Alternatively spliced neuronal nitric oxide synthase mediates penile erection K. Joseph Hurt*, Sena F. Sezen†, Hunter C. Champion‡, Julie K. Crone†, Michael A. Palese†, Paul L. Huang§, Akira Sawa*¶储, Xiaojiang Luo*, Biljana Musicki†, Solomon H. Snyder*储**††, and Arthur L. Burnett†,†† Departments of †Urology and ‡Internal Medicine, Division of Cardiology, and Departments of ¶Cellular and Molecular Medicine, *Neuroscience, **Pharmacology and Molecular Sciences, and 储Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and §Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129 Contributed by Solomon H. Snyder, December 30, 2005
A key role for nitric oxide (NO) in penile erection is well established, but the relative roles of the neuronal NO synthase (nNOS) versus endothelial forms of NOS are not clear. nNOS- and endothelial NOS-deficient mice maintain erectile function and reproductive capacity, questioning the importance of NO. Alternatively, residual NO produced by shorter transcripts in the nNOSⴚ/ⴚ animals might suffice for normal physiologic function. We show that the  splice variant of nNOS elicits normal erection despite a decrease in stimulus-response characteristics and a 5-fold increased sensitivity to the NOS inhibitor, L-NAME. Residual nNOS generates only 10% of the normal NO level in vitro but produces citrulline and diaphorase staining reflecting in vivo NOS activity in pelvic ganglion nerves that is comparable to WT animals. Thus, alternatively spliced forms of nNOS are major mediators of penile erection and so may be targets for therapeutic intervention. penis 兩 cavernous nerve 兩 NADPH diaphorase
N
itric oxide (NO) is synthesized by three major enzymes derived from distinct genes, inducible NO synthase (iNOS), endothelial NOS (eNOS), and several variants of neuronal NOS (nNOS) (1). Penile erection is mediated by NO produced by both nNOS and eNOS (2), the former initiating erection (3), and the latter providing sustained maximal erection (4). The relative contributions of different forms of NOS to penile erection are unclear, especially because erection is preserved in mice with targeted deletion of nNOS␣ (nNOS⫺/⫺) as well as in eNOS⫺/⫺ animals (5–7). In the present study, using nNOS⫺/⫺ (8), eNOS⫺/⫺ (9), and doubly mutant mice (10) (dNOS⫺/⫺), we show that alternatively spliced forms of nNOS mediate a major portion of penile erection. Results and Discussion We evaluated erectile function by monitoring intracavernous pressure (ICP) in response to electrical stimulation of the cavernous nerve at different voltages (Fig. 1A). Erection is maintained in all of the transgenic animals, and at the highest voltage tested (6 V), there is no significant difference among groups. However, nNOS⫺/⫺ and dNOS⫺/⫺ animals are less responsive to low-voltage stimulation. At 1 V, nNOS⫺/⫺ and dNOS⫺/⫺ animals have only 4% and 26% of the stimulated change in ICP seen in WT animals. In contrast, eNOS⫺/⫺ animals appear supersensitive to electrical stimulation, with 150–300% increased response at 1–4 V compared with WT. We wondered whether the retained erection in mutant mice reflects a residual NO-generating system or is unrelated to NO. We evaluated the influence of L-nitroarginine methylester (LNAME), which inhibits all forms of NOS nonselectively (11). Strikingly, all three mutant mice are supersensitive to inhibition of erection by L-NAME, with the doubly mutant mice 10 times more sensitive than WT and the nNOS⫺/⫺ and eNOS⫺/⫺ animals 5 and 2.5 times more sensitive, respectively (Fig. 1B). This supersensitivity may reflect the greater ease of L-NAME to inhibit smaller amounts of nNOS in mutant animals. At higher 3440 –3443 兩 PNAS 兩 February 28, 2006 兩 vol. 103 兩 no. 9
doses, L-NAME inhibition is similar among all groups. We also note striking differences in the patterns of stimulated erection in transgenic mice. dNOS⫺/⫺ mice, in particular, demonstrate an irregular and dysfunctional priapic activity (Fig. 1C). The preservation of electrically stimulated erectile function in nNOS⫺/⫺ animals suggests that the nerve fibers contain residual NOS. Accordingly, we compared total in vitro NOS activity in WT and transgenic penes (Fig. 2A). Total NOS activity is reduced 30–40% in eNOS⫺/⫺ tissue and almost 90% in nNOS⫺/⫺ and double-knockout preparations. Residual activity is completely inhibited by 500 M L-NAME. Because iNOS has not been detected in normal WT rat and mouse penile tissues (5), we wondered whether alternatively spliced forms of nNOS might maintain erectile activity. In certain brain regions, alternatively spliced and catalytically active nNOS has been described (12), whereas another alternatively spliced form, nNOS␥, also persists in the nNOS⫺/⫺ brain but lacks catalytic activity (13). We conducted in situ hybridization for the predominant ␣ form of nNOS and the catalytically active  form (Fig. 2B). In WT mice, both NOS␣ and - are prominent in the neuronal cell bodies of the pelvic ganglion, which give rise to the cavernous nerve innervation of the penis. Similar localizations are observed with a common probe that recognizes both ␣ and  forms. Staining for nNOS␣ mRNA is abolished in nNOS⫺/⫺ animals, but no reduction of nNOS is apparent, consistent with the design of the mutant mice in which exon-2 was deleted, leaving those forms that lack that portion of the N terminus of the protein unaffected. To investigate whether residual nNOS produces NO in nerve fibers, we stained penile tissue for citrulline, which reflects NOS activity in intact animals (14) (Fig. 2C). We observe citrulline staining in the cavernous and dorsal nerves of the penis as well as endothelial tissue lining the cavernous sinuses and penile blood vessels. Citrulline staining is not reduced in nNOS⫺/⫺ penile sections. The retention of apparent NOS activity in nNOS⫺/⫺ intact penes may reflect a greater capability of residual NOS to generate citrulline (and presumably NO) in vivo but not in vitro or might reflect diminished degradation of citrulline in the mutant animals. To distinguish these possibilities, we evaluated neuronal production of NO in fresh tissue. First, we measured Ca2⫹-dependent NOS activity from purified cavernosal nerve cells in transgenic animals transfected with a herpesvirus-lacZ marker (Fig. 3A), as described (15, 16). Although WT and eNOS⫺/⫺ animals have similar neuronal NOS activity, as expected, nNOS⫺/⫺ and dNOS⫺/⫺ animals have 60% and 57% of the WT activity. The activity is calcium-dependent and comConflict of interest statement: No conflicts declared. Abbreviations: NOS, NO synthase; nNOS, neuronal NOS; eNOS, endothelial NOS; iNOS, inducible NOS; dNOS, double (endothelial and neuronal) NOS; ICP, intracavernous pressure; L-NAME, L-nitroarginine methylester. ††To
whom correspondence may be addressed. E-mail:
[email protected] or aburnett@ jhmi.edu.
© 2006 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0511326103
Fig. 1. Transgenic mouse erectile responses to nerve stimulation. (A) Electrically stimulated erection as measured by increased ICP (22) is reduced at low voltages in nNOS⫺/⫺ and dNOS⫺/⫺ strains but becomes nearly the same as WT response at higher voltages. eNOS⫺/⫺ animals are more sensitive than WT at all voltages tested. *, P ⬍ 0.01; **, P ⬍ 0.05. (B) L-NAME more potently inhibits erection mediated by 4-V stimulation in nNOS␣- and dNOS-deficient mice, whereas eNOS⫺/⫺ strains are more resistant to inhibition. L-NAME IC50 values are: WT, 4.2 mM; eNOS⫺/⫺, 1.6 mM; nNOS␣⫺/⫺, 0.95 mM; and dNOS⫺/⫺, 0.44 mM. Data are mean ⫾ SEM for 3–15 animals per dose. (Inset) Magnification of the initial dose–response curves for the transgenic animals. (C) Representative tracings of 4-V stimulation of erection for 2 min (solid bar) followed by injection of 15 mM L-NAME in the mouse penis (at hatchmarks) and second stimulation at 4 V. The irregular unstable priapic response of the dNOS⫺/⫺ mouse is clearly shown. Peak change in ICP is measured as the highest continuous pressure peak maintained for at least 20 sec during the stimulus interval.
Hurt et al.
pletely abolished by 7-nitroindazole, a selective nNOS inhibitor. To further confirm the substantial nNOS activity in the transgenic mice, we performed diaphorase histochemical staining of isolated pelvic ganglia (17), which reflects NOS catalytic activity. This staining is similar in neuronal cell bodies for WT, eNOS⫺/⫺, nNOS⫺/⫺, and dNOS⫺/⫺ mice (Fig. 3B). In summary, our findings indicate that the alternatively spliced nNOS mediates a major portion of penile erection. Although NOS catalytic activity monitored in penile tissue extracts is reduced 90% in nNOS mutant mice, no substantial reduction in citrulline or NADPH diaphorase staining or NOS activity of cavernosal nerve lysates is evident in the mutants. nNOS generation of NO兾citrulline is also preserved in the brains of nNOS⫺/⫺ animals (14). Unique properties of nNOS may explain its robust production of NO to preserve erectile function in nNOS⫺/⫺ animals (18). nNOS lacks the N-terminal PDZ domain that binds to PSD95, the postsynaptic density protein that anchors nNOS␣ to neuronal membranes and links nNOS to the NMDA glutamate receptors (13). The lack of tethering to the plasma membrane for PNAS 兩 February 28, 2006 兩 vol. 103 兩 no. 9 兩 3441
PHYSIOLOGY
Fig. 2. Presence of enzymatically active alternatively spliced nNOS in transgenic mice. (A) Total NOS activity in penile homogenates measured in vitro by the conversion of arginine to citrulline as described (23) and compared with WT activity. Total NOS activity in WT animals was 367 ⫾ 73 pmol兾min per mg of protein. In controls, L-NAME added at 500 M blocked all activity. Data are mean ⫾ SEM for six to seven animals. (B) In situ hybridization of WT and nNOS⫺/⫺ mouse pelvic ganglia using sense and antisense probes to ␣ or  nNOS isoform-specific RNA or to a sequence common to all nNOS isoforms. Solid and broken arrows point to stained and unstained neuronal nuclei, respectively. (C) Citrulline staining in WT and nNOS⫺/⫺ penile tissue. Bracket marks the endothelial surface of the penile artery. Arrow marks a distinctly stained nerve fiber in the cavernous nerve. (Inset) Background staining in the absence of primary antibody.
Fig. 3. Neuronal localization of residual NOS activity in transgenic mice. (A) Calcium-dependent and 100 M 7-nitroindazole (7-NI) inhibited conversion of arginine to citrulline in isolated cavernosal nerve tissue is maintained in eNOS⫺/⫺ animals and reduced in nNOS⫺/⫺ and dNOS⫺/⫺ strains. Data are mean ⫾ SEM of three to five measurements. EGTA (5 mM) or L-NAME (100 M) also completely inhibit NOS activity. (B) Histochemical localization of diaphorase activity to large neuronal cell bodies is maintained in pelvic ganglia from all transgenic strains. Data are representative of three separate staining procedures for ganglia taken from two separate animals.
nNOS may provide greater in vivo than in vitro activity. Also, the absence in nNOS of the domain that binds to the protein inhibitor of NOS, an endogenous inhibitor of NOS (19), may also potentiate in vivo catalytic activity. Because of its unique properties, nNOS may be a novel therapeutic target for treating erectile dysfunction, cardiovascular disease, and other conditions with NO deficiency. A complete nNOS-deficient mouse lacking nNOS␣, -, and -␥ is infertile (20, 21). The nNOS␣⫺/⫺, eNOS⫺/⫺, and dNOS⫺/⫺ animals used in our studies are all fertile, indicating the crucial role of functional nNOS in normal reproductive function. Methods Physiologic Erection Studies and Inhibition. Age-matched adult
male C57BL6 (WT; The Jackson Laboratory), eNOS⫺/⫺, nNOS⫺/⫺, or dNOS⫺/⫺ (from the laboratory of P.L.H.) mice were used in ICP studies (4). Mouse penis was denuded of skin and fascia and the corpus cavernosum was monitored with a needle connected to a pressure transducer by using MATLAB Mathworks (Natick, MA) software. A bipolar electrode attached to a Grass Instruments (Quincy, MA) S48 stimulator was placed around the cavernous nerve and stimulation was conducted at 1, 2, 4, and 6 V. For inhibition studies, stimulation was performed at 4 V for 2 min after a stable baseline was obtained. Then 10 l of the indicated concentrations of L-NAME was injected directly into the penis followed by a 10-min stabilization phase and then stimulated again for 2 min. The percent inhibition is expressed as the ratio of ⌬ICPL-NAME兾⌬ICPcontrol. IC50 values were computed by using dose–response curve-fitting software from GRAPHPAD PRISM (GraphPad Software, San Diego). In Situ Hybridization. Digoxygenin in situ hybridization with
unique N- or C-terminal probes was conducted as published (14) and according to the manufacturer’s instructions (Calbiochem) by using common or NOS␣- or NOS-specific probes corresponding to residues 100–786 of mouse nNOS sequence. Citrulline Staining. Staining for glutaraldehyde-coupled citrulline
was performed as described (14) by using affinity-purified antibodies. Animals were killed and perfused with 5% glutaraldehyde兾0.5% paraformaldehyde, postfixed, and then cryoprotected. Free-floating sections of mouse penis were reduced with NaBH4, blocked with 4% goat serum, and permeabilized in 3442 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0511326103
0.1% Triton X-100 before incubating overnight with antiserum diluted to 1:5,000 in PBS. Staining was developed with the ABC Elite kit (Vector Laboratories). nNOS Activity from Isolated Cavernosal Nerve. Mouse penes were
transfected with a herpesvirus (HSVlacZ) encoding V5 and lacZ (25 ⫻ 107 plaque-forming units) by direct injection of the cavernosum by using a 30-gauge needle and proximal occlusion of penile venous return by using an elastic band, as described (15). Two days later, penes were harvested and purified by using anti-V5 antibody (Transduction Laboratories, Lexington, KY) and miniMACS separation unit (Miltenyi Biotec). Confirmation of the cavernosal nerve cell population was performed by f low cytometry by using both V5 and lacZ. Constitutive enzyme activity was assayed in total penile homogenates or isolated tissue, as described (22). Brief ly, samples were sonicated in a solution of Tris䡠HCl (250 mmol/liter, pH 7.4兾EDTA 10 nmol/liter兾EGTA 10 mmol/liter) and centrifuged at 12,000 ⫻ g for 10 min at 14°C. The supernatant was incubated in NADPH [10 mmol/liter)兾[3H]L-arginine (1 mCi/ ml)兾CaCl (6 mmol/liter)兾Tris䡠HCl (50 mmol/liter, pH 7.4)兾 tetrahydrobiopterin (6 mol/liter)兾f lavin adenine dinucleotide (2 mol/liter)兾f lavin mononucleotide (2 mol/liter)] for 60 min at 24°C. The reaction was stopped with Hepes (50 mmol/liter, pH 5.5) and EDTA (5 mmol/liter). The radioactivity eluted from a Dowex column was measured by liquid scintillation counting. Diaphorase Histochemical Staining. NADPH diaphorase staining
was performed on sections of mouse pelvic ganglia (17). Brief ly, 20- to 40-m sections were fixed in 4% paraformaldehyde and then developed in a reaction buffer containing NADPH and NBT. The sections were counterstained with Nuclear Fast Red and fixed in EtOH, then mounted for microscopy. Blanks were confirmed by omitting NADPH or nitroblue tetrazolium. This work was supported by U.S. Public Health Service Grants DA00266 and Research Scientist Award DA 00074 (to S.H.S.); by U.S. Public Health Service Grants DK64679 and DK67223 (to A.L.B.) and MH069853; by foundation grants from National Alliance for Research on Schizophrenia and Depression and the Stanley Foundation (to A.S.); and by the American Heart Association, the W. W. Smith Charitable Trust, and the Bernard Family Foundation (to H.C.C.). Hurt et al.
12. Lee, M. A., Cai, L., Hubner, N., Lee, Y. A. & Lindpaintner, K. (1997) J. Clin. Invest. 100, 1507–1512. 13. Brenman, J. E., Chao, D. S., Gee, S. H., McGee, A. W., Craven, S. E., Santillano, D. R., Wu, Z., Huang, F., Xia, H., Peters, M. F., et al. (1996) Cell 84, 757–767. 14. Eliasson, M. J., Blackshaw, S., Schell, M. J. & Snyder, S. H. (1997) Proc. Natl. Acad. Sci. USA 94, 3396–3401. 15. Bivalacqua, T. J., Usta, M. F., Champion, H. C., Adams, D., Namara, D. B., Abdel-Mageed, A. B., Kadowitz, P. J. & Hellstrom, W. J. (2003) J. Urol. 169, 1911–1917. 16. Champion, H. C., Bivalacqua, T. J., Hyman, A. L., Ignarro, L. J., Hellstrom, W. J. & Kadowitz, P. J. (1999) Proc. Natl. Acad. Sci. USA 96, 11648–11652. 17. Burnett, A. L., Tillman, S. L., Chang, T. S., Epstein, J. I., Lowenstein, C. J., Bredt, D. S., Snyder, S. H. & Walsh, P. C. (1993) J. Urol. 150, 73–76. 18. Gonzalez-Cadavid, N. F., Burnett, A. L., Magee, T. R., Zeller, C. B., Vernet, D., Smith, N., Gitter, J. & Rajfer, J. (2000) Biol. Reprod. 63, 704–714. 19. Jaffrey, S. R. & Snyder, S. H. (1996) Science 274, 774–777. 20. Gyurko, R., Leupen, S. & Huang, P. L. (2002) Endocrinology 143, 2767–2774. 21. Tranguch, S. & Huet-Hudson, Y. (2003) Mol. Reprod. Dev. 65, 175–179. 22. Champion, H. C., Bivalacqua, T. J., Takimoto, E., Kass, D. A. & Burnett, A. L. (2005) Proc. Natl. Acad. Sci. USA 102, 1661–1666. 23. Bredt, D. S. & Snyder, S. H. (1989) Proc. Natl. Acad. Sci. USA 86, 9030–9033.
PHYSIOLOGY
1. Boehning, D. & Snyder, S. H. (2003) Annu. Rev. Neurosci. 26, 105–131. 2. Burnett, A. L. (2004) Int. J. Impot. Res. 16 Suppl. 1, S15–S19. 3. Burnett, A. L., Lowenstein, C. J., Bredt, D. S., Chang, T. S. & Snyder, S. H. (1992) Science 257, 401–403. 4. Hurt, K. J., Musicki, B., Palese, M. A., Crone, J. K., Becker, R. E., Moriarity, J. L., Snyder, S. H. & Burnett, A. L. (2002) Proc. Natl. Acad. Sci. USA 99, 4061–4066. 5. Burnett, A. L., Nelson, R. J., Calvin, D. C., Liu, J. X., Demas, G. E., Klein, S. L., Kriegsfeld, L. J., Dawson, V. L., Dawson, T. M. & Snyder, S. H. (1996) Mol. Med. 2, 288–296. 6. Burnett, A. L., Chang, A. G., Crone, J. K., Huang, P. L. & Sezen, S. E. (2002) J. Androl. 23, 92–97. 7. Cashen, D. E., MacIntyre, D. E. & Martin, W. J. (2002) Br. J. Pharmacol. 136, 693–700. 8. Huang, P. L., Dawson, T. M., Bredt, D. S., Snyder, S. H. & Fishman, M. C. (1993) Cell 75, 1273–1286. 9. Huang, P. L., Huang, Z., Mashimo, H., Bloch, K. D., Moskowitz, M. A., Bevan, J. A. & Fishman, M. C. (1995) Nature 377, 239–242. 10. Son, H., Hawkins, R. D., Martin, K., Kiebler, M., Huang, P. L., Fishman, M. C. & Kandel, E. R. (1996) Cell 87, 1015–1023. 11. Boer, R., Ulrich, W. R., Klein, T., Mirau, B., Haas, S. & Baur, I. (2000) Mol. Pharmacol. 58, 1026–1034.
Hurt et al.
PNAS 兩 February 28, 2006 兩 vol. 103 兩 no. 9 兩 3443
