Ca2 + and Mg 2+ dependence of angiotensin II binding to isolated rat renal glomeruli

[ 125 I]angiotensin II binding to isolated rat renal glomeruli was clearly increased in the presence of either Ca 2 + or Mg 2 + (10 μM to 5mM for both). This effect was more marked at higher pH. In the absence of both Ca 2 + and Mg 2 + , [ 125 I]angiotensin II binding decreased with pH whereas at 2 mM of these two cations, the opposite was observed. There was a clear-cut interaction between the effects of pH and those of Ca 2 + or Mg 2 + . The increase in [ 125 I] angiotensin II binding was similar for the same molar concentrations of Ca 2 + or Mg 2 + . Effects of these two cations were additive at pH 8.5, but negatively interacted at pH 7.5. Mn 2 + had the same effects as Ca 2 + and Mg 2 + and interacted with them. Addition of ethyleneglycol bis ( β aminoethylether)- N , N ′tetraacetic acid or of ethylenediamine tetraacetic acid suppressed the stimulatory effects of Ca 2 + and Mg 2 + and decreased [ 125 I]angiotensin II binding even when these two cations were not introduced in the incubation milieu. The extent of increase in [ 125 I]angiotensin II binding at increasing concentrations of Ca 2 + or Mg 2 + was greater in the presence than in the absence of these chelating substances. Kinetics of [ 125 I]angiotensin II binding were modified in the presence of Ca 2 + and Mg 2 + . The slopes of the association curves were smaller and the heights of the equilibrium plateaus greater. Moreover the dissociation after addition of an excess of angiotensin II was slower and irregular and did not correspond to an exponential function. The constant of affinity estimated from association curves at two different hormonal concentrations or from binding experiments at equilibrium was unchanged. Addition of EDTA at equilibrium also produced dissociation of bound [ 12 5 I]angiotensin II whether or not Ca 2 + and Mg 2 + were present. Enhancement of [ 125 I]angiotensin II binding by Ca 2 + and Mg 2 + and interaction of these two cations with pH seem particular to the glomerular binding sites and suggest a role in the binding reaction for the electric charges of both angiotensin II and the glomerular structures.