Lecture 2 Nitric Oxide Synthase and Heme Oxygenase Knockout Mice Ð What Have We Learned?

International Journal of Impotence Research (2000) 12, Suppl 3, S42±S44 ß 2000 Macmillan Publishers Ltd All rights reserved 0955-9930/00 $15.00 www.nature.com/ijir Lecture 2 Nitric oxide synthase and heme oxygenase knockout mice Ð what have we learned? AL Burnett1* 1Department of Urology, The Johns Hopkins University School of Medicine and The James Buchanan Brady Urological Institute, The Johns Hopkins Hospital, Baltimore, MD 21287, USA The seemingly unthinkable involvement of gaseous transgenic technology or as a basic means to ensure molecules in basic biochemical mechanisms of sexual reproducibility of species. Given these concerns, do physiology only a few years ago seems an almost these mutant animals possibly yield useful informa- indisputable concept within this research ®eld today. tion regarding sexual function? The answer is that Major advances have occurred in the scienti®c under- they do. Early and ongoing investigation of these standing of penile erection and ejaculation, with animals has been informative with results suggest- experimental evidence amply supporting the roles of ing standard and apparently compensatory mechan- nitric oxide (NO) and carbon monoxide (CO) in these isms in¯uencing sexual function. This brief article sexual functions. NO has now been well characterized summarizes research ®ndings associated with the as a major peripheral mediator of penile erection, sexual function of transgenic mice with deleted released by a neurological stimulus upon its synthesis NOS and HO-2 genes, which clari®es roles of these involving neuronal NO synthase (NOS) contained in genes and their chemical products in mammalian the autonomic innervation of the penis and endothe- sexual physiology. lial NOS contained in the trabecular and vascular Predictably, mice lacking the neuronal NOS gene endothelium of this organ.1±3 A similar NO-depen- (nNOSn=n mice) would lend proof for the concept dent mechanism is thought to operate in the central that NO derives from intact neuronal NOS function control of erectile and ejaculatory responses at the and critically serves a neurotransmitter role in the levels of the paraventricular nucleus and medial nonadrenergic, noncholinergic mediation of penile preoptic area of the hypothalamus and in the erection.13 ± 15 However, the discrepancy between lumbosacral spinal cord.4±7 Heme oxygenase-2 (HO- the apparent absence of neuronal NOS using early 2), which catalyzes CO production, has been identi- available immunodetection techniques and persis- ®ed within the peripheral neuroanatomy involved in tence of erectile function in these mutant mice the ejaculatory response and has been implicated in demanded an explanation. Subsequent work the neuroregulatory control of this function.8,9 demonstrated that these animals maintain NOS- Transgenic mice engineered with deletion of dependent erections (as erections are inhibitable by speci®c genes required for the production of these administration of NOS enzyme inhibitors), which evanescent chemicals have ®gured prominently in indicated that NO remained responsible for regulat- this research progress.10 ± 12 In principle, the oppor- ing this function.16 Endothelial NOS was then tunity to study the behavior, physiology and proposed to be the candidate NOS gene to ®ll this molecular biology relating to the presumably altered role, with con®rmation of its expression and copulatory ability of these genetically altered ani- apparent upregulation in genitourinary structures mals may reveal the importance of the de®cient or of these mice.16 The explanation was thereby offered absent chemicals of interest as these relate to aspects that endothelial NOS compensatorily overcame the of the sexual response. Whether this purpose can neuronal NOS de®ciency and maintained regulatory actually be achieved has been challenged, most of control of penile erection. all because the ostensible reproductive success of More recent explanations for the retained erectile these animals implies preserved copulatory ability. function in nNOSn=n mice have turned to the The challenge takes the position that alternative probable roles played by neuronal NOS gene mechanisms exist to permit sexual function, variants. These variants are formed without expres- whether as a developmental biology effect of sion of exon 2 of the neuronal NOS gene, which was believed to contain the sole initiation region for translation of the neuronal NOS protein.10 The basic *Correspondence: AL Burnett, Department of Urology, The Johns Hopkins University School of Medicine and The James premise of targeting deletion of exon 2 in the mutant Buchanan Brady Urological Institute, The Johns Hopkins mice held that neuronal NOS expression would be Hospital, Baltimore, MD 21287, USA. abolished. Recent data, however, have established Nitric oxide synthase and heme oxygenase knockout mice AL Burnett S43 that, in addition to a primary exon 2-containing activity of the perineal musculature and decreased neuronal NOS variant (termed neuronal NOS- ejaculatory behavior.9 Erectile function is normal in alpha), there exist multiple functional neuronal these mutant mice.9 Based on these results, a NOS variants resulting from alternative mRNA plausible basis for HO-2 involvement in sexual splicing. Actually expressed neuronal NOS variants function relates to CO acting as a neurotransmitter (such as neuronal NOS-beta and neuronal NOS- involved in the regulation of neuromuscular re¯ex gamma) that begin alternative translation in exon 1 mechanisms responsible for ejaculation. in mice, unlike that occurring in humans in which this region is untranslated, have offered a basis for the persistent neuronal NOS function in select brain regions of the mutant mice.17,18 Similarly, more Conclusion recent molecular studies applied to the lower genitourinary tract of these mice and mutant mice The study of transgenic mice with deletions of lacking neuronal NOS and endothelial NOS in neuronal NOS-alpha, endothelial NOS and HO-2 combination (nNOSn=n, eNOS7=7 mice) have con- genes is worthy. Despite possible limitations asso- ®rmed expression of neuronal NOS variants.19 ciating genomic loss and physiologic effects, the Molecular evidence further points to a prominent results generally reinforce ®ndings from other genitourinary tissue-speci®c neuronal NOS variant investigations of the ®eld de®ning the roles of that persists in nNOSn=n mice as well.20 These data gaseous molecules that regulate sexual function. In indicate that the original design of neuronal NOS- some instances, research involving these mutant de®cient mice did not achieve complete neuronal mice has led to discoveries and hence guided the NOS protein de®ciency. More importantly, the data direction of the ®eld that may not have been easily reestablish the importance of NO in the erectile pursued with other scienti®c techniques. The pro- process and lend further insight into the molecular gress that has been gained in identifying neuronal basis of neuronal NOS regulation of penile erection. NOS gene variants and evaluating their potential Mice lacking endothelial NOS (eNOS7=7 mice) roles exempli®es this point. Moreover, these trans- have provided another tool to investigate NO genic mice may indeed provide valuable insight into regulation of sexual function, speci®c to the role of the physiologic impact of the changes resulting from endothelial NOS. These animals exhibit normal manipulation of NOS isoforms and HO-2 at the erectile function (Burnett et al, unpublished re- molecular level. Expectations are that lessons may sults). While the retained erectile function is not be learned from these mice to advance the develop- surprising, since the neuronal NOS gene is pre- ment of modulators of NOS and HO-2 gene function sumably intact and functional for this purpose, the as effective agents for the clinical treatment of contribution of endothelial NOS to the regulation of erectile and ejaculatory dysfunctions in man. penile erection should not be underestimated. NOS catalytic activity is reduced in eNOS7=7 mice compared with that of genetically intact animals, indicating that endothelial NOS may afford a full References complement of NOS function that maximally pre- serves erectile function (Burnett et al, unpublished 1 Burnett AL. Role of nitric oxide in the physiology of erection. 7=7 results). Further behavioral study of eNOS mice Biol Reprod 1995; 52: 485 ± 489. has recently revealed abnormalities in ejaculatory 2 Burnett AL. Nitric oxide in the penis: physiology and function, since these mice ejaculate after a shorter pathology. J Urol 1997; 157: 320 ± 324. latency and require less stimulation to ejaculate 3 Gonzalez-Cadavid NF, Rajfer J. Nitric oxide and other 21 neurotransmitters of the corpus cavernosum. In: Hellstrom compared with genetically intact mice. A likely WJG (ed). Textbook of Andrology: Relevant Issues in Male mechanism for endothelial NOS regulation of Infertility and Sexual Dysfunction. Springer: New York, 1997, ejaculation may involve NO antagonism of sympa- pp 425 ± 439. thetic nervous system activity that mediates the 4 Giuliano F et al. Stimulation of the medial preoptic area of the ejaculatory process.22 The absence of this endothe- hypothalamus in the rat elicits increases in intracavernosal pressure. Neurosci Lett 1996; 209:1±4. 7=7 lial NOS control mechanism in eNOS mice may 5 Melis MR, Succu S, Iannucci U, Argiolas A. Oxytocin thereby account for overactive sympathetically increases nitric oxide production in the paraventricular mediated

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