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. 1990 Aug 15;270(1):125–131. doi: 10.1042/bj2700125

Ca2(+)-stimulatable and protein kinase C-inhibitable accumulation of inositol 1,3,4,6-tetrakisphosphate in human platelets.

W G King 1, C P Downes 1, G D Prestwich 1, S E Rittenhouse 1
PMCID: PMC1131687  PMID: 2396972

Abstract

Thrombin-stimulated (10 s) human platelets produce Ins(1,4,5)P3 and an additional inositol trisphosphate (InsP3), in approximately a 1:20 ratio. The major InsP3 co-migrates with Ins(1,3,4)P3 on strong-anion-exchange h.p.l.c. To identify this species unequivocally, we treated putative Ins(1,3,4)P3 obtained from thrombin-stimulated myo-[3H]inositol-labelled platelets with NaIO4/NaBH4 or 4-phosphomonoesterase. The products indicate that the major InsP3 is at least 90% D-Ins(1,3,4)P3. D-[3H]Ins(1,3,4)P3 added to saponin-permeabilized platelets is hydrolysed to an InsP2 (7.8%) and phosphorylated by a kinase to yield an inositol polyphosphate (0.9%) in 5 min. The phosphorylation product co-migrates with Ins(1,3,4,6)P4 on Partisphere WAX h.p.l.c. Under similar conditions, L-[3H]Ins(1,3,4)P3 is dephosphorylated but not phosphorylated. Relative phosphatase:kinase ratios are 8.7:1 (Vmax. values) and 0.86:1 (Km values) with respect to D-Ins(1,3,4)P3. The kinase activity is predominantly cytosolic (96.8% of total activity) in freeze-thaw-disrupted platelets, and the accumulation of its product is Ca2(+)-dependent. The activity is identified as a 6-kinase on the basis of its product's insensitivity to 5-phosphomonoesterase, resistance to periodate oxidation and co-migration with standard Ins(1,3,4,6)P4 on h.p.l.c. Incubation of platelets with beta-phorbol dibutyrate (beta-PDBu, 76 nM), causing activation of protein kinase C, results in a 57.5% inhibition (reversible by the protein kinase C inhibitor staurosporine) of Ins(1,3,4,6)P4 accumulation. alpha-PDBu, which does not stimulate protein kinase C, has no effect. Stimulation of intact platelets with thrombin results in the production of Ins(1,3,4,6)P4 (1.4-fold rise in 30 s) and Ins(1,3,4,5)P4, with the latter being the major InsP4 species. Accumulation of Ins(1,3,4,6)P4 is slightly delayed in comparison with Ins(1,3,4)P3 and is relatively small. We propose that the major route of Ins(1,3,4)P3 metabolism in stimulated human platelets is via phosphatase action.

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Selected References

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  1. Balla T., Guillemette G., Baukal A. J., Catt K. J. Metabolism of inositol 1,3,4-trisphosphate to a new tetrakisphosphate isomer in angiotensin-stimulated adrenal glomerulosa cells. J Biol Chem. 1987 Jul 25;262(21):9952–9955. [PubMed] [Google Scholar]
  2. Banga H. S., Simons E. R., Brass L. F., Rittenhouse S. E. Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9197–9201. doi: 10.1073/pnas.83.23.9197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bansal V. S., Inhorn R. C., Majerus P. W. The metabolism of inositol 1,3,4-trisphosphate to inositol 1,3-bisphosphate. J Biol Chem. 1987 Jul 15;262(20):9444–9447. [PubMed] [Google Scholar]
  4. Batty I. R., Nahorski S. R., Irvine R. F. Rapid formation of inositol 1,3,4,5-tetrakisphosphate following muscarinic receptor stimulation of rat cerebral cortical slices. Biochem J. 1985 Nov 15;232(1):211–215. doi: 10.1042/bj2320211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  6. Connolly T. M., Bansal V. S., Bross T. E., Irvine R. F., Majerus P. W. The metabolism of tris- and tetraphosphates of inositol by 5-phosphomonoesterase and 3-kinase enzymes. J Biol Chem. 1987 Feb 15;262(5):2146–2149. [PubMed] [Google Scholar]
  7. Culty M., Davidson M. M., Haslam R. J. Effects of guanosine 5'-[gamma-thio]triphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets. Eur J Biochem. 1988 Feb 1;171(3):523–533. doi: 10.1111/j.1432-1033.1988.tb13821.x. [DOI] [PubMed] [Google Scholar]
  8. Downes C. P., Mussat M. C., Michell R. H. The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane. Biochem J. 1982 Apr 1;203(1):169–177. doi: 10.1042/bj2030169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  10. Hansen C. A., vom Dahl S., Huddell B., Williamson J. R. Characterization of inositol 1,3,4-trisphosphate phosphorylation in rat liver. FEBS Lett. 1988 Aug 15;236(1):53–56. doi: 10.1016/0014-5793(88)80284-9. [DOI] [PubMed] [Google Scholar]
  11. Hughes P. J., Hughes A. R., Putney J. W., Jr, Shears S. B. The regulation of the phosphorylation of inositol 1,3,4-trisphosphate in cell-free preparations and its relevance to the formation of inositol 1,3,4,6-tetrakisphosphate in agonist-stimulated rat parotid acinar cells. J Biol Chem. 1989 Nov 25;264(33):19871–19878. [PubMed] [Google Scholar]
  12. Inhorn R. C., Majerus P. W. Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+. J Biol Chem. 1987 Nov 25;262(33):15946–15952. [PubMed] [Google Scholar]
  13. Irvine R. F., Anggård E. E., Letcher A. J., Downes C. P. Metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in rat parotid glands. Biochem J. 1985 Jul 15;229(2):505–511. doi: 10.1042/bj2290505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Irvine R. F., Letcher A. J., Lander D. J., Berridge M. J. Specificity of inositol phosphate-stimulated Ca2+ mobilization from Swiss-mouse 3T3 cells. Biochem J. 1986 Nov 15;240(1):301–304. doi: 10.1042/bj2400301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. King W. G., Rittenhouse S. E. Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphosphates and tetrakisphosphate in human platelets exposed to thrombin. J Biol Chem. 1989 Apr 15;264(11):6070–6074. [PubMed] [Google Scholar]
  16. O'Rourke F. A., Halenda S. P., Zavoico G. B., Feinstein M. B. Inositol 1,4,5-trisphosphate releases Ca2+ from a Ca2+-transporting membrane vesicle fraction derived from human platelets. J Biol Chem. 1985 Jan 25;260(2):956–962. [PubMed] [Google Scholar]
  17. Rittenhouse S. E. Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP. Biochem J. 1984 Aug 15;222(1):103–110. doi: 10.1042/bj2220103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rittenhouse S. E., Sasson J. P. Mass changes in myoinositol trisphosphate in human platelets stimulated by thrombin. Inhibitory effects of phorbol ester. J Biol Chem. 1985 Jul 25;260(15):8657–8660. [PubMed] [Google Scholar]
  19. Shears S. B., Kirk C. J., Michell R. H. The pathway of myo-inositol 1,3,4-trisphosphate dephosphorylation in liver. Biochem J. 1987 Dec 15;248(3):977–980. doi: 10.1042/bj2480977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shears S. B., Parry J. B., Tang E. K., Irvine R. F., Michell R. H., Kirk C. J. Metabolism of D-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate. Biochem J. 1987 Aug 15;246(1):139–147. doi: 10.1042/bj2460139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shears S. B. The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase. J Biol Chem. 1989 Nov 25;264(33):19879–19886. [PubMed] [Google Scholar]
  22. Stephens L. R., Hawkins P. T., Barker C. J., Downes C. P. Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation. Biochem J. 1988 Aug 1;253(3):721–733. doi: 10.1042/bj2530721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stephens L., Hawkins P. T., Carter N., Chahwala S. B., Morris A. J., Whetton A. D., Downes P. C. L-myo-inositol 1,4,5,6-tetrakisphosphate is present in both mammalian and avian cells. Biochem J. 1988 Jan 1;249(1):271–282. doi: 10.1042/bj2490271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tarver A. P., King W. G., Rittenhouse S. E. Inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate are minor components of total mass of inositol trisphosphate in thrombin-stimulated platelets. Rapid formation of inositol 1,3,4-trisphosphate. J Biol Chem. 1987 Dec 25;262(36):17268–17271. [PubMed] [Google Scholar]
  25. Watson S. P., McConnell R. T., Lapetina E. G. The rapid formation of inositol phosphates in human platelets by thrombin is inhibited by prostacyclin. J Biol Chem. 1984 Nov 10;259(21):13199–13203. [PubMed] [Google Scholar]

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