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PubMed1983

Rapid changes in hepatocyte phosphoinositides induced by vasopressin.

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.

Animals↗
PubMed1984

Subcellular site and mechanism of vasopressin-stimulated hydrolysis of phosphoinositides in rat hepatocytes.

The intracellular site of vasopressin-induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 s of vasopressin treatment of hepatocytes prelabeled with 32Pi, the levels of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4,5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%, then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P] PI-P and [32P]PI-P2 were observed in the other subcellular fractions, and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol in the plasma membrane decreased by 15% after 45 s of vasopressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes, it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphosphoinositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (200 nM) of free calcium when assayed at low ionic strength, but the calcium requirement shifted to micromolar concentrations under isosmotic, intracellular-like, ionic conditions. Addition of vasopressin (200 nM) to the 32P-labeled plasma membranes stimulated the breakdown of 20% of the [32P]PI-P2 present in the plasma membranes in 1 min when assayed under isosmotic conditions in the presence of 2 nM MgCl2 and approximately 200 nM free calcium. This suggests that the phosphoinositide-specific phosphodiesterase is not active under normal cellular conditions, but is activated upon the addition of vasopressin to the intact cell.

Animals↗
PubMed1995

Inhibition of 5'-chloro-5'-deoxyadenosine on phosphoinositide and protein phosphorylation in swine platelets.

AIM: To study the effects of adenosine on the phosphorylation of phosphoinositides and proteins in platelets. METHODS: In the presence of Mg2+ and/or Ca2+, swine thrombocytic membranes were incubated with [gamma-32P]ATP at 30 degrees C for 3 min and the incorporations of 32P into phospholipids or proteins were measured. RESULTS: 5'-Chloro-5'-deoxyadenosine decreased the formation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate [IC50 71 and 75 (95% confidence limits 60-85 and 62-90) mumol.L-1, respectively], behaving as a competitive inhibitor to ATP, and inhibited the phosphorylation of pleckstrin (the major protein kinase C substrate) and myosin light chain [IC50 75 and 82 (95% confidence limits 62-90 and 66-102) mumol.L-1, respectively]. CONCLUSION: Adenosine affects the phosphoinositide signaling pathway in platelets, which helps to clarify the inhibition of adenosine on platelet activation.

Animals↗
PubMed1993

Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit.

Tyrosine-phosphorylated peptides based on the regions of polyoma virus middle t antigen and the platelet-derived growth factor receptor that bind phosphoinositide 3-kinase are shown to activate this enzyme 2-3-fold in vitro. The concentrations of the peptides required to activate the enzyme are at least 10-1000-fold higher than the dissociation constants of these peptides for the individual SH2 domains of the 85-kDa subunit (KD < 100 nM). Doubly phosphorylated peptides are more effective than singly phosphorylated peptides. The results suggest that a fraction of the cellular phosphoinositide 3-kinase has SH2 domains with relatively low affinity for phosphopeptides and that binding of phosphopeptides to these enzymes causes activation. Thus, SH2 domains may be involved not only in recruiting the enzyme but also in regulating activity.

Amino Acid Sequence↗
PubMed1993

Stem cell factor-induced signal transduction in rat mast cells. Activation of phospholipase D but not phosphoinositide-specific phospholipase C in c-kit receptor stimulation.

Stem cell factor (SCF), a hematopoietic growth factor for primitive hematopoietic stem cells, is also known as mast cell growth factor. SCF induced serotonin release from rat peritoneal mast cells, connective tissue-type mast cells. The treatment of rat peritoneal mast cells with SCF failed to produce inositol 1,4,5-trisphosphate, indicating the absence of involvement of phosphoinositide-specific phospholipase C pathway. 1,2-Diacylglycerol (1,2-DG) and phosphatidic acid, however, were increased after stimulation by SCF. Phosphatidylethanol formation catalyzed by phospholipase D (PLD) was observed, together with the release of choline but not phosphocholine. Propranolol, an inhibitor of phosphatidate phosphohydrolase, blocked the production of 1,2-DG. These results indicate that the phosphatidylcholine-specific PLD pathway is the main pathway for the production of 1,2-DG in SCF-stimulated rat peritoneal mast cells. Furthermore, treatment of cells with protein tyrosine kinase inhibitor, genistein, inhibited 1,2-DG formation and serotonin release dose-dependently. Taken together, SCF induces the activation of PLD through the protein tyrosine kinase pathway without activation of phosphoinositide-specific phospholipase C.

Animals↗
PubMed1994

Phosphoinositide-derived second messengers and the regulation of Ca2+ in vascular smooth muscle.

UNLABELLED: VASCULAR SMOOTH MUSCLE AND THE PHOSPHOINOSITIDE SIGNALLING PATHWAY: Vascular smooth muscle tone can be regulated by an array of agonists which act via receptor-mediated transmembrane signalling pathways to modify the concentrations of key intracellular second messengers. Following agonist stimulation the phosphoinositide signalling pathway initiates the contraction process in vascular smooth muscle, via the second messengers myo-inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol. DIVERSITY OF CALCIUM-REGULATORY MECHANISMS: The vascular smooth muscle cell apparently sustains the contraction with a number of diverse mechanisms, which act to increase intracellular Ca2+ by regulating both Ca2+ influx across the plasma membrane and Ca2+ release from intracellular calcium stores, or may act in the apparent absence of elevated cytosolic Ca2+ concentrations. FUTURE RESEARCH: The exact nature of these physiological interactions and their exact function are not yet fully understood. In particular, identification of the natural role of specific phospholipase C delta, phospholipase C gamma and protein kinase C isozymes and also the various ryanodine and myo-inositol 1,4,5-trisphosphate receptor subtypes present in vascular smooth muscle will prove critical to future understanding of the regulation of vascular smooth muscle tone in both the normal and the hypertensive phenotype.

Animals↗
PubMed1994

Endothelin receptors: binding and phosphoinositide breakdown in human myometrium.

Endothelin (ET) binding sites and phosphoinositide hydrolysis induced by ET peptides were studied in the nonpregnant human myometrium. Saturation binding experiments revealed that the proportion of [125I]ET-1 binding sites was 4-fold higher than that of [125I]ET-3 binding sites, whereas Kd values were not significantly different. In competition binding studies, unlabeled peptides displaced [125I]ET-1 binding with the following order of affinity ET-1 > sarafotoxin 6b > ET-3 >> sarafotoxin 6c, whereas very similar Ki values were obtained with the four peptides for the displacement of [125I]ET-3 binding. Approximately 75% of [125I]ET-1 binding sites exhibited high affinity to BQ 123 ([cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu)]), an ETA selective antagonist. ET-1 elicited a time-dependent accumulation of [3H]inositol phosphates in myometrial explants prelabeled with myo-[3H]inositol. All the peptides examined, ET-1, ET-3, sarafotoxin 6b and sarafotoxin 6c were able to induce phosphoinositide hydrolysis in a dose-dependent manner, ET-1 being more potent than ET-3 with corresponding EC50 values of 32 +/- 12 and 441 +/- 37 nM, respectively. Sarafotoxin 6c induced a moderate stimulation of inositol phosphates accumulation. ET-1- and ET-3-induced accumulation of [3H]inositol phosphates was (40-45%) inhibited in part by 100 microM BQ 123, whereas sarafotoxin 6c response was not affected by the ETA-antagonist. All these results indicate the presence of ETA and ETB receptors coupled to phospholipase C in human myometrium. Although ET-1 and ET-3 bind to both subtypes, sarafotoxin 6c only interacts with the ETB subtype.

Adult↗
PubMed1994

Effects of 3,4-diaminopyridine on myoplasmic calcium and phosphoinositide hydrolysis in frog sartorius muscle fibers.

Using Ca(2+)-selective microelectrodes, the effects of 3,4-diaminopyridine (DAP) on myoplasmic calcium ([Ca2+]i) were studied in frog satorius muscle fibers. DAP induced an increase of [Ca2+]i in a concentration-dependent manner. DAP 1 mmol.L-1 increased the [Ca2+]i from control 166 +/- 41 nmol.L-1 to 416 +/- 69 nmol.L-1 (n = 10). In the absence of external calcium, DAP still enhanced [Ca2+]i. [Ca2+]i of the fibers bathed in Ca(2+)-free Ringer's solution containing DAP 1 mmol.L-1 was 152 +/- 43 nmol.L-1, which was significantly higher than 77 +/- 35 nmol.L-1 of [Ca2+]i of the fibers in Ca(2+)-free Ringer's solution. In addition, DAP promoted the hydrolysis of phosphoinositides, and DAP-induced hydrolysis was more in the presence of external calcium. It is suggested that, through enhancing the hydrolysis of phosphoinositides, DAP released Ca2+ from intracellular Ca2+ store in frog sartorius muscle fibers.

4-Aminopyridine↗
PubMed1994

Signal transduction mechanisms in developmental neurotoxicity: the phosphoinositide pathway.

The importance of activation of second messenger systems which follows the interaction of neurotransmitters, hormones and growth factors with their receptors is increasingly recognized. The phosphoinositide/protein kinase C pathway has received particular attention in the past decade, particularly for its involvement in the control of intracellular calcium levels. Though new functions of this metabolic pathway in the nervous system are continuously discovered, little is still known about the possible role of phosphoinositide metabolism in mediating various aspects of neurotoxicity. Limited information also exists on various aspects of inositol metabolism during brain development and, as a consequence, on its possible role in developmental neurotoxicity. In this brief review, some of the current concepts of receptor--activated phosphoinositide metabolism, particularly during brain development, are summarized. Two examples are presented to indicate its possible involvement in developmental neurotoxicity. The acetylcholine muscarinic receptor--stimulated inositol metabolism is discussed as a potential target for the developmental neurotoxicity of ethanol. The possible contribution of the glutamate metabotropic receptors in the neurotoxicity of excitatory amino acid in the immature brain is also discussed.

Animals↗
PubMed1993

[Effects of Salvia miltiorrhizae compositae on phosphoinositides metabolism in acute myocardial ischemia].

The effects of Salvia miltiorrhizae compositae (SMC) on phosphoinositides metabolism of acute myocardial ischemia in rats were studied. The results showed that the function of the phosphoinositide signaling system was enhanced 10 min. after acute myocardial ischemia. Both of the accumulations of phosphatidylinositol-4,5-biphosphate (PIP2) and inositol-1,4,5-triphosphate (IP3) were higher than those of the non-ischemic control group (P < 0.01, n = 7). The rise of the levels of PIP2 and IP3 caused by acute myocardial ischemia was remarkably inhibited by the SMC (2g/kg body wt.), and the levels of PIP2 and IP3 were lower than those of the ischemic group (P < 0.01, n = 7).

Animals↗
PubMed1996

Endothelin-stimulated phosphoinositide turnover and protein kinase C translocation in rat synaptosomes.

The action of endothelin-1 (ET-1) on phosphoinositide metabolism was studied in rat synaptosomes. ET-1 caused an early and transitory decrease of 32P incorporation into phosphoinositides, concomitantly with an increase into phosphatidic acid (PA). This effect was time-dependent and was not found in the absence of exogenous calcium. Furthermore, ET-1 caused an increase in the generation of inositol phosphates and diacylglycerol (DAG). In addition, the peptide provoked a translocation of protein kinase C from the cytosol to membrane.

Animals↗
PubMedDate not supplied

[Phosphorylation of proteins and phosphoinositides in the axoneme of the bovine rod outer segments. The effect of structural factor].

The protein composition of microtubular structures of bovine retinal rod axoneme and phosphorylation of its proteins were studied. The axoneme was shown to consist of proteins of 25, 43, 51, 112, 145 and 240 kDa molecular mass. It was demonstrated that 32P is mainly incorporated into the 25, 43 and 51 kDa proteins. cGMP was found to enhance 32P. Incorporation into these proteins. In the presence of Ca(2+)-calmodulin the phosphorylation of the 43 and 51 kDa proteins was increased. The phosphoinositides were shown to be phosphorylated in axoneme. Phosphatidylinositol 4-monophosphate was found to be a principal substrate for phosphatidylinositol kinases to form phosphatidylinositol 4,5-bisphosphate. Ca2+, neomycin and colchicine inhibited 32P incorporation into phosphatidylinositol 4,5-bisphosphate indicating the dependence of phosphatidylinositol 4-monophosphate phosphorylation on structural state of axoneme. Phosphatidylinositol was found to diminish the axoneme ability for the polymerization of microtubules. Data was showed that kinases responsible for protein and phosphoinositides phosphorylation were tightly bound to axoneme structures.

Animals↗
PubMedDate not supplied

[The role of adenylate cyclase and phosphoinositide second messenger systems in regulation of acetylcholine secretion in synapses of the cerebral hemispheres and brain stem in rats].

Changes in acetylcholine secretion in the cholinergic synapses of the rat cerebral hemispheres and brain stem were studied in in vivo experiments against the background of modulation of the activity of the adenylate cyclase and phosphoinositide secondary messenger systems by injection of caffeine and lithium chloride. The level of mediator secretion was determined according to the content of bound acetylcholine fractions in the homogenate of the indicated parts of the animal's brain. It was established that secretion of the mediator in the rat hemispheres is regulated by postsynaptic N-cholinoceptors located on the body of cholinergic neurons which have a phosphoinositide system actiung as secondary mediators and, possibly are related to subtype M1. It is also possible that the secretion is also regulated by presynaptic autoreceptors connected with the adenylate cyclase system, which function according to the mechanism of negative feedback and is related to subtype M2. Acetylcholine secretion in the brain stem synapses is regulated according to the negative feedback mechanism by muscarine receptors linked with the adenylate cyclase system and probably related to subtype M4.

Acetylcholine↗
PubMed1998

Pharmacological characterization of an FP prostaglandin receptor on rat vascular smooth muscle cells (A7r5) coupled to phosphoinositide turnover and intracellular calcium mobilization.

An FP prostaglandin (PG) receptor on the A7r5 rat aorta smooth muscle cell line has been characterized by assays of phosphoinositide (PI) turnover and intracellular calcium mobilization stimulated by structurally diverse PGs. In the PI turnover assay, cloprostenol was the most potent PG tested, with a potency (EC50) of 0.84 +/- 0.06 nM (mean +/- S.E.M., n = 34), and was a full agonist. Other known FP receptor agonists tested in this assay had efficacies > or = 85% of the cloprostenol value and high potencies: 16-phenoxy PGF2 alpha (2.05 +/- 0.19 nM), 17-phenyl PGF2 alpha (2.80 +/- 0.59 nM), fluprostenol (4.45 +/- 0.19 nM), PGF2 alpha (30.9 +/- 2.82 nM) and PhXA85 (43.5 +/- 11.4 nM). Other classes of PGs evaluated (PGD2, enprostil, 17-phenyl PGE2, PGE2, sulprostone and U-46619) were less potent and less efficacious than the FP receptor agonists, or were inactive. For a large group of standard PGs evaluated in the PI turnover assay, both potencies and efficacies correlated well with those reported for the FP receptor of Swiss mouse 3T3 fibroblasts. The potencies of fluprostenol and PGF2 alpha as stimuli of intracellular calcium mobilization matched well their potencies in the PI turnover assay, but fluprostenol had twice the efficacy of PGF2 alpha. Both signaling responses stimulated by fluprostenol were significantly inhibited by U73122, a selective inhibitor of phosphoinositide turnover (IC50 = 1.25 +/- 0.16 microM for PI turnover), and by chelation of calcium in the medium. Together with the PI turnover data, these studies of intracellular calcium mobilization linked to activation of the FP receptor, provide additional characterization of the pharmacological properties of this receptor.

Animals↗
PubMed1998

[Participation of phosphoinositide-specific phospholipase Cgamma1 and STAT1 protein in the formation of latent complexes of signal proteins].

It is known that the growth factor activates appropriate membrane receptors which become starting points of cascades of protein-protein interactions leading to cellular response. Recent data suggest that different signalling pathways may cross-talk during the cellular response. Here we show that phosphoinositide-specific phospholipase C gamma 1, one of the key elements in phosphoinositide pathway of signal transduction, is physically associated with members of the STAT pathway. The precipitation of phospholipase C gamma 1, using polyclonal antibody in A-431 cells, leads to co-immunoprecipitation of STAT1 alpha and STAT1 beta, as well as STAT3. The formation of such complexes was observed in both unstimulated and EGF stimulated cells. The participation of SH3-domains in the formation of such complexes is discussed.

Cell Line, Transformed↗
PubMed1998

[The content of phosphoinositides in the biological membranes of different types of muscle fibers].

The content of phosphoinositids in biomembranes of slow (m.soleus) and fast (m. extensor digitorum longus) twitch muscular fibres (MF) was studied. Biochemical differences in different MF of fast and slow muscles were detected. The content of phosphotidylinosites in plasma membranes, mitochondrial and sarcoplasmic reticulum membranes of fast twitch MF was on average 1.28 times higher than in slow ones. The predominance of phosphatidylinositol-3,4,5-triphosphates in fast twitch MF over slow twitch MF was noted. The content of phosphatidylinoitol-3,4,5-triphosphates in plasma membranes, mitochondrial and sarcoplasmic reticulum membranes of slow twitch MF was 3, 2.35 and 1.25 times higher than in fast twitch MF. It was found that phosphoinositide content in biomembranes of different type MF was unequal which may be used to improve the expansion of understanding of the role of intracellular mediators in MF phenotype regulation.

Animals↗
PubMed2003

Nuclear translocation of 3'-phosphoinositide-dependent protein kinase 1 (PDK-1): a potential regulatory mechanism for PDK-1 function.

3'-Phosphoinositide-dependent protein kinase 1 (PDK-1) phosphorylates and activates members of the AGC protein kinase family and plays an important role in the regulation of cell survival, differentiation, and proliferation. However, how PDK-1 is regulated in cells remains elusive. In this study, we demonstrated that PDK-1 can shuttle between the cytoplasm and nucleus. Treatment of cells with leptomycin B, a nuclear export inhibitor, results in a nuclear accumulation of PDK-1. PDK-1 nuclear localization is increased by insulin, and this process is inhibited by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. Consistent with the idea that PDK-1 nuclear translocation is regulated by the PI3-kinase signaling pathway, PDK-1 nuclear localization is increased in cells deficient of PTEN (phosphatase and tensin homologue deleted on chromosome 10). Deletion mapping and mutagenesis studies unveiled that presence of a functional nuclear export signal (NES) in mouse PDK-1 located at amino acid residues 382 to 391. Overexpression of constitutively nuclear PDK-1, which retained autophosphorylation at Ser-244 in the activation loop in cells and its kinase activity in vitro, led to increased phosphorylation of the predominantly nuclear PDK-1 substrate p70 S6KbetaI. However, the ability of constitutively nuclear PDK-1 to induce anchorage-independent growth and to protect against UV-induced apoptosis is greatly diminished compared with the wild-type enzyme. Taken together, these findings suggest that nuclear translocation may be a mechanism to sequestrate PDK-1 from activation of the cytosolic signaling pathways and that this process may play an important role in regulating PDK-1-mediated cell signaling and function.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase.

Pyridinyl imidazole inhibitors, particularly SB203580, have been widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase (p38/HOG/SAPKII) in a wide array of biological systems. Studies by this group and others have shown that SB203580 can have antiproliferative activity on cytokine-activated lymphocytes. However, we recently reported that the antiproliferative effects of SB203580 were unrelated to p38 MAP kinase activity. This present study now shows that SB203580 can inhibit the key cell cycle event of retinoblastoma protein phosphorylation in interleukin-2-stimulated T cells. Studies on the proximal regulator of this event, the phosphatidylinositol 3-kinase/protein kinase B (PKB)(Akt/Rac) kinase pathway, showed that SB203580 blocked the phosphorylation and activation of PKB by inhibiting the PKB kinase, phosphoinositide-dependent protein kinase 1. The concentrations of SB203580 required to block PKB phosphorylation (IC(50) 3-5 microM) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC(50) 0.3-0.5 microM). These data define a new activity for this drug and would suggest that extreme caution should be taken when interpreting data where SB203580 has been used at concentrations above 1-2 microM.

3-Phosphoinositide-Dependent Protein Kinases↗