Uptake, metabolism and release of [³H]‐adrenaline by human platelets

Abstract

Summary1. Measurements were made of the uptake, metabolism and release of [3H]-adrenaline by human platelets in citrated plasma or in an artificial medium. 2. Radioactive adrenaline was not taken up at 0-2°C. At 370 C there was a slow uptake which continued for at least 5 hours.3. About half of the radioactivity in the platelets was intact adrenaline. The other half was an acidic metabolite from which adrenaline was released by acid hydrolysis. 4. The immediate uptake of adrenaline was proportional to its concentration in the plasma up to at least 1 x 1O-5M. Uptake measured after 1 h also increased linearly with concentration up to about 1 x 10-4M but less with higher concentrations. The highest concentration ratio was about 12. 5. The concentration of metabolite in the platelets increased with the concentration of added adrenaline only up to about 2 x 10-m. 6. The immediate uptake of adrenaline was partially inhibited by phentolamine and dihydroergotamine. Measurement of uptake both immediately and after 1 h showed that the inhibition produced was not increased beyond about 50% by these drugs or by (±)-propranolol, chlorpromazine or amitriptyline up to 1 x 10 'M. 7. Formation of the metabolite was inhibited by pyrogallol, 8-hydroxyquinoline, or tropolone. This inhibition was associated with a corresponding increase in the adrenaline accumulated intact. Formation of the metabolite was not inhibited by monoamine oxidase inhibitors. 8. Reserpine caused a small decrease in the uptake of adrenaline radioactivity in 1 h and a great increase in the proportion recovered as metabolite. 9. Thrombin caused the release from platelets of intact adrenaline but not of the metabolite. 10. Platelets of albino patients with spontaneous haemorrhages accumulated adrenaline radioactivity at the normal rate but this radioactivity was wholly accounted for by metabolite and not released by thrombin. 11. After taking up adrenaline, platelets resuspended in artificial medium at 370 C slowly released both adrenaline and its metabolite. At the same time, the intracellular adrenaline was slowly metabolized.

Content and Subcellular Localization of Catecholamines and 5‐hydroxytryptamine in Human and Animal Blood Platelets: Monoamine Distribution Between Platelets and Plasma

“…For instance, it was found that labelled CA injected into rabbits, were highly concentrated in a platelet fraction rich in 5-HT organelles (Da Prada & Pletscher, 1969b). In addition it has been shown that isolated platelets take up CA in vitro (Born et al, 1958;Weissbach et al, 1958;Weil-Malherbe & Bone, 1958;Born & Smith, 1970;Boullin & O'Brien, 1970) and that 5-HT organelles isolated from rabbit platelets are also able to concentrate labeled CA in vitro (Da Prada & Pletscher, 1969a). Born & Smith (1970) have demonstrated that reserpine, which reduces 5-HT uptake by acting at the level of the 5-HT granular membrane (Pletscher, Burkard & Tranzer, 1967;Da Prada & Pletscher, 1969b), also impairs Ad uptake in intact platelets.…”

“…Moreover, thrombin, which is known to release platelet 5-HT, ATP and calcium (typical constituents stored in the 5-HT organelles) was also able to release labelled Ad from platelets preloaded in vitro with this tritiated amine (Born & Smith, 1970). The present data obtained by measuring radioenzymatically the concentration of endogenous Ad, NA, dopamine and 5-HT in the subcellular structures isolated by subjecting rabbit platelet homogenates to density gradient centrifugation, directly demonstrate that 5-HT organelles are also the storage sites of the platelet CA.…”

“…In contrast to 5-HT, the CA exhibit a relatively low affinity for the platelet 5-HT carrier mechanism; therefore, it is generally believed that in vivo they cross the platelet membrane mainly by passive, nonsaturable transfer, even if at very high, unphysiological extracellular concentrations CA can be actively transported via the 5-HT carrier (Born & Smith, 1970;Gordon & Olverman, 1978;Pletscher, Laubscher, Graf & Saner, 1978). Thus it is likely that platelets accumulate substantial amounts of CA only in conditions of sustained and long-lasting high CA concentration in plasma.…”

“…Indirect experimental evidence indicates that 5-HT organelles might also be the storage sites for platelet CA (Born, Hornykiewicz & Stafford, 1958;Weissbach, Bogdanski & Udenfriend, 1958;Weil-Malherbe & Bone, 1958;Hughes & Brodie, 1959;Born & Smith, 1970;Boullin & O'Brien, 1970). However, the very low amounts of CA present in the platelets and the consequent methodological difficulties of their measurement account for the lack of data regarding content and localization of platelet adrenaline (Ad), noradrenaline (NA) and dopamine.…”

Relative activities on and uptake by human blood platelets of 5‐hydroxytryptamine and several analogues

“…Imipramine was much less inhibitory than methysergide and the inhibition was not competitive. Human platelets take up 5-HT by a mechanism which causes it to be highly concentrated in intracellular storage granules and which is highly specific for 5-HT as far as the naturally occurring amines are concerned (for reviews, see Pletscher, 1968;Born, 1969); adrenaline is also taken up but much more slowly and concentrated far less (Born & Smith, 1970). It was of particular interest, therefore, to see which of our structural analogues of 5-HT could be taken up by platelets by a process similar to that for 5-HT.…”