Characterization of histamine release in digitonin-permeabilized rabbit platelets

To manipulate the intracellular milieu of rabbit platelets, permeabilization was performed using digitonin. Permeabilized platelets showed dose-dependent release of histamine, which was stored in granules of rabbit platelets, in response to extracellular calcium ion. As PMA stimulated the release reaction in digitonin-permeabilized platelets, the protein kinase C system, which regulates metabolic processes and cell reactions in intact platelets, was revealed to be working. Cupric phenanthroline also released histamine from permeabilized rabbit platelets dose-dependently, and dithiothreitol inhibited the release strongly. Since cupric phenanthroline is a mild oxidant which catalyzes the formation of disulfide bridges, as in the case of Ca2+-ATPase of sarcoplasmic reticulum, the results suggested that protein cross-linking is implicated in the regulation of the release reaction in permeabilized rabbit platelets.     

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

ATP-dependent proteolysis in reticulocyte extracts is stimulated by ubiquitin, a polypeptide which is covalently conjugated to proteins. It has been proposed that ATP and ubiquitin act by repressing an inhibitor of an ATP-independent protease, rather than by conjugation to substrate proteins [Speiser, S. and Etlinger, J.D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3577-3580]. We find that the inhibitor preparation used by these authors contains a positively required factor of the ATP-ubiquitin proteolytic system, which can be separated from two types of protease inhibitors by gel filtration chromatography. The following observations indicate that the "inhibitors" are endogenous protease substrates which compete with the labeled substrate: (a) inhibition is competitive with exogenous substrate; (b) inhibition is abolished by a preincubation of "inhibitor" with protease prior to the addition of labeled substrate. These findings are not consistent with the notion that the inhibitors play a regulatory role in the ATP-ubiquitin proteolytic pathway.     

Phorbol esters modulate thrombin-operated calcium mobilisation and dense granule release in human platelets

Human alpha-thrombin-induced elevation of cytosolic free calcium ([Ca2+]i) and dense granule release was examined in platelets preincubated with either activators or an inhibitor of protein kinase C. 12-O-Tetradecanoylphorbol 13-acetate (TPA) or two 12-deoxy analogues of TPA, when added alone to platelets, did not elevate [Ca2+]i, as monitored by quin2 fluorescence, though small amounts of dense granule release were detected. Preincubation of the platelets with either TPA or 12-deoxyphorbol 13-phenylacetate, but not the parent, 4-beta-phorbol, produced a dose-dependent inhibition of the elevation of [Ca2+]i and 5-hydroxytryptamine release induced by human alpha-thrombin. Furthermore, this phorbol ester-mediated inhibition of human alpha-thrombin-induced activation could be prevented by H7 (1-[5-isoquinolinesulphonyl]-2-methylpiperazine), the recently described inhibitor of protein kinase C. These results suggest a role for protein kinase C as a modulator of receptor-operated calcium fluxes in human platelets.     

Triphosphoinositide breakdown and dense body release as the earliest events in thrombin-induced activation of human platelets

The activation by thrombin of human platelets prelabelled with 32P induced a 30-40% decrease in 32P-triphosphoinositides (TPI) in the first 10 sec; the decrease in the other 32P-labelled phosphoinositides occurred by 20-30 sec. At 10 sec., the intensity of these effects was maximum with 0.2-0.4 U/ml thrombin. Under these conditions, 53, 20 and 15% of the dense granule, alpha-granule and lysosome constituents, respectively were released and thromboxane B2 synthesis reached only 10% of its maximum. Together with experiments carried out with chlorpromazine - or PGE1 - treated platelets, our results suggest the existence of a close relationship between TPI-breakdown and dense body release which appear to be the earliest events resulting from the activation of human platelets by thrombin.     

Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP.

Elevation of intracellular cAMP is shown to increase the rate (V) and maximal extent of Ca2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt2-cAMP). The free concentration of Ca2+ in the dense tubular lumen ([Ca2+]dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca2+ concentration ([Ca2+]cyt) was monitored in parallel experiments with quin2. Both [Ca2+]cyt and [Ca2+]dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 microM external Ca2+, [Ca2+]cyt is approx. 50 nM and [Ca2+]dt is very low. When 2 mM external Ca2+ is added, [Ca2+]cyt rises to approx. 100 nM and the process of dense tubular Ca2+ uptake can be resolved. Forskolin (10 microM) and Bt2-cAMP increase the rate of dense tubular Ca2+ uptake (V) to 2.1 +/- 0.60 and 1.70 +/- 40 times control values (respectively). The agents also increase the final [Ca2+]dt to 1.70 +/- 0.21 and 1.72 +/- 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the Vm of the dense tubular Ca2+ pump. With [Iono] = 500 nM, [Ca2+]cyt was raised to greater than or equal to 1.0 microM and Vm of the dense tubular pump was elicited. (At [Iono] = 1.0 microM, the final [Ca2+]dt values were degraded 15% due to shunting of Ca2+ uptake.) Analysis showed that forskolin (10 microM) and Bt2-cAMP (1 mM) increase the Vm by a factors of 1.56 +/- 40 and 1.56 +/- 40, respectively. Analysis showed that neither agent changed the Km of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca2+ across the DT membrane (1.7 min-1).     

Calcium uptake and release by skeletal-muscle mitochondria

THE EGTA — ruthenium red quench technique was used to obtain initial-velocity plots of Ca²⁺ uptake by skeletal-muscle mitochondria. The K_m was 5 μM and the Hill coefficient 1.9 at both 0° and 10°C. Inorganic phosphate stimulated and Mg²⁺ inhibited initial rates of transport. In experiments on Ca²⁺ release, the $\text{Na}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{2+}}$ exchange was demonstrated. Factors influencing Ca²⁺ release during anaerobiosis include phosphate concentration and extent of Ca²⁺ loading. The results are discussed in relation to the possible participation of mitochondria in the calcium-ion regulation of muscle.     

Preparation and characterization of synthetic models for the dense bodies of human platelets

Calcium-pyrophosphate-nucleotide precipitates have been formed $\text{in}$$\text{vitro}$ with a composition which is essentially identical to that reported for the dense bodies (storage vesicles) of human platelets. The solution composition and effect of pH on precipitate formation, as well as the dissolution behavior and degree of protonation of the various species, were determined. The precipitates have a similar composition and form equally well at pH 5.7 or 7.4, or with various monovalent counter ions in the starting solutions. In all cases, the adenine nucleotides appear to be associated with fewer protons than the corresponding species in free solution, and all precipitate components can exchange or dissolve rapidly unless the precipitate is maintained in a solution relatively rich in calcium and adenine nucleotides. These precipitates may serve as useful models for examining the relative contributions of the dense-body membrane and core constituents to the uptake and storage of biogenic amines by human platelets.     

Galactose uptake by human platelets in vitro

Galactose transport by human platelets has been studied by measuring the cellular accumulation of the radiolabeled sugar during brief periods of suspension in varying concentrations of galactose. Weighted least-squares regression curves fitted to the measurements (initial velocity versus galactose concentration) indicate that a kinetic model with two saturable components is statistically more consistent with the data than a model based upon a single process ($\text{P < 0.001}$). For the two-component model $\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>1</mtext>}}\text{= 0.29}$ mM, $\text{V}{}_1\text{= 1.2}\text{mmol/min per 10}{}^{15}\text{platelets}$, $\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>2</mtext>}}\text{= 46}\text{mM}$, $\text{V}{}_2\text{= 117}\text{mmol/min per 10}{}^{15}\text{platelets}$. The fact that galactose metabolites did not accumulate during the initial phase of uptake indicates that the uptake process is not mediated by enzymatic catalysis. Surface binding also appears inadequate to explain the uptake. The most likely basis for the kinetic data, therefore, is membrane transport. The kinetics are consistent with transport by coexistent membrane structures as well as with transport by a single structure manifesting negative cooperativity.     

Nature and kinetic characteristics of L-DOPA uptake in rat renal proximal tubules

Summary The present study was performed with the aim to determine the kinetics and the caracteristics of cellular uptake of L-3,4-dihydroxyphenylalanine (L-DOPA) in rat renal proximal tubules. Incubation of renal tubules at 4°C in the presence of increasing concentrations of L-DOPA results in a linear and concentration-dependent accumulation of the substrate. In experiments carried out at 37°C, the accumulation of L-DOPA in renal tubules was found to be greater than that occurring at 4°C and showed a trend for saturation. The saturable component of L-DOPA uptake was derived from the total amount of L-DOPA accumulated in renal tubules at 37°C subtracted with the values obtained in experiments conducted at 4°C. The V_max and K_m values for the saturable component of L-DOPA uptake in renal tubules were, respectively, 241 ± 32 fmol µg protein^–1min^–1 and 567 ± 63 µM. Cyanine 863 (5 and 10 µM) was found to decrease the tubular uptake of L-DOPA, whereas probenecid (50 µM) did not change the rate of uptake of L-DOPA into renal tubules. The V_max and K_m values for the saturable component of L-DOPA uptake in renal tubules incubated in the presence of 10 µM cyanine 863 were, respectively, 97 ± 11 fmol µg protein^–1min^–1 and 160 ± 22 µM. It is suggested that the anionic L-DOPA may behave as an amphoteric substance, both hydroxyl groups in the aromatic ring determining the binding of the molecule to the organic cation transporter.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

An improved method for the isolation of dense storage granules from human platelets

Pretreatment of human platelets with the metabolic inhibitors rotenone and 2-deoxyglucose, before French press homogenization, has led to the isolation of dense storage granules in an overall yield of about 20%. The concentrations of serotonin, ATP and ADP were estimated in the dense granules. Serotonin was 40--60-fold enriched in the dense granules compared to the platelet homogenate. Stored ATP and ADP were also 40-fold enriched in the dense granules compared to the estimated storage nucleotide pool in intact platelets. The ATP to ADP ratio in the isolated dense granules was 0.68-0.70, the same as the ratio of the secreted ATP and ADP. In platelets prelabeled with [3H]adenine, the specific radioactivities of the ATP and ADP in the isolated dense granules and of the secreted ATP and ADP were both negligible, whereas the estimated specific radioactivity of the metabolically active ATP and ADP was 2,000 cpm/nmol. These results confirm that the ATP and ADP in the isolated dense granules are the same as the secreted ATP and ADP in terms of metabolic inactivity and their ATP to ADP ratios.     

Effects of cadmium treatment in vitro on the uptake and release of [3H]serotonin by human blood platelets

Effects of cadmium treatment on human platelets were studied with respect to uptake and release of 5-[3H]hydroxytryptamine (5-HT). The uptake of 5-[3H]HT in the presence of varying concentrations of CdCl2 (0.001-10 mM) was inhibited significantly with respect to control platelets and the inhibition was maximum at 1 mM CdCl2 concentration. From studies on the kinetics of 5-[3H]HT uptake a higher Km and significantly lower Vmax for CdCl2-treated platelets were observed. CdCl2 stimulated spontaneous release but inhibited thrombin-induced release of 5-[3H]HT. Spontaneous release of 5-[3H]HT induced by CdCl2 was not significantly altered in the presence of externally available CaCl2 (1 mM).