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Glucose-induced changes in protein kinase C and nitric oxide are prevented by vitamin E
1Section of Nephrology, Department of Medicine, Case Western Reserve University, Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106, USA. mbg4@po.cwru.edu
Abstract:
Changes in activity or expression of protein kinase C (PKC), reactive oxygen products, and nitric oxide (NO) may account for the alteration in cell behavior seen in diabetes. These changes have been proposed to be part of the pathophysiology of erectile dysfunction. We sought to ascertain if corpus cavernosal vascular smooth muscle cells (CCSMC) grown in a high glucose milieu exhibit changes in the activity and expression of PKC isoforms, NO, and reactive oxygen products and to find out if these changes are prevented by alpha-tocopherol. Rat CCSMC were grown in 5, 15, and 30 mM glucose concentrations for 3, 7, and 14 days. PKC isoform expression was assayed with isoform-specific antibodies. In CCSMCs grown in 30 mM glucose for 2-wk, PKC-beta(2)-isoform was upregulated (n = 4; P < 0.01), whereas the expression of alpha-, delta-, epsilon-, and beta(1)-isoforms was unchanged. NO as measured by nitrate-to-nitrite ratio was greatly diminished at 14 days in 30 mM (n = 4; P < 0.002) compared with 5 mM glucose. Reactive oxygen products were upregulated at 14 days when they were assayed by the fluorescent probe dichlorofluorescein diacetate bis(acetoxy-methyl) (DCFH-DA) (n = 5; P < 0.01). When these same cells were exposed to alpha-tocopherol for 14 days, there was a reduction of PKC-beta(2) (57.8%; P < 0.01; n = 4) and a reduction in reactive oxygen product formation (71.1%; P < 0.001; n = 4), along with an increase in nitrate-to-nitrite ratio (43.9%; P < 0.01, n = 4). These results suggest that there may be an interrelationship between PKC, NO, and reactive oxygen product formation in CCSMC exposed to a high glucose environment.
Insights
High glucose levels in diabetes alter protein kinase C (PKC) and nitric oxide (NO) in corpus cavernosal smooth muscle cells, contributing to erectile dysfunction. Alpha-tocopherol may mitigate these diabetes-related changes.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- Diabetes mellitus is associated with cellular changes impacting smooth muscle function.
- Erectile dysfunction (ED) is a common complication of diabetes, potentially linked to altered signaling pathways.
- Protein kinase C (PKC), nitric oxide (NO), and reactive oxygen species (ROS) are implicated in vascular smooth muscle cell (VSMC) behavior.
Purpose of the Study:
- To investigate the effects of high glucose on PKC isoforms, NO production, and ROS generation in corpus cavernosal smooth muscle cells (CCSMC).
- To determine if alpha-tocopherol can prevent or reverse high glucose-induced changes in CCSMC.
Main Methods:
- Rat CCSMC were cultured in varying glucose concentrations (5, 15, 30 mM) for up to 14 days.
- PKC isoform expression was analyzed using isoform-specific antibodies.
- Nitric oxide (NO) levels were assessed via the nitrate-to-nitrite ratio.
- Reactive oxygen products (ROS) were measured using the fluorescent probe DCFH-DA.
- Cells were also treated with alpha-tocopherol to evaluate its protective effects.
Main Results:
- High glucose (30 mM) for 14 days significantly upregulated PKC-beta(2) isoform expression in CCSMC.
- Nitric oxide (NO) levels were significantly diminished, and reactive oxygen products (ROS) were significantly upregulated under high glucose conditions.
- Alpha-tocopherol treatment reversed these effects, reducing PKC-beta(2) and ROS, while increasing NO levels.
Conclusions:
- High glucose environments induce detrimental changes in CCSMC, including PKC-beta(2) upregulation, NO reduction, and ROS increase, contributing to diabetic pathophysiology.
- Alpha-tocopherol demonstrates a protective role against high glucose-induced cellular damage in CCSMC.
- These findings suggest a potential therapeutic strategy targeting PKC, NO, and ROS pathways for diabetic complications like ED.