A. The History and Overview of cGMP Metabolism
Cyclic nucleotides play a variety of roles in the regulation of cellular functions in nearly all cell types. One of the most important, is that of an intracellular secondary messenger within vascular smooth muscle (Lincoln, 1989). Two of the most abundant cyclic nucleotides are adenosine 3', 5'- cyclic monophosphate (cAMP) and guanosine 3', 5'- cyclic monophosphate (cGMP). Cyclic AMP plays an important role in blood vessel relaxation as mediated by receptor systems coupled via G-proteins to adenylate cyclase. A large number of different agonists such as adenosine, prostaglandins, and other beta-adrenergic agonists can stimulate the production of cAMP. Cyclic GMP participates in blood vessel relaxation induced by such drugs as nitroglycerin, nitroprusside and agents that function by stimulating the release of an endothelium derived relaxing factor (EDRF). Although the existence of cGMP was first reported more than 25 years ago, not long after the discovery of cAMP, the role it plays as a secondary messenger remained mostly unexplored for 10 - 12 years (Goy, 1991). The majority of research conducted into cyclic nucleotides was focused on cAMP. The function of GMP was thought to be merely an antagonist or analogous to cAMP. Because cAMP was considered to be an important mediator of smooth muscle relaxation, it was then reasoned that cGMP must be a mediator of contraction. It was not until 1977 when several investigators (Katsuki and Murad, 1977)(Katsuki, et al., 1977)(Schultz, et al., 1977) showed that vasorelaxation induced by nitrovasodilators (nitrogen-oxide constituents) was associated with an increase in cGMP levels that new interest was generated in cGMP (Lincoln, 1989).
2. Overview of cGMP Metabolism
Over the last 20 years a more comprehensive picture of cGMP and its mechanisms of vasodilatation has emerged. Figure 1 is a simplified overview of the biochemical mechanisms most generally accepted throughout the literature to participate in the cGMP cascade leading to vasorelaxation. Under normal physiological conditions, the endothelium stimulates vascular smooth muscle relaxation through the release of EDRF. Since its discovery in the early 1980's (Furchgott and Zawadzki, 1980), EDRF has been shown to be nitric oxide (NO) (Hardman, 1984)(Ignarro, et al., 1987). The endothelium can also produce other factors which are capable of inducing relaxation, such as endothelium derived hyperpolarizing factor, but these factors do not involve in the stimulation of the cGMP cascade. Although the endothelium plays the most important role in the release of NO into the smooth muscle cells, there are other pathways for nitric oxide to enter the cell. Ignarro has reported the presence of small quantities of NO-synthase in the smooth muscle itself (Ignarro, et al., 1992).
Drugs such as nitroglycerin and nitroprusside do not require the presence of the endothelium, and function via biotransformation within the smooth muscle cell to release NO (Ignarro, et al., 1981)(Brien, et al., 1988). Also independent of the endothelium, many sympathetic neurons that innervate the vasculature utilize NO as a neurotransmitter (Cellek and Moncada, 1997). Still other drugs such as S-nitroso-N-acetyl-penacillamine (SNAP) release NO spontaneously upon hydration and can serve as an exogenous source of NO (Ignarro et al., 1981). Although NO has repeatedly been shown to be the major stimulus in the production of cGMP via soluble guanylate cyclase, several investigators have suggested that cGMP may also rise in direct response to increased Calcium-dependent protein kinase (PKC) activity stimulated by G-protein coupled receptor systems (Van Haastert, et al., 1986)(Louis, et al., 1993)(Barnett, et al., 1995).
No matter its source, nitric oxide directly stimulates soluble guanylate cyclase to convert the substrate guanosine triphosphate (GTP) (present within the vascular smooth muscule at an intracellular concentration of 0.1 - 0.3 mM) (Seager, et al., 1994), into cGMP. Once soluble guanylate cyclase is activated, cGMP levels quickly begin to rise. Although the exact mechanism of cGMP induced relaxation has not been fully elucidated, it is generally believed that cGMP binds directly with and activates cGMP-dependent protein kinase (G-kinase). Although the mechanics of G-kinase are not fully understood, it is known that once activated, G-kinase acts via phosphorylation of various protein substrates within the smooth muscle cell. It has been proposed that this phosphorylation affects at least three different pathways that regulate intracellular levels of Ca2+. It is these pathways, alone or combined, that lead to relaxation. It is well established that smooth muscle tone is dependent upon intracellular levels of Ca2+. High levels of Ca2+ induce contraction, whereas low levels are associated with relaxation. The first of these pathways leads to the direct reduction of Ca2+ within the cell by the activation of Ca2+-ATPase pumps, which pump Ca2+ out of the cell. The second pathway is capable of attenuating the polyphosphoinositide cycle, specifically inositoltriphosphate (IP3). This reduces the amount of stimulation by IP3 on the sarcoplasmic reticulum to extrude Ca2+, thus preventing intracellular levels of free Ca2+ from rising (Abdel-Latif, 1986). The third pathway effects the state of force produced by the myosin and actin filaments of the smooth muscle itself. G-kinase is capable of phosphorylation at no less than seven sites within the myosin-actin complex, thus altering the characteristics of the actin filaments which allow them to bind and interact with myosin. It has also been suggested that cGMP can activate proteins other than G-kinase to effect the myosin-actin complex. This change in the physical characteristics of actin and myosin (which would also include its sensitivity to Ca2+) leads to a change in the force produced by the vascular smooth muscle (Pearce and Harder, 1994)(Akopov, et al., 1997). Electrophysiological data has also suggested an additional pathway for vasorelaxation, which is independent of G-kinase.
Yao et al., (1995) have shown that cGMP can directly bind to certain vascular smooth muscle membrane voltage-gated potassium channels, causing a hyperpolarization of the vascular smooth muscle thus retarding the influx of Ca2+ into the vascular smooth muscle.
Ultimately, the magnitude and the duration of relaxation is directly linked to the amount of phosphorylation by activated G-kinase. If cGMP levels within the cell were to remain elevated above baseline for an extended period of time, nearly full and complete activation of G-kinase would occur. Conditions such as these would render the vascular smooth muscle incapable of maintaining tone. Therefore, the cGMP signal which controls the activation of G-kinase should be very short in duration. This is accomplished in vivo by a rapid and short pulse of cGMP. In order to achieve this rapid and short pulse, not only must rapid synthesis of cGMP by soluble guanylate cyclase occur, but rapid degradation of cGMP must also occur at roughly the same time. In adult sheep, the cGMP pulse has been shown to reach peak cGMP concentration by 60 sec. after NO stimulation, and then rapidly return to baseline by 100 sec. (Pearce, et al., 1994).
The family of enzymes responsible for this rapid degradation (hydrolysis of the 3'-phosphodiester bond) of cGMP, are known as cGMP specific phosphodiesterases. The hydrolysis of cGMP by phosphodiesterase simply yields guanosine monophosphate (GMP) which may be recycled to the intracellular nucleotide pool within the vascular smooth muscle.
In order for an organism to possess a dynamic vascular system capable of responding quickly and repeatedly to a changing environment, tight control over intercellular levels of cGMP must be maintained. To achieve this, synthesis and degradation of cGMP must be tightly linked. It is the balance of soluble guanylate cyclase and phosphodiesterases activity within the dynamic system which determines the total magnitude of the cGMP pulse, which in turn determines the amount of phosphorylation by G-kinase, and which finally determines the magnitude and the duration of vasodilatation.
A variety of different research disciplines have contributed to this model of cGMP induced vasodilatation. Although this model provides a basic understanding, it is in no way complete. At present, regulation involves short-term alterations of substrate or co-factor concentrations and phosphorylation states, whereas modulation is generally regarded as a long-term alteration of the protein components via transcriptional, translational and post-translational modifications. Whereas regulation of cGMP metabolism is important in terms of biochemical mechanisms, modulation of the cGMP metabolic cycle as a function of age is of considerable interest physiologically. As suggested above, two points for modulation of cGMP are its synthesis by soluble guanylate cyclase, and its degradation by phosphodiesterases. The majority of this modulation of this system is achieved using molecular alterations in the enzymes themselves. These alterations range from minor changes in the enzyme's amino acid composition which can drastically effect the enzyme's substrate specificity, to differential expression of the various enzyme isozymes in different tissue. Although post-translational modification is a common means of enzyme regulation in other systems, it has never been widely reported for soluble guanylate cyclase and only for a few isozymes of phosphodiesterases. Considering the importance of the cGMP synthesis/degradation relationship in the cascade of events leading to vasorelaxation, a better understanding of these molecular mechanisms is in order for each enzyme.