Sunil Joseph

Differential sensitivities of the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium in bovine aortic endothelial cells

1 Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nm) and endothelium‐derived relaxing factor (EDRF, NO; Kact = 0.7 nm) in a concentration‐dependent manner when exposed to bradykinin. 2 The bradykinin‐dependent release of PGI2 (but not EDRF) was inhibited by 1 μm isoprenaline or 5 μm forskolin, and the inhibitory effect of isoprenaline could be reversed by the β2‐adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PGI2 release stimulated by exogenous arachidonic acid. 3 Exposure of cells to bradykinin increased the cytosolic concentration of Ca2+ ions ([Ca2+]i; Kact = 4.8 nm), and the effect was inhibited by both 1 μm isoprenaline and 5 μm forskolin. 4 In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of that required for PGI2 biosynthesis and that these corresponded to a [Ca2+]i threshold of 350 nm for PGI2 release while that for EDRF release was less than 200 nm. 5 These differences in Ca2+ ion sensitivity explain the selective inhibition of bradykinin‐stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin‐dependent increases in [Ca2+]i.

Lack of inhibition of thrombin‐induced rise in intracellular Ca2+ levels and 5‐hydroxytryptamine secretion by 1‐oleoyl‐2‐acetylglycerol in human platelets

The effect of 1‐oleoyl‐2‐acetylglycerol (OAG) on the thrombin‐induced rise in intracellular Ca2+ levels ([Ca2+]i) and 5‐hydroxy[14C]tryptamine ([14C]5HT) secretion was studied. In washed human platelets prelabelled with [14C]5HT and quin 2, OAG (10–50 ) induced no significant aggregation, [14C]5HT secretion or rise in [Ca2+]i in the presence or absence of fibrinogen. However, addition of OAG (10–50 ) 10 s to 5 min before or 10–60 s after addition of threshold concentrations of thrombin ( < 0.03 ) resulted in a significant potentiation of aggregation and [14C]5HT secretion without any effect on the thrombin‐induced rise in [Ca2+]i. Both EGTA, which abolished the latter and creatine phosphate/creatine phosphokinase, the ADP scavenger, totally inhibited the aggregation but only partially reduced [14C]5HT secretion in response to thrombin plus OAG. At higher concentrations of thrombin, neither the rise in [Ca2+]i nor the extent of [14C]5HT secretion was significantly altered by OAG addition. The results demonstrate that, unlike phorbol esters, OAG has no inhibitory effect on thrombin‐induced [Ca2+]i mobilisation but can synergize with low concentrations of thrombin in potentiating [14C]5HT secretion even at basal [Ca2+]i.

The identity of IgE receptors (FcεRII) that mediate cellular activation of human macrophages : evidence against a role for CD23

Abstract There is now compelling evidence that macrophages bind IgE, and are involved in several IgE-dependent responses. The CD23 antigen mediates a mitogenic response in “primed” B-lymphocytes, although its expression is not confined to B cells, and CD23 is inducably expressed in many cells including macrophages. CD23 is also known to bind IgE, a property that leads to inhibition of the mitogenic response in B cells. In the present review, the possibility that CD23 mediates IgE-dependent responses in macrophages has been re-examined, and it is proposed that the functional receptor for IgE on macrophages may be quite separate from the CD23 antigen.

Thrombin signalling in U937 human monocytic cells is coupled to inositol phosphate formation but not to thromboxane B2 synthesis nor to inhibition of adenylate cyclase: distinct differences in thrombin signalling between U937 cells and platelets

The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic effects on mononuclear phagocytic inflammatory cells. In the present study, we have used the U937 human monocytic cell line to explore the signal transduction mechanisms utilised by thrombin in these cells. In U937 cells differentiated into a macrophage-like phenotype, thrombin stimulated the formation of inositol trisphosphate (IP3) and the mobilisation of intracellular Ca2+ [( Ca2+]i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx) B2 synthesis in the differentiated cells. In contrast, the chemotactic peptide N-formyl-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxB2 synthesis under conditions where it evoked increases in IP3 formation and [Ca2+]i mobilisation, via a pertussis toxin-sensitive mechanism, comparable in extent to those mediated by thrombin. Thrombin also failed to cause inhibitory guanine nucleotide binding protein (Gi)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. These results indicate that U937 cells express receptors for thrombin which are in part coupled via a pertussis toxin-sensitive guanine nucleotide binding protein to phospholipase C activation, the formation of IP3 and the mobilisation of [Ca2+]i. However, the failure of thrombin to stimulate TxB2 synthesis or cause Gi-mediated inhibition of adenylate cyclase in U937 cells contrasts with its effects in human platelets and other thrombin-responsive cells. These results suggest that the thrombin receptor or receptor-effector coupling mechanism(s) in mononuclear cells is functionally distinct from the thrombin receptor or receptor-effector coupling mechanism(s) present in other thrombin-responsive cells.