Intracellular calcium fluxes in human platelets

Abstract

1. Fluorescence changes and secretory responses have been measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membraneassociated Ca2+.2. When extracellular [Ca"] is decreased to less than 0.1 pM by addition of EGTA a linear correlation is observed between the extent of increase in cytosolic [Ca2 ' 1 and the extent of mobilisation of membrane-associated 3. Similar ECS0 values are observed for ATP secretion, the increase in cytosolic [Ca"] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve.4. Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca"] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence.5. The data provide further evidence supporting the proposal that chlortatracycline can be used as a probe to monitor mobilisation of membrane-associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2 + mobilisation and Ca2 + removal can under some conditions involve sites which are not monitored by this probe.It is generally accepted that changes in the cytosolic Ca2+ concentration ([Ca2+Ii) form an important part of the stimulus-response coupling pathway in human platelets [1] as in many other cells [2]. In platelets the Ca2+ ions required to sustain an increase in [Ca2+Ii can originate either from the extracellular fluid or from one or more intracellular pools [3 -51. The contribution of such intracellular pool(s) to the increase in [Ca2+Ii induced by several excitatory agonists, e.g. thrombin, can be demonstrated by observing increases in fluorescence from intracellularly trapped Quin 2 in the presence of EGTA as an extracellular Ca2+ chelating agent is, 61.Several storage sites exist within the platelet from which Ca2+ could be released to the cytosol including the dense tubular system, the amine storage granules and the mitochondria as well as a putative Ca2+ pool associated with the plasma membrane. Ca2+ sequestered in the amine storage granules does not appear to be in equilibrium with that present in the cytosol but rather is released to the extracellular fluid as a consequence of exocytosis following cellular activation [7 -91. Furthermore the decrease in chlortetracycline fluorescence induced by thrombin occurs more rapidly than the secretory response monitored as release of Ca2+ [3]. Although Ca2+ release can occur from mitochondria in vitro the conditions required to achieve such release do not appear compatible with participation in events at the time ...

1‐O‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phospho (N,N,N trimethyl) hexanolamine: an analogue of platelet‐activating factor with partial agonist activity

“…In contrast, the aggregation induced by hexanolamine PAF occurs independently of a detectable rise in [Ca2j]i. Adrenaline activates platelet a2-adrenoceptors (Grant & Scrutton, 1979) without increasing inositol phospholipid hydrolysis (Siess et al, 1984) or increasing Ca2+ uptake (Thompson & Scrutton, 1985;Siess & Lapetina, 1988). In a recent study, the PAF-receptor antagonists Ro-193704 and CV-3988, which are structurally related to PAF, were found to act as antagonists of e2-adrenoceptors (Schattner et al, 1989).…”

Ca2+‐dependent aggregation of rabbit platelets induced by maitotoxin, a potent marine toxin, isolated from a dinoflagellate

“…(for reviews: see Siess, 1989;Rink & Sage, 1990 (Rink et al, 1982;Thompson & Scrutton, 1985). However, MTX was reported to have no ionophore-like action on mitochondrial and liposomal membranes (Takahashi et al, 1983 (Ohizumi & Yasumoto, 1983a,b) and the marked prolongation of mean open time of Ca2+ channels in cardiac myocytes (Kobayashi et al, 1987a).…”

“…could activate platelets through the release of intracellular Ca2+ (for reviews: see Siess, 1989;Rink & Sage, 1990). Ionophores like ionomycin and A23187 elicit an increase in [Ca2+], of platelets in the absence of external Ca2+ (Rink et al, 1982; Thompson & Scrutton, 1985). However, MTX was reported to have no ionophore-like action on mitochondrial and liposomal membranes (Takahashi et al, 1983).…”

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

“…It is not possible to use the two probes simultaneously due to spectral interference (cf. Thompson & Scrutton, 1985). …”