Steve P. Watson

Collagen stimulates tyrosine phosphorylation of phospholipase C-γ2 but not phospholipase C-γ1 in human platelets

Collagen is an important primary stimulus of platelets during the process of hemostasis. As with many other platelet stimuli, collagen signal transduction involves the hydrolysis of inositol phospholipids; however, the mechanism which underlies this event is not well understood. Neither the collagen receptor nor the isoform of phospholipase C that is activated have been identified. We report that collagen‐activation of platelets induces tyrosine phosphorylation of phospholipase C‐γ2 but not phospholipase C‐γ1. We also show that the platelet low affinity Fc receptor (FcγRII), which mediates activation of platelets by immune complexes, and wheat germ agglutinin, which binds non‐specifically to glycoprotein, stimulate phospholipase C‐γ2 tyrosine phosphorylation. In contrast, we could not detect phospholipase C‐γ2 tyrosine phosphorylation in platelets stimulated by either thrombin or a stable thromboxane A2 analogue, U46619.

Non-peptide antagonists, CP-96,345 and RP 67580, distinguish species variants in tachykinin NK1 receptors.

1 The potency of the non‐peptide antagonists CP‐96,345 and RP 67580 on NK1 receptor‐stimulated [3H]‐inositol phosphate accumulation in cell lines or tissue from three different species has been examined. 2 We have used: UC11 cells, derived from a human astrocytoma, and rat LRM55 glial cells, both of which express large numbers of functional NK1 receptors, and the well characterized guinea‐pig ileum which expresses both NK1 and NK3 receptors. 3 RP 67580 has an ∼25 fold lower affinity for NK1 receptors in human UC11 cells (Kd = 194 nm) than in rat LRM55 cells (Kd = 7.9 nm), in contrast CP‐96,345 has an ∼200 fold lower affinity in rat LRM55 cells (Kd = 210 nm) relative to human UC11 cells (Kd = 0.99 nm). The pharmacological profile of CP‐96,345 and RP 67580 in guinea‐pig ileum was similar to that observed in human UC11 cells. 4 In conclusion, we have demonstrated that previously reported species differences in binding affinities for the non‐peptide NK1 antagonists, CP‐96,345 and RP 67580, are also observed in inhibition of NK1 receptor stimulated hydrolysis of inositol phospholipids.

Phosphorylation of JAK2 in thrombin-stimulated human platelets

We show the presence of the tyrosine kinase JAK2 in human platelets and demonstrate that it undergoes phosphorylation on tyrosine residues on challenge with the G protein receptor stimulus, thrombin, or the tyrosine phosphatase inhibitor, peroxovanadate. Thrombin‐induced phosphorylation of JAK2 is inhibited by two structurally distinct inhibitors of tyrosine kinases, staurosporine and the tyrphostin ST271. The protein kinase C (PKC) inhibitor, Ro 31‐8220, and intracellular Ca2+ chelator, BAPTA‐AM, also inhibit thrombin‐induced phosphorylation of JAK2, while the phorbol ester, phorbol dibutyrate (PDBu), and Ca2+ ionophore, A23187, induce tyrosine phosphorylation of JAK2. These results suggest that tyrosine phosphorylation of JAK2 stimulated by thrombin may be mediated downstream of phosphoinositide metabolism.

Fcγ receptor II stimulated formation of inositol phosphates in human platelets is blocked by tyrosine kinase inhibitors and associated with tyrosine phosphorylation of the receptor

We report that activation of phospholipase C (PLC) by cross‐linking of the platelet low‐affinity Fcγ receptor II (Fcγ RII) is inhibited by two structurally distinct tyrosine kinase inhibitors, staurosporine and ST271. This contrasts with PLC activation induced by thrombin and U46619, a thromboxane mimetic, whose receptors have seven transmembrane domains characteristic of G‐protein coupled receptors. Several proteins undergo phosphorylation on tyrosine on FcγRII cross‐linking upstream of protein kinase C (PKC), Ca2+ and aggregation, including the FcγRII itself. The role of FcγRII phosphorylation in the regulation of PLC is discussed.

The presence of NK3 tachykinin receptors on rat uterus

The NK3 agonist, senktide, induced a potent contraction of rat uterus in the presence of tetrodotoxin, atropine and indomethacin, or the tachykinin receptor antagonists L-659877 and [D-Pro4,D-Trp7,9,10]substance P (4-11). Additional contractile and radioligand binding studies with receptor selective agonists and antagonists confirmed the presence of NK3 receptors and also revealed the presence of NK1 and NK2 receptors. The rat uterus is the second peripheral tissue in which a post-synaptic, non-neuronal NK3 receptor has been identified.

Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C

1 The effect of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), on human platelets has been investigated. 2 Okadaic acid exerts a general increase in phosphorylation of platelet proteins but did not induce aggregation or secretion of 5‐hydroxytryptamine (5‐HT). Okadaic acid, however, did inhibit thrombin‐induced functional responses. 3 Maximally effective concentrations of prostacyclin, to elevate adenosine 3′:5′‐cyclic monophosphate (cyclic AMP), or phorbol dibutyrate, to activate protein kinase C, inhibited the formation of inositol phosphates by thrombin by approximately 60%. When used in combination, prostacyclin and phorbol dibutyrate reduced the levels of inositol phosphates induced by thrombin to 11%. 4 Okadaic acid (1 μm) decreased thrombin‐induced formation of inositol phosphates by approximately 55% and increased the inhibitory action of prostacyclin or phorbol dibutyrate. Okadaic acid had no further effect when prostacyclin and phorbol dibutyrate were used in combination. 5 These results suggest that protein kinases A and C act to inhibit phospholipase C by distinct mechanisms and that their action is reversed by PP1 and/or PP2A.

TiPS on nomenclature

In March the sixth edition of the TiPS Receptor and ion Channel Nomenclature Supplment (RNS), which serves as a guide to nomenclature in the journal, will be published. With the identification of new receptors each year, reappraisal of characterization and classification is necessary for each edition. After five years of publication we feel that it is timely to describe the criteria for classification in the RN’S and to outline the important changes that have been made to the 1995 edition.

Phenylarsine oxide inhibits tyrosine phosphorylation of phospholipase Cγ2 in human platelets and phospholipase Cγ1 in NIH‐3T3 fibroblasts

The sulphydryl reagent phenylarsine oxide (PAO) (1 μM) inhibited completely formation of inositol phosphates in human platelets induced by collagen or by cross‐linking of the platelet low affinity Fc receptor, FcγRIIA, but did not alter the response to the G protein receptor agonist thrombin. PAO also inhibited completely tyrosine phosphorylation of PLCγ2 in collagen and FcγRIIA‐stimulated cells, although tyrosine phosphorylation of other proteins including the tyrosine kinase syk was relatively unaffected. PAO (1 μM) also inhibited completely tyrosine phosphorylation of PLCγ1 induced by platelet derived growth factor (PDGF) in NIH‐3T3 fibroblasts but only partially reduced phosphorylation of the PDGF receptor. These results provide further evidence that collagen and FcγRIIA cross‐linking activate platelets through a pathway distinct from that used by thrombin and suggest that PAO may be a selective inhibitor of PLCγ relative to PLC β isozymes.

Lithium-induced decrease in spontaneous Ca2+ oscillations in single GH3 rat pituitary cells.

1 Measurement of [Ca2+]i in single rat pituitary GH3 cells by dynamic single cell imaging techniques demonstrated that under basal conditions there is a large variation in the temporal pattern of [Ca2+]i signalling between individual cells ranging from high frequency asynchronous oscillations to quiescence. 2 We have reported previously that treatment of GH3 cells with 1 mm Li+ (a concentration used therapeutically in the treatment of manic depression) for 7 days reduces basal and thyrotrophin‐releasing hormone (TRH)‐stimulated levels of mass inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]. In the present study, we show that this is associated with a reduction in the number of cells exhibiting basal Ca2+ oscillations over a sampling period of 60 s, whereas the maximum amplitude of oscillations is unaffected. 3 The pattern of [Ca2+]i responses to the agonist TRH varied considerably between individual cells, making quantitation of the responses difficult; however, data obtained from measurements made on a population of cells showed that increases in peak [Ca2+]i induced by high concentrations of TRH were reduced in cells treated with 1 mm Li+ for 7 days relative to control cells. 4 The sensitivity of the phosphoinositide pathway to [Ca2+]i was investigated by loading GH3 cells with BAPTA/AM at a concentration sufficient to lower ‘basal’ [Ca2+]i in a population of cells and to inhibit agonist‐stimulated increases in [Ca2+]i. Under these conditions, basal and TRH‐stimulated mass Ins(1,4,5)P3 levels were unaffected. 5 These results demonstrate that a 7‐day Li+ treatment leads to an alteration in Ca2+ signalling, in particular by reducing the number of cells exhibiting high frequency Ca2+ oscillations under basal conditions. The significance of these results to the clinical effectiveness of Li+ in the treatment of manic depression is discussed.