Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla.
1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H]-nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H]-nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]-nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L-type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown.
Ballesta
J J
JJ
Departamento de Neuroquímica, Universidad de Alicante, Facultad de Medicina, Spain.
Garcia
A G
AG
Gutierrez
L M
LM
Hidalgo
M J
MJ
Palmero
M
M
Reig
J A
JA
Viniegra
S
S
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D015220:Calcium Channels; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002838:Chromaffin System / Q000187:drug effects / Q000378:metabolism; D004110:Diltiazem / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D009568:Nitrendipine / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013237:Stereoisomerism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014700:Verapamil / Q000378:metabolism
D015220:Calcium Channels; D011978:Receptors, Nicotinic; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D009568:Nitrendipine; D014700:Verapamil; D004110:Diltiazem