Cyclic AMP-dependent phosphorylation of neuronalnitric oxide synthase mediates penile erectionK. Joseph Hurta, Sena F. Sezenb, Gwen F. Lagodab, Biljana Musickib, Gerald A. Rameauc, Solomon H. Snyderd,e,f,1,and Arthur L. Burnettb,1

aDepartment of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045; bDepartment of Urology, The James BuchananBrady Urological Institute, dThe Solomon H. Snyder Department of Neuroscience, ePharmacology and Molecular Sciences, and fPsychiatry and Behavioral Sciences,The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and cDepartment of Biology, Morgan State University, Baltimore, MD 21251

Contributed by Solomon H. Snyder, August 13, 2012 (sent for review May 2, 2012)

Nitric oxide (NO)generatedbyneuronalNOsynthase (nNOS) initiatespenile erection, but has not been thought to participate in thesustained erection required for normal sexual performance. We nowshow that cAMP-dependent phosphorylation of nNOS mediateserectile physiology, including sustained erection. nNOS is phosphor-ylated by cAMP-dependent protein kinase (PKA) at serine(S)1412.Electrical stimulation of the penile innervation increases S1412phosphorylation that is blocked by PKA inhibitors but not by PI3-kinase/Akt inhibitors. Stimulation of cAMP formation by forskolinalso activates nNOS phosphorylation. Sustained penile erectionelicited by either intracavernous forskolin injection, or augmentedby forskolin during cavernous nerve electrical stimulation, is pre-vented by the NOS inhibitor L-NAME or in nNOS-deleted mice. Thus,nNOS mediates both initiation and maintenance of penile erection,implying unique approaches for treating erectile dysfunction.

gasotransmitter | smooth muscle relaxation | endothelial NOS |cyclic GMP | phosphoantibody

Nitric oxide (NO) is well established as a mediator of penileerection (1, 2). Neuronal NO synthase (nNOS) is highly lo-

calized to the penile innervation (3, 4). Electrical stimulation of thecavernous nerve (CN) to the penis elicits penile erection, which isabolished with NOS inhibitors and markedly reduced in nNOS-α–deleted mice (nNOSα−/−) (5–7). Neuronal depolarization-induced production of NO reflects calcium entry activatingcalmodulin associated with nNOS to stimulate NO formation(8, 9). This activation is short-lived and thus is believed to accountonly for the initiation of penile erection (10, 11). Sessa and col-leagues (12) established that increased blood flow and associatedshear stress, acting via PI3-kinase, augments the activity of theserine protein kinase Akt, which phosphorylates vascular endo-thelial NOS (eNOS) at serine(S)1179, causing prolonged NO for-mation at resting intracellular calcium levels (13). The increasedpenile blood flow through cavernosal vessels initiated by nNOSactivation similarly stimulates penile Akt to phosphorylate eNOS,thereby promoting sustained maximal penile erection (14).nNOS possesses a phosphorylation consensus sequence at S1412

that closely resembles the sequence surrounding eNOS-S1179,and is thought to be a target for Akt in some systems (15–21). Wewondered whether nNOS phosphorylation at S1412 might regulatepenile erection.Using a highly selective antibody for phospho-S1412-nNOS (P-nNOS) we report electrically stimulated nNOS phos-phorylation via cAMP-dependent protein kinase (PKA) and notthrough Akt. P-nNOS activity contributes to sustained erection inconcert with phospho-eNOS stimulation, and the neuronal andendothelial NOS activities are independently regulated by sepa-rate signaling cascades. We propose a model integrating the post-translational phospho-stimulation of normal erectile physiology.

ResultsElectrical Stimulation of Penile Innervation Augments Phosphorylationof nNOS. We developed a C-terminal antibody that selectively rec-ognizes P-nNOS. Immunoreactivity is absent in cells overexpressing

nNOS in which S1412 is mutated to alanine (Fig. S1). We alsodeveloped an antibody selective for unphosphorylated nNOS (unP-nNOS) that shows decreased affinity for P-nNOS but can be usedat higher concentrations to detect total nNOS. A commercialN-terminus antibody that recognizes total nNOSα does not dis-criminate nNOS S1412 phosphorylation.Using an established physiologic model of erectile function,

we found electrical stimulation of the rat CN markedly increasesP-nNOS immunoreactivity but not unP-nNOS in penile lysates(Fig. 1A). With 6-V stimulation, P-nNOS increases 15- or 27-foldfollowing 1 or 5 min of stimulation, respectively (Fig. 1 B and C).Direct application of 1- or 6-V electrical stimulation to the ratmajor pelvic ganglion (MPG) increases P-nNOS in the neuronaltissue by two- or ninefold (Fig. 1 D and E).nNOS is alternatively spliced (22–24). The alternatively spliced

forms are designated nNOS-β and nNOS-γ, whereas the pre-dominant wild-type nNOS is designated nNOSα (25–27). Alter-natively spliced nNOS isoforms retain some catalytic activity butlack the N-terminal PDZ domain that links nNOS to PSD95 andNMDA-glutamate receptors (25, 28, 29). We wondered whethernerve stimulation would influence phosphorylation of S1412 in thealternatively spliced isoforms (Fig. 1F). We detect very low levelsof phosphorylated nNOSβ and nNOSγ in the mouse MPG, withno effect of electrical stimulation. Interestingly, unphosphorylatednNOSγ is increased in nNOSα−/− mice, perhaps reflecting somecompensatory response (30).The antibodies to P-nNOS and unP-nNOS are suitable for

immunohistochemistry. Both antibodies and the N-terminalnNOS antibody selectively stain MPG neuronal cell bodies andtheir processes in a pattern similar to the neuronal marker syn-aptophysin (Fig. 2). All three antibodies similarly stain cytosolicnNOS, with staining also evident at the plasma membrane.

Electrical Stimulation Provides Prolonged Activation of nNOS Phosphory-lation Mediated by PKA. Physiologic depolarization-dependent in-creases in intracellular calcium that maximally activate nNOS areshort lived, usually returning to baseline in seconds (31, 32). Theincrease of P-nNOS in rat MPG elicited by electrical stimulation ismaintained for over a minute and then declines at 3 and 10 min(Fig. 3 A and B), similar to other persistent calcium-dependentsignaling processes (33, 34). Although the proportion of P-nNOSincreases with electrical stimulation, total nNOS normalized toβ-actin is not significantly altered. Our prior work showed distincteNOS-dependent stimulation of erection by papaverine, with re-markably decreased intracavernosal pressure (ICP) responses in

Author contributions: K.J.H., S.F.S., S.H.S., and A.L.B. designed research; K.J.H., S.F.S., G.F.L.,B.M., G.A.R., and A.L.B. performed research; K.J.H., S.F.S., B.M., S.H.S., and A.L.B. analyzeddata; and K.J.H., S.H.S., and A.L.B. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: ssnyder@jhmi.edu or aburnett@jhmi.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1213790109/-/DCSupplemental.

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eNOS−/− versus wild-type or nNOSα−/− mice. Papaverine dilatesblood vessels, increasing flow and shear stress, which augmentsphospho-eNOS (12, 13) to produce penile erection. We investi-gated whether intracavernosal papaverine in the rat would alsoaugment P-nNOS. Although papaverine increases phospho-eNOS(Fig. 3C), there is no change in nNOS phosphorylation (14). Al-though papaverine is a nonselective phosphodiesterase inhibitor,it does not increase cAMP production in penile tissue (35, 36). Thisfinding suggests separate regulation of phosphorylation of eNOSandnNOSbydistinct signalingpathways innormalerectilephysiology.We used a pharmacologic approach to identify the kinase that

phosphorylates nNOS at S1412. Ten minutes after periganglionicinjection of inhibitors, the rat MPG and CN were electricallystimulated for 1 min, whereupon the ganglion-nerve preparationwas snap-frozen for subsequent analysis (Fig. 3 D and E).Electrical stimulation elicits three- to fourfold augmentation ofP-nNOS, which is prevented by treatment with PKA inhibitorsH89 and PKA inhibitor peptide (PKAI). In contrast, wortmannin(Wrt) and LY294002 (LY), well-established inhibitors of PI3-kinase, fail to alter P-nNOS levels. The response of phospho-Aktto electrical stimulation is markedly different. Electrical stimu-lation fails to alter phospho-Akt levels, which are reduced by Wrtalthough not significantly decreased by LY. Surprisingly, H89and PKAI significantly increase phospho-Akt.The ability of PKA inhibitors to abolish the increase in P-nNOS

with electrical stimulation strongly implies that physiologicphosphorylation of S1412 is uniquely mediated by PKA. The

sequence surrounding nNOS S1412 corresponds to a PKA con-sensus site (Fig. 4A), and although another PKA consensus se-quence is present at S370, direct phosphorylation of that site hasnot been reported. Full-length nNOS purified from transfectedHEK293 cells can be selectively phosphorylated with [32P]ATPand PKA. The phosphorylation is abolished in nNOS-S1412Dmutants (Fig. 4B and Fig. S1). Kemp and colleagues (37, 38)reported that both Akt and PKA phosphorylate eNOS at S1179,which is comparable to S1412 of nNOS and supports our findings.To investigate the role of PKA in S1412-nNOS phosphorylation

in the intact penis, we performed injections of forskolin (FSK),a potent and selective activator of adenylyl cyclase, beneath the ratMPG, and monitored P-nNOS in ganglion/CN preparations(Fig. 4C). Perigangliar FSK elicits a substantial increase inneuronal P-nNOS, but the inactive derivative deoxy-forskolin(dFSK) does not. No change in P-nNOS is detected with dFSKtreatment. We also examined penile P-nNOS levels followingintracavernosal injection of FSK or dFSK (Fig. 4D). FSK triplespenile P-nNOS, but dFSK has no effect. In contrast, FSK does notinfluence penile phospho-eNOS levels. The unexpected absence ofincreased P-eNOS in this preparation may reflect the shorter timecourse before tissue collection (30–90 s for FSK, and 5–7 min forpapaverine). Thus, in the MPG and penile tissue P-nNOS immu-noreactivity reflects PKA phosphorylation of nNOS at S1412.

Neurally Evoked Penile Erection Is Mediated by PKA Phosphorylationof nNOS. We explored whether PKA phosphorylation of nNOS isrequired for penile erection evoked by nerve stimulation. Firstwe examined the influence of FSK. As little as 0.25 μg FSKinjected intracavernosally in the mouse elicits a four- to fivefoldincrease in ICP, with only modest increase at higher doses (Fig. 5Aand Fig. S2), but the inactive dFSK has no effect. The effect of lowdoses of FSK (0.25–1 μg) is markedly reduced in nNOSα−/− miceor following treatment with the NOS inhibitor L-nitro-arginine-methylester (L-NAME). The increased ICP at high doses of FSK(2.5–5 μg) is not influenced by nNOS deletion or inhibition,consistent with known nNOS-independent actions of FSK medi-ating smooth muscle relaxation. The similar changes in ICP athigher doses of FSK suggest that different signaling pathwaysmediate low- and high-dose FSK effects and that the smoothmuscle contractile apparatus is not modified in the nNOSα−/−mice. The FSK effect on penile erection is evident in measurementsof both maximal ICP (Fig. 5A) and for the integrated total

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Fig. 1. Electrical stimulation increases P-nNOS in the penis and MPG. (A)Electrical stimulation of the rat CN causes voltage-dependent increase inpenile P-nNOS. (B) The electrically stimulated increase in P-nNOS is alsotime-dependent, but the total nNOSα detected with an N-terminal anti-body is unchanged, showing that nNOS protein is stable during stimula-tion. (C ) Quantification of P-nNOS in arbitrary units is performed bydensitometry. Each bar represents mean ± SE of P-nNOS/unP-nNOS expressedrelative to unstimulated sham control. (D) Direct electrical stimulation of theMPG also increases P-nNOS in isolated nerve lysates, quantified in E. n = 4–7animals per condition. *P < 0.05; **P < 0.001 compared with sham. (F) Rep-resentative blots show differential phosphorylation of nNOS isoforms inwild-type and double nNOS/eNOS- (dNOS−/−) deleted mice after directMPG stimulation.

Fig. 2. Immunohistochemical localization of P-nNOS in MPG. The rat MPGwas exposed and either sham or electrically stimulated (16 Hz, 4 V, 1 min),immediately dissected, and fixed. Paraffin-embedded serial sections wereprepared for staining of total nNOS (N-terminal antibody), P-nNOS, and unP-nNOS (Magnification: 100×). Basal is sham-treated. Synaptophysin stainingclearly identifies neuronal cell bodies in an untreated ganglion. No staining isseen when primary antibody is omitted. The differential phospho-staining wasconsistently apparent in multiple preparations from separate animals.

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pressure over time (area under the curve) (Fig. S3A). Previously,Andersson and colleagues (39) reported increased ICP elicitedby FSK with no alteration following treatment with L-NAME;however, their studies used a lower dose of L-NAME (50 mg/kg)than was used in our experiments (100 mg/kg).We explored the interaction between electrical stimulation and

FSK treatment (Fig. 5B and Fig. S3B). Modest electrical stimula-tion of the CN and MPG alone (5 Hz/1 V) elicits a nearly twofoldincrease in ICP. After injection of 5 μg intracavernosal FSK andreturn to ICP baseline, the response to low-voltage electricalstimulation is augmented fourfold. We cannot rule out the possi-bility that this synergy is the result of an effect on smooth muscle;however, the electrical stimulation is performed at a time pointwhen ICP has returned to baseline but P-nNOS remains elevated.In nNOSα−/− mice, or after treating with L-NAME, no FSK-dependent increase in penile erection is evident, suggesting thatP-nNOS may mediate the persistent effect of FSK. With maximalelectrical stimulation (16 Hz/4 V), ICP is similar to that elicited bymodest stimulation plus FSK, although no additive effect is ob-served. nNOS deletion and L-NAME treatment markedly reducepenile erection with maximal stimulation. As we previouslyreported, nNOSβ mediates the NO-dependent change in ICP withmaximal electrical stimulation in nNOSα−/− mice. However,

nNOSβ activity is not sufficient to produce erectile function withsubmaxmial electrical stimulation and nNOSβ is not phosphory-lated by PKA, so it cannot mediate the increased response afterFSK treatment seen in wild-type animals expressing nNOSα.

DiscussionIn the present study we demonstrate a major role for PKA phos-phorylation of nNOS at S1412 in mediating penile erection. Usinga highly specific antibody to phospho-nNOS-S1412, we showed thatelectrical stimulation of the CN markedly augments phosphoryla-tion of nNOS in both MPG and penile tissue preparations.Moreover, cAMP activation of PKA is responsible for that phos-phorylation, because FSK—which leads to generation of cAMPand PKA activation—markedly increases and PKA inhibitors de-crease P-nNOS levels. FSK-mediated penile erection includes acomponent mediated specifically by nNOS, as the effect is sim-ilarly abolished by treatment with NOS inhibitors and in nNOSα−/−animals. In vitro studies have shown increased NO production atresting calcium levels with nNOS serine-1412 phosphorylation (40).Previously we established two phases for penile erection. Psy-

chogenic- and reflex-mediated stimuli lead to depolarization of theCNs, causing increased intracellular calcium that binds calmodulinto activate nNOS (41). Because calcium entry is a brief and tightly

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Fig. 3. Phosphorylation of nNOS-S1412 is distinct from phospho-eNOS regulation in penile erection. (A) Representative immunoblot shows sustained P-nNOSafter electrical stimulation of rat MPG, quantified in B with mean ± SE. (C) P-nNOS is unchanged in rat penile lysates after intracavernosal injection ofpapaverine, but P-ser1179-eNOS is increased as previously reported. (D) Perigangliar injection of PKA inhibitors (H89, PKAI) decreases electrically stimulated P-nNOS in the MPG despite increasing Akt-S473 phosphorylation. PI3-kinase inhibitors (Wrt, LY) do not affect P-nNOS levels. (E) Mean ± SE for inhibitorexperiments. ES, electrical stimulation. *P < 0.05; **P < 0.01 for treatment groups compared with control.

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regulated event, this stimulation can only cause a brief increase inneuronal NO-dependent blood flow. This increased blood flow,however, activates endothelial PI3-kinase to stimulate Akt, phos-phorylate and activate eNOS, and provide persistent NO pro-duction and sustained penile erection (14, 42, 43). Our findingshere indicate that neuronal stimulation increases cAMP to ac-tivate PKA, which phosphorylates nNOS at S1412, stimulatingnNOS catalytic activity (Fig. 6). This covalent phospho-modi-fication can last substantially longer than the neuronal calciumtransient and so, in coordination with phospho-eNOS, phos-pho-nNOSmay contribute to sustained erection.We conjecture thatactivity dependent calcium-stimulated adenylyl cyclase couldmediate cAMP/PKA activation in the CN.Ways in which alterations of nNOS and eNOS interact in

coordinated neurovascular erectile physiology are not clear.Moreover, the influence of nNOS phosphorylation on its usualregulation by calcium/calmodulin is uncertain, although there isevidence that eNOS phosphorylation by Akt sensitizes it to theeffects of calcium (13, 44, 45) and eNOS also associates withcalmodulin. eNOS phosphorylation renders it more sensitive toresting intracellular calcium concentrations, providing a feed-forward augmentation of enzymatic activity. It is possible thatnNOS phosphorylation by PKA likewise increases its sensitivity toresting calcium/calmodulin, thereby prolonging nNOS activation(40). It is known that Akt can phosphorylate nNOS at S1412. Ourfindings establish that PKA also directly phosphorylates andactivates nNOS-S1412 in a physiologically meaningful way similarto endothelial PKA phosphorylation of eNOS-S1177 (37). Rameau

et al. (17, 21) used immunofluorescent staining techniques to showthat NMDA stimulation of cortical neuronal cultures enhancesphosphorylation of nNOS at S1412 in specific and localizedsynaptic signaling. Conceivably, Akt and PKA phosphorylationof nNOS interact in a regulated fashion. Further studies will de-termine the coordinated regulation of stimulatory and inhibitoryphosphorylation sites for cAMP-dependent penile NO production.FSK has heretofore been thought to stimulate penile erection by

activating PKA in smooth muscle of the penis (46–48), and that isalso the likely mechanism in our higher-dose FSK experiments.Our findings suggest that FSK also acts by stimulating nNOS, en-hancing the neurovascular coordination of sustained NO releaseand suggesting novel therapeutic approaches to erectile dysfunc-tion. Current therapeutic agents selectively inhibit forms of phos-phodiesterase that act primarily upon cGMP in the erectile smoothmuscle. Our findings imply that drugs inhibiting the metabolismof both cAMP and cGMP will act synergistically by respectivelyenhancing NO generation and preventing cGMP degradation.

Materials and MethodsReagents. FSK, dFSK, papaverine,Wrt, LY, H89, PKAI, L-NAMEwere from Sigma-Aldrich; commercial anti–nNOS-S1412 antibody were from Abcam; nNOS N-terminus antibody, anti-total Akt, anti-phospho-Akt-S473 were from CellSignaling Technology.

Animal Models. Male Sprague–Dawley rats (300–325 g; Charles River Breed-ing Laboratories) or 8- to 10-wk-old C57BL6/J (wild-type; Jackson Laborato-ries), and nNOSα−/−mice (S.H.S. laboratory, Johns Hopkins University, Baltimore,MD) were anesthetized by intraperitoneal injection of ketamine (50 mg/kg)/xylazine (5 mg/kg). MPG and CN were identified/isolated via midlinesuprapubic incision for electrical stimulation of the CN. To monitor ICP in

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Fig. 5. nNOS mediates the FSK-induced increase in ICP and enhances theneurogenic erectile response. (A) ICP increases with intrapenile injection of FSKin WT mice. FSK-stimulated ICP is significantly reduced by pretreatment withL-NAMEand innNOSα−/−mice. The inactive compound dFSKdoes not affect ICP.(B) Immediately following FSK or dFSK treatment, CN electrical stimulation wasperformed using low (5 Hz/1 V) or maximal (16 Hz/4 V) parameters. Electricallystimulated maximum ICP is similar in untreated wild-type mice and in micetreated with dFSK. FSK injection significantly increases the submaximal elec-trical stimulation response, but there is minimal response in animals treatedwith L-NAME or in nNOSα−/− animals. Data are mean ± SE for n = 6–9 animals.*P < 0.05 vs. wild-type FSK; **P < 0.001 vs. wild-type FSK. For 5 Hz vs. 16 Hzcomparisons, *P < 0.05 by Student’s t test. ns, not significant.

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mice, the penis was denuded of skin and right corpus cavernosum waspierced with 30-gauge needle attached to PE-50 tubing connected to apressure transducer (Harvard Apparatus) as previously described (49). Re-sponse parameters were recorded using a data acquisition (DI-190; DataqInstruments) and calculated using MATLAB software (Mathworks). All ex-periments were approved by the Johns Hopkins University InstitutionalAnimal Care and Use Committee (IACUC).

Electrically Induced Penile Erection Studies. In anesthetized animals, electricalstimulation of penile erection was performed by placing a bipolar platinumelectrode hook around the CN, as previously described (5). The electrode wasattached to a Grass Instruments S48 stimulator. Stimulation parameters areindicated in results for various experiments. Typical maximum stimulation inrats was 16 Hz at 6 V with square-wave duration of 5 ms. Except for time-course experiments, duration of electro-stimulation was 1 min. In dephosphory-lation experiments, rat CN was electrically stimulated at maximal parametersfor 1 min, then both the MPG and penis were collected 15 s to 10 min aftertermination of stimulation.

Preparation of Protein Extracts, Western Immunoblot, and Phospho-Labeling.Samples were prepared as previously described (14). Briefly, frozen tissue wasminced then homogenized (penis) or sonicated (MPG) in 8 vol of ice-coldbuffer containing 50 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 150 mM NaCl,1% Triton X-100, and 10% (vol/vol) glycerol, with phosphatase and proteaseinhibitors [50 mM NaF /5 mM sodium pyrophosphate/30 mM β-glycer-ophosphate (BGP)/1 mM sodium orthovanadate /2× Sigma-Aldrich phospha-tase inhibitor mixture/2 μg/mL aprotinin/10 μg/mL leupeptin/1 mM Pefabloc].After centrifuging at 16,000 × g for 30 min, soluble protein was de-termined by BCA assay (ThermoScientific). Protein (1–3 mg) was added to 40μL of packed 2′,5′-ADP-Sepharose for purification of penile NOS or 50–100 μgof total protein was used directly for MPG blots. For purification, after 3–4 hincubation, the beads were washed with PBS/400 mM NaCl/1% Triton X-100;PBS/2% Triton X-100; and finally PBS alone. Bound protein was directly elutedin 30 μL SDS loading buffer (62.5 mM Tris, pH 6.8/2% SDS/10% glycerol/2 mMEDTA) at 100 °C for 3 min. Samples were separated on 4–20% gradient gels(BioRad), then transferred to PVDF (Millipore), blocked with Superblok-PBS

(ThermoScientific), and probed for phospho-proteins overnight at 4 °C withthe indicated antibodies. Then, blots were stripped for 20 min at room tem-perature (Restore; ThermoScientific) and reprobed for total protein. Resultswere quantified by densitometry, and the ratio of phospho- to total orunphospho-protein was determined in arbitrary units expressed relative tothe ratio for sham-treated animals prepared and blotted at the same time.In phospho-labeling experiments, wild-type or S1412D nNOS from trans-fected HEK293 cells was purified on 2′,5′-ADP-Sepharose, eluted withNADPH, and incubated with PKA catalytic subunit in reaction buffer con-taining [32P]ATP. The reaction was stopped with SDS loading buffer and runon a gradient gel, as above, for Coomassie blue staining and autoradiog-raphy. A fraction of the same reaction was prepared for Western blot withP-nNOS antibodies, as described above.

Pharmacologically Induced Penile Erection Studies. In rats, papaverine hydro-chloride, FSK, or dFSK at the indicated concentration, was injected intra-cavernosally, as described previously (14, 50). Penes or MPG were collectedduring maximal ICP, snap-frozen, and prepared for Western blotting. In mice,FSK or dFSK was administered via a second 30-gauge needle inserted in the leftcorpus cavernosum; ICP was monitored as described above. Some animals werepretreated with L-NAME (100 mg/kg, i.p.) 30 min before FSK treatment. AfterFSK dose–response injections, and at least 5 min after ICP returned to baseline,the CN was electrically stimulated for 1 min at minimal parameters of 5 Hz/1 Vand then at 16 Hz/4 V with a 3-min rest between electro-stimulations. Thisprocess was done to confirm L-NAME effect and nNOS−/− phenotype, and toidentify changes in electrically stimulated erection after FSK injection.

Pharmacologic Inhibition of nNOS Phosphorylation with Perigangliar Injection.The CN and MPG were exposed and prepared as above. PI3-kinase inhibitors(1 μM Wrt or 50 μM LY), PKA inhibitors (30 μM H89 or 60 μM PKAI), or vehicle(0.1% dimethyl sulfoxide or ethanol) were injected directly beneath the MPGusing a 30-gaugeneedle attached to aHamilton syringe. After 10min, the CNwaselectrically stimulated at 16 Hz/6 V for 1 min. The MPG was then immediatelyexcised, rinsed in PBS, snap-frozen, and processed for phospho-protein analysis.

Immunohistochemistry. Whole MPGs with underlying prostate tissue were re-moved from adult rats immediately after electrical stimulation of CN (16Hz, 6 V,1min), and immediatelyfixed in ice-cold formalin (containing50mMBGP, 5mMsodium orthovanadate, 5 mM sodium pyrophosphate, and 1× Sigma-Aldrichphosphatase-inhibitormixture) for 15min, then1h at room temperature beforestoring in 70% ethanol. Tissue was paraffin-embedded, cut in 10-μm transversesections, and mounted on glass slides. Sections were baked at 60 °C, depar-affinized, and boiled in citrate buffer for 20 min, then permeabilized in 0.4%Triton X-100 for 15 min, quenched in 2% H2O2 in PBS for 15 min, and blockedwith PBS containing 5% goat serum/10 mM BGP/5% BSA for 2 h. Indicatedantibodies were incubated overnight at 4 °C PBS containing 0.2% BSA/1%normal goat serum/5 mM BGP:total nNOS (1:300), affinity-purified phospho-nNOS-S1412 (1:150) or unphospho-nNOS-S1412 (1:5), or synaptophysin (1:5).Staining was visualized using the Elite Vectastain kit (Vector Laboratories) andcounterstained with hematoxylin before dehydrating and cover-slipping. Con-trols without primary antibody were run for each set of slides. Stimulated andbaseline sections were stained and developed simultaneously on the same slide.

Statistical Evaluation. Analysis was performed with MATLAB or KaleidaGraph(Synergy Software) using one-way ANOVA followed by Holm–Sidak orNewman–Keuls multiple comparison or t test when appropriate. Data areexpressed as mean ± SE. n are listed within figure legends. P values < 0.05were considered significant.

ACKNOWLEDGMENTS. This work was supported by US Public Health ServiceGrants R01DK067223 (to A.L.B.) and MH018501 (to S.H.S.).

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NO ↑Relaxa�on↑ Shear Stress

NO

S�mulusPsychogenicReflexogenic Ini�a�on

Maintenance

eNOSP

PI3K/AktnNOSCa++ CaM

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nNOSP

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Fig. 6. A model integrating nNOS and endothelial NOS (eNOS) regulationin initiation and maintenance of erectile function. Initiating neural stimulipromote NO production by nNOS and nNOS sensitization by PKA phos-phorylation of S1412. Initial smooth muscle relaxation and increased penileblood flow stimulates PI3-kinase/Akt phosphorylation of eNOS-S1179.Phosphorylation and activation of nNOS and eNOS produces NO at restingintracellular calcium levels. Maintenance of maximum erectile response maybe sustained by synergistic NO production from P-nNOS and P-eNOS. Dottedlines represent the proposed contribution of P-nNOS in this model.

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