accumulation (-log EC50 (M), -4.87 +/- 0.07; P < 0.01; n = 4). A similar marked 8-9 fold discrepancy between these two values was observed for the 1S, 3S isomer, which elicited similar maximal responses to those caused by 1S, 3R-ACPD. 4. Significant differences were also observed with respect to the ability of (+/-)-MCPG (1 mM) to cause a rightward shift in the concentration-response relationships for 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 (5.59 +/- 0.24 fold shift) and [3H]-InsPx (3.04 +/- 0.34 fold shift; P < 0.01; n = 4) responses, giving rise to Kd values of 218 and 490 microM for (+/-)-MCPG antagonism of the respective responses. 5. The potency difference between the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 and [3H]-InsPx responses was reduced when experiments were performed in nominally calcium-free medium ([Ca2+]e = 2 - 5 microM) and EC50 values were almost identical when extracellular calcium was reduced further by EGTA addition ([Ca2+]e < or = 100 nM). Similarly, the Kd value for (+/-)-MCPG antagonism of the 1S, 3R-ACPD-stimulated [3H]-InsPx response decreased under [Ca2+]e-free conditions, approaching those obtained for the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 response in the presence of normal [Ca2+]e. 6. These data suggest that estimates of the activities of mGluR agonists and antagonists, derived by measuring phosphoinositide turnover, can differ significantly depending on whether Ins(1,4,5)P3 mass or [3H]-InsPx responses are measured. In particular, the possibility that the mGluR-mediated [3H]-InsPx response may not simply reflect direct receptor/G protein/phosphoinositidase C (PIC) activation, but may also be the consequence of stimulation of a facilitatory Ca2+-influx pathway is discussed.

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