BIOCHEMISTRY AND BIOPHYSICS EFFECT OF WATER-SOLUBLE ANTIOXIDANTS OF THE SCREENED PHENOL CLASS ON THE ~-ADRENERGIC SYSTEM OF RAT CARDIOMYOCYTE PLASMA MEMBRANES IN VITRO A~ P0 Khokhlov and K~ N. Yarygin UDC 616.128-008.939.15-39-02:615.272.014.425 KEY WORDS: synthetic antioxidants; ~-adrenoreceptors; rat cardiomyocytes; adenylate cyclase; lipid peroxidation Experimental and clinical data obtained in recent years suggest that changes in lipid peroxidation (LPO) in cell membranes play an important role in the pathogenesis of several diseases. For instance, cytotoxic effects in myocardial ischemia and hypoxia are largely due to depression of activity of the biochemical systems that protect the heart from active forms of oxygen, and in turn, this suggests that induction of LPO is a factor involved in cardiomyocyte injury [9, i0]. It has been suggested [13] that the entry of Ca++ into cells as a result of structural disturbances of their membranes, caused by activation of phospho- lipases and proteases, is an important factor in myocardial damage. The role of other con- sequences of LPO activation and, in particular, of modification of membrane receptor activity [3], is not clear. The possibility of using various substances to compensate the depressed activity of components of the antioxidant system of the heart in the treatment of ischemic disturbances in heart muscle has been discussed in the literature [i]. Synthetic antioxi- dants [4, 6], which are relatively nontoxic inhibitors of radical reactions [7], are promis- ing in this respect~ The aim of this investigation was to study the effect of changes in the level of LPO, induced by the action of synthetic antioxidants, on signal conduction in the $-adrenergic system of rat cardiomyocyte plasma membranes, i.e., in the receptor system which plays an important role in the pathogenesis of myocardial infarction [2], was studied. EXPERIMENTAL METHOD The followign water-soluble synthetic antioxidants beloning to the screened phenol class were used: phenosan [~-(4-hydroxy-3,5-di-tert-butylphenol)propionic acid] and its potassium salt (phenosan-iK). Both compounds were synthesized in V. V. Ershov's laboratory at the Institute of Chemical Physics, Academy of Sciencesof the USSRo LPO induction was monitored by measuring accumulation of conjugated dienes at 234 nm; absorption spectra were recorded on the Hitachi-557 spectrophotometer (Japan). Fractions of cardiomyocyte plasma membranes of Wistar rats were isolated by the method described pre- viously [5]. Adenylate cyclase activity was determined by the method in [12]. The incu- bation medium contained (in mmoles/liter): Tris-HCl (pH 7.5 at 37~ 50, cAMP I, MgCI 2 5, phosphocreatine 20, ATP 0.i, theophyl!ine 0.2~ and also 0.5 mg/ml of creatine phospho- kinase and 106 cpm of ~-32p-ATP (specific activity 3000 Ci/mmole, from AmershamCorporation, England). Samples were incubated for 15 min at 37~ and the final concentration of protein of the membrane preparation in the same was 0~ mg/mlo When complex formation of 3H-dihydroalprenolol (specific activity 78 Ci/mmole, Amersham) with cardiomyocyte ~-adrenoreceptors was investigated, specific binding was determined as the fraction of total binding which could be blocked by 1000-fold excess of L-alprenolol (from Sigma, USA). Cardiomyocyte membranes (500 Bg protein/ml) were incubated for 60 min at 25~ in medium of the following composition (in mmoies/liter): imidazole 20, Na2EDTA All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR. Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Smirnov.) Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. i02~ No. I0, pp. 415-417, October, 1986o Original article submitted March ii, 1986~ 0007-4888/86/0010-1355512.50 9 1987 Plenum Publishing Corporation 1355