Interactions Between Insulin and Angiotensin II on Calcium Mobilization in Skin Fibroblasts from Insulin Resistant Patients with Essential Hypertension
Objectives: Insulin attenuates angiotensin II (Ang II)-induced Cai2+ mobilization in insulin sensitive (IS) control subjects. Therefore we compared the interactions between Ang II and insulin on Cai2+ mobilization in skin fibroblasts from insulin resistant (IR) and insulin sensitive (IS) patients with essential hypertension (HT). Methods: Skin fibroblasts from 9 normotensives (NT) and 18 HT were cultured and used after 4 passages. Ca2+ was measured in monolayers of 24h serum-deprived cells using Fura-2 AM. mRNA expression of Gαi2 subunit was determined by RT-PCR using a 5 and 3 specific primers. Results: Resting and Ang II-stimulated (100nM) Cai2+ peak were higher in fibroblasts from HT than NT (76±3 vs 62±3 nM, p<0.01 and 225±8 vs 164±12 nM, p<0.01). In the absence of extracellular Ca2+, the Cai2+ response to Ang II was still significantly enhanced in fibroblasts from HT (212±8 vs 158±9 nM, p<0.01). Thapsigargin (500 nM), a Ca2+ -ATPase inhibitor, induced a Cai2+ rise that was similar in HT and NT (from 42±7 to 93±15 vs 52±5 to 106±8 nM). Pertussis toxin (20ng/ml, 6h), inhibitor of Gαi2 subunit, reduced Ang II stimulated Cai2+ peak both in NT (from 153±7 to 98±8nM, p<0.01) and HT (from 189±16 to 138±12, p<0.01). In IS HT, insulin (100 nM, 20 min) blunted the Ang II stimulated Cai2+ response (from 208±9 to 167±9 nM, p<0.01), while in IR HT it did not (from 205±16 to 208±13 nM, ns). Pertussis toxin attenuated AngII stimulated Cai2+ peak in IS HT (from 208±4 to 112±2 p<0.01), but not in IR HT (from 207±10 to 165±9). mRNA expression of Gαi2 subunit was increased in IR HT compared to IS HT. Conclusion: In human skin fibroblasts the inhibitory effect of insulin on Ang II - induced Cai2+ mobilization is blunted in IR HT by a pertussis toxin sensitive mechanism, probably involving a Gαi2subunit.