Effect of Glucose on Endothelin-1-induced Calcium Transients in Cultured Bovine Retinal Pericytes

Abstract

Published work has shown that endothelin-1-induced contractility of bovine retinal pericytes is reduced after culture in high concentrations of glucose. The purpose of the present study was to establish the profile of endothelin-1-induced calcium transients in pericytes and to identify changes occurring after culture in high concentrations of glucose. Glucose had no effect on basal levels of cytosolic calcium or on endothelin-1-induced calcium release from intracellular stores. However, influx of calcium from the extracellular medium after endothelin-1 stimulation was reduced in pericytes that had been cultured in 25 mM D-glucose. L-type Ca 2؉ currents were identified by patch clamping. The L-type Ca 2؉ channel agonist, (؊)-Bay K8644, caused less influx of calcium from the extracellular medium in pericytes that had been cultured in 25 mM D-glucose than in those cultured with 5 mM D-glucose. However, 3-O-methylglucose, a nonmetabolizable analogue of glucose which can cause glycation, had similar effects to those of high concentrations of glucose. The results suggest that reduced function of the L-type Ca 2؉ channel that occurs in bovine retinal pericytes after culture in high concentrations of D-glucose is probably due to glycation of a channel protein.In epidemiological studies hyperglycemia is the single factor most consistently associated with diabetic microangiopathy, including retinal microangiopathy (1). Longitudinal studies in diabetes have shown that alterations in capillary function and blood flow occur in advance of the structural changes seen in diabetic retinopathy such as pericyte loss, microaneurysm formation, and neovascularization (2). Pericyte tone normally regulates retinal capillary blood flow (3). Because retinal capillaries are devoid of extrinsic innervation (4), pericyte tone is controlled to a large extent by changes in the partial pressures of blood gases (5) and endothelium-derived agonists such as endothelin-1 (ET-1) 1 (6). We have previously shown (7) that exposure to high ambient glucose concentrations in vitro caused a reduction in the contractile response of the bovine retinal pericyte (BRP) to ET-1 without any reduction in either ET-1 binding or generation of inositol trisphosphate (Ins(1,4,5)P 3 ). The aim of the present study was 1) to establish the profile of ET-1-induced changes in cytosolic calcium concentrations ([Ca 2ϩ ] i ) in the cultured pericyte and 2) to determine whether prior exposure to raised concentrations of glucose would alter ET-1-induced calcium transients in these cells. EXPERIMENTAL PROCEDURESIsolation and Culture of Bovine Retinal Pericytes-BRP were isolated as described by Gitlin and D'Amore (8). Briefly, bovine retinal homogenates were subjected to microscopically controlled enzyme digestion, and the resultant microvessel fragments were trapped on a 53-m mesh. The microvessels were suspended in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 5 mM glucose, antibiotics (200 units/ml penicillin and 200 g/ml streptomycin, Sigma)...

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

“…Since our work used the same experimental model (STZ-induced diabetic rats) and diabetic duration (3 months) as the other studies, this difference appears to arise through properties intrinsic to retinal MVSM cells. The present observation is in contrast with a previous study on bovine retinal pericytes where endothelin produced a sustained increase in cell Ca 2+ that was sensitive to L-type Ca 2+ channel blockers [33]. This Ca 2+ increase was attenuated in high glucose.…”

Reduced Vasorelaxant Effect of Carbon Monoxide in Diabetes and the Underlying Mechanisms

“…After chronically culturing normal tail artery SMCs with 25 mmol/l glucose or 25 mmol/l 3-OMG, the sensitivity of K Ca channels in these SMCs to CO was consistently diminished. Because 3-OMG is a nonmetabolizable glucose analog, these results indicate that the glycation of K Ca channels rather than the metabolism of glucose by cultured vascular SMCs might be the mechanism for the altered K Ca channel functionality in diabetes (25). On the other hand, chronically culturing normal SMCs with 25 mmol/l mannitol did not alter the effect of CO on K Ca channels.…”

“…In these cells, CO (10 µmol/l) had no effect on the 3-min mean NPo of single K Ca channels. In a similar previous study, a 10-day culture period was applied for the study of the influence of high concentrations of glucose or 3-OMG on the cellular functions of pericytes (25). Also shown in Fig.…”

Diabetes-Induced Inhibition of Voltage-Dependent Calcium Channels in the Retinal Microvasculature: Role of Spermine

“…Interestingly, in contrast to our finding that diabetes inhibits VDCC function in feeder vessels whose diameters are Ͻ10 m, a study of retinal arterioles with diameters Ͼ30 m found that the VDCC current was not altered after 3 months of streptozotocin-induced diabetes. 36 These observations led us to postulate that VDCC function in the feeder vessel portion of the retinal vasculature is particularly vulnerable to diabetes.…”