Endogenous And Exogeneous Nitric Oxide Donors

The Compendium June 1996

Small Animal

THERAPEUTICS IN PRACTICE

V

Endogenous and Exogenous Nitric Oxide Donors*

A

survey of scientific material written from 1992 to 1995 revealed that almost 6000 articles on nitric oxide (NO) and the endothelium-derived relaxing factor (EDRF) have been published. The quantity of articles seems considerable for a small molecule whose major claim to fame before 1980 was as an environmental pollutant from the exhaust of cars, thus contributing to the formation of acid rain and destruction of the ozone layer.1 Endothelium-derived relaxing factor is believed to be nitric oxide or a very similar substance, and much of the literature uses the terms interchangeably. Thus, for the purpose of this presentation, any reference to nitric oxide should be interpreted as also referring *This column is derived from an indepth study that includes extensive referencing. A copy of the study, along with the references, is available from the author on request.

SERIES EDITOR

to endothelium-derived relaxing factor and vice versa. Although endogenous nitrovasodilators have been used pharmacologically for more than 100 years, the mechanism of their action was unknown until 1980. Nitrates were better known for their action as fertilizers and in the manufacturing of bombs. The discovery of the importance of nitric oxide in vascular, neural, phagocytic, and a myriad of other functions opened a vast unexplored realm of pathophysiology and pharmacology.

derived contracting factors (EDCFs), thromboxane-A2, and free radicals.4 Most relaxants are inhibitors of growth, and most constrictors are stimulators of growth.2 In response to some stimuli, the endothelium produces growth factors that, combined with platelet and macrophage growth factors, are responsible for vascular remodeling. The endothelium-derived relaxing factor is produced in the cytoplasm of vascular endothelial cells, neurovascular cells of

The Endothelium The vascular endothelium, previously believed to be no more than a cellular lining with a barrier role2 of maintaining the internal integrity of blood vessels, is now known to be a prodigious manufacturer of vasoactive chemicals. The endothelium exhibits all the characteristics of an endocrine gland.1 The vasorelaxants produced are the endotheliumderived relaxing factor, endothelium-derived hyperpolarizing factor (EDHF), prostacyclin, and C-type natriuretic peptide (CNP) (Table One). Vasoconstrictors produced are endothelin-1, 3 other endothelins, endothelium-

KEY POINTS

Gerard J. Rubin, DVM Diplomate, ACVIM (Cardiology and Internal Medicine) Animal Internal Medicine Clinic Dallas, Texas

most of the cells of the body.7,8 The factor is probably a complex that liberates nitric oxide and may contain a thiol.9 The major substrate is the amino acid L-arginine found in cellular cytoplasm. The dioxygenase enzyme nitric oxide synthase (NOS) on stimulation catalyzes oxidation of L-arginine, thereby producing nitric oxide and Lcitrulline.10 Citrulline also is produced from glutamine via glutamate and ornithine in the intestinal epithelial cells and is transported

■ Endothelium-derived relaxing factor is probably a complex that liberates nitric oxide or a very similar substance. ■ Some nonendothelial agents, such as exogenous nitrovasodilators, can mimic the effects of endothelium-derived relaxing factor. ■ Nitrovasodilators have many beneficial effects as treatment for canine and feline patients with congestive heart failure and no known incompatibility with other common cardiovascular drugs. ■ Dietary deficiency of arginine may be a contributory cause of hypertension and thrombosis in cats.

the central nervous system,5 vascular smooth muscle cells, myocardium, 6 endocardium, macrophages, and possibly

Susan E. Johnson, DVM, MS The Ohio State University

by the bloodstream to the kidneys. Citrulline is converted to arginine in the proximal tubules and made available for nitric

Small Animal

The Compendium June 1996

L -ornithine

TABLE ONE Comparison of Endothelium-Derived Relaxing Factor (EDRF) and Prostacyclin21 Characteristic

EDRF

Prostacyclin

Source

L-arginine

Arachidonic acid

Pathway

Nitric oxide synthase

Cyclooxygenase

Mechanism of action

↑ Cyclic guanine monophosphate

↑ Cyclic adenosine monophosphate

Half-life

Approximately 6 seconds

Approximately 30 seconds

Inhibitors

Oxyhemoglobin, methylene blue

Aspirin and other nonsteroidal antiinflammatory drugs

Stimulus for release

Acetylcholine, calcium ionophore A23187, wall stress, thromboxane A2, serotonin, thrombin

Acetylcholine, calcium ionophore A23187, wall stress, thrombin, hypoxia

Catabolism

Superoxide radicals

Hydrolysis

oxide synthesis in other tissue. Citrulline also is converted to arginine in endothelial cells and macrophages.11 The L-citrulline is recycled back to L-arginine by incorporation of one nitrogen from NH3.12 Cats cannot produce arginine in sufficient quantities from ornithine or citrulline and therefore require large amounts in the diet.13,14 Nitric oxide synthase occurs in two major forms, constitutive and inducible NOS (iNOS), and in three major isoforms. Inducible NOS, also referred to as isoform II, requires induction by immunologic stimulation.1 Constitutive nitric oxide synthases (cNOSs), isoform I and III, are present under normal physiologic or constitutional conditions, thus the designation constitutive. Isoform I is produced in neurovascular endothelial cells. 5

Isoform III is produced in cardiovascular endothelial cells, endocardium, and myocardium. The following formula helps to explain the process: L-arginine + NOS =

EDRF + L-citrulline

Inhibitors of Nitric Oxide Synthase Nitric oxide synthase is inhibited by L-arginine analogues7 (i.e., N G-monomethyl-L-arginine, N G-nitro-L-arginine methyl ester, and N-iminoethyl- L ornithine (Table Two). This inhibition can be overcome by the addition of L-arginine but not Darginine. 15 These inhibitory agents have been used to define the effects of nitric oxide by blocking nitric oxide production and observing the results. Examples are:

■ Induction of an endothelium-dependent constriction of rabbit aortic rings.16 ■ Inhibition of endotheliumdependent relaxation induced by acetylcholine and other relaxant substances.16 ■ Vascular smooth muscle constriction by acetylcholine.17 ■ Increased blood pressure accompanied by decrease in the glomerular filtration rate.18 ■ Induction of dose-related coronary vasoconstriction.19 Inducible nitric oxide synthase is also inhibited by glucocorticoid. L-canavanine inhibits nitric oxide synthase in macrophages but not in endothelial cells, platelets, and the brain.20 Aminoguanidine can be a selective inhibitor of inducible nitric oxide synthase.21 N-iminoethyl-

is a selective inhibitor of nitric oxide synthase in neutrophils.22

Nitrovasodilators Nonendothelial agents that mimic the effects of EDRF are exogenous nitrovasodilators, atrial natriuretic factor,23 bovine retractor penis inhibitory factor, prostacyclin,14 and β-adrenergic agonists (Table Three). The pharmacologic and biochemical effects of endothelium-dependent vasodilators and nitrovasodilators24 are almost the same.25 Nitrovasodilators function by releasing nitric oxide independent of L-arginine and nitric oxide synthase. Three groups of drugs are considered to be nitrovasodilators. The first group is compounds that contain a nitrate ester bond (R-O-NO2). Some of the drugs belonging to the nitrate ester group are isosorbide dinitrate (ISDN), isosorbide-5mononitrate (IS-5-MN), pentaerythritol tetranitrate, and mannitol hexanitrate. The second group is nitrocompounds, which have a carbogen-nitrogen bond (R-CNO2). Belonging to this group is nitroglycerin (chemical name: glyceryl trinitrate). The third group is nitricoxide–containing compounds, such as nitroprusside and molsidomine. Nitroprusside and molsidomine can release nitric oxide spontaneously, while other compounds require prior interaction with a thiol, such as cysteine.25 Organic nitrates (ISDN, IS-5MN, and nitroglycerin) are initially converted to nitric acid (HNO3) intracellularly and subsequently to S-nitrosothiol (SNO) by the addition of a sulfhydryl (SH) group. The S-nitrosothiols and

Small Animal

nitric oxide activate soluble guanylate cyclase (GC), which increases cyclic guanine monophosphate (cGMP). Removal of endogenous nitric oxide production or the endothelium enhances the sensitivity of the vasculature to exogenous nitrates and catecholamines,26 possibly because of the up-regulation of soluble guanylate cyclase.27 Nitrovasodilators have greater action on coronary arteries than on peripheral arteries, and veins are more sensitive than arteries. The latter effect may be attributable to arteries producing more endogenous nitric oxide than veins do.28 It has also been found that veins are more subject to developing tolerance to nitrovasodilators than are arteries.29 Of the three major nitrovasodilators, nitroglycerin is more potent than ISDN, which is more potent than IS-5-MN.30 Following are two formulas explaining the process: Organic nitrates → HNO3 + SH → SNO EDRF [SNO or NO] → ↑ GC → ↑ cGMP → ↓ CA++ → ↓ contraction Nitrovasodilators have many beneficial effects in a patient with congestive heart failure.31 They produce venous dilation and increase venous capacitance, thereby increasing peripheral pooling of blood.32 The result reduces left- and rightsided ventricular end-diastolic pressure and end-diastolic volume, thereby unloading the heart and reducing myocardial oxygen consumption. The dilation of coronary arteries also leads to an increase of oxygen supply to the heart. Afterload reduction by arterial dilation, reduction of arterial impedance,

The Compendium June 1996

and increased arterial compliance boost stroke volume and therefore cardiac output. As a result, wall stress is decreased and oxygen consumption is reduced. Nitrovasodilators exert a relaxing effect on the ventricular myocardium by increasing compliance and allowing dia-

stolic filling at a lower filling pressure without affecting systolic function.33 Nitrovasodilators have no known incompatibility with other commonly used cardiovascular drugs and may be used with them, some synergistically. Chronic nitrate therapy is

believed to improve the hemodynamic benefits of angiotensinconverting enzyme inhibitors.34 In Veterans Administration Cooperative Vasodilator–Heart Failure Trials,35 a combination of hydralazine and nitrates was shown to have a favorable effect on survival when com-

TABLE TWO Comparison of Forms of Nitric Oxide Synthase Constitutive Cytosolic

Inducible Cytosolic

a

NADPH dependent

NADPHa dependent

Requires tetrahydrobiopterin

Requires tetrahydrobiopterin

Dioxygenase

Dioxygenase

L-arginine analogues inhibit

L-arginine analogues inhibit

++

Ca and/or calmodulin dependent

Ca++ independent

Picomoles of nitric oxide released

Nanomoles of nitric oxide released

Short-lasting release

Long-lasting release

Unaffected by glucocorticoids

Inhibited by glucocorticoids

Isoform I and III

Isoform II

a

Nicotinamide adenine dinucleotide phosphate.

TABLE THREE Comparison of Endothelium-Derived Relaxing Factor (EDRF) and Nitrovasodilators22 Nitrovasodilator

EDRF

Stimulates cGMP

Stimulates cGMP

Exogenously administered

Endogenously released

Prolonged circulatory effect

Transient circulatory effect

Increased activity in coronary disease

Decreased activity in coronary artery disease

Systemically active

Locally active

Inhibits smooth muscle growth

Inhibits smooth muscle growth

Inhibits myocyte growth

Inhibits myocyte growth

Tolerance may develop

No tolerance demonstrated

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pared with a placebo. A study of the effect of drugs on ventriculoarterial coupling showed that nitrates delayed the peripheral wave reflection and improved the mechanical efficiency of the left ventricle while hydralazine increased the characteristic impedance and shortened wave reflection, thus decreasing the mechanical efficiency of the heart. 36 It is conceivable that trials using nitrates without hydralazine might result in a more favorable response. Some available forms of nitrovasodilators follow37: ■ Amyl nitrite is used by inhalation mainly for diagnostic purposes. ■ Nitroglycerin is available as a sublingual tablet, oral sustained-release tablet, 2% ointment, transdermal paste, and intravenous solution. ■ Nitroprusside is available for intravenous use only. ■ Isosorbide dinitrate is available as a sublingual tablet, chewable tablet, tablets of various sizes, oral spray, ointment, and intravenous solution. ■ Pentaerythritol tetranitrate is available as a sublingual tablet. ■ Erythrityl tetranitrate is available as a sublingual tablet and an oral tablet. ■ Molsidomine is a syndonimine that releases nitric oxide without requiring the presence of a thiol group and is less inclined to cause tolerance.8 Molsidomine is given orally but at this time is not available in the United States. ■ Nicorandil is a nicotinamide nitrate that has a dual cellular mechanism as a potas-

The Compendium June 1996

sium channel activator and a nitric oxide contributor. Nicorandil acts by dilating large coronary arteries and as a preload and afterload reducer. It causes less tolerance than do the nitrates.38 Nicorandil is given orally but at this time is not available in the United States.

Use in Veterinary Medicine I have used nitrates for almost 50 years in clinical practice, originally at the suggestion of another practitioner, Dr. Arthur Trayford, to treat old dog cough (by prescribing mannitol hexanitrate) and subsequently for various forms of diagnosed heart disease. The old dog cough was probably caused by pulmonary edema or mitral regurgitation. Although reports of the use of nitrovasodilators in veterinary medicine39–41 have been relatively sparse, a number of pathologic conditions may be improved by their use. Congestive heart failure, especially that attributable to mitral regurgitation, fits this category. Early studies42 of dogs have shown that intramural coronary arterial lesions, myocardial necrosis, and myocardial fibrosis (mostly attributable to ischemia) are common findings. Dogs with heart failure are at high risk for developing valvular endocardiosis, arterial lesions, and infarcts, which might be avoided with early treatment using nitrovasodilators. The vasoconstrictor effects of congestive heart failure also might be reversed. Inoperable congenital cardiac conditions, such as septal defects, patent ductus arteriosus with right- to left-sided shunts, aortic stenosis, and

The Compendium June 1996

valvular dysplasia, may benefit from the use of nitrovasodilators. Hypertrophic cardiomyopathy may be aided by the use of nitrovasodilators, but dilatory cardiomyopathy may not. Dogs with heartworm disease not only have pulmonary hypertension but have endothelial damage as well. It has been demonstrated that factors in serum from heartworm-diseased dogs intefered with endotheliumdependent relaxation. This decreased relaxation is corrected by addition of nitroglycerin.43 These patients may profit by adding nitrovasodilators to the treatment regimen. Cats are subject to hypertension as well as to cerebrovascular and caudal aortic thrombosis. Nitrovasodilators may be beneficial in these animals. Cats have a high dietary requirement for arginine because they cannot produce sufficient quantities from ornithine or citrulline.13,14 Dietary deficiency of arginine may possibly be a contributory cause of the hypertension and thrombosis diagnosed in cats. I have used nitrovasodilators without observing adverse effects in animals receiving cardioglycosides, diuretics, βadrenergic blockers, calcium blockers, and angiotensinconverting enzyme inhibitors. The dose of isosorbide dinitrate used is from 1⁄ 4 to 1 mg/kg twice daily in dogs and cats. The dose of 2% nitroglycerin ointment is from 1⁄4 to 1 inch rubbed into hairless parts of the body (inner thigh or inner ear pinna) twice daily. Although the benefits described here have been anecdotal, the results warrant further trials. The following trial groups are recommended for a thorough

Small Animal

pharmacologic study of animals with congestive heart failure: ■ Conventional therapy (i.e., digoxin and/or furosemide) ■ Conventional therapy plus angiotensin-converting enzyme inhibitors ■ Conventional therapy plus nitrovasodilators ■ Conventional therapy plus angiotensin-converting enzyme inhibitors and nitrovasodilators ■ Nitrovasodilators and/or angiotensin-converting enzyme inhibitors without conventional therapy.

Conclusion “The nitrates have not been included in most of the trials carried out in recent years, not because they have been found to be ineffective in the syndrome, but because they are generic drugs without adequate profit margin.”44 “Because nitrates are very old drugs and are no longer patented, it is unlikely that the pharmaceutical industry will be motivated to fund large placebo-controlled trials of nitrate therapy.”45 Endogenous and exogenous nitrovasodilators have been studied in detail, and there is little doubt of the efficacy of nitrovasodilators in treating cases of congestive heart failure.46 The issues that remain to be solved are the best method of use and the extent of nitric oxide toxicity under certain conditions.

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28. Miwa K, Toda N: The regional differences of relaxations induced by various vasodilators in isolated dog coronary and mesenteric arteries. Jpn J Pharmacol 38:313–330, 1985. 29. Ghio S, de Servi S, Perotti R, et al: Different susceptibility to the development of nitroglycerin tolerance in the arterial and venous circulation in humans: Effects of N-acetylcysteine administration. Circulation 86:798–802, 1992. 30. Toyoda J, Hisayama T, Takayanagi I: Nitro compounds (isosorbide dinitrate, 5-isosorbide mononitrate and glyceryl trinitrate) on the femoral vein and femoral artery. Gen Pharmacol 17:89–91, 1986. 31. Cohn JN: Role of nitrates in congestive heart failure. Am J Cardiol 60:39H–43H, 1984. 32. Schwarz M, Katz SD, Demopoulos L, et al: Enhancement of endothelium-dependent vasodilation by low-dose nitroglycerin in patients with congestive heart failure. Circulation 89:1609–1614, 1994. 33. Paulus WJ, Vantrimpont PJ,

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