AB73. Effect of human tissue kallikrein 1(hKLK1) on penile erectile function in the aged rat

Abstract: Human tissue kallikrein1 (hKLK1), as a pleiotropic agent in cardiovascular system, plays an important role in vasodilation, endothelial cell protection, anti-tissue fibrosis, etc. However, wheather hKLK1 has impact on erectile function is still unknown. This study was aim to explore the effect and the potential mechanisms of hKLK1 on age-related erectile dysfunction (ED) with the transgenic Sprague-Dawley (SD) rats harboring hKLK1 gene (TGR(hKLK1)). Both of male wild type SD rats and TGR(hKLK1) were fed to 4 month old and 27 month old and divided into four groups: young wild type rats (yWTR) as the control group; young TGR(hKLK1) (yTGR) group; aged wild type rats (aWTR) group and aged TGR(hKLK1) (aTGR) group. The existence of hKLK1 gene in each rat was identified at genome DNA, mRNA and protein level. The ratio of peak intracavernous pressure (ICP) to systemic mean arterial pressure (MAP) at various voltages of electrical stimulation to the cavernous nerve (CN) were measured to evaluate the erectile function of rats in all groups. Then, the expression of endothelial nitric oxide synthase (eNOS), phospho-eNOS(Ser1177), phospho-eNOS(Thr495), inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS), cyclooxygenase-2 (COX2) and prostaglandin I synthase (PTGIS) in corpus cavernosum (CC) were measured by real-time reverse transcriptase-PCR and/ or western blot. Afterwards, we assayed NO production and the nitric oxide synthase (NOS) activity in CC. The concentration of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) in CC were also examined by enzyme-linked immunosorbent assay (ELISA). Furthermore, the transforming growth factor β1 (TGFβ1) expression and the ratio of smooth muscle to collagen by masson’s trichrome staining within CC were analyzed to evaluate the degree of penile tissue fibrosis. Lastly, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was conducted to calculate the apoptosis index (AI) of cells in CC. All TGR (hKLK1) carried and expressed hKLK1 gene while wild type rats negatively contained it. No conspicuous diversity was found between yWTR and yTGR groups at all the rest of the tests. The peak ICP/MAP ratio of aWTR group was considerably decreased compared with the control group, but the erectile function was greatly improved in aTGR group relative to aWTR group at voltages of 2.5V, 5.0V and 7.5V. When compared to control group, the expression of eNOS, phospho-eNOS(Ser1177) and COX2 in CC were decreased while phospho-eNOS(Thr495) was increased significantly in aWTR group, but these changes were obviously restored in aTGR group. No difference of iNOS, nNOS and PTGIS protein content was found between four groups. The NO level and NOS activity in aTGR was markedly higher than that in aWTR, although it was still lower than yWTR. In accord with the above results, the cGMP and cAMP concentration were improved dramatically in CC of aTGR. Moreover, hKLK1 could prevent the increase of TGFβ1 mRNA and protein expression and smooth muscle/collagen ratio in CC of aWTR. Meanwhile, hKLK1 could attenuate the cell apoptosis in CC of aged rat. In summary, our results suggest that hKLK1 may improve penile erectile function in the aged rat via activating eNOS/NO/cGMP and COX2/cAMP pathways and anti-tissue fibrosis as well as anti-apoptosis effect.

Keywords: Human tissue kallikrein 1(hKLK1); erectile function; eNOS/NO/CGMP pathways; COX2/cAMP pathways