ABSTRACT
PnPP-19 peptide has a primary sequence design based on molecular modeling studies of PnTx2-6 toxin. It comprises the amino acid residues that are potentially significant for the pharmacological action of PnTx2-6. Ex vivo and in vivo experiments in normotensive, hypertensive, or diabetic murine models have shown a significant improvement in penile erection after administration of PnPP-19. Given the potential use of PnPP-19 in pharmaceutical formulations to treat erectile dysfunction and the lack of information concerning its mode of action, the present work investigates its activities on the nitrergic system. PnPP-19 induced a significant increase in nitric oxide (NO) and cGMP levels in corpus cavernosum (cc). These effects were inhibited by l-NAME, a non-selective inhibitor of nitric oxide synthase (NOS); were partially inhibited by 7- Nitroindazole, a selective inhibitor of neuronal NOS (nNOS); and were abolished by L-NIL, a selective inhibitor of inducible NOS (iNOS). This potentiating effect was not affected by atropine. PnPP-19 also led to changes in mRNA levels, protein expression and phosphorylation at specific sites of NOS, in cc. Assays using cavernous tissue from knockout mice to endothelial NOS (eNOS), nNOS or iNOS showed that PnPP-19 potentiates relaxation only in eNOS-knockout mice, which suggests an essential role for nNOS. Surprisingly, iNOS enhanced the potentiation of erectile function evoked by PnPP-19. Our results demonstrate that this new synthetic peptide potentiates erectile function via nitric oxide activation and reinforce its role as a new pharmacological tool for the treatment of erectile dysfunction.
Subject(s)
Erectile Dysfunction/drug therapy , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type I/metabolism , Peptides/pharmacology , Animals , Computational Biology , Erectile Dysfunction/metabolism , Male , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase Type I/deficiency , Nitric Oxide Synthase Type I/genetics , Nitric Oxide Synthase Type II/deficiency , Nitric Oxide Synthase Type II/genetics , Peptides/chemical synthesis , Peptides/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
During semen cryopreservation, the sensitivity of equine sperm to oxidative stress is increased by the eliminated seminal plasma. Thus, antioxidant addition to the semen extender can be helpful to the sperm survival after freezing and thawing. This work aimed to test whether coenzyme Q10 (CoQ10) added in different concentrations to the INRA 82 freezing extender has antioxidant function on equine sperm to improve its fertilizing ability. Semen samples from five stallions were frozen with the extenders: (T1) INRA 82, control, (T2) T1+ 5 µM CoQ10, (T3) T1+ 25 µM CoQ10, and (T4) T1+ 50 µM CoQ10. After sample thawing, sperm motility and kinetics characteristics were evaluated using a computer-assisted sperm analysis and sperm membrane functionality and integrity were evaluated with a hypo-osmotic swelling test and an epifluorescence microscopy, respectively. The nitrite (NO2-) and hydrogen peroxide (H2O2) concentrations of the semen samples were measured with spectrophotometry. There was no difference on the sperm characteristics among all treatments (P > .05). However, the 25 µM CoQ10 (T3) decreased NO2- concentration (6.7 ± 2.2 µM/µg protein) compared with the treatments T1, T2, and T4 (64.3 ± 3.7, 59.4 ± 5.3, 45.1 ± 8.6 µM/µg protein), respectively, as well H2O2 concentration (1.8 ± 0.3 µM/µg protein) compared with the control (4.6 ± 0.4 µM/µg protein) and 5 µM CoQ10 treatments (4.8 ± 0.2 µM/µg protein, P < .05). In conclusion, 25 µM CoQ10 plays a significant role as antioxidant to the frozen equine sperm, decreasing NO2- and H2O2 concentrations. Thus, its addition to the INRA 82 freezing extender may be beneficial to the fertilizing ability of equine semen.
