Calcium channel blockers increase the amount of nitrite production in rabbits without decreasing the responsiveness of platelets to collagen.

By virtue of their ability to increase nitric oxide (NO) production, it is thought that calcium channel blockers (CCBs) may be involved in the inhibition of platelet aggregation. In an attempt to compare the abilities of different groups of calcium antagonists to affect NO generation and platelet aggregation, single doses of the calcium antagonists verapamil, nicardipine and diltiazem were administered to rabbits. It was found that each of these drugs increased the levels of nitrite significantly. It was also found that these drugs had different time courses of action. Of the CCBs used in this study, verapamil was found to induce the greatest increase in nitrite production (about a 4-fold increase over basal levels), peaking at 90 min (P<0.001). Diltiazem and nicardipine (3.5-fold and 2.5-fold increase over basal levels, respectively) were both found to induce increases in NO which peaked at 150 min (P<0.001 and P<0.01 respectively). Each of the drugs was then given at double the original dose; however, nicardipine was the only drug that was seen to further increase nitrite production (P<0.001). Blood samples taken from the animals were analysed using whole-blood aggregometry in order to assess the amount of collagen-induced platelet aggregation. At the time point when NO release was expected to be maximal (150 min), it was found that the collagen-induced platelet responses were not significantly altered in platelets from rabbits that had been treated with either verapamil or nicardipine. In contrast, at the 150-min time point, diltiazem treatment made the platelets hypersensitive to collagen stimulation. The results of this study demonstrate that treatment with calcium channel antagonists increases the levels of NO produced in rabbits, however, this increase in NO production is not accompanied by a decrease in the reactivity of platelets to collagen.

5-Hydroxytryptamine-2A receptor gene (HTR 2 A) candidate polymorphism (T 102 C): Role for human platelet function under pharmacological challenge ex vivo.

Although the environmental and life-style factors influencing individual predisposition to acute myocardial infarction (AMI) have been well documented, little is known on the identity of genetic loci that may contribute to risk for AMI. Recently, genetic studies in patients with nonfatal AMI have suggested an association with the T 102 C polymorphism in the serotonin 5-HT(2A) receptor gene (HTR 2 A). Considering the significant role of the 5-HT(2A) receptor in serotonin-induced platelet responses and the contribution of platelet (patho)physiology to thromboembolic events, we postulated that the increased susceptibility to AMI in patients with the T 102 homozygosity may be attributable, in part, to altered serotonin-mediated platelet function. In a group of healthy volunteers recruited from the Eskisehir region in central Turkey (N=37), we investigated the functional consequences of HTR 2 A T 102 C polymorphism in relation to platelet pharmacodynamics ex vivo. The platelet shape change and aggregation response to serotonin were measured with use of the platelet aggregometry and expressed as aggregometer output (mm). Because the circulating catecholamine hormone epinephrine can augment platelet aggregation, pharmacodynamic response (aggregation and its inhibition by 5-HT(2A) receptor antagonist cyproheptadine) was measured in the presence of both serotonin and epinephrine, to mimic the clinical situation in patients. The mean platelet aggregation was higher by 38% in subjects with T 10 2 homozygosity (T/T genotype, N=13) when compared with the carriers of the 102 C-allele (T/C and the C/C genotypic groups, N=24) (39.5 mm+/-12.3 vs. 28.7 mm+/-16.8, respectively) (mean+/-SD) (p<0.05). On the other hand, neither the serotonin-induced platelet shape change nor the cyproheptadine inhibition of platelet aggregation was influenced by the HTR 2 A T 102 C genetic variation (p>0.05). These findings in healthy subjects may provide a mechanistic explanation for the previously reported genetic association between HTR 2 A and AMI. Further genetic association studies of the 5-HT(2A) receptor in patients with AMI in different populations are warranted.

Carboxyl terminal sequence of human phospholipase Cgamma2.

Phospholipase Cgamma2 (PLCgamma2), the predominant isoform of phospholipase C expressed in platelets, plays a major role in activation of platelets by collagen. Although PLCgamma2 has been shown to be tyrosine phosphorylated upon collagen-induced activation, the phosphorylation sites are yet to be determined. We have sequenced the 3' terminal cDNA of human phospholipase C-gamma-2 and found it different from the human PLCgamma2 cDNA sequence previously reported by Ohta et al. (Ohta S, Matsui A, Nazawa Y, Kagawa Y. FEBS Lett 1988; 242: 31-5). There is an extra guanosine at position 3723 which causes a shift in the reading frame. The new carboxyl terminal amino acid (aa) sequence beyond the frame shift is 88% identical to that of rat (21 out of 24 aa residues) which is considerably higher than the identity with published sequence (26% identity). The new deduced aa sequence contains two tyrosine residues at positions 1245 and 1264 which might be phosphorylated upon stimulation and hence might be important for the activation of the PLCgamma2.

Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the mu opioid receptor.

We examined whether a proposed spatial proximity between Asp114(2.50) and Asn332(7.49) affected the functional properties of the mu opioid receptor. The D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO and CTAP, but not for naloxone and [3H]diprenorphine; this mutation also abolished agonist-induced increase in [35S]GTPgammaS binding. The N332(7.49)D mutation eliminated detectable binding of either [3H]diprenorphine or [3H]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affinity binding for [3H]diprenorphine, CTAP and naloxone, and restored partially the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the residues are adjacent in space and that their chemical functionalities are important for ligand binding and receptor activation.