High glucose level inhibits capacitative Ca 2 + influx in cultured rat mesangial cells by a protein kinase C-dependent mechanism

Abstract

Diabetic glomerulopathy is a common long-term complication of insulin-dependent diabetes mellitus [1]. Hyperfiltration, resulting from a fall of afferent arteriolar resistances with increased glomerular capillary pressure, is an early sign of glomerular involvement [1,2]. Such haemodynamic changes are believed to result from reduced vascular sensitivity to vasoconstrictors [3]. Arteriolar relaxation could be explained by enhanced production of vasodilators, such as prostaglandins [4], or atrial natriuretic peptides [5]. Alternatively, reduced responsiveness of the contractile apparatus of the glomerulus should be considered [3,6]. Diabetologia (1997) 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 m mol/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 m mol/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]

Diabetes-induced activation of protein kinase C inhibits store-operated Ca 2+ uptake in rat retinal microvascular smooth muscle

“…In our study, the action of high K + was maintained in the retinal MVSM cells from diabetic rats suggesting that neither the VDCCs themselves nor their activation require- PKC activation (PMA). These findings are consistent with a similar mechanism for the glucose-induced inhibition of store-operated Ca 2+ channels in human glomerular mesangial cells [35] and contradict recent studies showing that PKCs open store-operated Ca 2+ channels in vascular myocytes [25] and glomerular mesangial cells [43]. These differences are explained by heterogeneity of store-depletion/receptor activation pathways and by tissue specificity in PKC isoform activation by diabetes/hyperglycaemia.…”

“…This accords with previous observations showing that hyperglycaemia can inhibit store-operated Ca 2+ influx in other tissues relevant to diabetic complications [23,35]. The functions of Ca 2+ entry through store-operated Ca 2+ channels include: (i) regulation of smooth muscle tone [36]; (ii) refilling of Ca 2+ stores following agonist stimulation [37,34] (iii) modulation of numerous Ca 2+ -dependent enzymes [38,39,40] and (iv) control of gene expression through calcineurin-induced dephosphorylation of the transcription factor NF-AT and calmodulin stimulation of various transcription factors [41,42].…”

Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-α stimulation, and effects of a phosphodiesterase inhibitor

“…On the other hand, an investigation using mesangial cells revealed a decrease in cytoplasmic Ca 2+ and changes in dynamics due to PKC activation (Mene et al, 1997;Rong et al, 2001). Furthermore, interaction between cAMP and Ca 2+ in various cells has been reported (Ahmed et al, 1995;Landa et al, 2005).…”

Protein Kinase Cα Phosphorylates the TRPC1 Channel and Regulates Store-operated Ca2+ Entry in Endothelial Cells

“…Although this effect of PKC␣ may be the result of activation of cellular Ca 2ϩ entry, the role of PKC␣ in the regulation of SOCs is unclear, as the results of studies in several cell types have been contradictory (27)(28)(29)(30)(31)(32)(33). Thus, we addressed the role of PKC␣ in signaling TRPC1 activation and thereby triggering Ca 2ϩ entry and endothelial barrier dysfunction.…”