Molecular and Functional Characterization of ORAI and STIM in Human Corporeal Smooth Muscle Cells and Effects of the Transfer of Their Dominant-Negative Mutant Genes into Diabetic Rats

Abstract

We investigated the molecular identity and functional activity of STIM1 and ORAI in human cavernous smooth muscle. We also determined whether transferring dominant negative mutants of the STIM1 or ORAI gene would correct diabetes related erectile dysfunction in a rat model. Reverse transcriptase-polymerase chain reaction was done to identify ORAI and STIM in human cavernous smooth muscle. For the in vivo study intracavernous pressure, blood pressure and their ratio were assessed after cavernous nerve stimulation to diabetic rats transfected with pcDNA encoding the ORAI1(DN) or the STIM1(DN) gene. ORAI (1, 2 and 3) and STIM (1 and 2) were identified in human cavernous smooth muscle cells. After [Ca(2+)] depletion by thapsigargin and cyclopiazonic acid we recorded store operated Ca(2+) entry in human cavernous smooth muscle cells. Entry was decreased by the store operated Ca(2+) channel blockers La(3+) and SKF96365. Mean ± SE intracavernous pressure/blood pressure in rats with ORAI1(DN) or STIM1(DN) gene transfer was 78.8% ± 2.2% and 77.1% ± 1.2% in 11 and 10, respectively. This result was significantly higher than that in 10 diabetic controls (51.0% ± 3.7%) and similar to that in 9 normal controls (85.8% ± 2.6%). Using reverse transcriptase-polymerase chain reaction we confirmed transgene expression in rat cavernous tissue. Transfer of ORAI(DN) or STIM1(DN) genes restored erectile function in diabetic rats. It might be applicable to develop new therapy for erectile dysfunction.