Pharmacological Research, Vol. 26, No. 2, 1992
187
NON-SEROTONERGIC 3H-KETANSERIN B I N D I N G S I T E S IN HUMAN
PLATELETS:
CHARACTERISTICS
AND INTERACTION WITH ANTAGONISTS
CALCIUM
DANIELA OLIVA*, FELICE POCCHIARI'~, LEOPOLDO ALLIEVIt, G. ENRICO ROVATI* and SIMONETTA NICOSIA*
*Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan and tCardiovascular Department, Zambon Research S.p.A., Via Cimabue 28, 20032 Cormano (Mi), Italy Received in final form 26 February 1992
SUMMARY Here we report the identification of binding sites for 3H-ketanserin in human platelet membranes. At 4~ 3H-ketanserin binding is saturable (Bmax= 0.58 pmol/mg protein), rapid (equilibrium being attained within 20 min) and reversible. The kinetics of the association and dissociation curves are consistent with the existence of a single class of binding sites, as confirmed also by computer-assisted analysis of the saturation curve. Specific binding is increased by Ca 2+ and Mg 2+. 3H-ketanserin binding is inhibited by serotonin (K,=48.5/.tM), unlabeled ketanserin (K,=3-15 nM), as well as by another antiserotonergic drug, methysergide (K,=32.6pM). However, other selective 5-HT2 ligands, such as ritanserin, spiperone and cyproheptadine fail to interact with 3H-ketanserin binding. On the contrary, tetrabenzine, a monoamine depleting agent, when preincubated at 30 ~ did inhibit the specific binding completely. 3H-ketanserin specific binding is inhibited in a dose-dependent fashion by some calcium blocking agents, with different potencies: verapamil (K,=2.25 pM), diltiazem (K,= 139/tM) and SIM6080, a new Ca2+-antagonist related to the phenylalkylamines (K,=5.22pM). Flunarizine inhibited 3H-ketanserin specific binding only at relatively high concentrations (IC50> 100/.tM), while nitrendipine did not show any inhibitory effect up to 20/tM. The present evidence indicates that all the sites labeled by 3H-ketanserin at 4 ~ might be coincident with the monoamino transporter identified in other systems, and that they might play a role in the modulation of platelet aggregation exerted by some calcium blocking agents. KEYWORDS:ketanserin, binding, human platelets, calcium antagonists, serotonin.
Address correspondence to: Prof. S. Nicosia. 1043-6618/92/060187-13/$08.00/0
9 1992 The Itahan Pharmacological Society
188
Pharmacological Research, Vol. 26, No. 2, 1992
INTRODUCTION Recently, much effort has been devoted to define the role of serotonin (5hydroxytryptamine, 5-HT) in nervous and non-nervous tissues. The resurgence of interest in serotonin is well documented by the proliferation of 5-HT receptor subtypes that have been identified either by the use of radioligand binding techniques or on the basis of functional and biochemical studies [1-3]. As for the peripheral effects of serotonin, it is well known that the monoamine causes platelet aggregation and shape change in various mammalian species including man, although with a marked species variability [4]. The above mentioned effects in human platelets are antagonized by ketanserin (a specific 5HT2 antagonist) [5] and other antiserotonergic drugs [1, 6] indicating that 5-HT interacts at human platelet level with the type 2 receptor subtype. However, although the existence of specific binding sites labeled by 3Hketanserin have been reported in cat blood platelets [7], no direct evidence for the existence in plasma membranes from human platelets of binding sites labeled by a specific 5-HT2 ligand had been reported until very recently. In fact, the only data available from the literature on the nature of serotonin receptors in human platelets by means of radioligand studies had been obtained with 3H-lysergic acid diethylamide (LSD) [8] and ~25I-LSD [9], which also labels 5-HT~ and dopamine receptor sites [10, 11], or using 3H-serotonin [12, 13], which can interact either with 5-HT2-sites associated with platelet aggregation or with 5-HT uptake sites [13]. Therefore, the aim of the present work was firstly to characterize the binding sites specific for 3H-ketanserin in membranes of human platelets and secondly to investigate their relationship with the calcium entry blockers, which inhibit platelet aggregation through unknown mechanisms.
MATERIALS AND METHODS Materials
3H-ketanserin (60-90 Ci/mmol) was obtained from New England Nuclear (Boston, USA). Unlabeled ketanserin was from Zambon Research (Cormano, Italy) and dissolved in 1% ethanol (1 mM) with 0.3% bovine serum albumin (BSA). Further dilutions were prepared with 50 mM Tris-HCl, pH 7.4 containing 0.3% BSA. The inclusion of BSA in the buffer was justified by the finding that 3Hketanserin was highly adsorbed to glass tubes and fibre filters, and BSA decreased this aspecific absorption. Fluoxetine and tetrabenazine were purchased from Eli, Lilly and Co. (Indianapolis, IN, USA) and Fluka (Buchs, Switzerland), respectively. Due to solubility problems, spiperone, (+)8-OH-DPAT (8-hydroxy2-(di-n-propylamino)-tetralin), flunarizine, nitrendipine (Zambon Research, Cormano, Italy) and prazosin (Sigma, USA) were dissolved in dimethylsulphoxide (DMSO) and diluted with the buffer to yield a final concentration of 2% DMSO in the sample. Clonidine, cyproheptadine, methiothepin maleate, diltiazem, verapamil and SIM6080 (Zambon Research, Cormano, Italy) were all dissolved in distilled water and the further dilutions were peformed in water containing 0.3%
Pharmacological Research, Vol. 26, No. 2, 1992
189
BSA. Ritanserin (Janssen Farmaceutici S.p.A., Latina, Italy) was dissolved in ethanol and further dilutions were prepared in buffer with 0.3% BSA (0.16% final ethanol concentration in the sample). The inclusion of 2% DMSO or 0.2% distilled water or 0.16% ethanol in the binding assay did not modify either the total or the non-specific binding.
Preparation of platelet membranes Blood was collected in citric acid/sodium citrate/sodium phosphate/dextrose and platelet concentrates from 4-5 healthy male individuals were pooled. A crtide membrane preparation (pellet at 27 000 g) of washed platelets was obtained as previously described [14].
Binding assay Standard assays were carried out in a total volume of 250 pl containing 50 mM Tris-HC1, pH 7.4, approximately 3-4 nM 3H-ketanserin (60-90 Ci/mmol), 1 mM CaCI2, 1 mM MgC12, 0.3% BSA (to prevent ketanserin adsorption to glass tubes and filters), unlabeled agents at the indicated concentrations and the membrane preparation (0.10-0.25 mg protein/sample). The inclusion of 0.3% BSA in the incubation buffer and its use for filter pretreatment and washing did not per se interfere with 3H-ketanserin binding to human platelet membranes. Unless otherwise stated, the incubation, started with the addition of the labeled ligand, was carried out at 4 ~ for 30 min. Bound 3H-ketanserin was separated from free ligand by filtration under vacuum through Whatman GF/C filters, pre-washed with 50 mM Tris-HCl, pH 7.4 containing 0.3% BSA, followed by a wash with 2x3 ml of the same buffer at 4 ~ Filtration and washing were completed in less than 8 s. Bound 3H-ketanserin was extracted from the filters with 10ml Filtercount (Packard) and measured in the same scintillation fluid. Non specific binding was obtained by addition of 3 pM ketanserin and represented approximately 15-25% of the total binding.
Computer analysis of binding curves The equilibrium data were fitted by an iterative program for non-linear regression analysis (LIGAND) [15] in order to obtain Bmax (maximal number of binding sites), Kd (dissociation constant for the radiolabeled ligand), K, (dissociation constant for unlabeled ligands) and N (non-specific binding). Curves of at least two experiments were analyzed simultaneously. The data were fitted both to a one- and a two-site model. The two-site model was discarded when the improvement in goodness-of-fit was not statistically significant (P>0.05) according to the F test used for the 'extra sum of squares' principle. In order to evaluate the statistical significance of the difference of the plateaus in the binding curves of 3H-ketanserin in the absence and presence of methysergide (see Figs. 5 and 6), the curves for the two ligands were fitted simultaneously with the ALLFIT program [16], both without any constraint on the parameter values and imposing the identity of either the lower or the upper plateau. The plateaus were considered significantly different when the constrained fitting was significantly worse (P
187
NON-SEROTONERGIC 3H-KETANSERIN B I N D I N G S I T E S IN HUMAN
PLATELETS:
CHARACTERISTICS
AND INTERACTION WITH ANTAGONISTS
CALCIUM
DANIELA OLIVA*, FELICE POCCHIARI'~, LEOPOLDO ALLIEVIt, G. ENRICO ROVATI* and SIMONETTA NICOSIA*
*Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan and tCardiovascular Department, Zambon Research S.p.A., Via Cimabue 28, 20032 Cormano (Mi), Italy Received in final form 26 February 1992
SUMMARY Here we report the identification of binding sites for 3H-ketanserin in human platelet membranes. At 4~ 3H-ketanserin binding is saturable (Bmax= 0.58 pmol/mg protein), rapid (equilibrium being attained within 20 min) and reversible. The kinetics of the association and dissociation curves are consistent with the existence of a single class of binding sites, as confirmed also by computer-assisted analysis of the saturation curve. Specific binding is increased by Ca 2+ and Mg 2+. 3H-ketanserin binding is inhibited by serotonin (K,=48.5/.tM), unlabeled ketanserin (K,=3-15 nM), as well as by another antiserotonergic drug, methysergide (K,=32.6pM). However, other selective 5-HT2 ligands, such as ritanserin, spiperone and cyproheptadine fail to interact with 3H-ketanserin binding. On the contrary, tetrabenzine, a monoamine depleting agent, when preincubated at 30 ~ did inhibit the specific binding completely. 3H-ketanserin specific binding is inhibited in a dose-dependent fashion by some calcium blocking agents, with different potencies: verapamil (K,=2.25 pM), diltiazem (K,= 139/tM) and SIM6080, a new Ca2+-antagonist related to the phenylalkylamines (K,=5.22pM). Flunarizine inhibited 3H-ketanserin specific binding only at relatively high concentrations (IC50> 100/.tM), while nitrendipine did not show any inhibitory effect up to 20/tM. The present evidence indicates that all the sites labeled by 3H-ketanserin at 4 ~ might be coincident with the monoamino transporter identified in other systems, and that they might play a role in the modulation of platelet aggregation exerted by some calcium blocking agents. KEYWORDS:ketanserin, binding, human platelets, calcium antagonists, serotonin.
Address correspondence to: Prof. S. Nicosia. 1043-6618/92/060187-13/$08.00/0
9 1992 The Itahan Pharmacological Society
188
Pharmacological Research, Vol. 26, No. 2, 1992
INTRODUCTION Recently, much effort has been devoted to define the role of serotonin (5hydroxytryptamine, 5-HT) in nervous and non-nervous tissues. The resurgence of interest in serotonin is well documented by the proliferation of 5-HT receptor subtypes that have been identified either by the use of radioligand binding techniques or on the basis of functional and biochemical studies [1-3]. As for the peripheral effects of serotonin, it is well known that the monoamine causes platelet aggregation and shape change in various mammalian species including man, although with a marked species variability [4]. The above mentioned effects in human platelets are antagonized by ketanserin (a specific 5HT2 antagonist) [5] and other antiserotonergic drugs [1, 6] indicating that 5-HT interacts at human platelet level with the type 2 receptor subtype. However, although the existence of specific binding sites labeled by 3Hketanserin have been reported in cat blood platelets [7], no direct evidence for the existence in plasma membranes from human platelets of binding sites labeled by a specific 5-HT2 ligand had been reported until very recently. In fact, the only data available from the literature on the nature of serotonin receptors in human platelets by means of radioligand studies had been obtained with 3H-lysergic acid diethylamide (LSD) [8] and ~25I-LSD [9], which also labels 5-HT~ and dopamine receptor sites [10, 11], or using 3H-serotonin [12, 13], which can interact either with 5-HT2-sites associated with platelet aggregation or with 5-HT uptake sites [13]. Therefore, the aim of the present work was firstly to characterize the binding sites specific for 3H-ketanserin in membranes of human platelets and secondly to investigate their relationship with the calcium entry blockers, which inhibit platelet aggregation through unknown mechanisms.
MATERIALS AND METHODS Materials
3H-ketanserin (60-90 Ci/mmol) was obtained from New England Nuclear (Boston, USA). Unlabeled ketanserin was from Zambon Research (Cormano, Italy) and dissolved in 1% ethanol (1 mM) with 0.3% bovine serum albumin (BSA). Further dilutions were prepared with 50 mM Tris-HCl, pH 7.4 containing 0.3% BSA. The inclusion of BSA in the buffer was justified by the finding that 3Hketanserin was highly adsorbed to glass tubes and fibre filters, and BSA decreased this aspecific absorption. Fluoxetine and tetrabenazine were purchased from Eli, Lilly and Co. (Indianapolis, IN, USA) and Fluka (Buchs, Switzerland), respectively. Due to solubility problems, spiperone, (+)8-OH-DPAT (8-hydroxy2-(di-n-propylamino)-tetralin), flunarizine, nitrendipine (Zambon Research, Cormano, Italy) and prazosin (Sigma, USA) were dissolved in dimethylsulphoxide (DMSO) and diluted with the buffer to yield a final concentration of 2% DMSO in the sample. Clonidine, cyproheptadine, methiothepin maleate, diltiazem, verapamil and SIM6080 (Zambon Research, Cormano, Italy) were all dissolved in distilled water and the further dilutions were peformed in water containing 0.3%
Pharmacological Research, Vol. 26, No. 2, 1992
189
BSA. Ritanserin (Janssen Farmaceutici S.p.A., Latina, Italy) was dissolved in ethanol and further dilutions were prepared in buffer with 0.3% BSA (0.16% final ethanol concentration in the sample). The inclusion of 2% DMSO or 0.2% distilled water or 0.16% ethanol in the binding assay did not modify either the total or the non-specific binding.
Preparation of platelet membranes Blood was collected in citric acid/sodium citrate/sodium phosphate/dextrose and platelet concentrates from 4-5 healthy male individuals were pooled. A crtide membrane preparation (pellet at 27 000 g) of washed platelets was obtained as previously described [14].
Binding assay Standard assays were carried out in a total volume of 250 pl containing 50 mM Tris-HC1, pH 7.4, approximately 3-4 nM 3H-ketanserin (60-90 Ci/mmol), 1 mM CaCI2, 1 mM MgC12, 0.3% BSA (to prevent ketanserin adsorption to glass tubes and filters), unlabeled agents at the indicated concentrations and the membrane preparation (0.10-0.25 mg protein/sample). The inclusion of 0.3% BSA in the incubation buffer and its use for filter pretreatment and washing did not per se interfere with 3H-ketanserin binding to human platelet membranes. Unless otherwise stated, the incubation, started with the addition of the labeled ligand, was carried out at 4 ~ for 30 min. Bound 3H-ketanserin was separated from free ligand by filtration under vacuum through Whatman GF/C filters, pre-washed with 50 mM Tris-HCl, pH 7.4 containing 0.3% BSA, followed by a wash with 2x3 ml of the same buffer at 4 ~ Filtration and washing were completed in less than 8 s. Bound 3H-ketanserin was extracted from the filters with 10ml Filtercount (Packard) and measured in the same scintillation fluid. Non specific binding was obtained by addition of 3 pM ketanserin and represented approximately 15-25% of the total binding.
Computer analysis of binding curves The equilibrium data were fitted by an iterative program for non-linear regression analysis (LIGAND) [15] in order to obtain Bmax (maximal number of binding sites), Kd (dissociation constant for the radiolabeled ligand), K, (dissociation constant for unlabeled ligands) and N (non-specific binding). Curves of at least two experiments were analyzed simultaneously. The data were fitted both to a one- and a two-site model. The two-site model was discarded when the improvement in goodness-of-fit was not statistically significant (P>0.05) according to the F test used for the 'extra sum of squares' principle. In order to evaluate the statistical significance of the difference of the plateaus in the binding curves of 3H-ketanserin in the absence and presence of methysergide (see Figs. 5 and 6), the curves for the two ligands were fitted simultaneously with the ALLFIT program [16], both without any constraint on the parameter values and imposing the identity of either the lower or the upper plateau. The plateaus were considered significantly different when the constrained fitting was significantly worse (P
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