Nitric Oxide

Function, Formation and Therapeutic Potential

by Michael A. Marletta, Ph.D. and Kirk M. Maxey, M.D.

In 1985, fewer than ten research papers devoted to the biological effects of nitric oxide (•NO) appeared in the published literature. This number increased to over 500 by the year 1990, and continues upward at an exponential rate today.¹ The intense interest and rapid pace of nitric oxide research can be attributed to several factors. One is the significant therapeutic potential associated with the rational control of •NO synthesis. Another is the complexity of the biochemical pathway that cells have evolved for the synthesis of •NO. Finally, •NO plays important physiologic roles in more than one area, including the brain and nervous system, the cardiovascular and the immune systems, making it relevant to many biomedical disciplines.

Formation of Nitric Oxide

Biosynthetic •NO is derived from the amino acid arginine in an unusual oxidative reaction that consumes molecular oxygen and reducing equivalents in the form of NADPH. The products of the reaction are •NO, NADP+, and citrulline. The enzyme responsible for •NO synthesis is Nitric Oxide Synthase (NOS), a remarkable enzyme requiring FAD, FMN, Heme, Ca²⁺, calmodulin and (6R)-tetrahydro-L-biopterin (H₄B) as cofactors.² Three NOS isoenzymes have been characterized and the salient features are summarized in Figure 1. Structurally, all NOS isozymes are similar in that they consist of a carboxy-terminal reductase domain which binds the flavin cofactors. A Ca²⁺/calmodulin binding domain lies in the center, followed by an oxidase domain with optical properties similar to P450 enzymes, where binding of Heme, O₂, H₄B and arginine substrate all take place. (See schematic in Figure 2) The cytosolic, neuronal form is a constitutive enzyme in both central and peripheral neurons. Its activity is regulated by intracellular Ca²⁺. The endothelial form is also constitutive and calcium dependent, but differs from the neuronal form by its smaller size and close association with the cell membrane. Endothelial NOS has been shown to be both myristoylated and palmitoylated at the N-terminus. Several cell types including macrophages express a third isoform of NOS in response to cytokine or endotoxin activation. This inducible NOS binds Ca²⁺/calmodulin so tightly that at normal physiologic levels its activity is Ca²⁺-independent.

Nitric Oxide Function

Cellular signaling with nitric oxide involves the highly regulated synthesis of •NO by NOS, diffusion of •NO into an adjacent cell, and activation of the soluble isoform of guanylate cyclase (sGC) leading to the synthesis of the second messenger cGMP. When •NO is synthesized in the vascular endothelium it causes relaxation of the adjacent smooth muscle. This relaxation is mediated by the transient increase in smooth muscle cellular cGMP, presumably through activation of a cGMP-dependent kinase. A similar mode of action has been proposed for cell to cell signaling in the brain. Although there is no uniform agreement on the function of •NO in the brain, evidence supports a role in long term potentiation and memory formation. In addition, most non-adrenergic, non-cholinergic (NANC) neurons are •NO-activated via the same cGMP-dependent mechanism. NANC nerves subserving important roles in gastrointestinal motility and penile erection are among those functioning through •NO signals.³

The immune system seems to have harnessed the toxic properties of •NO to kill invading organisms, pathogens and tumor cells. It is difficult to determine the exact mechanism of •NO cytotoxicity, but there is increasing evidence that •NO interferes with iron homeostasis and key cellular hemoproteins. •NO is an avid ligand for iron, and the mechanism of activation of sGC involves •NO binding to a pentacoordinate ferrous heme that appears to be uniquely tuned to interact with •NO. The elevated •NO concentrations generated by activated immune cells results in widespread disruption of other biologically important iron proteins. •NO also reacts extremely rapidly with superoxide (O₂^-) to form peroxynitrite, a potent oxidant with the potential to disrupt protein structures through the nitration of protein tyrosine residues. In support of this role, nitro-tyrosine has been identified both immunohistochemically and analytically in the debris of tissue damaged by inflammation.

Therapeutic Potential

An excess of •NO is clearly implicated in several clinical disorders. Inhibition of NOS, in particular isoenzyme selective inhibition, has great potential therapeutic value. This is especially evident in endotoxic shock where levels of •NO vastly exceed normal physiologic levels. The clinically observed extreme hypotension in shock has been linked to elevated levels of •NO and has been reversed by NOS inhibition in both animal models of shock and in humans. Post-ischemic damage to CNS neurons may also be due to elevated •NO, perhaps resulting from Ca²⁺ elevation, NMDA receptor stimulation and the resulting activation of neuronal NOS. Inhibitors selective for the neuronal NOS, such as the bis-amidine in Figure 3, may find a role in the treatment of stroke. Most of the research on selective NOS inhibition has focused on the inducible NOS that is associated with inflammation. Symetrical isothioureas such as 1,3-PBIT (Figure 1) have nearly a 200-fold selectivity for the inducible enzyme, and may have potential for the treatment of a variety of inflammatory conditions such as rheumatoid arthritis and ulcerative colitis, in addition to the acute inflammatory indications such as shock.

Interestingly, a relative deficit of •NO also seems evident in a number of clinical conditions. Although the explicit lack of NOS or •NO is not easy to demonstrate, clear clinical improvement occurs upon treatment with •NO or •NO-releasing drugs. While strategies to increase NOS activity in vivo are possible, the use of •NO-releasing drugs represents a more expedient approach toward resolving clinical problems related to low •NO levels. By far the most common example is oral nitroglycerin, which acts to augment •NO in the coronary arteries and increases blood flow to ischemic myocardium. Newly developed •NO-releasing agents such as the NONOates offer the potential to fine-tune the release of •NO over several hours or in a burst of a few minutes as the clinical situation warrants. A more recent development is the addition of •NO at very low levels (0.2-20 ppm) to the inspired air of acutely ill patients with ARDS, pulmonary hypertension and neonatal respiratory prematurity. Since •NO is a gas, it enters only those parts of the lung that are ventilated, dilating selectively the vessels of those areas and improving ventilation/perfusion ratios. In addition, •NO reacts rapidly with heme in the pulmonary blood, leading to inactivation of its vasodilatory effect before it can reach the systemic circulation. Such a selective pulmonary vasodilator has long been sought for the treatment of isolated and idiopathic pulmonary hypertension.

The impact of the last decade's discoveries about the biochemical role of •NO will be evident in the research laboratory and the hospital setting well into the next century. Cayman Chemical is committed to providing a broad range of •NO-related products to assist scientists working in this field. These products include:

• NOS Inhibitors
• NO-releasing compounds
• NOS enzymes
• NOS cDNA probes
• Nitrate/nitrite assay kits for the measurement of •NO production
• Antibodies to NOS isoenzymes and guanylate cyclase
• NOS co-factors including (6R)-tetrahydro-L-biopterin

References

1. Knowles, R.G. and Moncada, S. Biochem J. 298, 249-258 (1994).
2. Marletta, M.A., TIBS Letters 14, 488-492 (1989).
3. Schuman, E.M. and Madison, D.V. Ann. Rev.

Adhesion Molecules 101

by Pamela L.G. Balthazor

Recent increases in the interest of cellular adhesion have paved the way for a deeper understanding of the process and its implications for the study of inflammation and disease states. In this area, many scientists continue to focus on the form and function of adhesion molecules. Dramatic advances are being made by today's researcher toward the identification, characterization and comprehension of the role of this complex family of proteins. In light of these developments, Cayman Chemical Company introduces its new line of enzyme immunoassays for the measurement of the cellular adhesion molecules sL-Selectin, sE-Selectin, sVCAM-1 and sICAM-1.

What is an Adhesion Molecule?

Adhesion molecule is the general term which describes a specific class of transmembrane glycoproteins. Among other functions, these glycoproteins regulate the emigration of leukocytes at sites of inflammation or into the lymphatic tissue. Three classes of adhesion molecules are known to exist: selectins, integrins and the immuno-globulin superfamily. Each class possesses unique structural elements and characteristics.¹ Reports indicate that biologically active soluble forms of these adhesions are often found in blood and tissue fluids. Presence of these inflammation markers in soluble form allows nearly effortless measurement by immunometric assay methods.²

Adhesion Molecule Families

Selectins are calcium dependent lectins which bind to carbohydrate ligands on PMNs.⁴ Three selectins have been identified to date. P-selectin (GMP140) is stored in platelet a-granules and endothelial cell Weibel-Palade bodies.³ Within minutes of activation by thrombin and other agonists, it is redistributed to the cell surface. L-selectin (leukocyte adhesion molecule-1, LAM-1, LECAM-1) is expressed constitutively on various circulating leukocytes. Interestingly, L-selectin is down regulated by cytokines and shed from the cell membrane surface into the circulation within minutes of activation.^12,13 Along with P-selectin, L-selectin mediates the initial interaction of leukocytes with endothelial cells. E-selectin (endothelial leukocyte adhesion molecule-1, ELAM-1,CD62E) is expressed on endothelial cells three to four hours after activation by cytokines such as interleukin-1(IL-1) and tumor necrosis factor (TNF) or bacterial endotoxins (lipo-polysaccharide). Activation is short lived as decay is obvious after twenty four hours. Interaction of these selectin molecules with their ligands leads to the characteristic "rolling" of the leukocytes on the endothelium.

A second class, the integrins, are heterodimeric molecules that mediate cell-cell and cell-substratum adhesion. These molecules are composed of two non-covalantly associated a and b subunits. Specific conformational subfamilies based on the association of one subunit exist within the integrin family including: VLA (b1 integrins), LEUCAM (b2 integrins), and cytoadhe-sion (b3 integrins). Although constitutively expressed, b1 and b2 integrins require cellular activation by chemotactic cytokines (MIP-1b and GM-CSF) or lipids (PAF) for function.^6,7

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), belong to the third class, the immunoglobulin superfamily. These proteins act primarily as ligands for the b1 and b2 integrins. Both are widely distributed with VCAM-1 found on the endothelium, dendritic cells, kidney epithelium and macrophages and ICAM-1 located on endothelial cells, monocytes, epithelial cells, B and T cells and dendritic cells. Expression of these molecules is regulated by the cytokines, although some basal expression of ICAM-1 is apparent. In contrast to selectins, molecules from the immunoglobulin superfamily are expressed for prolonged periods of time. ICAM-1 peak expression occurs at 12-24 hours in vitro while maximum VCAM-1 levels are found at 6-10 hours post-activation. Induction is maintained beyond 36 hours.² The integrin-ligand interactions (LFA-1-ICAM-1 and VLA-4-VCAM-1) immobilize and flatten the leukocyte to the endothelium. Extravasation then takes place via a still unknown mechanism.

Soluble Adhesion Molecules

Presence of biologically active soluble forms of L-selectin, E-selectin, VCAM-1 and ICAM-1 have been confirmed in vivo and in vitro. These circulating proteins are of a distinct molecular weight from the membrane bound form as shown in various immunoprecipitation studies. Shedding appears to occur after expression on the cell surface and cytokine activation. The precise mechanism of the release of these proteins is not yet known although the observed molecular weights of the soluble forms are consistent with predicted weights assuming proteolytic cleavage at the membrane.^2,8 Measurable levels of these soluble proteins are present in the circulation, bronchial lavage and synovial fluid of healthy male and female subjects. sL-Selectin quantities of mg/ml and ng/ml levels for sE-Selectin, sVCAM-1, and sICAM-1 have been reported in serum samples when analyzed by non-isotopic immunometric assays.^2,11

Implications of Shedding

Shedding of these bound adhesion molecules into the circulation may represent a method of modulating and controlling cellular adhesion. Based on the observation that soluble L-Selectin will, at high concentrations, inhibit binding of lymphocytes to endothelium, it is speculated that the loss of surface L-selectin is required for leukocyte attachment to occur.^9,10 Purified soluble ICAM-1 and soluble VCAM-1 also retain their adhesion activity and may therefore bind the appropriate integrin ligands preventing firm leukocyte adhesion to the en-dothelial surface and extravasation. Further-more, recombinant soluble E-selectin has been shown to stimulate neutrophil chemotaxis and regulate integrin affinity. Another potential function of soluble adhesion molecules may be signal transmission and transduction. Research continues to probe these aspects of cellular adhesion in an effort to confirm these current hypotheses and explore other possible functions of these soluble proteins.⁸

Current Research Topics

Changes in the levels of soluble adhesion molecules have been studied in numerous chronic and acute inflammatory and immune disorders. High levels of soluble L-selectin were found to be present in patients with myeloid, lymphocytic and acute leukemia.^14,15 In contrast, reduced levels of soluble L-selectin have been tied to susceptibility of adult respiratory distress syndrome.¹⁶ Elevated levels of soluble E-Selectin were noted in studies of patients with septic shock, systemic lupus erythematosus, scleroderma, and other inflammatory vascular diseases.^17,18

Rhinoviruses are responsible for approximately fifty percent of common colds. Soluble ICAM-1 has been found to inhibit the binding of HRV54, a major group human rhinovirus strain, to cells. Antagonism of virus-receptor interactions may be a way of preventing rhinovirus infections.¹⁹ Circulating ICAM-1, E-Selectin and VCAM-1 have been measured in subjects possessing various malignancies. Both increases and decreases in the soluble forms of these molecules were reported, varying with the type of malignancy. For example, soluble ICAM-1 and VCAM-1 levels were elevated in myeloma patients while E-Selectin levels actually decreased.²⁰ Activation of the endothelium by cytokines has been shown to increase the adhesion of human melanoma and carcinoma cells in vitro. Tumor cell adhesion to the vessel wall may be the result of focal alterations in endothelial cells, involving the expression of specific cell surface molecules.²¹ Further studies are necessary to explore the diagnostic implications of these findings.

Summary

Cellular adhesion molecules appear to play a major role in the response to inflammation and associated pathological states. Further studies of the mechanistic actions of cellular adhesion molecules and their ligands will lead to enhanced understanding of these reactions and their implications. Measurement of sL-Selectin, sE-Selectin, sVCAM-1 and sICAM-1 via enzyme immunoassay will facilitate research and expedite progress in this area.

References

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5. Brandley, B.K, et al. Cell 63, 861-863 (1990).
6. McEver, R.P. Thrombosis and Haemostasis 65, 223-228 (1991).
7. Springer, T.A. Cell 76, 301 (1994).
8. Seth, R., et al. Lancet 338, 83-84 (1991).
9. Jutila, M.S., et al. J. of Immunol. 143, 3318-3324 (1989).
10. Kishimoto, T.K. et al. Science 245, 1238-1241 (1989).
11. Rothlein, R., et al. et al. J. of Immunol. 147, 3788-3793 (1991).
12. Griffin, J.D, et al. J. of Immunol. 145, 576-584. (1990).
13. Schleiffenbaum, B., et al. J. of Cell Biol. 119, 229-238 (1992).
14. Spertini, O., et al. Blood, 84, 1249-1256. (1994).
15. Zetterberg, E., et al. Eur. J. Haematol. 51, 113-119 (1993).
16. Marlin, S.D., et al. Nature 34, 70-74 (1990).
17. Newman, W., et al. J. of Immunol. 150, 644-654 (1993).
18. Carson, C.W., et al. J. of Rheumatol. 20, 809-814 (1993).
19. Donnelly, S.C., et al. Lancet 344, 215-219 (1994).
20. Banks, R.E., et al. Br. J. Cancer 68, 122-124 (1993).
21. Rice, G.E, et. al. Science 246, 1303-1305 (1989).