Curtis T M, Major E H, Trimble E R, Scholfield C N
Department of Ophthalmology and Vision Sciences, The Queen's University of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital, Grosvenor Road, Belfast, N. Ireland, UK.
Diabetologia. 2003 Sep;46(9):1252-9. doi: 10.1007/s00125-003-1178-5. Epub 2003 Jul 30.
AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells.
Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution.
Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine.
CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.
目的/假设：评估糖尿病诱导的蛋白激酶C（PKC）激活对视网膜微血管平滑肌细胞中电压依赖性和非电压依赖性Ca2+内流途径的影响。
使用fura-2显微荧光测定法，在链脲佐菌素诱导的糖尿病大鼠和非糖尿病大鼠新鲜分离的视网膜小动脉中估计胞质Ca2+。通过用100 mmol/L KCl测量[Ca2+]i的升高来测试电压依赖性Ca2+内流，通过在含5 μmol/L圆孢菌素酸的无钙培养基中孵育10分钟耗尽[Ca2+]i储存，随后添加2 mmol/L或10 mmol/L Ca2+溶液时测量Ca2+升高速率来评估储存依赖性Ca2+内流。
通过电压依赖性L型Ca2+通道的Ca2+内流不受糖尿病影响。相反，储存依赖性Ca2+内流减弱。在非糖尿病大鼠的微血管中，20 mmol/L D-甘露醇对储存依赖性Ca2+内流无影响。每天注射胰岛素的糖尿病大鼠的储存依赖性Ca2+内流速率与非糖尿病对照大鼠相似。糖尿病微血管中减少的Ca2+内流在暴露于100 nmol/L星形孢菌素（一种非特异性PKC拮抗剂）2小时后逆转，并且在非糖尿病大鼠的微血管中，暴露于PKC激活剂佛波醇肉豆蔻酸酯乙酸盐（100 nmol/L）10分钟后模拟了这种情况。特异性PKCβ拮抗剂LY379196（100 nmol/L）也逆转了Ca2+内流不良，尽管其作用不如星形孢菌素有效。
结论/解读：这些结果表明，在血糖持续升高的大鼠视网膜小动脉中，储存依赖性Ca2+内流受到抑制，并且PKCβ似乎在介导这种效应中起作用。
