Effect of 3-hydroxypyridines on cyclic nucleotide levels and inhibition of platelet aggregation in man

Abstract

eclass (3-HP), has recently been successfully used in clinical ophthalmology as an effective mode of treatment of intraocular hemorrhages of vascular genesis [3]. The molecular mechanisms of action of this compound have not been finally elucidated. The writers showed previously that 3-HP derivatives are able to inhibit phosphodiesterases (PDE) specific both for cGMP and for cAMP in platelets, and also to inhibit platelet aggregation [i]. On the basis of these results it has been suggested that one mechanism of action of emoxipine is by inhibiting platelet aggregation as a result of inhibition of PDE activity, with a consequent increase in the intracellular cyclic nucleotide concentration. To test this hypothesis experimentally, the action of several 3-HP derivatives was studied on the cyclic nucleotide levels in platelets and on platelet aggregation. EXPERIMENTAL METHOD Platelets were isolated from blood obtained form healthy blood donors [5], after which they were preincubated for 5 min in medium of the following composition: 15 raM Tris-HCl, 134 mM NaC1, 5 mM glucose (pH 7.4), in the presence of the test substances in different concentrations (Table i). The volume of the incubated mixture was 1 ml. Aggregation in the medium was initiated by addition of th~ombin (1.5 U/ml), and 1 min later the reaction was stopped by the addition of 1 ml of ethanol, followed by heating to 100~ on a water bath for 15 min. After heating, the samples were treated with a further 1 ml of ethanol and centrifuged (3000 rpm) and the supernatant was evaporated in vacuo at 50~ Cyclic nucleotides in the samples were assayed by means of the appropriate kits (cyclic AMP RIAKIT and Cyclic GMP RIAKIT; Amersham Corporation, England). To measure radioactivity, 1215 RackBeta II and Mark II counters were used. Aggregation was recorded on an aggregometer (Chrono Log, USA) connected to an automatic writer (LKB, Sweden). The cell concentration in the medium was 200,000/ml. The rate of aggregation was estimated quantitatively as the tangent of the angle of slope of the tangent to the region of the curve correpsonding to the rapid phase of aggregation. Inhibition of aggregation with different concentrations of 3-HP was expressed as a percentage of the control. 3-HP derivatives with substituents in different positions were synthesized at the Institute of Pharmacology, Academy of Medical Sciences of the USSR [2]. Preparations were used in the experiments in the form of bases, which were added to the incubation medium in the form of alcoholic solutions (the ethanol concent:ration in the incubation medium was 2%). The HCI, EDTA, and glucose were obtained from Sigma, USA and the remaining reagents from Reakhim (USSR), and were of the chemically pure grade.