Modulatory effect of insulin on release of calcium from human fibroblasts by angiotensin II

Background. Angiotensin II stimulates synthesis and deposition of collagen and might contribute to the vascular and cardiac dysfunction associated with arterial hypertension. Insulin attenuates angiotensin II-induced responses of intracellular Ca2+ concentration ([Ca2+]) in many cell types but this effect is less in insulin-resistant states. The mechanisms of the interaction between insulin and angiotensin II are still not known. Objective. To characterize the effects of angiotensin II on intracellular [Ca2+] and the effects of insulin on the angiotensin II-induced response of intracellular [Ca2+] in human skin fibroblasts. Methods. Spectrofluorophotometric measurements of intracellular [Ca2+] in monolayers of cultured human skin fibroblasts from 15 normotensive patients were performed using Fura-2 at 510 nm emission with excitation wavelengths of 340 and 380 nm. Results. Basal intracellular [Ca2+] in quiescent (24 h serum-deprived) human fibroblasts was 75 ± 3 nmol/l (n = 20). Administration of angiotensin II elevated intracellular [Ca2+] dose-dependently with a concentration for half-maximal effect of 20 nmol/l. Administration of 100 nmol/l angiotensin II stimulated a rapid and transient increase in intracellular [Ca2+] (from 75 ± 3 to 130 ± 2 nmol/l, n = 20). Removal of extracellular calcium did not change peak intracellular [Ca2+], but it did reduce the time to recovery of [Ca2+] (from 64 ± 4 to 48 ± 2 s, n = 10, P < 0.01), suggesting that an angiotensin II-induced transmembrane calcium influx had occurred. This hypothesis was confirmed by quenching studies with manganese. The angiotensin II-induced changes in intracellular [Ca2+] were completely blocked by administration of 100 nmol/l of the angiotensin II type 1 receptor inhibitor losartan but not by administration of 100 nmol/l of the angiotensin II type 2 receptor blocker CGP42112A. Acute (20 min) exposure to 100 nmol/l insulin did not alter basal intracellular [Ca2+] in quiescent fibroblasts, but significantly blunted angiotensin II-stimulated peak of [Ca2+] (to 101 ± 3 nmol/l, P < 0.01, n = 18) and delayed recovery of [Ca2+] (to 99 ± 5 s, P < 0.01). The inhibitory effect of insulin was observed both with and without extracellular Ca2+. Conclusions. Our results demonstrate that administration of angiotensin II increases intracellular [Ca2+] in human skin fibroblasts by release of Ca2+ from intracellular Ca2+ stores and by influx of Ca2+ and that administration of insulin attenuates the response of [Ca2+] to angiotensin II but prolongs the time to recovery of [Ca2+].