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Research Article

Elevated Ca²⁺ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells

¹University of Washington ²Children's Hospital Medical Center

Submitted 22 June 2009 ; revised 22 September 2009 ; accepted in final form 13 October 2009

Ca²⁺ sparklets are subcellular Ca²⁺ signals produced by the opening of L-type Ca²⁺ channels (LTCCs). In cerebral arterial myocytes, Ca²⁺ sparklet activity varies regionally, resulting in low and high activity, "persistent" Ca²⁺ sparklet sites. Although increased Ca²⁺ influx via LTCCs in arterial myocytes has been implicated in the chain of events contributing to vascular dysfunction during acute hyperglycemia and diabetes, the mechanisms underlying these pathological changes remain unclear. Here, we tested the hypothesis that increased Ca²⁺ sparklet activity contributes to higher Ca²⁺ influx in cerebral artery smooth muscle during acute hyperglycemia and in an animal model of non-insulin dependent, type 2 diabetes: the dB/dB mouse. Consistent with this hypothesis, acute elevation of extracellular glucose from 10 to 20 mM increased the density of low activity and persistent Ca²⁺ sparklet sites as well as the amplitude of LTCC currents in wild type cerebral arterial myocytes. Furthermore, Ca²⁺ sparklet activity and LTCC currents were higher in dB/dB than in control myocytes. We found that activation of PKA contributed to higher Ca²⁺ sparklet activity during hyperglycemia and diabetes. In addition, we found that the interaction between PKA and the scaffolding protein A-kinase anchoring protein (AKAP) was critical for the activation of persistent Ca²⁺ sparklets by PKA in cerebral arterial myocytes after hyperglycemia. Accordingly, PKA inhibition equalized Ca²⁺ sparklet activity between dB/dB and WT cells. These findings suggest that hyperglycemia increases Ca²⁺ influx by increasing Ca²⁺ sparklet activity via a PKA-dependent pathway in cerebral artery myocytes and contributes to vascular dysfunction during diabetes.

sparklets; PKA; total internal reflection fluorescence microscopy