Pharmacology of nitric oxide

We had seen earlier that both the sympathetic and the parasympathetic nervous system innervate blood vessel walls and regulate their contraction and wall tension (slide 6.13.1). The adrenergic nerve endings of the sympathetic nerve fibers are found in the muscular layer of the vessel walls. In contrast, cholinergic nerve endings are found both in the muscular and the endothelial layers.

The wall tension of the blood vessel is sustained by the muscular layer, not the endothelium; therefore, the cholinergic innervation of the endothelium may be surprising. As it turns out, however, it is needed for the vasorelaxation and vasodilation induced by the parasympathetic system.

This slide illustrates the experiment that led to the discovery of nitric oxide-mediated vasorelaxation.

Contraction and relaxation of the vascular smooth muscle cells in response to norepinephrine and acetylcholine were studied with aortic strips. In this technique, slices are cut from an animal’s aorta and then opened with a radial incision. The endothelium can be left in place or peeled away in order to study its effect on the muscular layer. The resulting intact or denuded aortic strips were mounted between two distending hooks to measure their contractile force.

Application of norepinephrine induced contraction in strips with or without attached endothelium, as would be expected from the innervation pattern of the sympathetic nerve fibers. In contrast, norepinephrine-induced contraction could be reversed by acetylcholine only if the strip retained the endothelium (top) but not when the endothelium had been peeled away.

Interestingly, relaxation of a denuded muscular strip could be restored if the endothelial side of an intact strip was strapped onto it (not illustrated). These observations showed that, in response to cholinergic stimulation, the endothelium releases a diffusible substance that enters the muscular layer and induces its relaxation. This “endothelium-derived relaxing factor” was subsequently isolated and identified as NO.

Nitric oxide synthase exists as a dimer. Each subunit contains a reductase domain and an oxidase domain. The reductase domain accepts electrons from NADPH and hands them over to the oxidase domain of the other subunit.

The reductase and oxidase domains are loosely connected by flexible hinges that, when extended, prevent the flow of electrons between them. The two domains are brought into close, productive interaction when calmodulin (CaM) binds to the hinges and changes their conformations. In the case of eNOS and nNOS, CaM binding only happens if the cytosolic level of Ca⁺⁺ is elevated, which occurs only transiently when triggered by action potentials or through activation of GPCRs, such as for example the muscarinic ones in endothelial cells.

In contrast, inducible NOS binds calmodulin very avidly regardless of the calcium concentration, and therefore is active even at the low resting level of Ca⁺⁺. Its activity is regulated not by short-term calcium signals but instead by transcriptional induction.

Like cAMP, cGMP targets multiple effector molecules inside the cell. The activation of cGMP-dependent protein kinases (cGK) is the most important single mechanism. Phosphodiesterase 5 (PDE) is activated, too, reducing the levels of both cAMP and cGMP. Actuation of cyclic nucleotide-gated cation channels affects the membrane potential and the cellular calcium level.

Membrane-bound receptor or “particulate” guanylate cyclases (pGC) provide an alternate means of cGMP production that is independent of NO but instead is controlled by several peptide hormones.

The relaxation of smooth muscle that occurs downstream of endothelial NO release is mediated by cGMP-dependent protein kinase. This kinase phosphorylates and thereby activates myosin light chain phosphatase. The phosphatase dephosphorylates myosin, which interrupts interaction of myosin with actin and induces relaxation.

cGK also phosporylates a regulatory subunit of the IP₃ receptor channel. As we have seen before (slide 5.3.2), this channel mediates the outflow of Ca⁺⁺ from the ER to the cytosol. Phosphorylation of the IP₃ channel reduces Ca⁺⁺ flow and therefore the calmodulin-dependent activation of myosin light chain kinase (MLCK).

It is worth noting that the effects of NO cut in below the GPCRs that mediate the effects of major vasoconstrictors such as angiotensin and norepinephrine. Therefore, NO-releasing drugs offer a means to interrupt the out-of-control signaling by such mediators that occurs in hemodynamic shock or hypertensive crisis.

One mechanism that may contribute to the selectivity of protein-S-nitrosylation in vivo is the transfer of nitrosyl groups between proteins, which is mediated by glutathione. In this way, the nitrosyl groups can travel around the cell before settling down on the proteins with the thermodynamically most favorable cysteine residues.

Since glutathione is the most abundant thiol in the cell, this also means that nitrosylated glutathione functions as a reservoir of S–N=O (nitrosothiol) groups in the cell. In fact, many in vitro studies on the regulatory effects of protein-S-nitrosylation have used nitrosylated glutathione as the source of nitrosyl group instead of free NO for simplicity.

To understand the regulatory effects of protein-S-nitrosylation, it is important to identify the proteins, and the specific cysteine residues within them, that actually become S-nitrosylated in vivo upon NOS activation. The biotin switch method is an experimental protocol that permits the identification of these cysteines by selectively attaching biotin to them. This procedure involves the following steps:

1. 1.After NOS has been activated and protein-S-nitrosylation has run its course, proteins are extracted from cells. Their remaining free SH groups are converted to disulfides using S-methyl-methanethiosulfonate.
2. 2.Ascorbic acid is used to selectively reduce S–NO groups, while leaving native or synthetic disulfide bonds intact.
3. 3.The reduced SH groups are labeled with a biotinylation reagent, via disulfide formation (as shown here) or other coupling chemistries.

After this procedure, biotin will be attached specifically to those cysteine residues that had been nitrosylated before. The biotinylated proteins can then be purified or selectively detected on a blot using the very strong and specific interaction between biotin and streptavidin.

This experiment illustrates the selectivity of protein-S-nitrosylation in brain tissue from mice due to activation of neuronal NOS. The role of nNOS was ascertained by comparing the extent of nitrosylation between wild-type and nNOS k.o. mice.

After extracting the proteins from homogenized tissue, S-nitrosyl groups were converted to biotin derivatives as outlined above, and selectively detected on blots using labeled streptavidin. The total amount of each protein—nitrosylated or not—was imaged with antibodies and seems indistinguishable between wild-type and nNOS k.o. mice in every case. With each protein, the extent of S-nitrosylation is represented by the intensity of the third sample relative to the first one.

In all cases, S-nitrosylation is caused by nNOS, as shown by its absence in the knockout mice. The extent of nitrosylation is particularly strong with the NMDA receptor, and indeed this receptor—one of the ligand-gated glutamate receptors (see section 6.11)—is known to be inhibited by S-nitrosylation. Glyceraldehyde-3-dehydrogenase (GAPDH) is also quite strongly affected. Intriguingly, this protein does not only serve in its catalytic role in glycolysis but moonlights as a signaling molecule in the control of apoptosis (programmed cell death). Figure adapted from [68].

Nitroglycerin and isosorbide dinitrate can be activated by multiple enzymes. In mice and several other species, an important enzyme is mitochondrial aldehyde dehydrogenase (ALDH). This slide shows the reaction mechanism—in vitro, the reaction requires the addition of a thiol-reducing agent such as dithiothreitol (DTT), and the reaction produces S-nitrosylated intermediates—as well as the effect of inhibiting ALDH with cyanamide on the relaxation of aortic strips ([69]; compare slide 8.2.2). Genetic knock-out of ALDH suggests an even greater contribution of ALDH to nitroglycerine bioactivation; however, this varies with the drug and the animal species in question.

While both nitroglycerin and isosorbide dinitrate are metabolized by cytochrome P450 enzymes, a more detailed analysis shows that the enzyme isoforms with the strongest activity toward each are somewhat different: CYP2E1 is highly active with nitroglycerin, whereas CYP3A4 is more active with ISDN. Both drugs are converted efficiently by CYP2J2, which is strongly expressed in blood vessels and is likely important in the prompt clinical effect of nitroglycerin.

As we have seen, cytochrome P450 enzymes contain heme. Like the heme in sGC, the one in cytochrome P450 may bind NO; this will inactivate the enzyme. Inactivation of cytochrome P450—and/or aldehyde dehydrogenase, which is subject to protein-S-nitrosylation—by NO released from drugs contributes to nitrate tolerance, which develops upon prolonged drug application and can only be broken by pausing the use of these drugs.67

In the experiment shown, the CYP isoforms were recombinantly expressed in yeast. Experimental systems such as this one are often used to study the susceptibility of novel drug molecules to metabolism during preclinical development. Figure prepared from original data in [70].

Molsidomine is an alternative NO-releasing drug that does not require metabolism by cytochrome P450 or aldehyde dehydrogenase, and therefore is less prone to the development of tolerance. The left panel shows the mechanism of NO release: Enzymatic hydrolysis of molsidomine yields linsidomine, which then decomposes spontaneously. The final step depends on O₂ and yields both NO and superoxide.

As we had seen above (slide 8.6), superoxide can combine with NO to form peroxynitrite. Accordingly, in order to render the NO released by linsidomine available for the activation of guanylate cyclase, superoxide must be scavenged. This is illustrated in the experiment in the right panel, which shows that appreciable amounts of cGMP are formed downstream of linsidomine decay only in the presence of scavenging agents such as superoxide dismutase (SOD) or glutathione (GSH). Figure prepared from original data in [71].

While NO-releasing drugs influence the contractile activity of smooth muscle cells by increasing cGMP, an equally viable approach should be the inhibition of cGMP degradation by phosphodiesterase. This rationale led to the development of sildenafil, which was originally intended as yet another cardiovascular drug for the elderly.

As it turned out, the drug proved very popular with the old folks alright, but not entirely for the intended reasons. Nevertheless, in addition to its well-known effect on male potency, the drug also does promote vasorelaxation, which can prove dangerous when used in combination NO-releasing drugs or similar.

Prompted by the risks of sildenafil use in cardiac patients engaging in strenuous exercise, some administrative districts (Kantons) in Switzerland have mandated the training of prostitutes in the use of defibrillators.