Characterization of [ 3 H]idazoxan binding sites on human platelets

Human platelets possess at least two non-adrenoceptor binding sites pharmacologically distinct from the f 2 -adrenoceptors. The effects of various imidazol(in)es on platelet aggregation have suggested that these compounds may interact with these non-adrenoceptor binding sites on platelets. [ 3 H]Idazoxan is an antagonist of the f 2 -adrenoceptors frequently used to characterize imidazoline I 2 receptors. We evaluated the binding of [ 3 H]idazoxan to human platelet membranes. In saturation experiments [ 3 H]idazoxan (1.25-32 nM) recognized a single, saturable binding site with high affinity. However, competition assays revealed the presence of f 2A -adrenoceptors and a non-adrenoceptor minor population (25-39%) recognized with high affinity by the imidazoline drug with low affinity for f 2 -adrenoceptors 2-BFI. After the addition of (-)adrenaline (5 w M) to mask f 2 -adrenoceptors, competition curves against [ 3 H]idazoxan binding were biphasic. The imidazoline I 1 receptor-selective drugs, efaroxan and rilmenidine, recognized the minor component with high affinity, whereas the imidazoline I 2 receptor-selective drugs, guanabenz and 2-BFI, bound with high affinity to the major component. Further masking experiments in the presence of efaroxan (2 w M) or guanabenz (1 w M) confirmed that [ 3 H]idazoxan labels two non-adrenoceptor binding sites pharmacologically compatible with imidazoline I 1 and I 2 receptors as well as f 2A -adrenoceptors in human platelets.     

The effect of thrombin on the uptake and transformation of arachidonic acid by human platelets

Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis.     

Increased arachidonic acid uptake by platelets from insulin-dependent diabetics and diabetic rats

14C-Arachidonic acid uptake was measured in platelets obtained from 15 insulin-dependent diabetic and 17 control subjects and in 12 streptozotocin-diabetic and 21 control rats. The 14C-arachidonic acid uptake, expressed as pmol/10(8) platelets/min, mean +/- SEM, was significantly higher in platelets from diabetic subjects (2.80 +/- 0.23) and diabetic rats (1.73 +/- 0.20) than in the control subjects (2.29 +/- 0.15) and the control rats (1.35 +/- 0.08). No significant correlations were found between arachidonic acid uptake and glucose, total cholesterol or triglyceride plasma levels in either rats or humans. Arachidonic acid uptake was inhibited by indometacin but not by nordihydroguaiaretic acid, in diabetic as well as in control subjects. The present results suggest that the increased arachidonic acid uptake by platelets from insulin-dependent diabetic patients and streptozotocin-diabetic rats depends on their increased platelet arachidonic acid utilization through the cyclooxygenase pathway.     

Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets

Arachidonic acid incubated with human platelets was converted into three compounds, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, 12L-hydroxy-5,8,10-heptadecatrienoic acid, and the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid. The formation of the two latter compounds from arachidonic acid proceeded by pathways involving the enzyme, fatty acid cyclo-oxygenase, in the initial step and with the prostaglandin endoperoxide, PGG(2), as an intermediate. The first mentioned compound was formed from 12L-hydroperoxy-5,8,10,14-eicosatetraenoic acid, which in turn was formed from arachidonic acid by the action of a novel lipoxygenase. Aspirin and indomethacin inhibited the fatty acid cyclo-oxygenase but not the lipoxygenase, whereas 5,8,11,14-eicosatetraynoic acid inhibited both enzymes. The almost exclusive transformation of the endoperoxide structure into non-prostaglandin derivatives supports the hypothesis that the endoperoxides can participate directly and not by way of the classical prostaglandins in regulation of cell functions. The observed transformations of arachidonic acid in platelets also explain the aggregating effect of this acid.     

Release of platelet-derived growth factor from human platelets by arachidonic acid

Platelet alpha-granules contain a factor that stimulates the proliferation of arterial smooth muscle cells and may play a role in atherogenesis. We have studied the role of arachidonic acid in mediating the release of the platelet-derived growth factor (PDGF) from human platelets. PDGF was assayed by stimulating of [(3)H]thymidine incorporation into DNA of mouse 3T3 cells. Platelet aggregation and the release of platelet factor 4,beta-thromboglobulin, and serotonin were also studied. A biphasic response pattern was observed when gel-filtered platelets were incubated with arachidonate over the concentration range 0.01-0.4 mM. At low arachidonate levels (approximately 0.025-0.1 mM), specific concentration-dependent aggregation and release of PDGF and of the other components were observed. This effect was not seen with any of five other fatty acids tested and was suppressed by indomethacin (25 muM). At higher arachidonate concentrations (approximately 0.15-0.35 mM), a concentration-dependent turn-off of both aggregation and release occurred. At these concentrations the platelets remained functional, and no release of lactate dehydrogenase was observed. A similar biphasic pattern of arachidonate-induced aggregation and release was observed with platelet-rich plasma, over a similar range of arachidonate to albumin mole ratios. These studies demonstrate that PDGF and other alpha-granule constituents can be released from platelets specifically by arachidonate via an indomethacin-sensitive pathway, most probably involving the platelet cyclooxygenase and conversion of arachidonate to prostaglandin metabolities. The mechanisms responsible for the turn-off of the specific arachidonate-mediated responses at higher arachidonate concentrations remain to be defined.     

Phorbol esters increase [(3)h]-dopamine uptake by human platelets

Dopamine is taken up by platelets by an energy and temperature dependent process that does not involve a known dopamine receptor. Whilst dopamine uptake by platelets has been shown to be altered in several disease states, little is known about factors controlling dopamine uptake by platelets which could cause such changes. As phorbol esters have been shown to affect dopamine functions in other tissues we examined the effect of phorbol esters on dopamine uptake by human platelets. Phorbol 12,13-dibutyrate increased [(3)H]-dopamine uptake by platelets in a dose dependent manner. Similarly, phorbol 12,13-didecanoate increased [(3)H-dopamine uptake by platelets but 4α phorbol 12,13-didecanoate, which does not affect protein kinase, did not. Staurosporin, a protein kinase inhibitor, reversed the effect of phorbol 12,13-dibutyrate. These data suggest protein kinases can modulate dopamine uptake by platelets.     

Selective labeling of serotonin receptors by d-[3H]lysergic acid diethylamide in calf caudate.

Since it was known that d-lysergic acid diethylamide (LSD) affected catecholaminergic as well as serotoninergic neurons, the objective in this study was to enhance the selectivity of [3H]LSD binding to serotonin receptors in vitro by using crude homogenates of calf caudate. In the presence of a combination of 50 nM each of phentolamine (added to preclude the binding of [3H]LSD to alpha-adrenoceptors), apomorphine, and spiperone (added to preclude the binding of [3H]LSD to dopamine receptors), it was found by Scatchard analysis that the total number of [3H]LSD sites went down to 300 fmol/mg, compared to 1100 fmol/mg in the absence of the catecholamine-blocking drugs. The IC50 values (concentrations to inhibit binding by 50%) for various drugs were tested on the binding of [3H]LSD in the presence of 50 nM each of apomorphine (A), phentolamine (P) and spiperone (S). With this combination, the IC50 for serotonin was 35 nM (compared to 1000 nM without it), indicating that [3H]LSD had become considerably more selectively displaceable by serotonin under these conditions whereas the effects of norepinephrine and dopamine on [3H]LSD binding were eliminated. Various ergots had approximately equal IC50 values against [3H]serotonin and [3H]LSD but tryptamines were much more selective against [3H]serotonin; the data may indicate the existence of the two types of serotonin receptors.     

Melatonin effect on arachidonic acid metabolism to cyclooxygenase derivatives in human platelets

The effect of melatonin on thrombin-induced [3H]-arachidonic acid (AA) metabolism to cyclooxygenase derivatives was determined in platelets obtained from normal volunteers at 0830 and 2030 h. Percent conversion of radioactive AA was generally greater at 2030 h than at 0830 h for every cyclooxygenase derivative analyzed. Micromolar or greater concentrations of melatonin decreased significantly the conversion of [3H]-AA to prostaglandin (PG) F2 and thromboxane (Tx) B2, and inhibited slightly the conversion to PGE2 and PGD2. After preincubation of platelets with 1 mM imidazole, the melatonin inhibitory effect was significant for PGF2 only. Melatonin (10(-6) M) showed a significant inhibitory influence on platelet ATP release induced by phorbol-12 myristate-13 acetate (PMA) at 2030 h, an effect inhibited by 1 mM aspirin. These results indicate that at pharmacological concentrations melatonin inhibits human platelet cyclooxygenase.     

Evidence for the generation of hydroxyl radical during arachidonic acid metabolism by human platelets

Reactive oxygen species, probably hydroxyl radicals (OH.), have been suggested to be generated during arachidonic acid (AA) metabolism and, once released, these species can modify the rate and extent of various reactions involved in AA metabolism. We have studied this phenomenon in washed human platelets. OH. generation was quantitated using 14C-benzoic acid as a specific trap in a continuous ionization chamber system. Resting platelets did not produce any detectable signal, whereas addition of AA resulted in gradual OH. production with peak values detected at approximately 20 min. Similar studies conducted under nitrogen or after boiling the platelets almost abolished OH. generation. Aspirin had no significant effect, whereas 5,8,11,14-eicosatetraynoic acid decreased the signal by greater than 90%, thus suggesting that OH. is produced primarily through the lipoxygenase pathway. Superoxide dismutase (SOD) and catalase had no effect and, as expected, phenol and mannitol decreased OH. production considerably, by greater than 50% and 90%, respectively. Azide and cyanide also reduced the OH. generation by about two thirds. We conclude that OH. is generated during AA metabolism by human platelets. It is primarily produced via the lipoxygenase pathway and may require a heme-dependent peroxidase. This highly reactive oxidant may play an important role in normal and abnormal hemostasis.     

Characterization of [(3)H]idazoxan binding sites on human platelets

Human platelets possess at least two non-adrenoceptor binding sites pharmacologically distinct from the alpha(2)-adrenoceptors. The effects of various imidazol(in)es on platelet aggregation have suggested that these compounds may interact with these non-adrenoceptor binding sites on platelets. [(3)H]Idazoxan is an antagonist of the alpha(2)-adrenoceptors frequently used to characterize imidazoline I(2) receptors. We evaluated the binding of [(3)H]idazoxan to human platelet membranes. In saturation experiments [(3)H]idazoxan (1.25-32 nM) recognized a single, saturable binding site with high affinity. However, competition assays revealed the presence of alpha(2A)-adrenoceptors and a non-adrenoceptor minor population (25-39%) recognized with high affinity by the imidazoline drug with low affinity for alpha(2)-adrenoceptors 2-BFI. After the addition of (-)adrenaline (5 microM) to mask alpha(2)-adrenoceptors, competition curves against [(3)H]idazoxan binding were biphasic. The imidazoline I(1) receptor-selective drugs, efaroxan and rilmenidine, recognized the minor component with high affinity, whereas the imidazoline I(2) receptor-selective drugs, guanabenz and 2-BFI, bound with high affinity to the major component. Further masking experiments in the presence of efaroxan (2 microM) or guanabenz (1 microM) confirmed that [(3)H]idazoxan labels two non-adrenoceptor binding sites pharmacologically compatible with imidazoline I(1) and I(2) receptors as well as alpha(2A)-adrenoceptors in human platelets.     

Nuclear uptake of 1,25-dihydroxy[3H]cholecalciferol in dispersed fibroblasts cultured from normal human skin

Because of the relative inaccessibility of known calciferol target tissues (i.e., intestine and bone), we examined fibroblasts derived from normal human skin and grown in tissue culture as a means of evaluating the interaction of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and its effector system. When dispersed, intact cells were used, nuclear uptake of 1,25-dihydroxy[23,24(n)3-H]cholecalciferol [1,25(OH)2[3H]D3) was temperature-dependent, optimal at 45 min at 37 degrees C, and saturable. In competition experiments with other calciferols, the 1,25(OH)2[3H]D3 uptake showed specificity indistinguishable from that reported for 1,25(OH)2D3 receptors from calciferol target tissues. Analysis of 1,25(OH)2[3H]D3 nuclear uptake in fibroblast strains from six normal adults (four male, two female) yielded an average binding capacity (R0) of 10,600 +/- 2,000 (SEM) nuclear sites per cell and an apparent dissociation contant (Kd) of 0.50 +/- 0.07 (SEM) x 10(-9) M. Donor sex, donor age, or anatomic site of origin of the cell line did not affect the characteristics of uptake. Similar nuclear uptake was demonstrable with cultured MCF-7 cells (derived from human breast cancer) when assayed in the same fashion. When hypertonic extracts of nuclei obtained from skin fibroblasts incubated with 1,25(OH)2[3H]D3 were subjected to centrifugation on sucrose gradients, a single peak of radioactivity sedimented at approximately 3 S; when excess 1,25(OH)2D3 was coincubated during the cellular uptake phase, this 3S peak was not observed. Molybdate was an essential buffer component for receptor stabilization during cell fractionation and sedimentation analysis. In summary, by using fibroblasts cultured from normal human skin, we have identified a process of nuclear uptake of 1,25(OH)2[3H]D3 with the affinity, saturability, and specificity characteristics of a steroid hormone--receptor interaction. This method should be useful in studying 1,25(OH)2D3 recept physiology in cells from normal persons as well as in cells from patients who have disorders in the responsiveness of calciferol target tissues.     

An alternative method for the synthesis of 11-[3H]ketotestosterone and 11β-[3H]hydroxytestosterone from [3H]cortisol

Experimental conditions for the synthesis of radioactive 11-ketotestosterone and 11β-hydroxytestosterone from cortisol of high specific activity are described. For the synthesis of 11-ketotestosterone, cortisol is first oxidized to cortisone with 0.1% aqueous chromic acid. The use of hydroxysteroid dehydrogenases for reduction of intermediates in the reaction sequence from C₂₁ to C₁₉ steroid provides consistently high yields of product.     

Arachidonic acid uptake by human platelets is mediated by CD36

The involvement of glycoprotein (GP) IV (CD36) in arachidonic acid uptake by human platelets was investigated using an anti-CD36 monoclonal antibody (MAB). The binding of [(14)C]arachidonic acid to MAB-treated platelets was significantly reduced compared with untreated platelets. The MAB also inhibited arachidonic acid-induced platelet aggregation and thromboxane A(2) synthesis in a dose-dependent manner. Pre-incubation of gel-filtered platelets with the MAB (10mg/I) inhibited arachidonic acid-induced platelet aggregation by 50% and collagen-induced platelet aggregation by 7-8% and the lag time was increased by 200%. Although the mechanism of platelet aggregation is not fully understood yet, the inhibition of arachidonic acid-induced platelet aggregation by the MAB could be the result of a reduced uptake of exogeneously added arachidonic acid by the MAB-treated platelets. Our data clearly indicate that arachidonic acid uptake by platelets is mediated, at least in part, by CD36.     

Arachidonic acid uptake by human platelets is mediated by CD36

The involvement of glycoprotein (GP) IV (CD36) in arachidonic acid uptake by human platelets was investigated using an anti-CD36 monoclonal antibody (MAB). The binding of [¹⁴C]arachidonic acid to MAB-treated platelets was significantly reduced compared with untreated platelets. The MAB also inhibited arachidonic acid-induced platelet aggregation and thromboxane A₂ synthesis in a dose-dependent manner. Pre-incubation of gel-filtered platelets with the MAB (10mg/I) inhibited arachidonic acid-induced platelet aggregation by 50% and collagen-induced platelet aggregation by 7-8% and the lag time was increased by 200%. Although the mechanism of platelet aggregation is not fully understood yet, the inhibition of arachidonic acid-induced platelet aggregation by the MAB could be the result of a reduced uptake of exogeneously added arachidonic acid by the MAB-treated platelets. Our data clearly indicate that arachidonic acid uptake by platelets is mediated, at least in part, by CD36.     

Interplexiform cell in cat retina: identification by uptake of gamma-[3H]aminobutyric acid and serial reconstruction.

After intravitreal injection of gamma-[3H] aminobutyric acid (GAB), 2% of the neurons at the outer margin of the inner plexiform layer were intensely labeled. Reconstructions of these neurons from serial electron microscope autoradiograms showed that they are interplexiform cells, which synapse on bipolar processes in the outer plexiform layer and on amacrine and bipolar processes in the inner plexiform layer.