The initial skepticism surrounding the discoveries showing that nitric oxide (•NO) is endogenously synthesized and plays a central role in cell-to-cell signaling as well in the host response to infection has largely disappeared, although central questions in all aspects of the biological function of •NO still remain. The skepticism was based on the fact that most investigators could not envision how a molecule as toxic as •NO could function in a biological setting. As the facts have unfolded over the last decade, it has become clear that •NO functions very effectively as a cell-to-cell signaling agent.¹ The chemical properties of •NO, namely a fast diffusion rate from the site of generation, the ability to cross cellular membranes and a chemical reactivity that allows it to function without the apparent need for a complicated system that acts to terminate the signal are well-suited to the appointed task (Figure 1). Critical for function is a highly regulated •NO biosynthetic machinery as well as a very sensitive receptor that will respond to non-toxic •NO levels. It is clear now that both these entities exist.

Although the main focus of this review is the mechanism of •NO cellular signaling, it is also without question that •NO plays a crucial role in the host response to infection. In fact, it appears that the immune system has harnessed the toxic properties of •NO to induce cytostasis in invading microorganisms as well as in tumor cells. Presumably •NO acts in a non-specific fashion by interfering with the function of key metalloproteins. Ferrous iron centers are particularly susceptible to •NO-induced inhibition because •NO typically binds tightly to proteins of this type, thus interfering with their functions.

Figure 1: Mechanism of •NO Production

Nitric Oxide Synthase (NOS)

The constitutive eNOS and nNOS have an absolute requirement for Ca²⁺ and calmodulin; therefore, Ca²⁺ plays a critical role in •NO signal transduction. In fact, given the relative toxicity of •NO, a tightly regulated synthetic machinery is required for signaling to function safely. The other critical aspect of signaling with •NO concerns the receptor sensitivity (discussed below). iNOS, on the other hand, binds calmodulin tightly and is not regulated by Ca²⁺.^16,17 Presumably, since this isoform is involved in non-specific cytostasis/cytotoxicity and is generated at the site of infection, post-transcriptional regulation of the activity is not needed. Ca²⁺ and calmodulin regulate activity by controlling the required electron transfer from NADPH through the bound flavins and then finally to the P450-type heme.^18,19 Recent reports have also shown the existence of alternatively spliced forms of NOS, the significance of which is not clear.^{20, 21} All isoforms exist as homodimers and most likely are only active in the dimeric form. Although there will be some isoform-specific differences, the major aspects of catalysis are likely to be quite similar, since all isoforms contain the same cofactors and have the same co-substrate requirements.

As mentioned above, critical to the capacity of •NO to act as a signaling agent is that it function below toxic levels. Strict control over the catalytic activity has emerged as one of the most important aspects of this signal transduction system. In fact, the unregulated activity that occurs because of the high Ca²⁺ levels present after a stroke is thought to lead to high and damaging levels of •NO.²² Under normal conditions, the tight control of intracellular Ca²⁺ concentrations leads to a similar tight control over NOS activity.

•NO In Solution
After formation by NOS, •NO freely diffuses across cellular membranes and into adjacent cells. Although reactive with molecular oxygen, •NO is a remarkably unreactive free radical, evidenced by the inability to dimerize except under pressure. The lack of reaction with unsaturated lipids is also important in the ability of •NO to signal adjacent cells. The solution decomposition leading primarily to NO₂ − is governed by the following rate law: v = k [•NO]²[O₂]. The concentration of •NO generated under signaling conditions is in the picomolar to nanomolar range, therefore, the solution lifetime is likely to be in the order of minutes if the decomposition was solely dependent on the reaction with O₂. However, •NO also reacts with a number of other important cellular constituents. For example the reaction of protein-bound iron is significant as well as the reaction with oxyhemoglobin (oxyHb). The products of the latter reaction are metHb and NO3 −. •NO also forms very tight complexes with some iron proteins including deoxyHb and deoxyMb where the ferrous nitrosyl complexes formed have very slow off-rates. Also quantitatively important is the reaction of superoxide with •NO, yielding NO₃−. While not the only reactions contributing to the loss of •NO from solution, these are the most important reactions.²³

Soluble Guanylate Cyclase
(sGC) Soluble guanylate cyclase (sGC) is the only definitively accepted receptor for •NO. sGC catalyzes the conversion of guanosine 5'- triphosphate (GTP) to cyclic guanosine 3',5'-monophosphate (cGMP). The newly-formed cGMP then acts in the cardiovascular system (e.g., in the regulation of vascular tone and platelet function) and in the nervous system (e.g. in neurotransmission and, possibly, long-term potentiation and depression). sGC is a heterodimeric hemoprotein composed of a and b subunits. •NO binds to the sGC heme and then activates the enzyme ~400-fold. The activation of sGC and the subsequent rise in cGMP concentration are what allow sGC to transmit an •NO signal to the downstream elements of the signaling cascade—cGMP-dependent protein kinase, cGMP-gated cation channels and cGMP-regulated phosphodiesterase. Although the past 30 years have provided a general understanding of sGC structure and function, many facets, especially at the molecular level, remain to be discovered.

Figure 2: Role of cGMP in Mediating the Biological Effects of Nitric Oxide

Since the initial elucidation of the •NO/cGMP system, many good examples of signal transduction by this pathway have come to light, some better characterized than others. They include: smooth muscle relaxation and blood pressure regulation²⁴; platelet aggregation and disaggregation²⁵; and neurotransmission both peripherally, in non-adrenergic, non-cholinergic (NANC) nerves ²⁴, and centrally, in the processes of long-term potentiation and depression.²⁶

There are several proposed paths for •NO signal transduction which do not rely on the production of cGMP by sGC, although they are less clearly defined than the •NO/cGMP pathway. These targets include: activation of calcium-dependent potassium channels in vascular smooth muscle²⁷, inhibition of NF-kB^28,29, activation of G-proteins (such as protooncogene p21^ras) by enhancing the rate of nucleotide exchange, leading to an increase in the GTPbound form. In fact, a p21^ras cysteine residue (C118) was shown to be S-nitrosylated in the presence of •NO.^30,31 Additionally, caspases, a group of cysteine proteases involved in apoptosis, were shown to be inhibited by S-nitrosylation of the active-site cysteine.³² Finally, both hemoglobin and the cardiac calcium release channel (i.e., the ryanodine receptor) were shown to be S-nitrosylated in vivo.^33-35 While there is no doubt that S-nitrosylation can occur in vivo, the extent to which it does and the attendant change in biological function are still under study.

The diversity of target proteins allows cGMP to have wide-ranging effects differing by cell and tissue type. There are three known targets for cGMP which mediate the transmission of the •NO/cGMP pathway signal downstream of guanylate cyclase: cGMP-dependent protein kinase³⁶, cGMP-regulated phosphodiesterase^37,38 and cGMP-gated ion channels.³⁹ The first of these, cGMP-dependent protein kinase, phosphorylates target proteins in response to an increase in cGMP concentration. For example, in smooth muscle cells, cGMP-dependent protein kinase phosphorylates the inositol 1,4,5-trisphosphate receptor, leading to a decrease in Ca²⁺ concentration and, ultimately, smooth muscle relaxation. The second, cyclic nucleotide phosphodiesterase, catalyzes the hydrolysis of the 3’- phosphodiester bond of cAMP and cGMP to yield AMP and GMP, respectively. Of the various PDE families, three (PDEII, PDEV and PDEVI) are allosterically regulated by cGMP and one (PDEIII) is inhibited by the binding of cGMP in its substrate site. Phosphodiesterases allow crosstalk between cGMP and cAMP signaling pathways because they cause the concentration of one cyclic nucleotide to influence the degradation of the other. The third target for cGMP, cyclic nucleotide-gated ion channels, are non-specific cation channels found in a variety of tissues. Most notably, these channels are found in the retina and in the olfactory epithelium where they are involved, respectively, in visual phototransduction and in olfaction.

Since the original identification of sGC, a great deal of information about sGC genetics is now known. Thus far, a total of twelve sGC cDNAs have been cloned from five different species. The first were cloned on the basis of protein sequence from purified bovine and rat a₁b₁ heterodimer. The cDNA sequences for bovine and rat b₁ subunits, respectively were reported in 1988^40,41, followed by the bovine and rat a₁ subunits in 1990.^42,43 Subsequently, a₁b₁ pairs were cloned from Homo sapiens (originally called a₃ and b₃)^44,45, Drosophila melanogaster,^10,46,47 and the fish Oryzias latipes.⁴⁸ Putative sGC sequences exist in the C. elegans genome, but these have yet to be confirmed with any empirical studies beyond the sequencing itself. In addition to the cloning of a₁b₁ pairs, the cDNAs for two additional sGC subunits have been cloned: b₂ from rat kidney⁴⁹ and a₂ from human fetal brain.⁵⁰

Although the existence of other isoforms of sGC besides the a₁b₁ has been suggested by cloning and expression experiments, only the a₁b₁ has been purified from mammalian tissue. In the past, a number of different sources for sGC, including lung, liver, and brain, were used for purifications. Although there has been some debate in the past, it is clear now that sGC binds one protoporphyrin IX-type heme per heterodimer. The hypothesis that the sGC heme is ligated by a histidine residue, much like the well-studied heme proteins hemoglobin and myoglobin, is not new; however, it has been only within the last several years that this suspicion has been confirmed and the ligand identified as HIS¹⁰⁵ of the bovine, human, and rat b₁. Electronic absorption spectroscopy on pure sGC revealed a spectrum that was entirely consistent with a histidine-ligated heme, namely, a Soret peak at 431 nm and a broad a/b band at 562 nm.^51-53 Additional observations with electronic absorption spectroscopy⁵⁴, magnetic circular dichroism spectroscopy⁵⁴, resonance Raman spectroscopy (RR)^55,56 and Fourier transform infrared spectroscopy (FTIR)⁵⁷ confirmed that the heme ligand was histidine. Subsequent observations definitively identified b₁-His¹⁰⁵ as the heme ligand: the NH₂-terminal portion of the b₁ subunit (1-385) expressed and purified from E. coli was capable of binding heme with spectral characteristics virtually identical to the heterodimeric enzyme⁵⁸; mutants of His¹⁰⁵ did not bind heme⁵⁹; and heme-binding to the H105G mutant could be rescued by including imidazole during growth and purification.⁵⁹ The majority of groups studying sGC purify the enzyme in a five-coordinate, high-spin histidyl complex that resembles deoxyHb and deoxyMb in terms of ligation, oxidation state, and spectral characteristics. Unlike hemoglobin and myoglobin, however, sGC has an extremely low affinity for oxygen, which fails to bind to the sGC heme even under 100% O₂.⁵¹ This property enables sGC to act as a receptor for •NO in the presence of much higher concentrations of O₂. Upon •NO binding, the Soret maximum shifts to 399 nm indicative a 5-coordinate nitrosyl complex. Deactivation involves the loss of •NO and the reformation of the HIS¹⁰⁵ ligation to the ferrous iron. The mechanism for deactivation remains poorly understood, but clearly stands as a critical pharmacological question in NO signal transduction.