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

• Published in: Diabetologia 1997-05, Vol.40 (5), p.521-527
• Main Authors: MENE, P, PUGLIESE, G, PRICCI, F, DI MARIO, U, CINOTTI, G. A, PUGLIESE, F
• Format: Article
• Language: English
• Subjects: Animals; Arginine Vasopressin - pharmacology; Associated diseases and complications; Biological and medical sciences; Calcium - metabolism; Cells, Cultured; Diabetes Mellitus - metabolism; Diabetes. Impaired glucose tolerance; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Enzyme Activation; Fura-2; Glomerular Mesangium - drug effects; Glomerular Mesangium - metabolism; Glucose - pharmacology; Medical sciences; Models, Biological; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Rats; Staurosporine - pharmacology; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0012-186X; 1432-0428
• DOI: 10.1007/s001250050710

In cultured mesangial cells (MC), capacitative Ca2+ influx via store-operated channels (SOC) is potentiated by agents that release Ca2+ 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 Ca2+ signalling in response to vasoconstrictors, probably due to downregulation by elevated PKC activity. Since SOC might be relevant to this phenomenon, we assessed Ca2+ 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 Ca2+ to NG cells equilibrated in Ca(2+)-free media induced an immediate Ca2+ influx with a free cytosolic Ca2+ ([Ca2+]i) plateau of 155 +/- 50 and 318 +/- 114 nmol/l, respectively. Basal influx was reduced to 88 +/- 8 and 145 +/- 17 nmol/l [Ca2+]i (1-10 mmol/l Ca2+, p < 0.01) by 30 mmol/l D-glucose. This effect of HG was confirmed by Mn2+ quenching of fura-2, indicating reduced entry of divalent cations via the capacitative pathway. Equimolar L-glucose had no effect on Ca2+ influx, consistent with a non-osmotic mechanism. Arginine vasopressin (10 mumol/l) elicited weaker release of stored Ca2+ and subsequent influx in HG cells (191 +/- 33 vs 153 +/- 24 nmol/l, 400 +/- 76 vs 260 +/- 33 nmol/l, 1-10 mmol/l Ca2+, NG/HG, p < 0.05). To examine the involvement of PKC in the effect of HG on capacitative Ca2+ influx, the enzyme was activated or downregulated by treatment with 0.1 mumol/l phorbol myristate acetate (PMA) for 3 min or 24 h, respectively. PMA acutely inhibited Ca2+ 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 Ca2+ influx via SOC by HG is one mechanism of the reduced MC [Ca2+]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.