Alternatively spliced form enables process in mice lacking the primary enzyme
By Ishani Ganguli | February 15, 2006
The relatively rare, alternatively spliced beta form of neuronal nitric oxide synthase can trigger penile erections in mice lacking the primary form, underscoring the essential role of nitric oxide in reproduction, according to a new study in the Proceedings of the National Academy of Sciences.
This research "really puts the final stamp on the whole issue that NO is the final mediator," study author Arthur Burnett told The Scientist.
Fourteen years ago, Burnett and his colleagues at the James Buchanan Brady Urological Institute of Johns Hopkins first implicated the gaseous messenger molecule nitric oxide as the primary mediator of penile erection. In subsequent work with mice, they found that neuronal NO synthase (nNOS) is required to initiate the response, while endothelial NOS (eNOS) sustains maximal erection.
Consequently, he and other researchers were surprised to find that mutant mice knocked out for eNOS, the predominant form of nNOS, or both, still had functional erections in response to electrical stimulation. When Burnett's team subjected the knockout mice to a nonselective NOS inhibitor, all three types were supersensitive to it, confirming NO's pivotal role in the process. The researchers hypothesized that there was a residual mechanism for generating the molecule.
In what R. Clinton Webb at the Medical College of Georgia, who was not a study author, called "a very novel observation," the team used in situ hybridization to identify a rare, catalytically active beta form of nNOS in the pelvic ganglion of mice, the same enzyme researchers already identified in rat brains. In the earlier experiments, Burnett and his team had removed only exon 2 of the nNOS gene from the knockout mice - a step that silenced the alpha form of nNOS, but left the beta form unaffected.
In vitro, residual nNOS beta alone was only able to generate 10% of normal NO levels, but in vivo staining methods showed NO production in the knockout mice was comparable to wild type. What's more, a selective nNOS inhibitor completely abolished NO production. Though the alpha form of nNOS is "90% of the total gene factory," the beta form mediates a significant portion of penile erection, Burnett said, "at least enough to jumpstart the engine."
"There's a will to succeed here, to persist with erections," explained Wayne Hellstrom at Tulane University School of Medicine's Urology Department, who did not participate in this study. The finding represents an interesting new "redundancy in the system" that is highly adaptive, he said.
While previous studies have found that mice lacking nNOS alpha, beta, and the non-catalytic gamma forms are infertile, even the double nNOS alpha/eNOS knockouts in this study were fertile, indicating the crucial role nNOS plays in reproduction.
Structurally, the beta form lacks the N-terminal PDZ domain that can help tether the alpha form to neuronal membranes and thus impede NO production. According to the authors, this difference may explain the beta form's robust ability to make NO in vivo, even without the help of nNOS alpha.
In addition, the binding domain for an inhibitory protein that targets nNOS alpha is missing from the beta form, he said, making it "much less restricted." The variant's "different ways of being controlled and regulated" is an important topic for future research, Burnett said.
Indeed, the beta variant's unique structural and regulatory properties may also make it a promising therapeutic target for erectile dysfunction and other conditions marked by NO deficiency, such as cardiovascular disease, Webb told The Scientist.
Links within this article
K.J. Hurt et al., "Alternatively spliced neuronal nitric oxide synthase mediates penile erection," PNAS, published online February 13, 2006.
Arthur L. Burnett
A.L. Burnett et al, "Nitric oxide: a physiologic mediator of penile erection," Science, July 17, 1992.
M. Greener, "Now you're signaling, with gas," The Scientist, September 13, 2004
A.L. Burnett et al, "Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase," Molecular Medicine, May 1996.
R. Clinton Webb
M.A. Lee et al., "Tissue- and development-specific expression of multiple alternatively spliced transcripts of rat neuronal nitric oxide synthase," Journal of Clinical Investigation, September 15, 1997.
B.A. Wilson and R. Lewis, "Erectile dysfunction: Serious research for a serious problem," The Scientist, October 12, 1998.