Transport of 5-hydroxytryptamine by platelet dense granules

Dense granules, the subcellular storage organelles for 5hydroxytryptamine in the blood platelet have been isolated and were shown to transport 5-hydroxytryptamine via two mechanisms: : (1) a carrier-mediated process perdominating at low substrate concentrations and (2) a diffusion-mediated process predominating at higher substrate concentrations. The apparent Km for the carrier-mediated process was 3.3uM and its Vmax was 0.79 nmole 5-hydroxytryptamine/min/img protein. 5-hydroxytryptamine transport was sensitive to temperature and studies revealed an apparent activation energy of 14.9 kcal/moie for the mediated process. Transport was inhibited by several structural analogs of 5-hydroxytryptamine and was also inhibited by reduction of the osmotic pressure of the incubation medium below 300m0sM. 5hydroxytryptamine transport was sensitive to changes in pH of the incubation medium. This effect was apparently due to a change in the transmembrane ph gradient (Aph). It was shown by direct measurements using [¹⁴c]-methylamine that decreasing the pH of the incubation medium decreased Aph from a value of 1.l pH unit (acid inside) at pH 7.0 to values near zero at pH 6.0 and produced a concomitant decrease in the initial rate of S-hydrcxytryptamine transport by intact granules.

Ghosts of dense granules were also prepared by hypotonic lysis. Measurements showed that ghosts were depleted of 95% of the original endogenous 5-hydroxytryptamine content. Ghosts prepared in the above iv

way transported exogenously added 5-nydroxytryptamine with a time course similar to intact granules when incubated in the presence of ATp and Mg+t. Transport in the absence of ATP and Mg++ was low, reaching approximately 30% of the steady state levels found in their presence. ATP-stimulated transport was sensitive to addition of nigericin, an ionophore which catalyzes electroneutral exchange between K+ or Na+ and H+. It was found that pH gradients formed in the presence of ATP and Mg++ were alse reduced substantially by such additions of nigericin. Ghosts were found to swell upon the addition of ATp and Mgtt as measured by light scattering techniques. This swelling was inhibited by agents which were shown to inhibit the Mgtt- stimulated ATPase and was therefore attributed to inwardly-directed proton translocation by the ATPase. Swelling in the presence of ATp and Mg++ was stimulated by addition of nigericin or the combination of valinomycin and carbonyl cyanide chlorophenylhydrazone. This effect was apparently due to increased anion transport in the presence of these uncouplers. Addition of increasing concentrations of the anion transport inhibitor, SITS (4-acetamido-4--isothiocyanostilbene-2,2- disulfonic acid) inhibited swelling induced by nigericin in the presence of ATp and Mg+t however SITS had no effect on swelling in the presence of ATP alone. This effect suggests that SITs acts at anion transport sites on the ghost membrane and does not inhibit ghost ATPase activity. Transport of 5-hydroxytryptamine was stimulated by ATp in a concentration-dependent manner and was also stimulated by the establishment of an artificial Aph across the ghost membrane.

In summary, the results presented here demonstrate that isolated dense granules transport 5-hydroxytryptamine via a mechanism which is dependent upon the presence of a transmembrane Aph (acid inside). Ghosts prepared from dense grarules also transport 5-hydroxytryptamine in response to ApH. The transmembrane ApH appears to be generated by an inwardly-directed proton-translocating ATPase in the granule membrane. The results presented here along with those obtained by others using amine storage organelles such as chromaffin granules from adrenal medulla and synaptic vesicles from brain suggest a generalized mechanism for amine transport by subce!lular biogenic amine storage organelles.