Excitatory and inhibitory modulation of intracellular free calcium in human platelets by hormones and drugs.
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Biomedical subjects
Publications and source records attributed to H Affolter.
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In maize mitochondria the effect of the herbicide amiprophosmethyl was studied on massive and limited Ca2+-loading and on mitochondrial energy transduction. Under massive Ca2+-loading conditions amiprophosmethyl inhibited the Ca2+ transport system directly without a significant effect on the respiratory chain, the membrane potential or Ca2+ efflux. Under limited Ca2+-loading conditions an increased Ca2+ efflux was noted, which could be partially responsible for the inhibition of the net Ca2+ accumulation.
Blood platelets show specific, high affinity binding of 3H-5-hydroxytryptamine, 3H-ketanserin and 3H-D-lysergic acid diethylamide. 5-HT-antagonists are considerably more potent than agonists regarding both the displacement of specifically bound 3H-ketanserin and the shape change reaction mediated by the 5-HT-receptor. The latter depends on a rise of free intracellular Ca2+. The binding site for 3H-ketanserin and the site at which the 5-HT-induced shape change is triggered show the characteristics of a 5-HT2-receptor whose intracellular mediator seems to be Ca2+. The 5-HT2-receptor of platelets may be used as a partial model for that in neurons; however, it remains to be elucidated whether Ca2% is a mediator of the latter.
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1 The effects of K(+), NaCN and the ionophores monensin, nonactin and carbonyl-cyanide-p-trifluoro-methoxyphenylhydrazone (FCCP) on the contents of [(3)H]-5-hydroxytryptamine ([(3)H]-5-HT), [(3)H]-dopamine and [(3)H]-noradrenaline ([(3)H]-NA) in guinea-pig synaptosomes preloaded with these amines were measured.2 In the presence of Ca(2+), K(+) markedly reduced the amine content of the synaptosomes, indicating an acceleration of spontaneous amine release. In the absence of Ca(2+), K(+) had much less effect.3 Monensin, nonactin and FCCP caused a release of all the three labelled amines. This release was considerably faster and more marked than that induced by K(+) and showed no dependence on Ca(2+). The ionophores did not release lactate-dehydrogenase from synaptosomes.4 NaCN, a blocker of oxidative energy production, did not enhance the spontaneous release of [(3)H]-5-HT nor did it influence the monensin-induced release of [(3)H]-5-HT.5 It is concluded that (a) the intragranular storage of 5-HT, dopamine and NA is dependent on the maintenance of a pH-gradient across the granular membrane as well as on the granular membrane potential; (b) the ionophores cause a non-exocytotic release of granular amines, and (c) blood platelets are partial models for aminergic brain neurones as far as intragranular amine storage is concerned.
Alterations in rheo-optical signals obtained from suspensions of human blood platelets treated in various ways (drugs, storage in the cold, stirring, etc.) were monitored. A self-normalizing instrument measuring scattered light and relative amplitude of rapid oscillation of light intensity (noise level) at different angles was used. The following results have been obtained: 1. The light scattered at an angle of 40 degrees was decreased with considerable selectivity by pseudopod formation, if aggregation was inhibited. 2. The noise levels at 0 degree and 40 degrees were selectively diminished by the transition from the discoid to the spheroid shape of the platelets. 3. At 40 degrees but not at 0 degree, the noise level responded without delay and was not influenced by aggregate formation. Using this method alterations in platelet form, i.e. spheroid transformation and pseudopod formation, can be specifically and continuously monitored. In addition, the noise level at 40 degrees is a definite quantitative measure of the discoid state. This allows the determination of platelet activation without manipulations such as changes in stirring rates or addition of drugs.
Synaptosomes from guinea-pig brain were compared with blood platelets (partly from previous experiments) regarding the action of imipramine, the benzoquinolizine derivative Ro 4-1284, tyramine and p-chlormethamphetamine (PCMA) on the contents of stored radio-labelled 5-hydroxytryptamine (5HT), dopamine (DA) and noradrenaline (NA). Normal and reserpinized preparations were used in order to differentiate between granular and extragranular (reserpine-resistant) sites. Imipramine, in concentrations greater than those inhibiting 5HT- and NA-uptake released the three amines from granular and extragranular sites and showed the same order of potency in synaptosomes as in platelets. The drug Ro 4-1284 acted on the granular amines only, but its action on NA was less potent in synaptosomes than in platelets. Tyramine and PCMA also caused an exclusive release of granular amines in synaptosomes, whereas in platelets the drugs released extragranular 5HT (and DA) as well as the granular amines. Tyramine was more potent in synaptosomes than in platelets, and PCMA showed a preferential effect on 5HT in synaptosomes but not in platelets. It is concluded that, regarding the action of monoamine-releasing drugs, platelets are only partial models for synaptosomes. However, they may be superior to synaptosomes in predicting the pattern of amine release in brain in vivo as seen with Ro 4-1284.
The actions of ionophores with different ion specificities and of thrombin on the release of 14C-labeled 5-hydroxytryptamine, [3H]noradrenaline, and endogenous ATP were measured in human platelets suspended in media with various K+ and Na+ concentrations. Besides thrombin, those ionophores [monensin, nigericin, and the combination of carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) with nonactin and/or valinomycin] which cause a rapid collapse of H+ gradients induced a fast and virtually total release of 14C-labeled 5-hydroxytryptamine and [3H]noradrenaline into the various media. FCCP alone, which causes an inversion of the membrane potential to inside negative values, induced a considerably slower amine release. Changes in the K+ and Na+ gradients did not lead to amine release, nor did interference with energy transduction by antimycin A with or without glycolysis inhibitors. Monensin and FCCP did not release ATP, whereas thrombin, added before or after incubation of platelets with FCCP and monensin, caused a marked liberation of the nucleotide. It is concluded that in intact human platelets (a) the intragranular storage of 5-hydroxytryptamine and noradrenaline mainly depends on the proton gradient across the granular membrane, and (b) ionophores causing a collapse of H+ gradients induce non-exocytotic release of 5-hydroxytryptamine and noradrenaline from intracellular storage granules.
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The use of a fast-responding Ca2+-selective electrode has allowed the simultaneous measurement of the true Ca2+-activity in the medium and of the Ca2+-transport rate, eliminating the uncertainties connected with the use of indirect methods of estimating Ca2+ transport rates (e.g. O2-stimulation, K+-efflux). The results on the kinetics of Ca2+ uptake by rat liver mitochondria obtained in this study are qualitatively similar to those obtained by Bragadin et al. [Bragadin, M., Pozzan, T. & Azzone, G. F. (1979) Biochemistry, 18, 5973--5978] by monitoring the K+-efflux from mitochondria. In the valinomycin-driven Ca2+ uptake, the Ca2+ translocase is the limiting factor, whereas in the system driven by succinate extraneous limiting factors play a role. Thus, liver mitochondria, when studied under appropriate non-limiting conditions, exhibit a hyperbolic dependence on the Ca2+ uptake rate on Ca2+-activity analogous to that of mitochondria from other tissues. The sigmoidal dependence of the uptake rate on Ca2+-activity in liver mitochondria, observed under conditions which produce hyperbolic kinetics in mitochondria from other tissues, can thus be attributed to factors which are extraneous to the Ca2+ transport system per se.
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A study has been carried out of the interaction of dog heart mitochondria and sarcoplasmic reticulum with Ca2+ bound to troponin or to its Ca2+-binding component (troponin-C). Both organelles are able to release more than 80% of the Ca2+ bound to the two proteins. However, sarcoplasmic reticulum is only able to do so in the presence of oxalate, whereas mitochondria are active also in the absence of permeant Ca-complexing anions. On the other hand, calcium uptake in heart mitochondria is severely inhibited by Mg2+. The results are discussed with reference to the problem of the control of Ca2+ in heart sarcoplasm.
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