title: Modulatory effect of insulin on release of calcium from human fibroblasts by angiotensin II.
abstract: 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+].
authors.affiliation: Department of Clinical and Experimental Medicine, University of Padova Medical School, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Ceolotto
authors.forename: A C
authors.initials: AC
authors.lastname: Pessina
authors.forename: E
authors.initials: E
authors.lastname: Iori
authors.forename: A
authors.initials: A
authors.lastname: Monari
authors.forename: R
authors.initials: R
authors.lastname: Trevisan
authors.forename: P
authors.initials: P
authors.lastname: Winkleswski
authors.forename: A
authors.initials: A
authors.lastname: Semplicini
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004347:Drug Interactions; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D002118:Calcium