Abstract
BackgroundDiminished calcium (Ca2+) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. However, the mechanism responsible was unclear.Methodology/Principal FindingsVSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP3R) and the SR Ca2+ pumps (SERCA 2 and 3). Generally, a decrease in IP3R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP3R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca2+ in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca2+ sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca2+ in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca2+ in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca2+ responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.Conclusions/SignificanceThis work demonstrates that the previously-reported reduced Ca2+ signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP3R Ca2+ channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.
Highlights
Diminished calcium (Ca2+) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals
The body weight of control and diabetic Diabetes Resistant Bio-Breeding (DR-BB) rats were comparable but in the STZ diabetic rat model, the diabetic rats demonstrated a significant decline in weight over time (Table 1, p < 0.001)
Diabetes Attenuated the Ca2+ responses in Freshly Dispersed SMCs Freshly dispersed VSMC from arteries of control and diabetic rats were evaluated for agonist-induced Ca2+ transients
Summary
Diminished calcium (Ca2+) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. Cardiovascular disease accounts for a significant percentage of deaths of individuals inflicted with type 1 diabetes [3,4,5,6,7]. Cardiovascular events such as stroke are higher in people with type 1 diabetes than in those without diabetes [8]. As the duration of life with diabetes increases, so increases the risk of cardiovascular events and mortality [9]. While the majority of research effort concerning diabetic vascular disease has been focused on changes in the endothelial cells, alterations in the vascular smooth muscle cells (VSMCs) are known to occur [11,12,13]. Many of them have a common upstream pathway that includes the release of calcium (Ca2+) from intracellular stores, including the sarcoplasmic reticulum (SR)
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