Abstract
1 [(3)H]-clonidine binds reversibly to membranes prepared from regions of guinea-pig kidney.2 Higher levels of binding were obtained in the membranes prepared from renal cortex (2.15 +/- 0.27 pmol/g wet wt.) than renal medulla (0.53 +/- 0.07 pmol/g wet wt.) or papilla (0.14 +/- 0.06 pmol/g wet wt.; n = 4).3 Scatchard analysis performed by addition of unlabelled clonidine (1 to 30 pmol) gave figures for the dissociation constant (K(d)) for the binding of [(3)H]-clonidine to renal cortical membranes of 9.0 +/- 0.8 nM and B(max) of 21.6 +/- 1.7 pmol/g wet wt. (n = 4). Hill plots of these data gave gradients close to unity, indicating a lack of co-operative site interactions.4 The monovalent cations, sodium and potassium, and the divalent cation, calcium, produced concentration-dependent decreases in [(3)H]-clonidine binding to membranes prepared from renal cortex, the EC(50)s being respectively 25 mM, 37 mM and 23 mM.5 At low concentrations the divalent cations, magnesium (1 mM) and manganese (0.1 mM), produced enhancement of [(3)H]-clonidine binding. At higher concentrations (>10 mM) both divalent cations inhibited binding.6 Scatchard analysis of [(3)H]-clonidine binding performed in the presence of sodium (100 mM), magnesium (1 mM) or manganese (0.1 mM) revealed that the alterations in binding are primarily due to changes in apparent affinity rather than a change in the number of binding sites. Sodium (100 mM) produced a change in the K(d) from 7.0 +/- 0.4 nM (n = 8) to 42.3 +/- 27.5 nM (n = 3), whereas magnesium (1 mM) decreased the K(d) to 6.0 +/- 0.9 nM and manganese (0.1 mM) to 4.0 +/- 1.0 nM (n = 3).7 The results indicate that [(3)H]-clonidine labels a binding site that has properties resembling an alpha(2)-adrenoceptor, located in the renal cortex. The changes produced by the addition of monovalent and divalent cations are entirely due to changes in the apparent affinity of [(3)H]-clonidine binding.