Abstract
Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca2+ spark and STOC activities, measured by confocal microscopy and patch-clamp technique, respectively, occur in isolated CASMCs of an experimental model of type-2 diabetes (db/db mouse). We found that mean Ca2+ spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca2+ released through Ca2+ sparks in db/db CASMCs, though Ca2+ spark frequency remained. Interestingly, the frequency of large-amplitude Ca2+ sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from −50 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel β1/α subunit ratio found in db/db vascular tissues. Taken together, Ca2+ spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK β1 subunit/α subunit ratio which underlies the significant reduction of Ca2+ spark/STOC coupling in CASMCs of diabetic animals.
Highlights
More than 65% of patients with diabetes die from cardiovascular disease or stroke [1]
Since impaired function of Ryanodine Receptors (RyRs) might contribute to the vascular alterations in the cerebral arteries of diabetic db/db mice [8,9,33], our first aim was to study the in situ activity of vascular RyR in single, freshly isolated cerebral artery smooth muscle cells (CASMCs) of diabetic mice
We found that the db/db CASMCs produced similar Ca2+ spark frequency to control cells (in Hz: 0.860.1 vs. 0.860.1; or in events?s21?mm21: 0.05360.008 vs. 0.04460.006, in 41 db/db cells and 43 control cells, respectively (Fig.1B), with no further modifications in the number of firing sites per cell, eager site probability and the maximum number of events within a same site (Figure S1)
Summary
More than 65% of patients with diabetes die from cardiovascular disease or stroke [1]. Investigators have demonstrated impairment of endothelium-independent dilation in the presence of nitric oxide donors: in coronary arterioles and aorta of db/db mice in the presence of sodium nitroprusside [17,20], and in arteries of type 2 diabetic patients after administration of glycerin trinitrate [24,25]. All these data suggest that smooth muscledependent mechanisms are responsible for the vascular dysfunction associated with type-2 diabetes. The disease appears to alter functional responses of resistance arteries at endothelial level and in active smooth muscle layers
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