High glucose level inhibits capacitative Ca2 + influx in cultured rat mesangial cells by a protein kinase C-dependent mechanism

Summary

In cultured mesangial cells (MC), capacitative Ca^{2 +} influx via store-operated channels (SOC) is potentiated by agents that release Ca^{2 +} from intracellular stores, and inhibited by protein kinase C (PKC). Cells grown under high glucose conditions, as a model of the diabetic microenvironment, display reduced Ca^{2 +} signalling in response to vasoconstrictors, probably due to downregulation by elevated PKC activity. Since SOC might be relevant to this phenomenon, we assessed Ca^{2 +} influx by microfluorometry of fura-2-loaded rat MC cultured for 5 days in normal (5.5 mmol/l, NG) or high glucose (30 mmol/l, HG). The addition of 1–10 mmol/l Ca^{2 +} to NG cells equilibrated in Ca^{2 +} -free media induced an immediate Ca^{2 +} influx with a free cytosolic Ca^{2 +} ([Ca^{2 +} ]_i) plateau of 155 ± 50 and 318 ± 114 nmol/l, respectively. Basal influx was reduced to 88 ± 8 and 145 ± 17 nmol/l [Ca^{2 +} ]i (1–10 mmol/l Ca^{2 +} , p < 0.01) by 30 mmol/l d-glucose. This effect of HG was confirmed by Mn^{2 +} quenching of fura-2, indicating reduced entry of divalent cations via the capacitative pathway. Equimolar l-glucose had no effect on Ca^{2 +} influx, consistent with a non-osmotic mechanism. Arginine vasopressin (10 μmol/l) elicited weaker release of stored Ca^{2 +} and subsequent influx in HG cells (191 ± 33 vs 153 ± 24 nmol/l, 400 ± 76 vs 260 ± 33 nmol/l, 1–10 mmol/l Ca^{2 +} , NG/HG, p < 0.05). To examine the involvement of PKC in the effect of HG on capacitative Ca^{2 +} influx, the enzyme was activated or downregulated by treatment with 0.1 μmol/l phorbol myristate acetate (PMA) for 3 min or 24 h, respectively. PMA acutely inhibited Ca^{2 +} influx in NG cells, while PKC downregulation restored it in HG cells. Similarly, the PKC inhibitors staurosporin or H-7 normalized SOC activity in HG cells. In summary, impairment of Ca^{2 +} influx via SOC by HG is one mechanism of the reduced MC [Ca^{2 +} ]_i responsiveness to vasoconstrictors. This event is mediated by PKC and may contribute to the glomerular haemodynamic changes in the initial stages of diabetes mellitus. [Diabetologia (1997) 40: 521–527]