Nitric oxide synthase in cardiac muscle: isoform characterization and functional role

The nomination of nitric oxide as Science’s molecule of the year 1992 was a consecration for a seemingly common gas otherwise known as a component of cigarette smoke or atmospheric pollutant. Its more recent prestigious career was started by the discovery of its role as s key signaling molecule in as diverse physiological functions as neurotransmission in central and peripheral nervous systems, immune defense against microorganisms and modulation of vascular tone (Nathan, 1994).
The latter functional effect of nitric oxide on blood vessel tone is the basis for the empirical observation, more than a century ago, of transient headaches following application on the tongue of nitroglycerine first synthetised in 1857 by Sobrero. The therapeutic use of nitroglycerine and some of its derivatives was proposed by Lord Brunton, a British physician who first used amyl nitrite for the relief of anginal pain. It wasn’t until about eighty years later, however, that nitric oxide radical was clearly identified as the active molecule released by organic nitrates, following the work of Ignarro and Murad starting in the early 1970’s (Gruetter et al., 1979 ; Murad et al., 1978). Both investigators also identified soluble guanylyl cyclise as a main intracellular target for the action of nitric oxide and Murad definitively established the link between smooth muscle relaxation and NO-dependent increases in intracellular cyclic GMP (Katsuki et al., 1977).
While this pathway was still being characterized, Furchgott and Zawadzki discovered the obligatory role of the endothelium in the muscarini cholinergic-dependent relaxation of rabbit aorta, through the release of a diffusable substance later termed “endothelium-derived relaxing factor” or EDRF (Furchgott and Zawadzki, 1980 ; Cherry et al., 1982). After much speculation about the chemical identity of EDRT, Ignarro’s and Furchgott’s groups simultaneously made a number of observations (Martin et al., 1985 ; Ignarro, 1986) leading these authors to propose, as early as July 1986, the similarity between the pharmacologic profiles of EDRF and nitric oxide. Two full papers from Salvador Moncada’s group and Louis Ignarro’s groutp, both published in 1987, reported the initial identification of EDRF as nitric oxide, as detected from isolated tissues with either chemiluminescence or spectrophotometric detection techniques (Ignarro et al., 1987 ; Palmer et al., 1987). Thereafter, Moncada’s group provided the key evidence for endogenous synthesis of nitric oxide by endothelial cells form the amino acid L-arginine (Palmer et al., 1988), stimulating a worldwide quest for the characterization and identification of the responsible enzymatic system subsequently known as nitric oxide synthase. The availability of somewhat specific pharmacological tools for the detection of nitric oxide synthase activity rapidly expanded the list of tissues and cells capable of producing endogenous nitric oxide beyond blood vessels where EDRF had first been characterized. The discovery of a robust enzyme activity in parts of the brain (where muscarinic cholinergic agonists had long been known to induce potent increases in intracellular cyclic GMP) led Bredt and Snyder to the purification to near homogeneity of a protein that were subsequently used to screen a rat brain cDNA library from which they isolated a full length cDNA encoding the first member of the NOS family, now termed nNOS (for neuronal constitutive nitric oxide synthase) (Bredt et al., 1991). Using similar approaches, members of Carl Nathan’s and Thomas Michel’s laboratories subsequently isolated the full length cDNA’s for two additional members of the family, iNOS and eNOD, in murine macrophages and bovine aortic endothelial cells, respectively (Xie et al., 1922 ; Lamas et al., 1993).At the same tome, efforts in many laboratories led to a more complete understanding of NOS enzymology as well as physiological function in many different systems, which have been described in more than 10.000 publications in the filed since the year 1990. This historical perspective on the discovery of a key signaling role for a molecule that had been empirically used for years from exogenous sources somehow recapitulates a common theme in pharmacology for the characterization of endogenous receptor and ligand systems. Likewise, the characterization of opioid receptors in the brain for example, had followed more than 20 centuries of empirical use of exogenous opium extracts. As in the brain, there is no doubt that future work on the nitric oxide synthase pathway in the cardiovascular system will fill some of the remaining gaps in the understanding of the molecular events linking receptor activation to a specific physiological effect, such as the eNOS-mediated endothelial-dependent smooth muscle relaxation. In the present work, we would like to propose a physiological role for both iNOS and eNOS in modulating the responsiveness of cardiac myocytes to both adrenergic and muscarinic cholinergic stimulation. The evidence provided is based on the molecular identification of these two NOS isoforms in various cell types comprising cardiac muscle in both rat and human species, as well as the characterization of the functional role of the NO synthase pathway in the heart using classical pharmacological approaches