Shared signaling pathways among gasotransmitters

Gasotransmitters, including NO, CO, and H₂S, are a group of endogenously produced gas molecules that are membrane-permeable and share similar molecular targets (1). Gasotransmitters may antagonize or potentiate each other’s cellular effects at three levels: their production, their downstream molecular targets, and the direct chemical interaction among themselves. The study published in PNAS by Coletta et al. (2) presents an interesting example of how the interaction of gasotransmitters converges at the same downstream molecular target. Coletta et al. found that the proangiogenic effect of H₂S on angiogenesis and wound healing was completely absent in endothelial NO synthase KO mice, and that eliminating H₂S production by silencing cystathionine-γ-lyase (CSE) abolished NO-stimulated angiogenesis (2). This mutually dependent relationship between H₂S and NO in vascular endothelial cells is rooted in the regulation of the cellular levels of cGMP. NO activates soluble guanylyl cyclase (sGC) to generate cGMP, whereas H₂S inhibits phosphodiesterase-5 (PDE5) to slow down the degradation of the existing cGMP (2). A net increase in cGMP level leads to the activation of protein kinase G (PKG) and its downstream effector, vasodilator-stimulated phosphoprotein. Lack of NO might curtail the production of cGMP so that H₂S-inhibited cGMP degradation becomes meaningless. Conversely, rapid degradation of the existing cGMP in the absence of H₂S would render NO powerless to elicit angiogenesis.

The activation of sGC by NO and CO has been well documented (3). The affinity of NO to sGC is approximately 30 to 100 times greater than that of CO (4). Hitherto, there has been no evidence that H₂S actually interacts with sGC. It had been suggested, however, that endogenous H₂S might increase cGMP level by inhibiting PDE activity in the heart (5). This suggestion was later verified by Bucci et al. with cultured rat aortic smooth muscle cells (SMCs) (6). It is worth pointing out that H₂S appears to inhibit different subtypes of PDE without specificity. As such, H₂S may inhibit the degradation of both cAMP and cGMP in different types of cells. Depending on whether it is cAMP or cGMP level that is being affected, the cellular effects of H₂S may be quite different.

Compared with their effects on angiogenesis, individual and combined effects of NO and H₂S on endothelium-dependent vasorelaxation are more complex. After being synthesized in, and released from, the endothelium, NO activates sGC in vascular SMCs to induce vasorelaxation (3), whereas H₂S opens intermediate- and small-conductance calcium-sensitive K channels in the endothelium as an endothelium-derived hyperpolarizing factor (7), and stimulates K_ATP channels in vascular SMCs to relax blood vessels (8). A mechanism was introduced by Coletta et al., who ascribed endothelium-dependent and H₂S-induced vasorelaxation partially to the inhibition of PDE5 in vascular SMCs (2). This proposed mechanism is not undisputed, as, under resting conditions, total cGMP content in rat aortic rings was not significantly decreased by silencing CSE, or increased by addition of NaHS (a H₂S donor) (2).

Another important consideration for NO- or H₂S-induced endothelium-dependent vasorelaxation is the roles played by individual gasotransmitters in different types of blood vessels. It has been proposed that NO may play a more important vasorelaxant role in conduit artery (e.g., aorta) as an endothelium-derived relaxing factor but that H₂S may be more critical in regulating the relaxation of peripheral resistance arteries (e.g., mesenteric arteries and coronary arteries) as an endothelium-derived hyperpolarizing factor (7, 9). Previous studies have shown that H₂S is approximately five times more potent in relaxing the rat mesenteric artery vs. the rat aortic artery (9). In CSE-KO mice, the methacholine-induced endothelium-dependent vasorelaxation was significantly decreased (10), which may or may not be related to changes in cGMP level, as the blood vessel under investigation is a classical resistance mesenteric artery (10), whereas the proposed inhibition of PDE5 by H₂S was derived from studies on rat aortic tissues (2).

Whether H₂S-induced vasorelaxation is mediated by the cGMP pathway, wholly or partially, is still unsettled. NO-induced vasorelaxation was abolished by ODQ or NS- 2028. However, these two sGC inhibitors neither had effect on, nor potentiated, H₂S-induced relaxation of rat aortic tissues (11). Also tested on rat aortic tissues, NaHS-induced relaxation was mostly inhibited by the blockade of K_ATP channels and to a much lesser extent by ODQ (12). The vasorelaxant effect of NaHS on rat coronary arteries is also unaffected by Nω-nitro-L-arginine methyl ester or ODQ (13). The precontracted chicken ductus arterious rings were relaxed by Na₂S (another H₂S donor) or by authentic CO. The vasorelaxant effect of CO is inhibited by Nω-nitro-L-arginine methyl ester or ODQ, but that of Na₂S is not affected (14). Differences in these vasoactive studies regarding the mediation of H₂S effect by cGMP level cannot yet be readily explained. At any rate, it is too early to conclude that the vasorelaxant effects of H₂S and NO converge at the cGMP-PKG node. Alterations in K_ATP channel openings, intracellular free calcium levels, oxidative stress status, and other signaling events may all be involved in the vasoactive effects of H₂S, irrespective of changes in cGMP level in different vascular preparations or in the same type of blood vessels from different species.

Cellular effects of gasotransmitters, and the signaling mechanisms for the same, are cell type-specific. Endothelial cells and SMCs make this case in point. We can start with the production of gasotransmitters. NO production is virtually a patented function of eNOS in endothelial cells. H₂S, however, can be produced in both endothelial cells and SMCs. As such, the direct effect of endogenous H₂S on SMCs would unlikely be affected by endogenous NO in the absence of the endothelial cell. Coletta et al. showed that exogenous NO failed to increase cGMP level in the absence of CSE (2). This phenomenon observed in vascular endothelial cells, however, cannot be fully realized in vascular SMCs (2). Here we see that the dependency of H₂S effect on NO varies among different types of cells. The contrast is even sharper when one further explores the proliferative responses of endothelial cells and vascular SMCs to gasotransmitters. H₂S inhibits the proliferation of vascular SMCs but stimulates that of endothelial cells (15).

Signal transductions in cells are intertwined and orchestrated. Gasotransmitters act together in the same network fashion. Elevation of cGMP and activation of PKG in a set of cells link together the cellular effects of NO, H₂S, and CO as mentioned earlier. There are many other converging targets for gasotransmitters’ cellular effects that have been shown. As an example of synergistic convergence, both NO and CO increase the opening probability of big-conductance calcium-sensitive K (BK_Ca) channels in vascular SMCs. CO specifically interacts with the α-subunit of BK_Ca channel, whereas the stimulatory effect of NO depends on the presence of β-subunit of BK_Ca channel complex (16). In addition to activating different types of K_Ca channels, H₂S

Signal transductions in cells are intertwined and orchestrated. Gasotransmitters act together in the same network fashion.

appears mostly to enhance the opening of K_ATP channels in vascular SMCs. By activating different potassium channels, these three gasotransmitters hyperpolarize cell membrane and decrease intracellular free calcium level. Changes in calcium levels at this convergence site lead to vasorelaxation. In fact, changes in cGMP levels induced by NO, CO, and H₂S would also lead to changes in intracellular calcium levels to add another layer of convergence (Fig. 1).

Fig. 1.

Signaling convergence of gasotransmitters in vascular SMCs. ER, endoplasmic reticulum; IK_Ca/SK_Ca, intermediate- and small-conductance calcium-sensitive K channel; K_ATP, ATP-dependent K channel.

S-nitrosylation and S-sulfhydration mechanisms often create an antagonistic convergence between NO and H₂S. H₂S-induced S-sulfhydration may convert cysteine–SH groups to hydropersulfide, leading to increased protein activity. The covalent modification of the same cysteine can be induced by NO, i.e., S-nitrosylation, but the functions of the modified protein may be decreased. GAPDH is an enzyme in the glycolytic pathway whose S-nitrosylation (17) and S-sulfhydration (18) can both occur at Cys-150. Direct interaction of NO with purified GAPDH abolishes its catalytic activity (17), but the interaction of H₂S increases the activity of GAPDH.

When H₂S met NO, there was no longer “That’s Mine, This Is Yours.” These two gasotransmitters would have entered into a complicated and variable relationship dictated by their surroundings, in or out, love or hate. This converged relationship affects the destinies of different cells, organs, and systems. This is only the story of two gasotransmitters. Could this also be true for other gasotransmitters, neurotransmitters, or any other signaling events in our body?