The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y1 receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y1 receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y1 antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 microM) totally inhibited the [Ca2+]i rise induced by ADP (0.1 microM) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 microM) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 microM). A3P5P (1 mM) reduced the number of [33P]2MeSADP binding sites on control rat platelets from 907 +/- 50 to 611 +/- 25 per platelet. After clopidogrel treatment, binding of [33P]2MeSADP decreased to 505 +/- 68 sites per platelet and further decreased to 55 +/- 12 sites in the presence of A3P5P (1 mM). In summary, these results demonstrate that the platelet P2Y1 receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Labeling of A2A adenosine receptors in human platelets by use of the new nonxanthine antagonist radioligand [3H]SCH 58261

The present study describes the binding to human platelet A2A adenosine receptors of the new potent and selective antagonist radioligand [3H]5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine ([3H]SCH 58261). Saturation experiments revealed that [3H]SCH 58261 labels a single class of recognition sites with high affinity (Kd = 0.85 nM), limited capacity (apparent Bmax = 85 fmol/mg of protein) and good specific binding (about 60%). [3H]SCH 58261 binding was not modulated by either the divalent cation Mg(+2) or guanine nucleotides. In competition experiments, a series of both adenosine agonists and antagonists inhibited [3H]SCH 58261 binding to A2A platelet receptors with rank order of potency and affinity similar to those observed in rat striatal membranes with the same radioligand. This confirms that the platelet A2A receptor is similar to that labeled in the brain striatum. Binding data were also found to be in good agreement with the results from functional studies such as A2A agonist-induced stimulation of adenylate cyclase or platelet aggregation inhibition. The present findings indicate that [3H]SCH 58261 is the first radioligand available for the characterization of the A2A receptor subtype in platelets.     

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.     

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI2) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI2 (Kd1 = 9.1 +/- 2.0 nM; n1 = 170 +/- 32 sites per cell vs. Kd1 = 7.2 +/- 1.1 nM; n1 = 23 +/- 8 sites per cell), with no significant change in the low-affinity receptors (Kd2 = 1.9 +/- 0.1 microM; n2 = 1,832 +/- 232 sites per cell vs. Kd2 = 1. 6 +/- 0.1 microM; n2 = 1,740 +/- 161 sites per cell) as determined by Scatchard analysis of the binding of [3H]PGE1. The loss of high-affinity PGI2 binding led to the failure of PGI2 to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI2 to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [3H]PGE1 to the high-affinity PGI2 receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [3H]PGE1 to the high-affinity platelet PGI2 receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI2 binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.     

Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D

In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.     

A novel regulatory epitope defined by a murine monoclonal antibody to the platelet GPIIb-IIIa complex (alpha IIb beta 3 integrin)

We characterized a murine monoclonal antibody, PT25-2 (IgG1), raised against washed human platelets. The antibody and its Fab fragments were both capable of inducing platelet aggregation in a fibrinogen-dependent manner and induced 125I-fibrinogen binding to unstimulated platelets (120,000 molecules/platelet at a 100 nM IgG concentration). The antibody immunoprecipitated the alpha IIb beta 3 complex from lysates of iodinated platelets but did not react with the respective subunits when complex formation was disrupted by treatment with 5 mM EDTA at 37 degrees C for 30 min. However, simply removing the extracellular divalent cation with EDTA had no effect on antibody binding indicating that the antibody's epitope depends upon a conformational structure maintained by alpha beta subunit association. Antibody binding to unstimulated, washed platelets yielded binding parameters (Kd = 40 nM, Bmax = 100,000 molecules/platelet), which were found to be virtually unchanged when binding was performed using thrombin or RGDS-peptide-stimulated platelets. Thus, the PT25-2 antibody defines a novel regulatory epitope expressed by the alpha IIb beta 3 integrin on unstimulated, quiescent platelets.     

Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.     

Multiple alpha 2 adrenergic receptor subtypes. I. Comparison of [3H]RX821002-labeled rat R alpha-2A adrenergic receptors in cerebral cortex to human H alpha2A adrenergic receptor and other populations of alpha-2 adrenergic subtypes

In the present study, we examined the binding of the alpha-2 adrenergic receptor (AR) antagonist [3H]-(2-(2-methoxy-1,4-benzodioxan- 2yl)-2-imidazoline ([3H]RX821002) to alpha-2 AR in rat cerebral cortex (CC) and compared the properties of these sites to those of rat alpha-2A (R alpha-2A) AR in submaxillary gland (SMG), human alpha-2A (H alpha-2A) AR in human platelets and alpha-2B AR in neonatal rat lung. In the presence of guanidinium phosphate, [3H]RX821002 bound with high affinity to a large and homogeneous population of sites in CC (Kd = 0.30 +/- 0.03 nM and Bmax = 271 +/- 7 fmol/mg of protein), SMG (Kd = 0.7 and Bmax = 274), human platelets (Kd = 0.6 nM and Bmx = 189) and neonatal rat lung (kd = 0.9 and Bmax = 161). A total of 34 chemically diverse AR ligands monophasically inhibited the binding of [3H]RX821002 from each site with, for the CC, the most potent ligand being atipamezole (Ki = 0.2 nM). For all ligands, and at each site, Hill coefficients did not differ significantly from unity. Although the profiles of inhibition of [3H]RX821002 were virtually identical in rat CC and SMG, these populations revealed several marked differences to human platelets; the alkaloids, rauwolscine and yohimbine, as well as the benzodioxane, [2-(2,6- dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane] (WB 4101), displayed about 10-fold lower affinity for R alpha-2A as compared to H alpha-2A sites, whereas the benzopyrrolidines, fluparoxan and des-fluorofluparoxan, showed about 10-fold greater affinity for R alpha-2A sites. Further, whereas the calculation of potency ratios for selected pairs of ligands, as well as of correlation coefficients, revealed virtual identity between R alpha-2A AR in CC and SMG, these analyses revealed that each of these populations of R alpha-2A AR clearly differed to H alpha-2A AR in human platelets. In addition, both R alpha-2A AR in rat CC and SMG as well as H alpha-2A AR in human platelets markedly differed to alpha-2B AR in neonatal rat lung; thus, they showed 20-fold higher affinity for [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5- dihydroimidazoline] (BRL 44408), oxymetazoline, guanfacine and guanabenz yet 10- to 100-fold lower affinity for [2-(2-4-o- methoxyphenyl)piperazine-1-yl)-ethyl)-4,4-dimethyl-1,3-(2H,4H)- isoquinolinedione] (ARC 239) prazosin, chlorpromazine and corynanthine. Similar differences in R alpha-2A and H alpha-2A sites to alpha-2C sites were apparent upon analysis of literature data.(ABSTRACT TRUNCATED AT 400 WORDS)     

XV454, a novel nonpeptide small-molecule platelet GIIb/IIIa antagonist with comparable platelet alpha(IIb)beta3-binding kinetics to c7E3

XV454 demonstrated high potency (IC50 = 14-25 nM) in inhibiting human platelet aggregation induced by adenosine diphosphate (ADP, 10 microM), thrombin receptor agonist peptide (TRAP) (10 microM), or collagen (20 microg/ml). XV454 exhibited a high degree of selectivity for platelet alpha(IIb)beta3 in comparison with c7E3, which is a nonspecific antagonist for both alpha(IIb)beta3 and alpha(v)beta3. Both XV454 and c7E3 bind with high affinity to either activated (A) or unactivated (U) human, baboon, or canine platelets. XV454 binds with a relatively higher affinity [Kd = 0.5 nM (A), 0.6 nM (U)] as compared with c7E3 [Kd = 9.1 nM (A), 9.2 (U) nM]. XV454 demonstrated a tight association with human, baboon, and, to a lesser extent, with canine platelets (t(1/2) of dissociation = 110 +/- 6, 80 +/- 10, and 23 +/- 2 min, respectively). Both c7E3 and XV454 associate tightly with a slower dissociation rate with unactivated human platelets: t(1/2) of 42 and 116 min, respectively. In non-human primates, oral (0.1 mg/kg, p.o.) and intravenous (0.05 mg/kg, i.v. bolus administration of XV454 methyl ester pro-drug resulted a long-lasting maximal antiplatelet efficacy for < or = 72 h with significant but reversible prolongation of bleeding time and without effects on platelet count, clinical chemistry, or hemodynamic profile. In conclusion, XV454 represents a potent antiplatelet agent in inhibiting platelet aggregation along with a high affinity and relatively slow dissociation rate from human platelet GPIIb/IIIa receptors that allow a long-lasting antiplatelet efficacy after single i.v. or oral administration.     

Slide-binding characterization and autoradiographic localization of delta opioid receptors in rat and mouse brains with the tetrapeptide antagonist [3H]TIPP

Slide-binding and autoradiographic studies were performed on cryostat sections from brains of adult Sprague-Dawley rats and BALB C mice to describe the binding characteristics of the tetrapeptide [3H]TIPP, an antagonist with high specificity and affinity for the delta opioid receptors. Steady-state binding of [3H]TIPP to cryostat sections of brain paste was reached in 120-180 min of incubation. Specific [3H]TIPP binding resulted in maximal numbers of binding sites (Bmax) of 15.59 and 23.91 fmol/mg protein, and dissociation constants (Kd) of 0.46 and 0.85 nM for rat and mouse brain paste sections, respectively. TIPP displayed the highest affinity for delta opioid receptors in inhibiting specific [3H]TIPP binding, with IC50 values of 0.82 nM and 0.14 nM in rat and mouse brain sections, respectively. While DPDPE was also effective in displacing the specific binding of [3H]TIPP (IC50 = 3.18 +/- 0.53 nM and 0.63 +/- 0.42 nM in rat and mouse brain paste sections, respectively), other subclass-selective or nonopioid ligands were much less effective, or ineffective. Autoradiographic localization of [3H]TIPP binding revealed the characteristic distribution of delta opioid receptors in both species. In consequence of its antagonistic nature, and of its unnatural amino acid residue, which makes this ligand more resistant to biodegradation, [3H]TIPP is a superior ligand for evaluation of the binding characteristics and autoradiogaphic distribution of the delta opioid receptors.     

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [3H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT7 receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT ([3H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 microM) displayed a pharmacological profile similar to the recombinant 5-HT7 receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT recognition sites also resembled, pharmacologically, the 5-HT7 receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [3H]5-CT binding to residual, possibly, 5-HT1A sites. Competition for this [3H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT7 receptor. Saturation studies also indicated that (+/-)-pindolol (10 microM)/WAY 100635 (100 nM)-insensitive [3H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (Bmax=33.2+/-0.7 fmol mg(-1) protein, pKd=8.78+/-0.05, mean+/-S.E.M., n=3). The development of this 5-HT7 receptor binding assay will aid investigation of the rat native 5-HT7 receptor.     

On the susceptibility of human platelets to aggregation by concanavalin A and the effect of this lectin on their response to ADP

Concanavalin A aggregated gel-filtered platelets in 0.9% NaCl solution signifying cross-bridging by the lectin. Aggregation of these platelets by concanavalin A was temperature dependent; it did not occur at 0-4 degrees C unless the platelets were previously trypsinized. The level of aggregation of trypsinized platelets by concanavalin A at 0-4 degrees C was similar to that of untreated platelets at 37 degrees C. It is suggested that trypsin facilitates platelet aggregation by concanavalin A at 0-4 degrees C by causing a configurational change in membrane glycoproteins which orientates concanavalin A receptor sites into positions that favour lectin cross-bridging. Concanavalin A failed to aggregate platelets in plasma. Radioisotope studies showed that the amount of [3H]concanavalin A which combined with platelets in plasma was extremely low compared with gel-filtered platelets in saline. The aggregation of Ehrlich ascites cells by concanavalin A was considerably reduced when platelet-free plasma was added to the medium suggesting that it was due to the presence of concanavalin A-reactive components in the plasma. Concanavalin A inhibited the ADP-induced aggregation of platelets suspended in plasma or in a salts solution supplemented with calcium and fibrinogen, although the inhibitory effect was more conspicuous in the latter case. The results suggests that concanavalin A produces its inhibitory effect on ADP-induced platelet aggregation by interacting with membrane glycoproteins, and this further suggests their involvement in aggregation.     

Platelet sialic acid and platelet survival after aggregation by ADP

Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP-induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.     

Rat platelet aggregation by ATP. Aggregometrical and ultrastructural comparison with aggregations induced by ADP and collagen

This paper describes the aggregation of rat platelets by adenosine triphosphate (ATP). The aggregometry of ATP-induced aggregation and the ultrastructure of ATP-aggregated platelets were compared and contrasted with those of adenosine diphosphate (ADP)-treated and collagen-treated samples. Human platelets were also studied alongside with rat specimens. Several lines of evidence indicate that the ATP-induced aggregation of rat platelet-rich plasma (PRP) is not a result of contaminating ADP in the ATP preparation. ATP did not cause aggregation of human platelets; it inhibited ADP- and collagen-induced human platelet aggregation. ATP pretreated with a creatine phosphate/creatine phosphokinase system caused similar rat platelet aggregation as did ATP not treated with this system. The aggregometry of ATP-induced aggregation of rat PRP was similar to that of collagen-induced aggregation but markedly different from that of ADP-induced aggregation. However, the nature of ATP-induced aggregation was similar to that induced by ADP. Both ATP- and ADP-induced rat platelet aggregations were not affected by adenosine, adenosine monophosphate, or acetylsalicylic acid. The ultrastructure of ATP-aggregated platelets was similar to that of ADP-aggregated ones. It appears that either platelets of rats possess specific ATP receptors or the rat plasma contains a material, lacking or insufficiently present in human plasma, that converts ATP to ADP in a fashion similar to the release of ADP from platelet storage granules.     

Characterization and distribution of binding sites for [3H]-SR 141716A, a selective brain (CB1) cannabinoid receptor antagonist, in rodent brain

SR 141716A belongs to a new class of compounds (diarylpyrazole) that inhibits brain cannabinoid receptors (CB1) in vitro and in vivo. The present study showed that [3H]-SR 141716A binds with high affinity (Kd=0.61 +/- 0.06 nM) to a homogenous population of binding sites (Bmax=0.72 +/- 0.05 pmol/mg of protein) in rate whole brain (minus cerebellum) synaptosomes. This specific binding was displaced by known cannabinoid receptor ligands with the following rank order of potency SR 141716A > CP 55,940 > WIN 55212-2 = delta9-THC > anandamide. Apart from anandamide, all these compounds were found to interact competitively with the binding sites labeled by [3H]-SR 141716A. On the other hand, agents lacking affinity for cannabinoid receptors were unable to displace [3H]-SR 141716A from its binding sites (IC50 > 10 microM). In addition, the binding of [3H]-SR 141716A was insensitive to guanyl nucleotides. Regional rat brain distribution of CB1 cannabinoid receptors detected by [3H]-SR 141716A saturation binding and autoradiographic studies, showed that this distribution was very similar to that found for [3H]-CP 55,940. In vivo, the [3H]-SR 141716A binding was displaced by SR 141716A with ED50 values of 0.39 +/- 0.07 and 1.43 +/- 0.29 mg/kg following intraperitoneal and oral administration, respectively. Finally, the [3H]-SR 141716A binding sites remained significantly occupied for at least 12 hr following oral administration of 3 mg/kg SR 141716A. Taken together, these results suggest that SR 141716A in its tritiated form is a useful research tool for labeling brain cannabinoid receptors (CB1) in vitro and in vivo.     

Identification and characterization of a lysophosphatidic acid receptor

A specific binding site for 1-[3H]stearoyl-lysophosphatidic acid (stearoyl-LPA) was identified and characterized in membranes prepared from rat brain and Swiss 3T3 fibroblasts. Specific binding of [3H]LPA to these sites was protein dependent, was saturable, reached equilibrium in 15 min, and was displacable by the addition of excess unlabeled LPA. Scatchard analysis of saturation binding experiments indicated that these sites had affinities of 2.0 +/- 0.5 nM and 5.4 +/- 2.6 nM and densities of 19 +/- 3 fmol/micrograms of protein and 38 +/- 6 fmol/micrograms of protein in rat brain and 3T3 cell membranes, respectively. Various LPAs, with different acyl groups in the sn-1-position, competed with [3H]LPA for these binding sites, with a rank order of potency of 1-oleoyl-LPA > 1-stearoyl-LPA = 1-palmitoyl-LPA > 1-myristoyl-LPA. Phosphatidic acid also bound to these sites, but with lower affinity than any LPA tested. Neither lysophosphatidylcholine, lysophosphatidylethanolamine, nor any free fatty acid competed with [3H]LPA for these binding sites. Binding of [3H]LPA to these sites was regulated by nonhydrolyzable guanine nucleotides in both rat brain and 3T3 cell membranes. Furthermore, in 3T3 cells, these sites were regulated by cell density. It was subsequently determined that LPA induced a transient increase in intracellular Ca2+ levels in 3T3 cells. The concentrations required for this response, as well as the rank order of potency of the various LPAs and phosphatidic acid, correlated with the affinity of these compounds for the [3H]LPA binding site. These results suggest that the specific, high affinity, binding sites for [3H]LPA are G protein-coupled receptors.     

3H]naltrindole: a potent and selective ligand for labeling delta-opioid receptors

Naltrindole (NTI) is a selective and potent delta-opioid antagonist which preferentially antagonizes a subset of selective delta-opioid agonists. The purpose of this study was to evaluate whether [3H]NTI, the first radiolabeled delta-opioid antagonist, could selectively label delta-opioid receptors in a synaptosomal preparation. Increasing temperature and protein concentration (0.1-1.6 mg protein) increased the specific binding of [3H]NTI. Monovalent and divalent cations (0.01-100 mM) had minimal effects on the binding properties of [3H]NTI, in contrast to their effects on binding of the delta agonists [3H]DPDPE and [3H]DSLET. Subfractionation of rat brain homogenates revealed that [3H]NTI and [3H]DSLET primarily labeled binding sites in synaptosomal and microsomal fractions, whereas [3H]DPDPE labelled half as many sites in synaptosomal fraction. The Bmax determined for [3H]NTI in crude synaptosomal fraction was 95 +/- 12 fmol/mg. The dissociation constant (Kd) was determined from three different methods to be 0.08 +/- 0.02 nM (Scatchard analysis), 0.07 +/- 0.02 nM (competition study) and 0.03 +/- 0.005 nM (kinetic analysis). [3H]NTI binding was not significantly inhibited by mu- or kappa-opioid ligands or by nonopioid compounds. These results demonstrate that [3H]NTI is a potent and selective radioligand for delta-opioid receptors in rat brain preparations.     

Effects of 2,2'-dipyridyl and related compounds on platelet prostaglandin synthesis and platelet function

2,2'-dipyridyl, a chelator of ferrous iron and inhibitor of platelet aggregation, was studied together with several similar compounds to determine the mechanism of their effects on platelets. All of these compounds were more potent inhibitors of arachidonic-acid-mediated aggregation (IC50, 0.17-1.8 mM) than of ADP-mediated aggregation (IC50, 7.6-19.7 mM). At low concentrations required to inhibit arachidonic-acid-mediated aggregation, 2,2'-dipyridyl, 4,4'-dipyridyl and 2-chloropyridine specifically inhibited the platelet cyclo-oxygenase. The mechanism of inhibition of ADP-induced aggregation was investigated, but was not explained. At concentrations needed to inhibit ADP-induced aggregation, 2,2'-dipyridyl did not alter cell ultrastructure, serotonin or nucleotide content or interfere with release of [14C]arachidonic acid or calcium movements. Therefore, our results indicate that 2,2'-dipyridyl and related compounds have two effects on platelets, both due to the unprotonated form. The inhibition of cyclo-oxygenase by low concentrations of these compounds is not due to bidentate iron chelation, since 4,4'-dipyridyl was almost as effective as 2,2'-dipyridyl, but is compatible with binding of these inhibitors to the iron in the heme of the cyclo-oxygenase.     

Serotonin modulation of low-affinity ouabain binding in rat brain determined by quantitative autoradiography

Previous results showed that Na+/K+-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na+/K+-ATPase activity and [3H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which alpha isoform is involved in the Na+/K+-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [3H]ouabain binding to rat brain slices was employed at different [3H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [3H]ouabain binding was sensitive to serotonin 10(-3) M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [3H]ouabain concentrations, strongly suggests the participation of the alpha2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K+ handling by the glial alpha2 isoform of the pump.     

Platelet activation by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate is mediated by its binding to a putative ADP receptor, aggregin

Platelet responses induced by ADP are mediated by a unique P21-purinergic receptor. Although a variety of ADP analogs, substituted at C2, have been used to delineate pharmacological properties of the ADP-binding site(s), the identity of the receptor protein has not been firmly established. 2-(4-Bromo-2,3-dioxobutylthio)- ADP [2-BrCH2(CO)2CH2-S-ADP], a well-characterized ADP analog, has been previously used as an affinity label to examine the structure/function relationship of ADP-requiring enzymes [Kapetanovic, E., Bailey, J.B. & Colman, R.F. (1985) Biochemistry 24, 7586-7593]. We found that it induced platelet shape change, aggregation, exposure of fibrinogen binding sites, secretion and mobilization of intracellular calcium, but was less potent than ADP. Under non-stirring conditions, incubation of platelets with this analog for longer time periods blocked ADP-induced shape change, aggregation, and the ability to ADP to antagonize the rise in intracellular levels of cAMP induced by iloprost (a prostaglandin I2 analog). Of a variety of agonists examined, only ADP-induced aggregation was almost completely inhibited in platelets irreversibly modified by the analog. An autoradiogram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[3H], showed the presence of a single radiolabeled protein band at 100 kDa. Platelets incubated first with either ADP, ATP, or 2-methylthio-ADP were not labeled by 2-BrCH2(CO)2CH2S-ADP and NaB[3H]4-8-BrCH2(CO)2CH2-S-ADP was previously shown by us to irreversibly antagonize ADP-induced platelet responses by selectively modifying aggregin. Incubation of platelets with 2-BrCH2(CO)2CH2S-ADP completely blocked labeling of aggregin in platelets by 8-BrCH2(CO)2CH2S-[32P]ADP. These results show that 2-BrCH2(CO)2CH2S-ADP initially interacts reversibly with aggregin (100kDa), a putative ADP receptor, and induces platelet shape change and aggregation, and at longer periods of incubation reacts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6-BrCH2(CO)2CH2S-ADP was found to be a weak and reversible inhibitor of ADP-induced platelet aggregation. Prior incubation of platelets with the latter analog reduced labeling of aggregin by 8-BrCH2(CO)2CH2S-[32P]ADP. Taken together, the results further show that substitution by the BrCH2(CO)2CH2 group at the C2 and C8 positions is tolerated, while the presence of a free amino function at the C6 position is essential for its interaction with aggregin.