September 2003 - More than eight million men are at risk for erectile dysfunction (ED) induced by Type II (insulin resistant) diabetes. While the exact mechanism(s) involved in diabetes mellitus induced erectile dysfunction (DMED) is not yet understood, a team of researchers has hypothesized that certain proteins may regulate penile vascular tone increasing sensitivity to the action of vasoconstrictor agents. Their findings suggest that protein kinase C (PKC) may contribute to an enhanced vasoconstriction of the penile circulation and reduced erectile response.
Constriction of the penile vasculature prevents erection and is largely mediated by two agents: �-adrenergic agonists or endothelin (ET-1). These agents cause vasoconstriction by activating phospholipase C (PLC) and result in the generation of inositol triphosphate (IP3) and diacylglycerol (DAG). This pathway is believed to recruit PKC in the constrictor response. Researchers have tested the hypothesis that in diabetic-obese Zucker rats, there is a depressed erectile response caused by increased action of the vasoconstrictor pathway involving PKC in a key sensitization process.
A New Study
The authors of a new study entitled "Altered Penile Vascular Reactivity and Erection of the Zucker Rat: A Role for PKC Ca2+ Sensitization," are Christopher J. Wingard, Delores Young, Katherine Lane and Shadhid Husain, all of the Department of Physiology, the Medical College of Georgia, Augusta, GA. They will present their findings during the upcoming scientific conference, Understanding Renal and Cardiovascular Function Through Physiological Genomics, a meeting of the American Physiological Society (APS) (www.the-aps.org), being held October 1-4, 2003 at the Radisson Riverfront Hotel and Convention Center, Augusta, GA.
Methodology
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The researchers examined the erectile response (ICP/MAP) to pelvic ganglion stimulation using lean and obese-diabetic Zucker rats. Their methodology included:
- Erectile Response Measurements: Lean or obese-diabetic male rats between 15-18 weeks of age were used. The animals were anesthetized and the left carotid artery cannulated to continuously monitor the mean arterial pressure (MAP). The right corpus cavernosum was cannulated to permit continuous monitoring of intra-cavernosal pressure (ICP) and the left corpus cavernosum was cannulated to allow for administration of vasoactive compounds. Bipolar electrodes were positioned on the right major pelvic ganglion (MPG) and, during the experiment, stimulatory voltages applied to the MPG ranged from 1 to 6 volts delivered in 5 msec pulses at a frequency of 12 Hz. The duration of stimulation was 1 minute with rest periods of 5 minutes between subsequent stimulations.
- Isolated Cavernosal Tissue Force Measurements: Cavernosal strips were bathed in a physiological salt solution and gassed with breathing air. Strips were mounted at lengths that allow maximal force generation during potassium-depolarization. Cumulative dose response curves for the �-adrenergic agonist phenylephrine, (PE) 0.1-10 mM and ET-1 (0.01-10 mM) were preformed. Cumulative dose-response protocols were completed either in the absence (Control) or presence of PKC inhibitor Chelerythrine (10 mM). Tissues were incubated with the inhibitor for 30 minutes prior the completion of the dose-response protocol.
- Western Blotting: Equal amounts of proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes followed by incubation with anti-PKC and Rho-kinase isoforms or RhoA antibodies for 3 h at 20 �C. After washing, the membranes were incubated with secondary antibodies for 1 h at 20 �C. For chemiluminescent detection, the membranes were treated with enhanced chemiluminescent (ECL) reagent and subsequently exposed to ECL hyperfilm.
- Quantification of Protein of Interest: The densities of the protein bands were determined by scanning with a densitometer. Specific immunoreactive bands were expressed as arbitrary units (AU), which were calculated from the area of peak of selected band scanned by the densitometer. The density values were normalized to the protein content of b-actin and expressed relative to those determined from the lean tissues. Data were analyzed using analysis of variance (ANOVA) with post hoc comparisons; statistical significance was set at P < 0.05.
Results
The researchers observed that:
- erectile response of the obese animals was suppressed by >30 percent at voltages >3;
- maximal contractile response of tissues from obese-diabetic animals was increased by 25% for PE and 35% for ET-1 stimulations. However, there was no significant shift in the sensitivity to these agonists when comparing calculated EC50's for lean and diabetic-obese tissues;
- PKC inhibitor Chelerythrine inhibited more than 70 percent of the force generated by ET-1 in tissues from the obese-diabetic animals while only blocking 30 percent of the phenylephrine induced force generation;
- obese-diabetic corpus cavernosum showed increased protein expression of PKC isozymes a, d and Rho-kinase b.
Conclusions
These results suggest that PKCs may contribute to a vasoconstriction of the penile circulation, and to reduced erectile response in the diabetic-obese Zucker rat. Future research is aimed at identifying the specific elements in the signaling pathway involving PKC and controlling the constrictive behavior of the penile vasculature. Such findings, by adding to the understanding of how constrictor agonists play a role in DMED, will eventually make a significant contribution to the treatment methods for those who display the hallmarks of obesity-induced hypertension and diabetes.
Source: American Physiological Society
