Organ specificity of the inhibitor- and substrate binding site of the adeninenucleotide translocator (ANT)

69 187 ORGAN SPECIFICITY OF THE INHIBITOR- AND SUBSTRATE BINDING SITE OF THE ADENINE NUCLEOTIDE TRANSLOCATOR (ANT). H.-P. Schultheiss, P. S c h w i r ~ c k , H.-D. Bolte. Dept. of Medicine, Klinikum Grosshadern, University of Munich, W. Germany. A further iranunochemical characterization of the ANT, an auto-antigen in dilated cardiomyopathy, is described. For the precise characterization of the transport associated antigenic determinants we measured the influence of antibodies on the nucleotide transport by the back exchange with the inhibitor stop method, the binding of 3 H - carboxyatractylate (CAT) to the cytosolic side of the ANT and of 3HBongkrekate (BKA) to the matrix side of the ANT. Previously the organ specificity of the antibodies was determined by a radioinm~/no-assay and i~mauno-absorption studies. Results: The binding of the anti-ANT antibodies to the translocator protein caused I. an inhibition of the substrate transport, 2. a reduction of the CAT-binding and 3. a reduction of the BKA-binding indicating an inhibition of the conformational change from the "c"- to the "m"-conformation. Although a partial crossreactivity between the translocator protein from heart, kidney and liver was seen, the inhibition of the ligand binding and the substrate transport by the antibody was only seen on heart mitochondria. Conclusions: These results indicate an organ-specificity not only of the ANT but of the "translocational apparatus" itself and give new evidence for the assumption that the inhibitor- and substrate binding site is identical!

188 EFFECTS OF THE IONOPHORE GRISORIXIN ON MYOCARDIAL FUNCTION AND METABOLISM IN THE NORMOXIC AND HYPOXIC PERFUSED WORKING RAT HEART. F. Chollet, N. Moins, M. Renoux, P. Gachon. U195 INSERM, Facult~ de M~decine, 63000 Clermont-Ferrand, France. Grisorixin is a monocarboxylie ionophore, preferentially potassium carrier, which exhibits in u~vo potent cardiotonic and coronary vasodilator properties in dogs. Its direct effects on myocardium function and metabolism were determined using a model of working rat heart perfused with a suspension of washed fresh pig erythrocytes (I0% hematocrit) in a modified Krebs-Henseleit bicarbonate buffer. Treatment with grisori ~ xin (2.5 pM) induced a rapid and transient increase in heart work, expressed by an increase in heart rate (+21%) and in aortic flow (+]7%), and maximum at 5 min. The coronary dilatation was maximum at 10 min (+47%). Myocardial creatine phosphate level dropped during the first two min only while the ATP level remained almost unchanged during the 60 min of the experiment except for a slight decrease during the first lO min. The evolution of lactate production by the myocardium was biphasic : it first increased by 82 % at the 5th min, then dropped to the control value (IOth min) and increased again till the 45th min (+2]1%), suggesting a cardiac metabolic drift towards anaerobic glycolysis due to an inhibition of the mitochondrial respzratzon by grisorixin. A slight decrease in O^ consumption was also noted after the lOth min. The effects of grisorixin have als~ been tested on hearts in hypoxic conditions and the modifications of the ultrastructure of left ventricular tissue have been examineg

189 RELATIONSHIP OF CARDIAC PERFORMANCE,FREE ENERGYOF ATP-HYDROLYSISAND ENERGYREQUIREMENT OF ION-TRANSPORTPROCESSESUNDERHYPOXIC STEADY STATE CONDITIONS.H. Kammermeier, M. Griese. Dept. Physiology, Med. Faculty, RWTHAachen, F.R.G. In early hypoxic f a i l u r e free energy of ATP-hydrolysis drops rapidly, though myocardial ATP-levels decrease only l i t t l e . The rather rapid decrease in both parameters in p a r a l l e l shown previously under anoxic perfusion (J. Mol. Cell. Cardiol. 14:267 (1982)) suggest causal relationship. However, steady state conditions are desirable to exclude accidental correlation. Therefore, in a new series of experiments the contract i l e performance and the free energy of ATP-hydrolysis were compared under hypoxic ( r e l a t i v e ) steady state conditions (pO2 40-700 mmHg) in isolated perfused hearts. The results e x h i b i t a close relationship between both parameters in the range of AG ATP of 55-50 kJ/mol. Calculation of the free energy of ion gradients indicates that the regular function of the Na/K-ATPase, the sarcolemmal and the sarcotubular Ca§ require an energy level of 45-50 kJ/mol ATP as long as the resting potential is unchanged. Thus cardiac f a i l u r e can be caused by e i t h e r of the energy requiring mechanisms and t h e i r interplay, where nevertheless, free energy of ATP-hy~rolysis seems the determinant factor. Supported by the Deutsche Forschungsgemeinschaft - Ka 337/5-7