ENHANCED EFFECTS OF INSULIN AND ANGIOTENSIN-II ON INTRACELLULAR PH AND FREE CYTOSOLIC CALCIUM IN FIBROBLASTS FROM MICROALBUMINURIC PATIENTSWITH NON-INSULIN-DEPENDENT DIABETES-MELLITUS

1. Whether an alteration in cell membrane cation transport after expos ure to insulin and angiotensin II (two important growth promoters that have been shown to be involved in the pathogenesis of atherosclerosis and hypertension) is present in cells from non-insulin-dependent diab etes patients with microalbuminuria, a known risk factor for cardiovas cular and renal disease, is unknown. We therefore examined intracellul ar pH and calcium changes after acute exposure to insulin and angioten sin II in cultured skin fibroblasts from eight non-insulin-dependent d iabetes patients with and eight others without microalbuminuria and fr om a group of seven matched, normal control subjects, 2. Cultured fibr oblasts were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluores cein acetoxymethyl ester or fura 2-acetoxymethyl ester for continuous monitoring of intracellular pH and free calcium concentrations respect ively, 3. In quiescent growth-arrested cells, both intracellular pH an d free calcium concentrations were similar in the three groups of subj ects, Acutely, insulin induced a gradual alkalinization in all groups of patients, The pH increase was significantly greater in non-insulin- dependent diabetes mellitus patients with microalbuminuria (Delta pH 0.24+/-0.04 pH units) than in normoalbuminuric patients with non-insul in-dependent diabetes mellitus (0.08+/-0.02; P<0.01) and normal contro l subjects (0.05+/-0.01; P<0.01). Although the alkalinizing effect of angiotensin II was smaller than that obtained by insulin, intracellula r pH increase after angiotensin addition was more pronounced in non-in sulin-dependent diabetes mellitus patients with microalbuminuria (Delt a pH +0.14+/-0.04 pH units) than in those without (0.08+/-0.02; P<0.01 ) and in normal control subjects (0.02+/-0.02; P<0,01), That the incre ase in intracellular pH was mediated by the sodium-hydrogen antiport w as demonstrated by its dependence on the presence of sodium in the med ium and its inhibition by amiloride. Whereas insulin addition did not evoke any significant increase in intracellular free calcium levels in fibroblasts from the three groups studied, angiotensin II evoked a fa st and transient rise in intracellular free calcium that was higher in fibroblasts from microalbuminuric patients with non-insulin-dependent diabetes mellitus than in cells from normoalbuminuric patients with n on-insulin-dependent diabetes mellitus and control subjects, In the wh ole population of patients with non-insulin-dependent diabetes mellitu s, the increase in intracellular pH after exposure to angiotensin II w as positively correlated with intracellular free calcium increase (r=0 .53; P<0.05), suggesting a possible role of intracellular free calcium levels in the activation of the sodium-hydrogen antiport, 4. In concl usion, we have described an association between increased agonist-indu ced responsiveness of sodium-hydrogen antiport activity and the presen ce of microalbuminuria in with non-insulin-dependent diabetes This inc reased responsiveness, persisting in cultured fibroblasts after severa l passages in vitro, suggests that in vitro phenotypic characteristics of fibroblasts are likely to be genetically determined and to be, at least in part, independent of the degree of metabolic control in vivo.