UC Berkeley

The biochemical signaling pathways that involve gaseous primary signaling molecules have only been characterized in the last three decades, due to the small diversity of diatomic gases and the lack of other known physiological functions of these gases (apart from O2 as the terminal electron acceptor in oxidative phosphorylation). In general, certain events must take place for any process to be considered biochemical signaling: the primary signaling molecule must be synthesized and excreted from the generator cell or be present or absent in the environment, that signaling molecule must come in contact with its specific receptor molecule within the receiving cell, and the receptor molecule must transduce the primary singling molecule into a secondary signal which activates downstream signaling pathways. Only a pathway that is comprised of signal generation, the primary signal, and signal reception constitutes a complete biochemical signaling pathway. In spite of the relatively recent discovery, gaseous signaling molecules control critical signaling pathways in many aspects of organismal physiology.

In the 1980s, two different lines of research serendipitously came together to describe the first gaseous biochemical signaling pathway. The first was pharmacologists’ observation that vasculature that had the endothelium removed would constrict, and thus there must be an Endothelium Derived Relaxation Factor (EDRF) that maintained appropriate vasculature tone. Curiously, EDRF had the exact same chemical properties as nitric oxide (NO), however there was no known biotic source of this toxic gas. The second line of research centered on the observation that excreted nitrates spiked in concentration when sickness was incurred, even when nitrate intake remained the same. This led to the discovery that macrophages synthesize the cytotoxic agent NO to attack invading pathogens, and thus a biotic source of NO had been discovered. The EDRF and NO were shown to be one and the same, and this coupled with the fact that soluble guanylate cyclase had been known to be stimulated by NO since the 1970s provided the receptor of the first gaseous biochemical signaling pathway.

The enzyme responsible for the generation of NO is nitric oxide synthase (NOS) (EC 1.14.13.39). NOS catalyzes the 5-electron oxidation of arginine to citrulline and NO. There are three different isoforms of NOS in Homo sapiens, inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal (nNOS). Nitric oxide synthases are activated by calcium-bound calmodulin (Ca2+-CaM). iNOS has a high affinity for Ca2+-CaM, and thus is regulated at the transcriptional level in macrophages where it synthesizes NO at sites of infection or inflammation. eNOS and nNOS have lower affinity for Ca2+-CaM and are constitutively expressed, which allows calcium gradients in neurons and endothelial cells to control the activation of these isoforms. For example, when endothelial cells hyperpolarize due to high blood pressure, intracellular concentrations of Ca2+ increase up to ten-fold which in turn increases Ca2+-CaM concentration and thus activates eNOS.

Activated eNOS generates NO at nanomolar concentrations, which diffuses to nearby smooth muscle cells that express the mammalian NO receptor, soluble guanylate cyclase (sGC) (EC 4.6.1.2). sGC is a heterodimeric hemeoprotein which can sense NO at nanomolar concentrations. When NO binds to sGC, the enzyme increases its catalytic activity of cyclizing 5′-guanosine triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP). The molecular mechanisms by which NO activates sGC are not completely understood. In particular, it is clear from experiments both in vitro and in cells that one equivalent of NO does not activate sGC to its maximal activity but only to a low activity state. Excess NO must be present in order to completely stimulate the enzyme. If that excess NO is removed either with a buffer exchange or a chemical trap, the enzyme returns to a low activity state. The increase in cellular concentration of cGMP activates a variety of downstream signaling proteins, including cGMP-dependent protein kinases, cGMP-regulated ion channels, and phosphodiesterases that mediate the downstream cell signaling cascade. The resultant phenotype of these cellular signaling molecules is the relaxation of the smooth muscle cells, the dilation of the vasculature, and the decrease of overall blood pressure. Thus, any malfunctions of this pathway can result in too high of blood pressure and increased risk of cardiovascular diseases.

The NOS-NO-sGC-cGMP signaling pathway has been the target for stimulating therapeutics to treat forms of high blood pressure. A screen of small molecules that induce platelet aggregation found that the chemically synthesized benzylindazole YC-1 was able to stimulate sGC in a heme-dependent manner. Significant efforts in medicinal chemistry in the following two decades yielded riociguat, the first FDA-approved sGC stimulator that is used to treat certain forms of pulmonary hypertension. However, the specific binding site of YC-1 derived compounds on sGC and the reason for the specificity of these types of molecules against the soluble form of guanylate cyclases has not been elucidated. Additionally, atypical soluble guanylate cyclases have been described that sense O2 instead of NO. Thus, the possibility exists for gases other than NO and O2 to function as signaling molecules in eukaryotic organisms. These outstanding questions will be answered in the following thesis entitled: The Structure and Activation of Soluble Guanylate Cyclase.