Activation Of Phosphoinositide Phospholipase Research Articles

Tetanus toxin modulates serotonin transport in rat-brain neuronal cultures.

As has been previously described, tetanus toxin (TeTx) and its H(C) fragment inhibit the sodium-dependent 5-hydroxytryptamine (5-HT) uptake in rat-brain synaptosomes, probably through a kinase mechanism affecting the 5-HT transporter. Now, the inhibition of 5-HT uptake in neurons in primary culture by TeTx in a dose-dependent manner is described in this work. This effect is also produced by the nontoxic C-terminal fragment of the TeTx heavy chain (H(C)-fragment), indicating that 5-HT uptake inhibition is a consequence of the toxin binding to the plasmatic membrane and not to its catalytic activity. This conclusion is supported by the fact that the 5-HT accumulation was not inhibited by the light chain of TeTx or the toxoid, and was even potentiated by botulinum neurotoxin A. These results correlate with the activation of phosphoinositide-phospholipase C activity in the cultures used in this study, this activity only being enhanced by TeTx and by its Hc-fragment. On the other hand, the use of tyrosine phosphorylation modulators indicates that both Na3VO4 and basic fibroblast growth factor (bFGF) produce an enhancement of 5-HT uptake in this system, which is also sensitive to TeTx inhibition. On the other hand, genistein alone is able to reduce the 5-HT transport in cultured neurons, and this effect did not appear to be additive to that elicited by TeTx. This result suggests that TeTx and genistein may share some events in their respective mechanisms of action. Furthermore, the incubation at different concentrations of 12-O-tetradecanoylphorbol 13-acetate (TPA) confirms the involvement of protein kinase C (PKC) in 5-HT transport modulation in rat-brain neuronal primary cultures. In summary, we shall demonstrate in this work that TeTx induces, through its Hc fragment, an inhibition of both basal and stimulated serotonin uptakes in primary neuronal cultures, in parallel to the activation of phosphoinositide-phospholipase C activity and PKC activation.

Dissociation of tyrosine phosphorylation and activation of phosphoinositide phospholipase C induced by the protein kinase C inhibitor Ro-31-8220 in Swiss 3T3 cells treated with platelet-derived growth factor

Platelet-derived growth factor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP 2) via phospholipase C- γ1 (PLC- γ1) in Swiss 3T3 cells. Treatment of cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly decreased PDGF-induced tyrosine phosphorylation of PLC- γ1, but paradoxically enhanced the production of inositol phosphates (InsPs). The inhibitor also caused an increase of PDGF receptor tyrosine phosphorylation at later times. The changes in phosphorylation of the receptor were correlated with alterations in PLC- γ1 translocation to the particulate fraction. Thus, although activation of PLC- γ1 was associated with phosphorylation of the receptor and translocation of the enzyme to the particulate fraction, it was dissociated from its tyrosine phosphorylation. A non-receptor-associated, cytosolic tyrosine kinase also was found to phosphorylate PLC- γ1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment decreased the tyrosine phosphorylation of PLC- γ1 induced by PDGF and slowed the translocation of PLC- γ1, but Ro-31-8220 produced further effects. The effect of Ro-31-8220 to enhance the production of InsPs could not be attributed to inhibition of PKC since InsPs production with PDGF was decreased in PKC-depleted cells and a stimulatory effect of the inhibitor was still evident. Interestingly, Ro-31-8220 decreased the radioactivity in phosphatidylinositol and increased that in phosphatidylinositol 4-phosphate and PtdInsP 2 in cells labeled with myo[ 3H]inositol. The increased synthesis of PtdInsP 2 could contribute to the increased production of InsPs induced by Ro-31-8220. In summary, these results support the conclusion that the activation of PLC- γ1 in response to PDGF requires autophosphorylation of the receptor and membrane association of PLC- γ1, but not phosphorylation of the enzyme. Furthermore, the effects of Ro-31-8220 on the tyrosine phosphorylation and activity of PLC- γ1, and on PtdInsP 2 synthesis cannot be attributed to inhibition of PKC. © 1997 Elsevier Science B.V. All rights reserved.

P2U Agonists Induce Chemotaxis and Actin Polymerization in Human Neutrophils and Differentiated HL60 Cells

Human neutrophils or HL60 cells express P2U receptors and respond to micromolar concentrations of ATP, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), or UTP with immediate increases in intracellular Ca2+ through activation of phosphoinositide phospholipase C (Cowen, D. S., Lazarus, H. M., Shurin, S. B., Stoll, S. E., and Dubyak, G. R. (1989) J. Clin. Invest. 83, 1651-1660). P2U agonists reportedly induce limited enzyme secretion and enhance the respiratory burst in response to chemotactic factors. We demonstrate here that P2U agonists are chemotactic for neutrophils or differentiated HL60 cells. Rhodamine phalloidin staining indicates that ATPgammaS treatment induces actin polymerization and shape changes similar to those seen when these cells are treated with chemotactic peptide fMet-Leu-Phe. Although undifferentiated HL60 cells fail to mount a rise in Ca2+ when challenged with fMet-Leu-Phe, they increase Ca2+ in response to P2U agonists. However, functional expression of phospholipase C-coupled receptors is not sufficient for chemotaxis since HL60 cell migration in response to these agonists or to fMet-Leu-Phe occurs only after exposure to differentiating agents such as BT2cAMP. In addition to the well known G protein-linked receptors for lipid or peptide chemotactic factors, neutrophils apparently also can utilize G protein-linked purino/pyrimidino receptors to recognize nucleotides as chemoattractants. High concentrations of ATP and UTP generated at sites of platelet aggregation and tissue injury could thus be important mediators of inflammation.

Growth hormone increases phosphoinositide turnover in rat adipocytes that are sensitive to the insulin-like action of the hormone

The effect of pituitary human growth hormone (hGH) on the 32P-labelling of phosphoinositides and phosphatidic acid was studied in noradrenaline-stimulated rat adipocytes which were either responsive or non-responsive to the antilipolytic (insulin-like) effect of hGH. In cells responsive to the insulin-like effect of hGH, hormone treatment resulted in a marked increase of the 32P-labelling of phosphatidic acid and phosphatidyl inositol in the plasma membrane, high density microsomal, and low density microsomal fractions. The increased 32P-labelling most likely reflects an activation of phosphoinositide phospholipase C. IP3 - Myo-Inositol 1,4,5-Triphosphate. BSA - Bovine Serum Albumin, HEPES - N-2-Hydroxyethylpiperazine-N'-2-Ethanesulphonic Acid, hGH - Human Pituitary Growth Hormone, LPC-Lysophosphatidylcholine, LPE - Lysophosphatidylethanolamine, LPI - Lysophosphatidylinositol, PA - Phosphatidic Acid, PC - Phosphatidylcholine, PE - Phosphatidylethanolamine, PI - Phosphatidylinositol, PIP - Phosphatidylinositol-4-Phosphate, PIP2-Phosphatidylinositol-4,5-Diphosphate, PS-Phosphatidylserine.

The separation of [ 32P] inositol phosphates by ion-pair chromatography: Optimization of the method and biological applications

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in 32P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the 32P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P 3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and [ 3H] inositol labeling: (i) 32P labeling is less expensive and more efficient than 3H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.