Inositol Phosphate Formation In Cells Research Articles

Regulation of histamine H1 receptor coupling by dexamethasone in human cultured airway smooth muscle.

1. The regulation of histamine-induced [3H]-inositol phosphate and intracellular calcium responses in human cultured airway smooth muscle cells was studied. 2. Histamine induced concentration-dependent [3H]-inositol phosphate formation (EC50 4 microM). This response was inhibited by a range of selective H1 receptor antagonists but not by the H2-selective antagonist, tiotidone or the H3 receptor-selective antagonist, thioperamide, indicating that an H1 receptor is involved in this response in human cultured airway smooth muscle cells. 3. Preincubation of human cultured airway smooth muscle cells with concentrations of dexamethasone > 10 nM for 22 h produced concentration-dependent inhibition of histamine-induced inositol phosphate formation. The maximum inhibition observed was 45% of the response in control cells. The inhibitory effect of dexamethasone was itself reversed by prior exposure to the glucocorticoid receptor antagonist, RU38486 (10 microM). Preincubation for 22 h with 1 microM dexamethasone produced inhibition of the inositol phosphate response to histamine to all concentrations of histamine inducing significant inositol phosphate formation in these cells. In contrast, the response to the G protein activator, NaF (0.1-20 mM) was unaltered by preincubation with dexamethasone. 4. Preincubation of human airway smooth muscle cells with 1 microM dexamethasone for time periods of < 6 h failed to inhibit histamine-induced inositol phosphate formation in human airway smooth muscle cells. 5. Histamine also induced concentration-dependent elevation of intracellular calcium levels in Fura 2-loaded human airway smooth muscle cells. This response was inhibited by preincubation with 1 microM dexamethasone. 6. We conclude that signal transduction through the H1 receptor in human airway smooth muscle is subject to regulation by dexamethasone and that this may in part account for the protective effect of dexamethasone against spasmogen-induced contractile responses in the airways.

In human monocytes interleukin-1 stimulates a phospholipase C active on phosphatidylcholine and inactive on phosphatidylinositol

Interleukin-1 (IL-1) can initiate the synthesis of prostaglandins which in turn act as endogenous modulators of IL-1 production. The human monocyte/macrophage synthesizes various eicosanoids through the activation of the cellular phospholipase system. Cell stimulation results in the activation of phospholipase A 2 (PLA 2) whose major substrate is phosphatidylcholine (PC) and the release of the eicosanoid precursor arachidonic acid (AA) from PC. Another pathway is the stimulation of a phospholipase C (PLC) mainly active on phosphoinositides and the resulting formation of inositol phosphates (IPs) and diacylglycerol (DAG). Phospholipids other than phosphoinositides can also be hydrolysed by PLC to give rise to DAG. Studies have shown that IL-1 does not activate the IP pathway, but it primarily stimulates a PLC linked to phosphatidylethanolamine in cultured rat mesangial cells, and a PLC linked to PC in Jurkart cells. We have stimulated human monocytes with IL-1 and calcium ionophore A23187 and we have observed their effect on the phospholipase system. The results indicate that IL-1 does not activate the formation of IPs in cells labeled with [ 3H] myo-inositol. In contrast, in cells labeled with [ 3H]AA, IL-1 causes the formation of DAG associated with the hydrolysis of PC. Moreover, after stimulation with IL-1 there is no accumulation of free AA which would indicate that there has been no activation of PLA 2, which occurs instead with A23187 stimulation. These data suggest that, in monocytes, IL-1 does not directly stimulate a PLA 2 or a PLC active on phosphatidylinositol; instead it primarily stimulates a PLC active on PC.

Platelet-activating factor synergizes with phorbol myristate acetate in activating phospholipase D in the human promonocytic cell line U937. Evidence for different mechanisms of activation.

The activation of phospholipase D by platelet-activating factor (PAF) in the human promonocytic cell line U937 has been investigated. In cells prelabeled with [3H]palmitic acid, addition of PAF or phorbol 12-myristate 13-acetate (PMA) induced the synthesis of [3H]phosphatidylethanol, indicating phospholipase D activation. When U937 cells were preincubated for 5 min with PMA, and then stimulated with PAF, formation of phosphatidylethanol was greatly enhanced. In contrast, under the same experimental conditions PMA treatment blocked completely the PAF-induced inositol phosphates formation in cells prelabeled with [3H]inositol. Thus, PMA treatment demonstrates that phospholipase D activation can occur independently from phosphoinositide-specific phospholipase C activation during PAF stimulation in U937 cells. On the other hand, the data herein presented suggest that influx of external calcium is required for phospholipase D activation by PAF, as assessed by complete inhibition of the enzyme activity by chelation of extracellular calcium or by treatment with the calcium channel blocker verapamil. Based on these findings, a hypothetical model for phospholipase D activation is discussed.

Pharmacological modulation of oscillations in the blowfly salivary gland

lation counting and expressed as d.p.m./culture well. (ii) Alternatively, the various inositol phosphates within the sample were separated by h.p.1.c. on a Partisil Sax column eluted with a 0-3 M-ammonium formate/O.l M-formic acid gradient at a flow rate of 1.25 ml/min. Fractions (0.5 min) were collected and radioactivity in them determined as above expressed as d.p.m./fraction. Treatment of 3T3 cultures with FGF stimulates total inositol phosphate formation in cells. Total [3HH]inotisol phosphates in control 3T3 cultures was 12.5 1 f 36 d.p.m./culture well; in cultures exposed to basic FGF (50 ng/ml) for 2 h it was 2 126 f 77 d.p.m./culture well. Table 1 shows different inositol phosphates produced during this response analysed by h.p.1.c. By 10 s of stimulation, inositol trisphosphate levels are raised, as are levels of inositol tetrakisand pentakisphosphate. After 10 min, the inositol trisand tetrakis-phosphate levels have fallen to control levels while the raised inositol pentakisphosphate levels are maintained. By 30 min of stimulation, although still higher than controls, the inositol pentakisphosphate levels are falling towards control values. This observation of a rapid and transient inositol trisphosphate signal accompanied by a much more persistent inositol pentakisphosphate signal suggests a multiple signalling pathway coupled by phospholipase C and activated by FGF. What mechanisms the two intracellular signals are targeting remain to be established, but it might be that the more highly phosphorylated inositol derivatives are involved in mediating the long-term regulation of cell growth. The responses to FGF in vitro can be divided into very early events, which include ionic fluxes, protein kinase activation and jntracellular pH changes, and later events such as gene transcription and protein synthesis (Gospodarowicz, 1087). It is interesting to speculate that the transient and persistent inositol phosphate signals observed here may be linked t o early and late responses to FGF. Overall these data establish that FGF does stimulate phosphoinositide hydrolysis, at least in Balb/c 3T3 fibroblast cells. However, the relative importance of this signalling pathway for mitogenesis has still to be defined.