Effect of large conductance Ca2+-activated K+ channel current and cytosolic calcium concentrations in retinal artery smooth muscle cells on diabetic retinal artery tension

Background

Diabetic retinopathy (DR) is a common microvascular complications of the retina, retinal vascular smooth muscle cells of large conductance calcium-activated potassium channels (BK) is a major factor in regulating vasomotor and hemodynamic.Currently, functional changes of BK channel in retinal artery smooth muscle cells (RASMCs) and its role in DR were rarely reported.

Objective

This study was to investigate the early vascular damage mechanisms in DR by detecting the changes of BK channels current, calcium concentration and open probability (NP₀) of BK channel with different calcium concentration in RASMCs of normal and diabetic rats.

Method

Fifty SPF SD 8-12 weeks old rats were randomly divided into normal control group and diabetic model group.Forty diabetic rats was intraperitoneally injected with 60 mg/kg streptozotocin to form type 1 diabetic model, 10 rats (the normal control group) were injected sodium citrate solution with the same manner.Fluorescent probe was applied to detect calcium concentration in rat RASMCs; RASMCs were isolated by using enzyme digestion, and BK-channel electric currents and calcium concentrations in the RASMCs were measured by whole-cell patch clamp technique and fluorescence assay, respectively.The NP₀ of BK channel was measured by single patch clamp technique.

Results

Diabetic models were successfully established in 36 rats with the success rate 90%.When stimulation voltage is greater than 60 mV, the current density of BK channel in RASMCs of diabetic model group decreased; when stimulating voltage was 100 mV, the BK channel currents of RASMCs in the normal control group and diabetic model group were (100±23)PA/PF and (50±7)PA/PF, the difference was statistically significant (t=19.80, P<0.05). After adding specific BK channel blocker African scorpion toxin 100 nmol, the BK channel current in the normal control group significantly reduced, and that in the diabetes model group was not significantly changed; the calcium ion concentrations in RASMCs were (123±11)nmol/L and (255±10)nmol/L in the normal control group and diabetic model group, the difference was statistically significant (t=32.50, P<0.05). When stimulation voltage was 60 mV, with increasing calcium ion concentration, the NP₀ of BK channel increased (F=15.28, P<0.05).

Conclusions

The electric current and NP₀ of BK-channel are obviously reduced and the calcium concentration is evidently elevated in RASMCs of diabetic rats, suggesting that the abnormal of BK-channel is probably one of the important causes of retinal artery abnormal contraction in diabetic rats.

Diabetes mellitus, experimental; Retinal arterioles; Muscle, smooth, vascular/metabolism; Large conductance calcium-activated potassium channel; Calcium/physiology; Animals, rats; Patch-clamp