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The study of biological nitric oxide
NEUROPROTOCOLS: A Companion Vol. 1, No. 2, October, pp. 99-107,
to Methods 1992
in Neurosciences
INTRODUCTION
The Study of Biological Nitric Oxide James N. Bates Department
of Anesthesia,
University
of Iowa
College
of Medicine,
Iowa
Prior to 1977 nitric oxide played no role in mammalian biology-or so went the conventional wisdom. Bacteria made and decomposed nitric oxide (NO) as part of the nitrogen cycle, and mammals were exposed to NO as an air pollutant, but there appeared to be nothing to suggest that mammals made NO or had any systems that recognized NO for intercellular communication. The conventional wisdom began to change in the mid 1970s when it was discovered that NO would activate soluble guanylate cyclase (1, 2). By that time it was widely believed that 3’: 5’-cyclic GMP was a major component of intracellular regulation and signaling, acting as a “second messenger” in a manner very similar to its more widely studied and better understood analog 3’:5’-cyclic AMP. Like cyclic AMP, cyclic GMP was present in virtually every cell looked at. The enzyme that made cyclic GMP from GTP, guanylate cyclase, existed in two isozymes, a soluble or cytoplasmic form and a particulate or membrane-bound form. One or both of these enzymes occurred in most cells (3). Unlike cellular systems that used cyclic AMP as a second messenger, the identity of the “first messenger” for cells using cyclic GMP was proving more elusive. Then it was discovered that a class of drugs, now known as nitrovasodilators, would activate the soluble isozyme of guanylate cyclase to increase its production of cyclic GMP and would do so by generating nitric oxide (2). Subsequently it was discovered that soluble guanylate cyclase contained a heme group, and the binding of NO to the heme induced activation of the enzyme (4-7). Nitric oxide liberation by nitrovasodilators provided a very useful tool for modulating cGMP levels and explained much of the pharmacology of some widely used cardiovascular drugs. The relevance of activation of guanylate cyclase by nitric oxide was unknown but it was regarded by some as a pharmacological curiosity that NO just happened to mimic some as yet unidentified biological modulator of the enzyme. Although NO had some interesting pharmacological properties, it was still not believed to have any role in normal mammalian biology. 1058~6741/92 $5.00 Copynght 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
City,
Iowa
52242
Simultaneously with the elucidation of the mechanism of nitrovasodilator actions, another major discovery in nitric oxide biology was developing. Furchgott and Zawadski (8) observed that endothelial cells generated an endothelium-derived relaxing factor (EDRF) that caused vascular smooth muscle to relax. Through the early 1980s much effort was spent trying to identify EDRF. It was recognized to be a very labile but potent compound that was released from endothelial cells and diffused to the vascular smooth muscle cells, where it caused relaxation. By 1986 it was recognized that EDRF was rapidly destroyed by superoxide or hemoglobin, and either directly or indirectly caused changes in the cGMP levels in vascular smooth muscle (9-13). It was proposed in 1986 that EDRF might in fact be NO (14, 15), and in 1987 it was demonstrated that effluent from endothelial cells stimulated to produce EDRF also contained NO or its oxidation product, inorganic nitrite (16). Although some physiological systems showed some dissimilarity between EDRF and free NO (17-22), and there has been debate about whether these dissimilarities represent true components of EDRF that are different from NO or whether they are artifacts of the experimental systems, it was widely agreed that EDRF was NO or an NO-releasing compound. The discoveries that EDRF is produced by the oxidation of the guanidino nitrogen of arginine (23-25) and that inhibition of this oxidation with structural analogs of arginine completely inhibits EDRF production (26, 27) essentially sealed t,he argument that EDRF was NO or a closely related nitrosyl compound that captured and then released NO. The biosynthesis of nitric oxide from L-arginine is summarized in Fig. 1. This reaction sequence is carried out by a single enzyme, nitric oxide synthase, which exists in several isozymes. The enzyme, which is related to cytochrome P450, oxidizes arginine by way of the shown proposed intermediates to citrulline and NO. The first intermediate, p-hydroxyL-arginine, has been prepared and demonstrated to be an 99
JAMES N. BATES
100
active substrate for nitric oxide synthase, and it has been detected among the products of arginine conversion by the enzyme (28). Two of the competitive inhibitors, Nomethyl-L-arginine and No-nitro-L-arginine, are shown below. They are analogs of arginine, or more correctly, analogs of Nc-hydroxy-L-arginine, that are not readily oxidized to citrulline and NO. EDRF could be considered the endogenous nitrovasodilator that like other nitrovasodilators generated or existed as NO which activated soluble guanylate cyclase. Figure 2 shows the nitric oxide-guanylate cyclase pathway in endothelial cells and vascular smooth muscle. This pathway was the earliest and best-described model for the role of NO in intercellular communication. The NOproducing cell, in this case an endothelial cell, responds to one or more hormones with an increased cytoplasmic calcium concentration. This, in turn, activates nitric oxide synthase to synthesize NO from arginine. In some other cells NO production occurs by a calcium-independent mechanism using a different isozyme of nitric oxide synthase. NO diffises to the target cell, in this case a vascular smooth muscle cell, where it binds soluble guanylate cyclase and activates production of cyclic GMP. In smooth muscle cells the rise in cyclic GMP alters the cascade involved in muscle contraction and leads to relaxation. Some cells may also contain other target enzymes for NO in addition to guanylate cyclase that act by pathways such
NH;
NH+ II * C-NHOH I NH NADPH,
~-NH* NH
NADPH,
H&i
O2
-HAli
$
NH2
C-NO
q=o
AH
O2
+A
+ F +Ny-CH-COO-
as the ADP-ribosylation of proteins (29-31). Some portion of the NO or its oxidized intermediates may be sequestered as nitrosyl compounds that can regenerate NO. Similarly, various nitrovasodilators, such as sodium nitroprusside, nitroglycerin, other organic nitrates, and nitrites, can decompose to generate NO that will act in the same manner as endothelium-derived nitric oxide. So, unlike the conventional wisdom, NO did have a biological role, and the nitrovasodilators were causing vasodilation by exogenously producing the same end product, NO, that endothelial cells delivered to the vascular smooth muscle. In the short span of 5 years the substrates and cofactors necessary for that production of NO have been largely determined, and the enzyme for its production, or at least several of the isozymes of nitric oxide synthase, has been purified and its cDNA cloned and sequenced (32). This in itself was a remarkable turn of events, but an examination of the facts already known suggested that NO might play an even wider role in cell communication. Cyclic GMP has long been widely suspected to be a second messenger in many cell types, and it could be found in almost all cell types. The soluble isozyme of guanylate cyclase is certainly present in very many cell types (3). The only known activator of soluble guanylate cyclase in physiological systems is nitric oxide (some other compounds, such as protoporphyrin IX can activate partially
7 NADPH,
AH
+Hh
+NO
HAi
H&i
HdH
&
HA
IAH +NH;dH-COO-
+N$H-COO-
+N$H-COO-
L-citrulline
L-arginine NH;
;“: C-NHCH,
~-NH-NO*
AH
AH
H&i
HAI
HdH I+&
H&l
+NH;dH-COO-
+Nt$H-COO-
NG-methyl-L-arginine FIG. 1. structures
4
Biosynthesis of nitric oxide from L-arginine. Proposed intermediates of two of the widely used competitive inhibitors of the biosynthesis.
HAI
NEnitro-L-arginine in the conversion
of arginine
to citrulline
are shown,
as are the
INTRODUCTION
__-
purified enzyme, but the physiological relevance of this process has not been demonstrated) (3). These facts would suggest that other systems in addition to the vasculature might use NO in intercellular communications, and this is now known to be the case. Demonstration of NO as an important component in many different types of cellular communication has proven to be quite easy. It is easy because there are several readily available analogs of arginine that are quite potent reversible inhibitors of NO synthase. The first of these to be found quite useful was L-p-methylarginine (LNMA or L-NMMA), where a methyl group is present on one of the two guanidino nitrogens of L-arginine. This compound binds well to the active site but is poorly converted to NO and therefore acts as a competitive inhibitor for arginine. fl-dimethyl-L-arginine (either with both methyl groups on the same nitrogen or with one methyl on each nitrogen), p-nitro-L-arginine (LNNA), the methyl ester of fl-nitro-L-arginine (L-NAME), fl-amino-L-arginine (LNAA), and several other related analogs have all been used to inhibit NO production. These compounds are generally quite easy to work with and are relatively specific for inhibition of nitric oxide synthesis. In principle, it is very easy to add these inhibitors to virtually any physiological preparation and observe whether the normal behavior of that system is altered by the inhibitors. If it is, and if the behavior can be returned to normal by an excess of L-arginine to overcome the competitive inhibition, then nitric oxide is implicated in the normal physiology of that
101
system. Proving that NO participates in the normal physiology of the system usually involves more complicated experiments such as the actual detection of NO or its decomposition products or the application of NO directly or through nitrovasodilators to elicit the normal response. Nonetheless, the simplicity of inhibitor studies and the apparent widespread occurrence of NO have led to a predictable flood of reports that show that NO is likely involved in a very large number of diverse physiological processes. Some of the systems that appear to use NO as a component in intercellular communication are shown in Table 1. This list is certainly incomplete and continually growing. Some entries, like the role as a second messenger to the glutamate receptor, could be appropriately broken down further. Nitric oxide has been repeatedly associated with the N-methyl-D-aspartate (NMDA) class of glutamate receptors, while in some, but not all, systems NO synthesis has been linked to stimulation of other types of glutamate receptors by kainate, quisqualate, and CYamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). The study and measurement of nitric oxide are complicated by the chemical properties of the molecule. Structurally, it is a small, diatomic, gaseous molecule. Recognition by an antibody is improbable because of the small size of the molecule. Likewise, the molecule cannot be “tagged” with a fluorescent dye or spin label, etc. The molecule itself is paramagnetic but is difficult to detect
c A23107
-b
r
ENDOTHELIUM 1
Acetvlcholine)
SMOOTH
MUSCLE
;ca*+ :z;: contractlon
L-Arginlne
Bradykin1n-W
GTP
Acetylchollne+
cGMP
Nltroprusside/ Nltroglycerln
FIG.
2.
The
nitric
oxide-guanylate
cyclase
pathway
in endothelial
smooth muscle
cell communication
with
vascular
smooth
muscle.
JAMES
102
directly by electron paramagnetic resonance spectroscopy. A useful (for most purposes) radioactive form of the molecule cannot be made because the only radioisotopes of nitrogen or oxygen have half-lives of 10 min or less. Nitric oxide cannot be extracted into a lipid phase and concentrated, but it can be readily extracted from solution into a gas phase because of its low solubility in water. This property has been exploited in some assays for NO, but in many physiological preparations it is difficult to perform such an extraction. NO is very susceptible to oxidation, but knowledge of the chemistry of its oxidation is incomplete. In the gas TABLE Some
Physiological
BATES
phase it reacts with oxygen to form NOp. At low concentrations such as below 10 ppm, it has a half-life in air at 25°C of several hours. At equivalent concentrations in water equilibrated with oxygen at 37°C it has a half-life of seconds or fractions of a second. Clearly, in aqueous solution there is a fundamental difference in the chemistry of oxidation of NO that is so far unexplained. In the gas phase the kinetics of oxidation follow thirdorder kinetics as would be expected from the reaction 2N0 + O2 --t 2N02.
PI
However, the actual reaction probably occurs by sequential second-order reactions such as
1
Responses That NO Production
N.
Appear
to Require
Endothelium-dependent vasodilation (16, 35, 36) Flow-mediated vasodilation (37) Hypoxic pulmonary vasoconstriction (38,39) Transition from fetal to adult circulation (40) Autoregulation of cerebral blood flow (41-45) Coronary blood flow (46-48) Hepatic blood flow (49-51) Renal blood flow (52) Renal papillary blood flow and sodium excretion (53) Renin release (54,55) Regulation of insulin production in islet cells (56) Neurotransmitter in the nonadrenergic, noncholinergic autonomic nervous system regulating of smooth muscle tone in the Esophagus (57,58) Stomach (59-62) Duodenum (63,64) Small bowel (65-69) Colon (70-72) Rectum and anus (72-75) Urethra and bladder (76-78) Bronchi (79-81) Vasculature to corpus cavernosum effecting penile erection (82-87) Gastric acid secretion (88) Inhibition of hepatocyte protein synthesis (89-91) Inhibition of platelet aggregation (92-95) Inhibition of leukocyte adhesion (96) Macrophage-mediated cytotoxicity of Mycobacterium tuberculosis (97,98) Leishmania major (99-105) Entamoeba histolytica (106) Trichmonas uaginulis (107) Toxoplasma go&ii (108, 109) Schistosomu mansoni (109) Francisella tukzrenais (110) cryptococcw neoformuns (111) Tumors (112,113) Lymphocyte-mediated cytotoxicity (114, 115) Lymphocyte proliferation (114, 116-119) Edema formation at sites of inflammation (120) Activation of splenocytes (121, 122) Neurotransmitter in the CNS implicated in Pain perception (123-126) Long-term depression (127-129) Long-term potentiation (130-134) Second messenger to the glutamate receptor (44, 127, 135-149)
2N0 + Nz02
PI
Nz02 + O2 + 2N02
[31
or more likely NO+02+N0, NO3 + NO + 2N02.
[41
PI
Reaction [5] has been studied and has fast kinetics (33, 34); thus, in the latter reaction sequence the slow overall rate at low NO concentrations would be due to very low concentrations of NOa. In solution the reaction sequence is even less clear. NOa might react with water or dissociate into ions (such as NO+ and 0; or HNO; and OH-) which could alter the reaction sequence and accelerate the overall oxidation. Similarly, NOB could be in a different isomeric form in the gas phase versus that in the aqueous phase. In one form it could be the nitrate radical, NOB, with each oxygen bonded to the nitrogen. The alternative structure ONOO, the peroxynitrite radical, is also possible and is the logical initial reaction product of NO + Oz. If the isomerization of ON00 and NOB was altered in solution so that the aqueous mixture was more reactive toward NO or dissociated back to NO and O2 more slowly, then the kinetics of NO oxidation at low concentrations would be faster. Knowledge of the chemical properties of nitric oxide is important because the study of nitric oxide and its role in neurobiology, or any aspect of physiology, will involve measuring and manipulating the concentration of NO. A variety of methods are available to accomplish this. The most straightforward of the manipulations, the inhibition of NO biosynthesis with analogs of arginine, is not discussed in this issue. Over 400 articles in the past 4 years have dealt with the use of No-monomethyl-L-arginine, Nc-nitro-L-arginine, or one of the other closely related compounds. These provide abundant examples of various
INTRODUCTION.~___
experimental designs that use arginine analogs to inhibit nitric oxide synthase. In this issue we concentrate on methods of measuring nitric oxide or nitric oxide synthase directly. The first of these methods is the original method used in the study of NO, even before it was recognized to be NO. “The Detection and Quantification of Nitrosyl Compounds Using Biological Assays” by Paul R. Myers and Janet L. Parker details the vascular ring preparation that is essentially the preparation in which EDRF was discovered and in which much of its characterization was described. Using vessel rings that have been denuded of endothelium and preconstricted with the appropriate vasoconstrictor, the system provides a profound and reproducible response to levels of nitric oxide that are physiological and are at or below the lower limit of sensitivity of many chemical assays. The major limitation of the assay is that it is not specific for NO, with other nitrovasodilators producing the same response and a variety of other vasodilators or vasoconstrictors potentially interfering with the assay. Another biological assay for NO uses rat fetal lung fibroblasts and involves the measurement of cyclic GMP accumulation in these cells after exposure to possible sources of NO. This technique is described in “Bioassay for EDRF/NO by Accumulation of Cyclic GMP in RFL6 Fetal Rat Lung Fibroblasts” by Timothy Warner, Ulrich Forstermann, Kunio Ishii, Jane A. Mitchell, Masaki Nakane, Jennifer S. Pollock, Harald H. H. W. Schmidt, Hong Sheng, and Ferid Murad. In principle, it has many of the same advantages and problems that a vascular ring assay has, such as the advantage of accurate measurements at the biologically relevant concentrations and the disadvantage of interference by other nitrovasodilators and other compounds. It differs from the vascular ring assay in that it is more easily adapted to large numbers of samples and small volumes of NO-donating sources. There are five articles on methods of direct chemical detection of NO or its oxidation products. “Spectrophotometric Detection of Nitrogen Oxides Using AZO Dyes” by W. Ross Tracey is a discussion of the reaction of nitric oxide oxidation products, especially nitrite, with aromatic amines to give azo dyes that can be measured spectrophotometrically. This is the oldest of the chemical detection methods for nitrite. It is easy to perform, can be automated so that measurement of a large number of samples is relatively easy, and uses equipment readily available to most laboratories. Its major limitation is that it has a lower limit of detection that is in the upper range of biologically relevant concentrations of nitrite. It is a very convenient assay for some biological applications where nitrite levels are relatively high, but some systems produce too little NO to be readily detected by this assay. “Spectrophotometric Detection of Nitric Oxide Using Hemoglobin” by E. Noack, D. Kubitzek, and G. Kojda
103
describes another spectrophotometric technique that is based on the reaction hemoglobin + NO + O2 + methemoglobin + NO,. This reaction is very fast, hemoglobin has a very high affinity for NO, and the high extinction coefficient of the difference spectrum between hemoglobin and methemoglobin allows measurement of NO down to concentrations less than 10m8M. It is a sensitive assay that requires careful control of temperature, pH, hemoglobin concentration, and protection from interfering substances like other heme proteins and redox reagents. Nonetheless, it does not require equipment more specialized than a difference spectrophotometer and represents probably the most readily available technique for chemical measurement of NO at physiological concentrations. Measurement of NO with a microelectrode is described in the article “Detection of Nitric Oxide by an Electrochemical Microprobe” by Katsuei Shibuki. The principle employed in this measurement is the same as that used in the measurement of oxygen in the Clark electrode. NO is detected by its electrochemical oxidation after it diffuses across a thin polymer membrane. The use of a microprobe offers the great advantage that the detector can be taken to the tissue and used in situ, rather than bringing the sample to the detector as in most of the other techniques. It requires sensitive electronics, has a tendency for baseline drift that complicates measurements made over longer time frames, and is subject to interference by other reducible gases such as carbon monoxide and hydrogen. These problems are largely offset by the great sensitivity and small size of the electrode, and because most of the problems are technical ones that might reasonably be improved with time and experience, this technique holds great promise. Probably the most sensitive of the available methods for detecting nitric oxide is based on the chemiluminescence of the reaction of nitric oxide with ozone as described in the article “Nitric Oxide Measurement by Chemiluminescence Detection” by James N. Bates. This is a gas-phase measurement that is highly specific for NO and can measure lower concentrations of NO than any of the other techniques available. It has the problem that even though it will measure lower concentrations of NO, it cannot measure total amounts of NO smaller than those measured with many of the other techniques, and with small samples it is often the total amount of NO rather than the concentration that limits the technique. Additionally, the NO must be extracted into the gas phase for measurement, which may introduce considerable difficulties in some preparations. Unlike any of the other techniques described above, the chemiluminescence technique is not subject to interference from compounds likely to be encountered in most biological experiments. The chemical detection technique that uses the most complex instrumentation is explained in “Quantification of Nitric Oxide in Biological Samples by Electron Spin
104
JAMES N. BATES
Resonance Spectroscopy” by Alexander Miilsch, Peter Mordvintcev, and Anatol Vanin. Nitric oxide is paramagnetic, and strongly paramagnetic complexes are formed with some iron compounds, including iron-containing proteins. When NO binds to heme proteins or iron-sulfur compounds (including some proteins), their electron spin resonance (ESR) signals can change appreciably. Various studies of the biological function of NO have involved ESR measurements of these Fe-NO complexes. In particular, the compound iron-diethyldithiocarbamate (Fe-DETQ,) binds NO tightly in a stable complex and gives a strong ESR signal that can be readily distinguished from Fe-DETC, without bound NO. All ESR measurements require the necessary equipment for electron spin resonance spectroscopy, which may not be readily available to some researchers, and the technique may be difficult for those without experience in ESR spectroscopy. In return one can measure quite small amounts of NO and often study the interactions of NO with cellular components with a sensitivity and specificity not readily attainable using other techniques. Because of the instability of NO in biological tissue, it is sometimes more useful to measure its production by means other than direct detection of NO. Since all NO in mammalian tissue seems to be derived from the conversion of arginine to citrulline, it is possible to measure the activity of nitric oxide synthase by following citrulline formation from arginine rather than NO formation from arginine. This method offers two major advantages: First, citrulline is metabolized or destroyed much more slowly than NO, so its isolation and measurement are much easier. The second advantage is that citrulline contains both carbon and nonionizing hydrogen, making it possible to use radiolabeled (14C or 3H) arginine and measure the appearance of radiolabeled citrulline. This gives the technique great sensitivity, and the presence of functional nitric oxide synthase activity can sometimes be detected even when NO itself cannot. Details of the technique are discussed in the article “Determination of Nitric Oxide Synthase Activity by Measurement of the Conversion of L-Arginine to L-Citrulline” by Appavoo Rengasamy and Roger Johns. Another technique for measuring nitric oxide synthase in neural tissue, one that is not discussed in this issue, is the localization of the enzyme nitric oxide synthase with antibodies. Multiple isozymes of nitric oxide synthase exist, and antibodies to one isozyme may not cross-react well with other isozymes, but with the appropriate antibodies the sites of potential NO synthesis can be determined. This is a measurement of potential NO production rather than actual NO production, but in trying to understand the physiologic role of NO, the localization of which cells are capable of producing it is very important. Now that several different isozymes of nitric oxide synthase have been genetically cloned and sequenced, and antibodies that are highly selective for nitric oxide syn-
thase are available, one can expect this to be an important tool. All of the techniques described in this issue have their limitations. With time, however, we can expect that some of these techniques will be improved and that new techniques will appear. What kinds of new techniques might we expect? Predicting the future is always a risky venture, but several developments seem to be only slightly beyond our present capabilities. Noticeably absent at present are techniques using antibodies or fluorescent dyes. A molecule that could penetrate cells without affecting their viability, bind NO with an affinity similar to that of guanylate cyclase, and become fluorescent only after binding NO would be ideal. Nitric oxide-containing cells would literally light up, making fluorescence microscopy a powerful analytical tool in the study of NO and its physiological function. No such molecule is known at present, but it might be composed of a heme-like or iron-sulfur binding site and a dye moiety that changes its absorption or emission spectrum or quantum yield when NO is bound. Another kind of fluorescent dye might be an analog to the azo dyes described in the article “Spectrophotometric Detection of Nitrogen Oxides Using Azo Dyes” by W. Ross Tracey, where NO or its oxidation products react with nonfluorescent amine or aniline precursors to form a fluorescent dye. Such a system would not likely be reversible and would be used in essentially the same kinds of experiments that the current azo dyes are, but the use of fluorescence rather than absorbance would likely add at least 2 or 3 orders of magnitude more sensitivity to the method. It seems unlikely that an antibody to NO could be developed, but it might be possible to develop an antibody that distinguishes between a complex that contains NO (such as NO-Fe-DETCz, where DETC is diethyldithiocarbamate) and the corresponding complex without NO (e.g., Fe-DETQ. With fluorescent antibodies of this type, fluorescence microscopy could be used in a manner similar to that described above. It might also allow the development of a radioimmunoassay for NO with the sensitivity inherent in such assays. As molecular biology becomes more utilitarian, perhaps an altered form of soluble guanylate cyclase that produces an analog of 3’:5’-cyclic GMP that is not hydrolyzed by phosphodiesterases can be formed. After application of the modified enzyme, the cyclic GMP analog would accumulate at rates proportional to the level of NO. Apart from these futuristic possibilities, how does one choose a method of studying NO? That, of course, is determined by the limitations of the system being studied, the requirements of the various measurement techniques, and the availability of the necessary equipment. It is our hope that this collection of techniques will give the reader information that is helpful in deciding the appropriate method of NO measurement in any given physiological
INTRODUCTION
system and the technical information the measurements.
needed to perform
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INTRODUCTION
The Study of Biological Nitric Oxide James N. Bates Department
of Anesthesia,
University
of Iowa
College
of Medicine,
Iowa
Prior to 1977 nitric oxide played no role in mammalian biology-or so went the conventional wisdom. Bacteria made and decomposed nitric oxide (NO) as part of the nitrogen cycle, and mammals were exposed to NO as an air pollutant, but there appeared to be nothing to suggest that mammals made NO or had any systems that recognized NO for intercellular communication. The conventional wisdom began to change in the mid 1970s when it was discovered that NO would activate soluble guanylate cyclase (1, 2). By that time it was widely believed that 3’: 5’-cyclic GMP was a major component of intracellular regulation and signaling, acting as a “second messenger” in a manner very similar to its more widely studied and better understood analog 3’:5’-cyclic AMP. Like cyclic AMP, cyclic GMP was present in virtually every cell looked at. The enzyme that made cyclic GMP from GTP, guanylate cyclase, existed in two isozymes, a soluble or cytoplasmic form and a particulate or membrane-bound form. One or both of these enzymes occurred in most cells (3). Unlike cellular systems that used cyclic AMP as a second messenger, the identity of the “first messenger” for cells using cyclic GMP was proving more elusive. Then it was discovered that a class of drugs, now known as nitrovasodilators, would activate the soluble isozyme of guanylate cyclase to increase its production of cyclic GMP and would do so by generating nitric oxide (2). Subsequently it was discovered that soluble guanylate cyclase contained a heme group, and the binding of NO to the heme induced activation of the enzyme (4-7). Nitric oxide liberation by nitrovasodilators provided a very useful tool for modulating cGMP levels and explained much of the pharmacology of some widely used cardiovascular drugs. The relevance of activation of guanylate cyclase by nitric oxide was unknown but it was regarded by some as a pharmacological curiosity that NO just happened to mimic some as yet unidentified biological modulator of the enzyme. Although NO had some interesting pharmacological properties, it was still not believed to have any role in normal mammalian biology. 1058~6741/92 $5.00 Copynght 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
City,
Iowa
52242
Simultaneously with the elucidation of the mechanism of nitrovasodilator actions, another major discovery in nitric oxide biology was developing. Furchgott and Zawadski (8) observed that endothelial cells generated an endothelium-derived relaxing factor (EDRF) that caused vascular smooth muscle to relax. Through the early 1980s much effort was spent trying to identify EDRF. It was recognized to be a very labile but potent compound that was released from endothelial cells and diffused to the vascular smooth muscle cells, where it caused relaxation. By 1986 it was recognized that EDRF was rapidly destroyed by superoxide or hemoglobin, and either directly or indirectly caused changes in the cGMP levels in vascular smooth muscle (9-13). It was proposed in 1986 that EDRF might in fact be NO (14, 15), and in 1987 it was demonstrated that effluent from endothelial cells stimulated to produce EDRF also contained NO or its oxidation product, inorganic nitrite (16). Although some physiological systems showed some dissimilarity between EDRF and free NO (17-22), and there has been debate about whether these dissimilarities represent true components of EDRF that are different from NO or whether they are artifacts of the experimental systems, it was widely agreed that EDRF was NO or an NO-releasing compound. The discoveries that EDRF is produced by the oxidation of the guanidino nitrogen of arginine (23-25) and that inhibition of this oxidation with structural analogs of arginine completely inhibits EDRF production (26, 27) essentially sealed t,he argument that EDRF was NO or a closely related nitrosyl compound that captured and then released NO. The biosynthesis of nitric oxide from L-arginine is summarized in Fig. 1. This reaction sequence is carried out by a single enzyme, nitric oxide synthase, which exists in several isozymes. The enzyme, which is related to cytochrome P450, oxidizes arginine by way of the shown proposed intermediates to citrulline and NO. The first intermediate, p-hydroxyL-arginine, has been prepared and demonstrated to be an 99
JAMES N. BATES
100
active substrate for nitric oxide synthase, and it has been detected among the products of arginine conversion by the enzyme (28). Two of the competitive inhibitors, Nomethyl-L-arginine and No-nitro-L-arginine, are shown below. They are analogs of arginine, or more correctly, analogs of Nc-hydroxy-L-arginine, that are not readily oxidized to citrulline and NO. EDRF could be considered the endogenous nitrovasodilator that like other nitrovasodilators generated or existed as NO which activated soluble guanylate cyclase. Figure 2 shows the nitric oxide-guanylate cyclase pathway in endothelial cells and vascular smooth muscle. This pathway was the earliest and best-described model for the role of NO in intercellular communication. The NOproducing cell, in this case an endothelial cell, responds to one or more hormones with an increased cytoplasmic calcium concentration. This, in turn, activates nitric oxide synthase to synthesize NO from arginine. In some other cells NO production occurs by a calcium-independent mechanism using a different isozyme of nitric oxide synthase. NO diffises to the target cell, in this case a vascular smooth muscle cell, where it binds soluble guanylate cyclase and activates production of cyclic GMP. In smooth muscle cells the rise in cyclic GMP alters the cascade involved in muscle contraction and leads to relaxation. Some cells may also contain other target enzymes for NO in addition to guanylate cyclase that act by pathways such
NH;
NH+ II * C-NHOH I NH NADPH,
~-NH* NH
NADPH,
H&i
O2
-HAli
$
NH2
C-NO
q=o
AH
O2
+A
+ F +Ny-CH-COO-
as the ADP-ribosylation of proteins (29-31). Some portion of the NO or its oxidized intermediates may be sequestered as nitrosyl compounds that can regenerate NO. Similarly, various nitrovasodilators, such as sodium nitroprusside, nitroglycerin, other organic nitrates, and nitrites, can decompose to generate NO that will act in the same manner as endothelium-derived nitric oxide. So, unlike the conventional wisdom, NO did have a biological role, and the nitrovasodilators were causing vasodilation by exogenously producing the same end product, NO, that endothelial cells delivered to the vascular smooth muscle. In the short span of 5 years the substrates and cofactors necessary for that production of NO have been largely determined, and the enzyme for its production, or at least several of the isozymes of nitric oxide synthase, has been purified and its cDNA cloned and sequenced (32). This in itself was a remarkable turn of events, but an examination of the facts already known suggested that NO might play an even wider role in cell communication. Cyclic GMP has long been widely suspected to be a second messenger in many cell types, and it could be found in almost all cell types. The soluble isozyme of guanylate cyclase is certainly present in very many cell types (3). The only known activator of soluble guanylate cyclase in physiological systems is nitric oxide (some other compounds, such as protoporphyrin IX can activate partially
7 NADPH,
AH
+Hh
+NO
HAi
H&i
HdH
&
HA
IAH +NH;dH-COO-
+N$H-COO-
+N$H-COO-
L-citrulline
L-arginine NH;
;“: C-NHCH,
~-NH-NO*
AH
AH
H&i
HAI
HdH I+&
H&l
+NH;dH-COO-
+Nt$H-COO-
NG-methyl-L-arginine FIG. 1. structures
4
Biosynthesis of nitric oxide from L-arginine. Proposed intermediates of two of the widely used competitive inhibitors of the biosynthesis.
HAI
NEnitro-L-arginine in the conversion
of arginine
to citrulline
are shown,
as are the
INTRODUCTION
__-
purified enzyme, but the physiological relevance of this process has not been demonstrated) (3). These facts would suggest that other systems in addition to the vasculature might use NO in intercellular communications, and this is now known to be the case. Demonstration of NO as an important component in many different types of cellular communication has proven to be quite easy. It is easy because there are several readily available analogs of arginine that are quite potent reversible inhibitors of NO synthase. The first of these to be found quite useful was L-p-methylarginine (LNMA or L-NMMA), where a methyl group is present on one of the two guanidino nitrogens of L-arginine. This compound binds well to the active site but is poorly converted to NO and therefore acts as a competitive inhibitor for arginine. fl-dimethyl-L-arginine (either with both methyl groups on the same nitrogen or with one methyl on each nitrogen), p-nitro-L-arginine (LNNA), the methyl ester of fl-nitro-L-arginine (L-NAME), fl-amino-L-arginine (LNAA), and several other related analogs have all been used to inhibit NO production. These compounds are generally quite easy to work with and are relatively specific for inhibition of nitric oxide synthesis. In principle, it is very easy to add these inhibitors to virtually any physiological preparation and observe whether the normal behavior of that system is altered by the inhibitors. If it is, and if the behavior can be returned to normal by an excess of L-arginine to overcome the competitive inhibition, then nitric oxide is implicated in the normal physiology of that
101
system. Proving that NO participates in the normal physiology of the system usually involves more complicated experiments such as the actual detection of NO or its decomposition products or the application of NO directly or through nitrovasodilators to elicit the normal response. Nonetheless, the simplicity of inhibitor studies and the apparent widespread occurrence of NO have led to a predictable flood of reports that show that NO is likely involved in a very large number of diverse physiological processes. Some of the systems that appear to use NO as a component in intercellular communication are shown in Table 1. This list is certainly incomplete and continually growing. Some entries, like the role as a second messenger to the glutamate receptor, could be appropriately broken down further. Nitric oxide has been repeatedly associated with the N-methyl-D-aspartate (NMDA) class of glutamate receptors, while in some, but not all, systems NO synthesis has been linked to stimulation of other types of glutamate receptors by kainate, quisqualate, and CYamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). The study and measurement of nitric oxide are complicated by the chemical properties of the molecule. Structurally, it is a small, diatomic, gaseous molecule. Recognition by an antibody is improbable because of the small size of the molecule. Likewise, the molecule cannot be “tagged” with a fluorescent dye or spin label, etc. The molecule itself is paramagnetic but is difficult to detect
c A23107
-b
r
ENDOTHELIUM 1
Acetvlcholine)
SMOOTH
MUSCLE
;ca*+ :z;: contractlon
L-Arginlne
Bradykin1n-W
GTP
Acetylchollne+
cGMP
Nltroprusside/ Nltroglycerln
FIG.
2.
The
nitric
oxide-guanylate
cyclase
pathway
in endothelial
smooth muscle
cell communication
with
vascular
smooth
muscle.
JAMES
102
directly by electron paramagnetic resonance spectroscopy. A useful (for most purposes) radioactive form of the molecule cannot be made because the only radioisotopes of nitrogen or oxygen have half-lives of 10 min or less. Nitric oxide cannot be extracted into a lipid phase and concentrated, but it can be readily extracted from solution into a gas phase because of its low solubility in water. This property has been exploited in some assays for NO, but in many physiological preparations it is difficult to perform such an extraction. NO is very susceptible to oxidation, but knowledge of the chemistry of its oxidation is incomplete. In the gas TABLE Some
Physiological
BATES
phase it reacts with oxygen to form NOp. At low concentrations such as below 10 ppm, it has a half-life in air at 25°C of several hours. At equivalent concentrations in water equilibrated with oxygen at 37°C it has a half-life of seconds or fractions of a second. Clearly, in aqueous solution there is a fundamental difference in the chemistry of oxidation of NO that is so far unexplained. In the gas phase the kinetics of oxidation follow thirdorder kinetics as would be expected from the reaction 2N0 + O2 --t 2N02.
PI
However, the actual reaction probably occurs by sequential second-order reactions such as
1
Responses That NO Production
N.
Appear
to Require
Endothelium-dependent vasodilation (16, 35, 36) Flow-mediated vasodilation (37) Hypoxic pulmonary vasoconstriction (38,39) Transition from fetal to adult circulation (40) Autoregulation of cerebral blood flow (41-45) Coronary blood flow (46-48) Hepatic blood flow (49-51) Renal blood flow (52) Renal papillary blood flow and sodium excretion (53) Renin release (54,55) Regulation of insulin production in islet cells (56) Neurotransmitter in the nonadrenergic, noncholinergic autonomic nervous system regulating of smooth muscle tone in the Esophagus (57,58) Stomach (59-62) Duodenum (63,64) Small bowel (65-69) Colon (70-72) Rectum and anus (72-75) Urethra and bladder (76-78) Bronchi (79-81) Vasculature to corpus cavernosum effecting penile erection (82-87) Gastric acid secretion (88) Inhibition of hepatocyte protein synthesis (89-91) Inhibition of platelet aggregation (92-95) Inhibition of leukocyte adhesion (96) Macrophage-mediated cytotoxicity of Mycobacterium tuberculosis (97,98) Leishmania major (99-105) Entamoeba histolytica (106) Trichmonas uaginulis (107) Toxoplasma go&ii (108, 109) Schistosomu mansoni (109) Francisella tukzrenais (110) cryptococcw neoformuns (111) Tumors (112,113) Lymphocyte-mediated cytotoxicity (114, 115) Lymphocyte proliferation (114, 116-119) Edema formation at sites of inflammation (120) Activation of splenocytes (121, 122) Neurotransmitter in the CNS implicated in Pain perception (123-126) Long-term depression (127-129) Long-term potentiation (130-134) Second messenger to the glutamate receptor (44, 127, 135-149)
2N0 + Nz02
PI
Nz02 + O2 + 2N02
[31
or more likely NO+02+N0, NO3 + NO + 2N02.
[41
PI
Reaction [5] has been studied and has fast kinetics (33, 34); thus, in the latter reaction sequence the slow overall rate at low NO concentrations would be due to very low concentrations of NOa. In solution the reaction sequence is even less clear. NOa might react with water or dissociate into ions (such as NO+ and 0; or HNO; and OH-) which could alter the reaction sequence and accelerate the overall oxidation. Similarly, NOB could be in a different isomeric form in the gas phase versus that in the aqueous phase. In one form it could be the nitrate radical, NOB, with each oxygen bonded to the nitrogen. The alternative structure ONOO, the peroxynitrite radical, is also possible and is the logical initial reaction product of NO + Oz. If the isomerization of ON00 and NOB was altered in solution so that the aqueous mixture was more reactive toward NO or dissociated back to NO and O2 more slowly, then the kinetics of NO oxidation at low concentrations would be faster. Knowledge of the chemical properties of nitric oxide is important because the study of nitric oxide and its role in neurobiology, or any aspect of physiology, will involve measuring and manipulating the concentration of NO. A variety of methods are available to accomplish this. The most straightforward of the manipulations, the inhibition of NO biosynthesis with analogs of arginine, is not discussed in this issue. Over 400 articles in the past 4 years have dealt with the use of No-monomethyl-L-arginine, Nc-nitro-L-arginine, or one of the other closely related compounds. These provide abundant examples of various
INTRODUCTION.~___
experimental designs that use arginine analogs to inhibit nitric oxide synthase. In this issue we concentrate on methods of measuring nitric oxide or nitric oxide synthase directly. The first of these methods is the original method used in the study of NO, even before it was recognized to be NO. “The Detection and Quantification of Nitrosyl Compounds Using Biological Assays” by Paul R. Myers and Janet L. Parker details the vascular ring preparation that is essentially the preparation in which EDRF was discovered and in which much of its characterization was described. Using vessel rings that have been denuded of endothelium and preconstricted with the appropriate vasoconstrictor, the system provides a profound and reproducible response to levels of nitric oxide that are physiological and are at or below the lower limit of sensitivity of many chemical assays. The major limitation of the assay is that it is not specific for NO, with other nitrovasodilators producing the same response and a variety of other vasodilators or vasoconstrictors potentially interfering with the assay. Another biological assay for NO uses rat fetal lung fibroblasts and involves the measurement of cyclic GMP accumulation in these cells after exposure to possible sources of NO. This technique is described in “Bioassay for EDRF/NO by Accumulation of Cyclic GMP in RFL6 Fetal Rat Lung Fibroblasts” by Timothy Warner, Ulrich Forstermann, Kunio Ishii, Jane A. Mitchell, Masaki Nakane, Jennifer S. Pollock, Harald H. H. W. Schmidt, Hong Sheng, and Ferid Murad. In principle, it has many of the same advantages and problems that a vascular ring assay has, such as the advantage of accurate measurements at the biologically relevant concentrations and the disadvantage of interference by other nitrovasodilators and other compounds. It differs from the vascular ring assay in that it is more easily adapted to large numbers of samples and small volumes of NO-donating sources. There are five articles on methods of direct chemical detection of NO or its oxidation products. “Spectrophotometric Detection of Nitrogen Oxides Using AZO Dyes” by W. Ross Tracey is a discussion of the reaction of nitric oxide oxidation products, especially nitrite, with aromatic amines to give azo dyes that can be measured spectrophotometrically. This is the oldest of the chemical detection methods for nitrite. It is easy to perform, can be automated so that measurement of a large number of samples is relatively easy, and uses equipment readily available to most laboratories. Its major limitation is that it has a lower limit of detection that is in the upper range of biologically relevant concentrations of nitrite. It is a very convenient assay for some biological applications where nitrite levels are relatively high, but some systems produce too little NO to be readily detected by this assay. “Spectrophotometric Detection of Nitric Oxide Using Hemoglobin” by E. Noack, D. Kubitzek, and G. Kojda
103
describes another spectrophotometric technique that is based on the reaction hemoglobin + NO + O2 + methemoglobin + NO,. This reaction is very fast, hemoglobin has a very high affinity for NO, and the high extinction coefficient of the difference spectrum between hemoglobin and methemoglobin allows measurement of NO down to concentrations less than 10m8M. It is a sensitive assay that requires careful control of temperature, pH, hemoglobin concentration, and protection from interfering substances like other heme proteins and redox reagents. Nonetheless, it does not require equipment more specialized than a difference spectrophotometer and represents probably the most readily available technique for chemical measurement of NO at physiological concentrations. Measurement of NO with a microelectrode is described in the article “Detection of Nitric Oxide by an Electrochemical Microprobe” by Katsuei Shibuki. The principle employed in this measurement is the same as that used in the measurement of oxygen in the Clark electrode. NO is detected by its electrochemical oxidation after it diffuses across a thin polymer membrane. The use of a microprobe offers the great advantage that the detector can be taken to the tissue and used in situ, rather than bringing the sample to the detector as in most of the other techniques. It requires sensitive electronics, has a tendency for baseline drift that complicates measurements made over longer time frames, and is subject to interference by other reducible gases such as carbon monoxide and hydrogen. These problems are largely offset by the great sensitivity and small size of the electrode, and because most of the problems are technical ones that might reasonably be improved with time and experience, this technique holds great promise. Probably the most sensitive of the available methods for detecting nitric oxide is based on the chemiluminescence of the reaction of nitric oxide with ozone as described in the article “Nitric Oxide Measurement by Chemiluminescence Detection” by James N. Bates. This is a gas-phase measurement that is highly specific for NO and can measure lower concentrations of NO than any of the other techniques available. It has the problem that even though it will measure lower concentrations of NO, it cannot measure total amounts of NO smaller than those measured with many of the other techniques, and with small samples it is often the total amount of NO rather than the concentration that limits the technique. Additionally, the NO must be extracted into the gas phase for measurement, which may introduce considerable difficulties in some preparations. Unlike any of the other techniques described above, the chemiluminescence technique is not subject to interference from compounds likely to be encountered in most biological experiments. The chemical detection technique that uses the most complex instrumentation is explained in “Quantification of Nitric Oxide in Biological Samples by Electron Spin
104
JAMES N. BATES
Resonance Spectroscopy” by Alexander Miilsch, Peter Mordvintcev, and Anatol Vanin. Nitric oxide is paramagnetic, and strongly paramagnetic complexes are formed with some iron compounds, including iron-containing proteins. When NO binds to heme proteins or iron-sulfur compounds (including some proteins), their electron spin resonance (ESR) signals can change appreciably. Various studies of the biological function of NO have involved ESR measurements of these Fe-NO complexes. In particular, the compound iron-diethyldithiocarbamate (Fe-DETQ,) binds NO tightly in a stable complex and gives a strong ESR signal that can be readily distinguished from Fe-DETC, without bound NO. All ESR measurements require the necessary equipment for electron spin resonance spectroscopy, which may not be readily available to some researchers, and the technique may be difficult for those without experience in ESR spectroscopy. In return one can measure quite small amounts of NO and often study the interactions of NO with cellular components with a sensitivity and specificity not readily attainable using other techniques. Because of the instability of NO in biological tissue, it is sometimes more useful to measure its production by means other than direct detection of NO. Since all NO in mammalian tissue seems to be derived from the conversion of arginine to citrulline, it is possible to measure the activity of nitric oxide synthase by following citrulline formation from arginine rather than NO formation from arginine. This method offers two major advantages: First, citrulline is metabolized or destroyed much more slowly than NO, so its isolation and measurement are much easier. The second advantage is that citrulline contains both carbon and nonionizing hydrogen, making it possible to use radiolabeled (14C or 3H) arginine and measure the appearance of radiolabeled citrulline. This gives the technique great sensitivity, and the presence of functional nitric oxide synthase activity can sometimes be detected even when NO itself cannot. Details of the technique are discussed in the article “Determination of Nitric Oxide Synthase Activity by Measurement of the Conversion of L-Arginine to L-Citrulline” by Appavoo Rengasamy and Roger Johns. Another technique for measuring nitric oxide synthase in neural tissue, one that is not discussed in this issue, is the localization of the enzyme nitric oxide synthase with antibodies. Multiple isozymes of nitric oxide synthase exist, and antibodies to one isozyme may not cross-react well with other isozymes, but with the appropriate antibodies the sites of potential NO synthesis can be determined. This is a measurement of potential NO production rather than actual NO production, but in trying to understand the physiologic role of NO, the localization of which cells are capable of producing it is very important. Now that several different isozymes of nitric oxide synthase have been genetically cloned and sequenced, and antibodies that are highly selective for nitric oxide syn-
thase are available, one can expect this to be an important tool. All of the techniques described in this issue have their limitations. With time, however, we can expect that some of these techniques will be improved and that new techniques will appear. What kinds of new techniques might we expect? Predicting the future is always a risky venture, but several developments seem to be only slightly beyond our present capabilities. Noticeably absent at present are techniques using antibodies or fluorescent dyes. A molecule that could penetrate cells without affecting their viability, bind NO with an affinity similar to that of guanylate cyclase, and become fluorescent only after binding NO would be ideal. Nitric oxide-containing cells would literally light up, making fluorescence microscopy a powerful analytical tool in the study of NO and its physiological function. No such molecule is known at present, but it might be composed of a heme-like or iron-sulfur binding site and a dye moiety that changes its absorption or emission spectrum or quantum yield when NO is bound. Another kind of fluorescent dye might be an analog to the azo dyes described in the article “Spectrophotometric Detection of Nitrogen Oxides Using Azo Dyes” by W. Ross Tracey, where NO or its oxidation products react with nonfluorescent amine or aniline precursors to form a fluorescent dye. Such a system would not likely be reversible and would be used in essentially the same kinds of experiments that the current azo dyes are, but the use of fluorescence rather than absorbance would likely add at least 2 or 3 orders of magnitude more sensitivity to the method. It seems unlikely that an antibody to NO could be developed, but it might be possible to develop an antibody that distinguishes between a complex that contains NO (such as NO-Fe-DETCz, where DETC is diethyldithiocarbamate) and the corresponding complex without NO (e.g., Fe-DETQ. With fluorescent antibodies of this type, fluorescence microscopy could be used in a manner similar to that described above. It might also allow the development of a radioimmunoassay for NO with the sensitivity inherent in such assays. As molecular biology becomes more utilitarian, perhaps an altered form of soluble guanylate cyclase that produces an analog of 3’:5’-cyclic GMP that is not hydrolyzed by phosphodiesterases can be formed. After application of the modified enzyme, the cyclic GMP analog would accumulate at rates proportional to the level of NO. Apart from these futuristic possibilities, how does one choose a method of studying NO? That, of course, is determined by the limitations of the system being studied, the requirements of the various measurement techniques, and the availability of the necessary equipment. It is our hope that this collection of techniques will give the reader information that is helpful in deciding the appropriate method of NO measurement in any given physiological
INTRODUCTION
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needed to perform
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