cAMP‐dependent post‐translational modification of neuronal nitric oxide synthase neuroprotects penile erection in rats

Abstract

Objectives To evaluate nNOS phosphorylation, nNOS uncoupling, and oxidative stress in the penis and major pelvic ganglia (MPG), before and after the administration of the cAMP-dependent protein kinase A (PKA) agonist colforsin in a rat model of bilateral cavernous nerve injury (BCNI) which mimics nerve injury following prostatectomy. Materials and Methods Adult male Sprague–Dawley rats were divided into BCNI and sham groups. Each group included 2 subgroups: vehicle and colforsin (0.1 mg/kg/day i.p.). After 3 days, erectile function (intracavernosal pressure) was measured and penes and MPG were collected for molecular analyses of phospho(P)-nNOS (Ser-1412 and Ser-847), total nNOS, nNOS uncoupling, binding of neuronal nitric oxide synthase (PIN) to nNOS, gp91phox subunit of NADPH oxidase, active caspase 3, PKA catalytic subunit alpha (PKA-Cα) (by Western blot) and oxidative stress (hydrogen peroxide [H2O2] and superoxide by Western blot and microdialysis method). Results Erectile function was decreased 3 days after BCNI and normalized by colforsin. nNOS phosphorylation on both positive (Ser-1412) and negative (Ser-847) regulatory sites, and nNOS uncoupling, were increased after BCNI in the penis and MPG and normalized by colforsin. Hydrogen peroxide and total ROS productions were increased in the penis after BCNI and normalized by colforsin. Protein expression of gp91phox was increased in the MPG after BCNI and was normalized by colforsin treatment. Binding of PIN to nNOS was increased in the penis after BCNI and was normalized by colforsin treatment. Protein expression of active Caspase 3 was increased in the MPG after BCNI and was normalized by colforsin treatment. Protein expression of PKA-Cα was decreased in the penis after BCNI and normalized by colforsin. Conclusion Collectively, BCNI impairs nNOS function in the penis and MPG by mechanisms involving its phosphorylation and uncoupling in association with increased oxidative stress, resulting in erectile dysfunction. PKA activation by colforsin reverses these molecular changes and preserves penile erection in the face of BCNI.

Phosphodiesterase type 5 in men with vasculogenic and post‐radical prostatectomy erectile dysfunction: is there a molecular difference?

“…By contrast, in post‐RP ED erectile tissue, nNOS phosphorylation on Ser‐1412 was increased and nNOS was uncoupled. The latter data mimic our recent findings in the rat penis 3 days after cavernous nerve damage, which, similarly to men with post‐RP ED, showed increased nNOS phosphorylation on Ser‐1412 and nNOS uncoupling . While the mechanism of increased nNOS phosphorylation on its activation site in the neuropathic erectile tissue is unknown, it conceivably indicates a deleterious effect of over‐activated nNOS, shown previously in the central and peripheral nervous systems ; overactivated, but uncoupled, nNOS further contributes to decreased NO bioavailability.…”

“…Penile tissue was homogenized as reported previously . NO synthases (NOSs) were partially purified as described previously .…”

“…Penile tissue was homogenized as reported previously . NO synthases (NOSs) were partially purified as described previously . Membranes with partially purified NOSs were probed with anti‐phospho‐neuronal (n)NOS (Ser‐1412) antibody (polyclonal rabbit, kindly provided by Dr Solomon Snyder, Johns Hopkins University, Baltimore, MD, USA) at 1:6 000 dilution or anti‐phospho‐endothelial (e)NOS (Ser‐1177) antibody (polyclonal rabbit; Cell Signaling Technology, Beverly, MA, USA) at 1:450 dilution (for phospho‐nNOS and phospho‐eNOS analyses) .…”

“…NO synthases (NOSs) were partially purified as described previously . Membranes with partially purified NOSs were probed with anti‐phospho‐neuronal (n)NOS (Ser‐1412) antibody (polyclonal rabbit, kindly provided by Dr Solomon Snyder, Johns Hopkins University, Baltimore, MD, USA) at 1:6 000 dilution or anti‐phospho‐endothelial (e)NOS (Ser‐1177) antibody (polyclonal rabbit; Cell Signaling Technology, Beverly, MA, USA) at 1:450 dilution (for phospho‐nNOS and phospho‐eNOS analyses) . After probing for phosphoforms, membranes were stripped and probed with antibodies against nNOS (polyclonal rabbit, 1:9 000 dilution; Dr Solomon Snyder) or eNOS (monoclonal mouse, 1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA,).…”

“…phospho‐nNOS (Ser‐1412) and phospho‐eNOS (Ser‐1177) densities were normalized relative to those of nNOS and eNOS, respectively, in partially purified samples. Dimeric and monomeric forms of nNOS and eNOS were measured by low temperature SDS electrophoresis, as described previously , using anti‐nNOS antibody (polyclonal rabbit, 1:1 000 dilution; Cell Signaling Technology) and anti‐eNOS antibody (monoclonal mouse, 1:500 dilution; Santa Cruz Biotechnology). For analysis of total nNOS (polyclonal rabbit antibody, 1:1 000 dilution; Cell Signaling Technology), total eNOS (monoclonal mouse antibody, 1:500 dilution; Santa Cruz Biotechnology), ROCKα (monoclonal mouse antibody, 1:1 000 dilution; BD Transduction Laboratories, San Jose, CA, USA), ROCKβ (monoclonal mouse antibody, 1:1 000 dilution; Santa Cruz Biotechnology), phospho‐myosin phosphatase target subunit 1 (MYPT1; Thr‐696; polyclonal rabbit antibody, 1:1 000 dilution; Santa Cruz Biotechnology), PDE5 (monoclonal mouse antibody, 1:450 dilution; BD Transduction Laboratories), 4‐hydroxy‐2‐nonenal (4‐HNE; polyclonal rabbit antibody, 1:2 000 dilution; Alpha Diagnostic International, San Antonio, TX, USA), phospho‐Akt (Ser‐473; polyclonal rabbit antibody, 1:1 000 dilution; Cell Signaling Technology), and Akt (polyclonal rabbit antibody, 1:1 000 dilution; Cell Signaling Technology), a separate set of homogenates (70–100 μg) was used without purification, and standardized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; monoclonal mouse antibody, 1:1 000 dilution; Santa Cruz Biotechnology) .…”