Effects of media of low osmolarity on the dense bodies of human platelets.

Exposure of human platelets to buffer at 30 milliosmoles (mOs) caused marked swelling of the cells (a volume increase of almost 300% as estimated from electronic sizing) without a statistically significant loss of dense bodies or a change in the measured diameter of the dense-body core (as examined electron-microscopically in air-dried whole mounts). Even biochemically isolated dense bodies maintained the integrity of their electron-opaque cores in 30 mOs buffer. However, exposure of platelets to 30 mOs buffer reduced the platelet content of cytoplasmic 14C-labeled nucleotides and the vesicular content of [3H]5-hydroxytryptamine (5-HT) by 90%. It thus seems likely that the dense-body membrane displays a sufficiently low permeability to water to prevent swelling and dissolution of the core, but that exposure to hypotonic media allows stored 5-HT to cross the vesicular membrane. Impermeability of this membrane to 5-HT under normal conditions may, therefore, play an important role in the maintenance of vesicular 5-HT stores, rather than the presence of an intact dense-body core. The data also suggest that an "osmotic lysis" or "chemiosmotic" model of secretion may not account for the exocytotic release of human platelet dense bodies.

Reserpine as an uncoupler of oxidative phosphorylation and the relevance to its psychoactive properties.

Many drugs differing widely in chemical structure uncouple mitochondrial oxidative phosphorylation in vitro. This observation has led to the hypothesis that in vivo uncoupling is the basis of their pharmacological activity. Serpasil, a parenteral preparation of reserpine, recently has been shown to uncouple oxidative phosphorylation in vervet monkey kidney mitochondria. Although the drug exhibits some properties of a "classical" uncoupler, our studies show that it has a dual effect on energy conservation. Reserpine released respiratory control in rat liver mitochondria only when dissolved in organic solvents (as in Serpasil) or when deprotonated. Reserpine also released the oligomycin-induced respiratory control in beef heart submitochondrial particles, and inhibited energized uptake of Ca2- by rat liver mitochondria. Reserpine had a dual effect on mitochondrial ATPase: It (a) enhanced ATP hydrolysis by intact liver mitochondria, and (b) inhibited ATP hydrolysis by submitochondrial particles of beef heart. On a molar basis, reserpine was less effective than carbonyl cyanide 3-chlorophenylhydrazone in all bioenergetic reactions examined. Homogenates and mitochondria isolated from brain and liver of rats stuporous from intraperitoneally injections of Serpasil exhibited no detectable abnormalities in respiratory states and responded to known uncouplers in the expected manner. There was no evidence of in vivo uncoupling of oxidative phosphorylation as a basis of the pharmacological activity of reserpine, although interference with energy transfer may be involved in toxic manifestations of the drug. The results indicate the need for caution in interpreting the action of drugs formulated in complex pharmaceutical preparations and based solely on in vitro experiments.

Preferential accumulation of lithium in the dense bodies of human platelets.

From 50 to 73% of the lithium contained in platelets of patients receiving oral therapy with lithium carbonate was released by brief thrombin treatment. Similarly, about 50% of the lithium in platelets of normal volunteers incubated with lithium chloride was thrombin-releasable. The data indicate that an amount of lithium approximately equal to 10% of the calcium content was sequestered in the dense bodies (amine storage organelles) of human platelets. Electron microprobe analysis of dense bodies suggests that the addition of lithium did not change the phosphorus content but produced a loss of about 10% of the dense-body calcium. Nevertheless, synthetic solid analogues of the dense-body core incubated with lithium chloride did not sequester lithium preferentially over potassium and failed to exchange calcium for lithium. Thus, the mechanism responsible for the observed changes in platelet dense bodies may be related to selective membrane permeability properties rather than to binding of lithium to nucleotides or pyrophosphate in the dense-body core.

Comparison of serotonin uptake by dense bodies inside and outside human platelets.

A technique has been developed for quantitating the absolute number of dense bodies present in solution following isolation from human platelets. The amount of [3H]-5HT accumulated per dense body was measured following either sonication alone or sonication plus isolation utilizing a Metrizamide density gradient; the dense bodies in each case were washed and resuspended in sodium or potassium-rich buffer. Uptake per dense body following removal from the cell was less than 10% of the amount of uptake per dense body by intact platelets. It thus seems possible that residence of dense bodies inside intact platelets is required for 5HT transport into dense bodies to proceed at a maximal rate.

Quinacrine and basic amines in human platelets: subcellular compartmentation and effects on serotonin.

Human platelets appear to accumulate quinacrine both in a thrombin-releasable compartment (dense bodies or amine storage vesicles) and in another compartment from which it is released by agents known to collapse pH gradients (possibly lysosomes with an acidic interior). Approximately 61% of the total amount of quinacrine present in human platelets resides in dense bodies and 14+ in lysosomes, with the remainder probably present in the cytoplasm. Other basic amines are accumulated in the three compartments to widely varying extents, suggesting that several factors besides the existence of pH gradients act to determine the distribution of these substances within the cell. The fluorescence emission of quinacrine excited with 420 nm light is completely quenched for quinacrine inside both dense bodies and lysosomes, and the absorption of 440 nm light is decreased by approximately 25%. Quinacrine added to dense bodies at 37 degrees C induces the efflux of 5-hydroxytryptamine (5HT) from the bodies. There is, however, no 5HT loss following quinacrine entry at 0 degree C, and the relationship between the two types of amine movement varies according to incubation time at 0 degree C and 37 degrees C. This action of quinacrine therefore does not appear to be associated with stoichiometric exchange of 5HT and quinacrine, but rather to modulation of the passive permeability of the dense body membrane for 5HT.

Elemental composition of bacterial metachromatic inclusions determined by electron microprobe X-ray analysis.

Electron microscopy and microprobe X-ray analysis were used to study metachromatic inclusions of Spirillum itersonii , Corynebacterium diphtheriae, and Micrococcus luteus. In situ metachromatic inclusions were electron dense and contained phosphorus and divalent cations. Metachromatic inclusions isolated by anion-exchange column chromatography and by isoosmolar Metrizamide density gradient centrifugation were similar in composition to in situ inclusions.