International Journal of Impotence Research (2004) 16, 459–469 & 2004 Nature Publishing Group All rights reserved 0955-9930/04 $30.00 www.nature.com/ijir Review Nitric oxide–cyclic GMP pathway with some emphasis on cavernosal contractility IF Ghalayini1* 1Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan Nitric oxide (NO) is formed from the conversion of L-arginine by nitric oxide synthase (NOS), which exists in three isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). nNOS is expressed in penile neurons innervating the corpus cavernosum, and eNOS protein expression has been identified primarily in both cavernosal smooth muscle and endothelium. NO is released from nerve endings and endothelial cells and stimulates the activity of soluble guanylate cyclase (sGC), leading to an increase in cyclic guanosine-30,50-monophosphate (cGMP) and, finally, to calcium depletion from the cytosolic space and cavernous smooth muscle relaxation. The effects of cGMP are mediated by cGMP dependent protein kinases, cGMP-gated ion channels, and cGMP-regulated phosphodiesterases (PDE). Thus, cGMP effect depends on the expression of a cell-specific cGMP- receptor protein in a given cell type. Numerous systemic vasculature diseases that cause erectile dysfunction (ED) are highly associated with endothelial dysfunction, which has been shown to contribute to decreased erectile function in men and a number of animal models of penile erection. Based on the increasing knowledge of intracellular signal propagation in cavernous smooth muscle tone regulation, selective PDE inhibitors have recently been introduced in the treatment of ED. Phosphodiesterase 5 (PDE5) inactivates cGMP, which terminates NO-cGMP-mediated smooth muscle relaxation. Inhibition of PDE5 is expected to enhance penile erection by preventing cGMP degradation. Development of pharmacologic agents with this effect has closely paralleled the emerging science. International Journal of Impotence Research (2004) 16, 459–469. doi:10.1038/sj.ijir.3901256 Published online 1 July 2004 Keywords: nitric oxide; cGMP; soluble guanylate cyclase; phosphodiesterase; corpus cavernosum Introduction NO and cGMP together comprise an especially wide-ranging signal transduction system when one considers the many roles of cGMP in physiological Nitric oxide (NO) was first described by Stuehr and regulation, including smooth muscle relaxation, 1 Marletta (1985) as a product of activated murine visual transduction, intestinal ion transport, and macrophages. Also, the substance known as en- platelet function.5 dothelium-derived relaxing factor (EDRF), described Erectile dysfunction (ED) is defined as the con- 2,3 by Furchgott and Zawadzki (1980), has been sistent inability to achieve or maintain an erection identified as NO. sufficient for satisfactory sexual performance, and is Soluble guanylate cyclase (sGC), responsible for considered to be a natural process of aging.6 Studies the enzymatic conversion of guanosine-5-tripho- have shown that ED is caused by inadequate sphate (GTP) to cyclic guanosine-30,50-monopho- relaxation of the corpus cavernosum with defect in sphate (cGMP), was first identified as a constituent NO production.7 4 of mammalian cells almost three decades ago. It is clear that NO is the predominant neurotrans- mitter responsible for cavernasal smooth muscle relaxation and hence penile erection. Its action is mediated through the generation of the second *Correspondence: IF Ghalayini, MD, FRCS, PO Box messenger cGMP. Neurally derived NO has been 940165, Amman 11194, Jordan. established as a mediator of smooth muscle relaxa- E-mail: [email protected] tion in the penis, and it is thought that constitutive Received 25 October 2003; revised 25 May 2004; accepted forms of nitric oxide synthase (NOS) work to 3 June 2004 mediate the erection.8 Released NO activates sGC, Nitric oxide–cyclic GMP pathway IF Ghalayini 460 which catalyzes the conversion of GTP to the and release of NO by these cells.14 Bivalacqua et al intracellular second messenger cGMP in smooth (2001)15 found in their study that in vivo adenoviral muscle cells. An increase in cGMP modulates gene transfer of CGRP can physiologically improve cellular events, such as relaxation of smooth muscle erectile function in the aged rat, while others cells.9 reported that intracavernosal injections of CGRP in This review will describe current knowledge of combination with adrenomedullin (ADM) or pros- cellular events involved in cavernosal relaxation taglandin E1 (PGE1) induce penile erection by and the range of putative factors involved in NO- activating different receptors.16,17 mediated relaxation. The combination of molecular oxygen and the amino acid arginine in the presence of reduced nicotinamide adenine dinucleotide phosphate Synthesis of NO (NADPH) and NO synthase (NOS) yields citrulline and NO, through a 5-electron oxidation of the 18 guanidine nitrogen of L-arginine (Figure 1). Recent observations suggest that the main site of NO L-citrulline can be converted by arginine synthase biosynthesis in human corpus cavernosum is within (AS) to form L-arginine, the precursor for NO. Each the terminal branches of cavernosal nerves supply- of these enzymes, cofactors, or transport systems ing the erectile tissue. It is strongly suggested that could be an eventual target of pharmacologic NO released from nonadrenergic–noncholinergic intervention in the NO cascade. (NANC) neurons increases the production of cGMP, Oral administration of L-arginine in high doses which in turn relaxes the cavernous smooth mus- seems to cause significant subjective improvement cle.10,11 Endothelial-derived NO plays a major role in sexual function in men with organic ED only if in the sustained erectile response and is a major they have decreased production of plasma and urine source of NO in the penis.8 Some suggest that NO nitrite and nitrates, which are stable metabolites of is highly labile, therefore, it cannot be stored as a NO.19 preformed neurotransmitter.12 Alternatively, an- There are at least three isoforms of NOS (neuronal, other neurotransmitter such as vasoactive intestinal endothelial, and macrophage)18,20 (Table 1). A polypeptide (VIP) may interact with either endothe- constitutive form of NOS is found in endothelium lial or smooth muscle cells in the corpus caverno- and neurons, and is calcium dependent.21 The sum to trigger the local formation of NO.13 Other constitutive NOS found in endothelial and smooth proerectile mediators, such as acetylcholine, calci- muscle cells has been named NOS-3, whereas the tonin gene-related peptide (CGRP) or substance P, constitutive NOS found in neural and epithelial act via endothelial cells by promoting the synthesis tissue has been named NOS-1. The latter has also Figure 1 NO generation from L-arginine and NO donors and the formation of cGMP. L-citrulline can be converted by arginine synthase(AS) to form L-arginine. Nitric oxide synthase (NOS), in the presence of O2 and the cofactors converts arginine to NO, with the formation of citrulline. Cofactors include reduced nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin (BH4), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). AS, NOS, cofactor, and L-arginine availability are all possible sites of pharmacologic intervention in this pathway. International Journal of Impotence Research Nitric oxide–cyclic GMP pathway IF Ghalayini 461 Table 1 Properties of the three isoforms of nitric oxide synthase (NOS) Property Isoform I Isoform II Isoform III Name bNOS, cNOS, nNOS iNOS, mNOS eNOS Tissue Neuronal, epithelial, skeletal, Macrophages, smooth muscle Endothelial, smooth muscle cardiac muscle cells cells cells Expression Constitutive Transcriptional induction Constitutive Calcium requirement Yes No Yes Chromosome 12 17 7 Approximate mass of protein 150–160 kDa 125–135 kDa 133 kDa bNOS ¼ brain NOS, cNOS ¼ constitutive or Ca þ regulated NOS, nNOS ¼ neuronal NOS, iNOS ¼ inducible NOS, mNOS ¼ macrophage NOS, eNOS ¼ endothelial NOS. been found in a number of cell types including skeletal and cardiac muscle.22 An inducible form of NOS, now designated iNOS, is calcium indepen- dent.23 It is induced within 4–24 h of the appro- priate stimulus and can produce NO in a 100-fold greater amount than can constitutive NOS. Neuronal NOS has multiple regulator sites, in- cluding binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin monoucleotide Figure 2 Modular structure of neuronal nitric oxide synthase (FMN). All of these are cofactors for the synthesis showing approximate locations of prosthetic groups and co- of NO.24 These cofactors bind to a reductase domain factors. to process electron transfer. This is then linked to a heme and tetrahydrobiopterin (BH4)-containing catalytic oxygenase domain by calcium–calmodulin complex25 (Figure 2). The complete enzyme con- centrally from the brain, or peripherally by means verts L-arginine to L-citrulline and NO in the of the dorsal nerve spinal reflex, is assumed to be presence of molecular oxygen. In addition to the exerted through the activation of PnNOS activity.30 various protein modules or domains of neuronal This mechanism occurs mainly by Ca2 þ binding to NOS, which are involved in electron transfer, calmodulin by means of a Ca2 þ flux through the N- substrate binding, oxygen activation, and calcium methyl-D-aspartate receptor (NMDAR). Both the binding, a four-amino-acid motif (glycine–leucine–