Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2‐loaded human platelets

Abstract

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated CAMP on the activation of washed human platelets loaded with the fluorescent Ca 2+ indicator quin2. In the presence of 10 PM isobutylmethylxanthine forskolin inhibited rises in [Ca2+], evoked by thrombin and platelet-activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape-change persisted, even in the absence of a measurable rise in [Caz+],. Forskolin did not affect the rise in [Cazf], evoked by CaZ+ ionophore; aggregation was suppressed but shape-change persisted.

cAMP reduces the affinity of Ca2+‐triggered secretion in platelets

“…The recordings of quin2 fluorescence after six different ionomycin additions to either control or forskolin-treated cells were practically identical under both conditions, indicating that, at least during the period under study, elevated intracellular cAMP levels did not interfere with Ca 2+ homeostasis. This result contrasts with previous reports, in which forskolin treatment completely abolished the intracellular Ca 2+ rise induced by thrombin, collagen [11] or PAF [12]. This difference suggests that cAMP affects only the Ca 2+ entry and release mediated by receptor-operated mechanisms and not by an artificial increase of Ca z+ permeability.…”

Characterization of platelet‐activating factor‐induced elevation of cytosolic free calcium concentration in eosinophils

“…The concentrations of PAF needed to elicit the functional effects and to increase [Ca2+]i in eosinophils are similar with an effect starting at I0 pM and reaching a maximum at 10/~M, indicating that the transduction signal generated by PAF-receptors in eosinophils may involve an elevation of [CaZ+]i. A similar relation between the PAF-induced cellular response and [Ca2+]i have been described for peritoneal macrophages from mice [17] and guinea pig neutrophils [19] as well as for platelets [20].…”

Deliberate quin2 overload as a method forIn Situ characterization of active calcium extrusion systems and cytoplasmic calcium binding: application to the human platelet

“…The table shows that all three loading conditions gave essentially the same resting [Ca2+]cyt. The values are comparable to those obtained with "indicator" levels of quin2 reported in the literature (Rink & Sanchez, 1984;Sage & Rink, 1985;. This tends to rule out adverse effects, such as Ca2+-Mg2+-ATPase inhibition, attributable to high fluorophore loading.…”

“…Studies using the fluorescent chelate probes quin21 Rink & Sanchez, 1984;Maclntyre, Bushfield & Shaw, 1985;Sage & Rink, 1985) and fura-2 (Rao, Peller & White, 1985;Pollock, Rink & Irvine, 1986) have shown that resting platelets maintain their [Ca2+]cy t in the 100 nM range. Since the internal Ca 2+ storage sites in platelets have a finite capacity, this must ultimately be achieved by limiting influx and actively extruding the ion across the plasma membrane.…”