Neurobiofogy [ 3 H ] Nitrendipine - labeled calcium channels discriminate inorganic calcium agonists and antagonists ( nifedipine / verapamil / receptor / hypertension / angina )

3H]Nitrendipine binds with high affinity to brain membranes with a drug specificity indicating association with sites mediating the pharmacologic actions of dihydropyridine slow-calcium-channel antagonist drugs. In brain membranes, [3Hjnitrendipine binding is absolutely dependent on the presence of calcium ions. Interactions of cations with [3H]nitrendipine binding sites correlate with their physiologic actions at voltage-dependent calcium channels. Ions such as strontium and barium, which mimic calcium physiologically, share the action of calcium in enhancing [3H]nitrendipine binding. Ions such as lanthanum and cobalt, which block the effects of calcium, can inhibit [3H]nitrendipine binding and block the stimulating actions of calcium. The ability to monitor the influence of ions on an agonist-antagonist continuum at [3H]nitrendipine binding sites provides a molecular probe to explore the regulation of cellular function by calcium and other cations. The entry of calcium into cells via voltage-dependent channels regulates muscle contraction and neuronal discharge (1, 2). Drugs that block these voltage-dependent calcium channels have considerable therapeutic importance in treating angina, hypertension, and other vasoconstrictive disorders (3-5). Various organic slow-calcium-channel blocking agents seem to act differently, with the pharmacologic actions of the dihydropyridine agents differing somewhat from those of drugs such as verapamil, D-600, and diltiazem (6). Recently, we (7) and others (8-10) have labeled the calcium channel binding sites for dihydropyridine drugs in brain and heart, using [3Hlnitrendipine. While the affinities of the dihydropyridines for these binding sites correlate well with their pharmacologic properties, verapamil, D-600, and diltiazem have 'low affinities for these sites, indicating that the latter drugs act through different receptors. Some inorganic ions, such as barium and strontium, mimic the ability of calcium to pass through the voltage-dependent calcium channels, while cations such as lanthanum and cobalt are calcium antagonists (11). We now report that [3H]nitrendipine binding to 'brain membranes has an absoluterequirement for physiological concentrations ofcalcium. Moreover, inorganic cations interact with calcium in the regulation of [3H]nitrendipine binding in an agonist-antagonist continuum. MATERIALS AND METHODS Male Sprague-Dawley rats were killed by cervical dislocation and decapitation. Brains were removed immediately and washed with 0.9% ice-cold NaCl, and the cerebral cortices were separated. Where indicated, brains were dissected into distinct anatomical regions and binding to these regions was assayed. Tissue was homogenized using a Brinkmann Polytron in 10 vol of 50 mM Tris HCl (pH 7.7) and then centrifuged at 20,000 rpm for 10 min in a Sorvall SS-34 rotor. The-pellet-was washed three times with ice-cold 50 mM Tris-HCI (pH 7.7) and suspended to a final concentration of 1-4 mg (wet weight)/ml in 50 mM Tris HCl. In those experiments exploring the ionic regulation of [3H]nitrendipine binding, cerebral cortex or heart was homogenized in 10 vol of 50 mM Tris HCl, pH 7.7/10 mM EDTA and immediately centrifuged at 20,000 rpm for 10 min. The pellet was suspended in 50 mM Tris-HCl, pH 7.7/10 mM EDTA and incubated on ice for 30 min. After centrifugation as before, the pellet was suspended in 50 mMM Tris'HCl, pH 7.7/ 10mM EGTA and again incubated on ice for 30 min. The pellet obtained after centrifuging was then washed three times with 10 ,uM EGTA/50 mM Tris-HCl, pH 7.7, and finally resuspended at 4 mg (wet weight)/ml in this EGTA/Tris buffer. Mixtures were incubated at 25°C as described in the figures and tables. Incubations were terminated by rapidly filtering through GF/B glass fiber filters (Whatman). The filters were washed with three 3-ml portions of 50 mM Tris HCl (pH 7.7), and the radioactivity retained was determined by 'liquid scintillation spectrometry using NEN-947. [3H]Nitrendipine (85 'Ci/mmol; 1 Ci = 3.7 x 1010 becquerels) and oxidized [3H]nitrendipine (85 Ci/mmol) were obtained from New England Nuclear. Nifedipine (Pfizer), nimodipine, nisoldipine, nitrendipine (Miles), felodipine (Hassle, Molndal, Sweden), diltiazem (Tanabe, Osaka, Japan), verapamil, and D600 (gifts ofJohn Daly) were first dissolved in absolute ethanol to 1mM and then diluted to the appropriate concentrations with 50 mM Tris HCI (pH 7.7). All other chemicals were obtained from standard commercial sources.