[3H]-Nimodipine and [3H]-nitrendipine as tools to directly identify the sites of action of 1,4-dihydropyridine calcium antagonists in guinea-pig tissues. Tissue-specific effects of anions and ionic strength

Abstract

We have performed a comparative binding study with tissues from the guinea-pig with the tritiated calcium antagonists [3H]-nimodipine (isopropyl-[2-methoxy-ethyl]-1,4-dihydro-2,6-dimethyl-4- [3-nitrophenyl]-3,5-pyridinedicarboxylate, Bay e 9736) and [3H]-nitrendipine (1,4-dihydro-2,6-dimethyl-4-[3-nitrophenyl]-3,5-pyridine carboxylic acid, 3-ethyl-5-methyl ester, Bay 3 5009). These compounds are potent nifedipine analogues. Binding of both tritiated calcium antagonists to heart, kidney, lung and brain membranes was evaluated under four different buffer conditions, namely TrisCl and TrisNO3, present at low and high ionic strength (50 and 500 mmol/l). Effects of anions, independent of ionic strength, were observed in brain membranes. In the lung membranes no [3H]-nitrendipine binding in excess above 10 mumol/l unlabelled calcium antagonist was observed at low ionic strength in either TrisCl or TrisNO3. The pharmacological profile of [3H]-nimodipine binding in brain membranes was that expected of a potent 1,4-dihydropyridine calcium antagonist. The apparent dissociation constant (KD) of [3H]-nimodipine for binding sites in brain membranes, determined in TrisNO3 buffer (50 mmol/l, pH = 7.4), was 0.3-0.4 nmol/l at 37 degrees C. The maximum number of binding sites (Bmax) was 300-350 fmol/mg of protein and is in the same range as is commonly observed for neurotransmitters, hormones or channel toxins.