1 BioCurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543; 3 Departments of Biology and Ophthalmology and Visual Science, University of Illinois at Chicago, Chicago, Illinois 60607; and 2 Environmental and Occupational Health Sciences Institute, Rutgers University and University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nitric oxide (NO) fluxes released from the surface of individual
activated macrophages or cells localized in small aggregates were
measured with a novel polarographic self-referencing microsensor. NO
fluxes could be detected at distances from the cells of 100-500 µm. The initial flux and the distance from the cells at which NO
could be detected were directly related to the number of cells in the
immediate vicinity of the probe releasing NO. Thus, whereas NO fluxes
of ~1 pmol · cm2 · s
1
were measured from individual macrophages, aggregates composed of
groups of cells varying in number from 18 to 48 cells produced NO
fluxes of between ~4 and 10 pmol · cm
2 · s
1. NO fluxes
required the presence of L-arginine. Signals were significantly reduced by the addition of hemoglobin and by
N-nitro-L-arginine methyl ester. NO fluxes were
greatest when the sensor was placed immediately adjacent to cell
membranes and declined as the distance from the cell increased. The NO
signal was markedly reduced in the presence of the protein albumin but
not by either oxidized or reduced glutathione. A reduction in the NO
signal was also noted after the addition of lipid micelles to the
culture medium. These results demonstrate that NO can be detected at
significant distances from the cell of origin. In addition, both
proteins and lipids strongly influence the net movement of free NO from macrophages. This suggests that these tissue components play an important role in regulating the biological activity of NO.
nitric oxide flux; nitric oxide synthase; self-referencing electrode
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN RESPONSE TO AN
ARRAY of inflammatory cytokines, most notably, interferon
(IFN)-, activated macrophages produce nitric oxide (NO), a highly
reactive nitrogen intermediate that plays a critical role in
nonspecific host defense, tumor cell cytostasis, and inflammation
(21). Excessive or inappropriate production of NO by
activated macrophages is also thought to be a key factor mediating
tissue injury associated with inflammation and some degenerative
diseases (7). To initiate biological effects, NO must
diffuse from the activated macrophage to adjacent cells and tissues.
Half-life measurements ranging up to several seconds have been reported
for NO in aqueous media, suggesting that this free radical may persist
for sufficient time to diffuse to and interact with sensitive targets
(7, 10, 21). However, direct quantitation of the distances
NO diffuses away from macrophages has not been determined.
To predict biological activity in tissues, previous investigators
(12, 13) have developed mathematical models based largely
on the rapid rate of diffusion of NO and its chemical reactivity.
However, in tissues, NO can react with cellular antioxidants and
oxidants as well as with other free radicals, which may alter its
diffusion pattern and biological activity (10, 21).
Certain oxidation products of NO retain NO-like biological activity;
some, such as peroxynitrite, are highly cytotoxic, whereas others,
including nitrate and nitrite salts, may be slower to initiate
biological effects (10, 21). In addition, the composition
of proteins such as hemoglobin, unsaturated lipids, and other
sulfhydryl, iron, or ion-sulfur moieties within the inflammatory
microenvironment surrounding the macrophage may modify NO activity.
The critical parameters required for evaluating the beneficial and deleterious effects of NO released from macrophages include the direct quantification of the amounts of NO released from the cell, the distances over which it persists, and the length of time the macrophages continue to release this free radical. In the present studies, we describe a novel self-referencing NO-selective probe system that we have used to monitor NO release from cultured macrophages. The system allows real-time measurements of NO flux and exhibits sufficient sensitivity to detect NO release from isolated individual cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells and treatments.
RAW 264.7 macrophages (24) were plated at varying
densities (0.3-0.5 × 106 cells) onto
polylysine-coated, glass-bottom 35-mm petri dishes (MatTek,
Ashland, MA) in phenol red-free DMEM with 10% fetal calf serum. At low
density, cells grow as single isolated cells, whereas at higher
densities, cells grow as a confluent monolayer and in small
distinct aggregates. The aggregates ranged in cell number from
~10 to 75 cells growing in random arrays, with cells overlapping as
well as adjacent to one another. The number of cells in an aggregate
was determined with fluorescence microscopy after fixation and staining
with propidium iodide (5). To stimulate NO production, cells were treated with mouse IFN- (100 U/ml; kindly provided by
Sidney Pestka, University of Medicine and Dentistry of New Jersey, Piscataway, NJ) in serum-free medium. BSA,
N-nitro-L-arginine methyl ester
(L-NAME), and liposomes (Sigma, St. Louis, MO) were added
directly to the culture medium. Recordings were suspended during the
10-15 s required to apply the drugs. Time was recorded continuously during the addition of reagents. Liposomes were prepared from kits according to the manufacturer's directions. Negatively charged liposomes were prepared from egg yolk
L-
-phosphatidylcholine, dicetyl phosphate, and
sterylamine in a molar ratio of 7:2:1. Positively charged liposomes
were prepared from egg yolk
L-
-phosphatidylcholine, sterylamine, and cholesterol in
a molar ratio of 7:2:1. Neutral liposomes were prepared from
L-
-phosphatidylcholine,
-oleoyl-
-palmitoyl, and
cholesterol in a molar ratio of 1.5:1. Nitrite assays,
immunofluorescence, and Western blotting were performed as previously
described by our laboratories (7, 9). In experiments
examining the arginine dependence of the NO signal, cells were cultured
for 1-4 h in a modified Hanks' balanced salt solution that
consisted of (in mM) 118 NaCl, 5.33 KCl, 1.26 CaCl2, 0.5 MgCl2, 0.4 MgSO4, 5.6 glucose, and 25 Na-HEPES;
the pH of the solution was adjusted to 7.3 with NaOH.
Preparation of the NO microelectrode.
A basic carbon fiber electrode was modified with
o-phenylenediamine (OPD) and Nafion; a schematic drawing of
the electrode is shown in Fig.
1A. The electrode was
constructed by modifying and combining two previously described
protocols (2, 6). Briefly, this involved pulling a 5-µm
carbon fiber (Amoco, Greenville, SC) into a glass microcapillary tube
with a Brown-Flaming P-97 micropipette puller. When the glass was
heated and pulled, it was drawn down to make contact with the fiber.
The fiber was stabilized and sealed in the pulled-glass pipette with
Epoxylite (Epoxylite, Westerville, OH). After being cured (110°C for
5-10 h), the electrode was backfilled with a graphite-epoxy paste
(PX-grade Graphpoxy, Dylon Industries, Cleveland, OH). A copper wire
was inserted into the probe to make electrical contact with the carbon
fiber through the graphite-epoxy paste. The probe was then cured, and
excess carbon fiber was trimmed with a scalpel and beveled to 30°C.
|
Calibration and flux measurements in an artificial NO gradient. The NO electrodes were calibrated with a standard 2 mM NO solution that was prepared by bubbling HEPES solution with argon for 30 min and then with compressed NO gas (Praxair, Danbury, CT) for 45 min in a fume hood. The saturation concentration for NO in an aqueous buffer solution at 22°C is 2 mM (6). Although the sensitivity of the NO electrodes for sodium nitrite was 1.43 ± 0.16 pA/mM (n = 6 measurements), the sensitivity for NO was 0.73 ± 0.11 pA/µM (n = 6 measurements). Therefore, the NO electrodes were ~510 times more sensitive to NO than nitrite. Before and after use, the electrode was calibrated against known concentrations of NO and tested for specificity against ascorbic acid at 37°C (6). Figure 1B confirms that the signal from the electrode increased linearly with increasing concentrations of NO regardless of the electrode sensitivity.
A problem commonly encountered with NO and other polarographic-style electrodes is an inherent and random drift of the electrical output with time. This drift greatly limits the useful sensitivity of the electrode. The key to significantly improving both the stability and sensitivity of these electrodes is to use them in a self-referencing format (27). In this method, the electrode is moved alternately from a position close to the membrane of a cell to a measured distance away in the medium but within the gradient. By subtracting signals from the two different points, a differential current can be obtained and converted to a directional measurement of the flux of NO. An important assumption underlying this method is that the translational movement of the electrode is fast relative to the rate of drift but not fast enough to mechanically disturb the diffusional gradient of NO by mixing. When these conditions are met, the self-referencing format permits interference caused by random drift and noise to be effectively filtered out of the signal, rendering a current resolution of the electrode on the order of several femptoamperes. This is at least two orders of magnitude more sensitive than typical applications of NO-selective electrodes. The validity of the NO electrode for a gradient measurement was tested by measuring the release of NO from S-nitroso-N-acetylpenicillamine (SNAP) in a source micropipette (11, 14, 25). The micropipette (diameter 10 µm) was filled with a 0.5% agar solution containing 10.0 mM SNAP in phosphate buffer (pH 7.4). The diffusional profile for NO release from the source pipette was determined with the electrode in a normal static (i.e., nonoscillating) mode (Fig. 1C). The NO gradient from the artificial source showed an exponential decay over distance. We developed a model of the NO gradient to compare with the measured gradient. Assuming that NO is stable and spherically diffuses, the steady-state concentration gradient around a finite spherical source of radius Ro with the boundary conditions C = Co at r = Ro and C = 0 at r = infinity is
![]() |
(1) |
![]() |
(2) |
![]() |
(3) |
![]() |
(4) |
![]() |
(5) |
![]() |
(6) |
Self-referencing electrode system. Culture dishes with adherent cells were placed on a Zeiss inverted microscope fitted with a stage plate on which the head stage and translational motion control system were mounted. The latter consisted of Newport 310 series translation stages arranged in an orthogonal array and driven by size 23 stepper motors. This arrangement provided nanometer resolution of movement of the head stage in either an oscillating (square wave at 0.3 Hz) or static mode (27). Control over movement was achieved by computer with IonView software. Software, motion controllers, and amplifiers were obtained from the BioCurrents Research Center (Woods Hole, MA). The entire assembly was mounted on an antivibration table and housed in a Faraday box equipped with a temperature control system; in this study, all data with cells were obtained at 37°C. Data were collected at a rate of 1,000 events/s and signal-averaged to 10 values consisting of 166 data points/position. Values obtained over the period of electrode movement were excluded (first third discarded). Unless otherwise specified, data are presented as means ± SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In initial studies, we characterized NO production and the
expression of NO synthase (NOS) in RAW 264.7 macrophages. NO production by the cells was quantified by measuring the accumulated levels of
nitrite and nitrate in the culture medium (9). As
expected, these stable salts, formed by the oxidation of NO, were only
observed in cultures treated with IFN- (100 U/ml for 24 h; Fig.
2A). Accumulation of nitrite
and nitrate increased continually with time and was inhibited by the
NOS inhibitor L-NAME. Western blot analysis demonstrated that NO production in response to IFN-
was correlated with
expression of the inducible isoform of the enzyme (NOS2; Fig.
2B). L-NAME had no effect on the expression of
NOS2. These findings were confirmed by immunofluorescent staining of
the cells with antibodies specific for NOS2. In addition, we found that
NOS2 expression was restricted to the cytoplasm of individual cells.
Moreover, the enzyme was expressed homogeneously within the cell
population (Fig. 2, C-F).
|
Figure 3A illustrates an
individual macrophage with a NO-selective electrode placed directly
above the cell. In our experiments, the probe was initially placed as
close as possible to the cell membrane without physically touching it
(~1 µm). Maximal NO flux was then determined. The probe was then
oscillated in a vertical plane. A typical recording obtained from an
IFN--stimulated macrophage is shown in Fig. 3B. When the
electrode was placed close to the cell membrane and then oscillated a
distance of 10 µm at a rate of 0.3 Hz, a differential current could
be detected, corresponding to a NO flux of ~1
pmol · cm
2 · s
1. Assuming
the half-life of NO is 1 min in the given situation and with the use of
Einstein's equation [l = (2Dt)1/2, where l is the travel
distance], it takes 0.015 s for NO to travel 10 µm. During this
period, only 0.02% NO will turn into nitrite. Therefore, given the
preferential selectivity of the electrode for NO (see MATERIALS
AND METHODS), the contribution from the conversion of NO to
nitrite will be negligible in the flux measurements. Placing the
electrode at a position ~1 mm away from the cell (background) and
oscillating over the same 10 µm resulted in complete loss of the
signal. Returning the electrode to the cell membrane restored the
differential signal. Significantly, the addition of 5 mg/ml of
oxyhemoglobin (Hb), a NO scavenger, to the dish when the electrode was
next to the cell eliminated the signal. Replacement of the
Hb-containing medium with fresh medium restored the measurable flux of
NO. In three similar experiments, we obtained signals from isolated
single cells ranging from 0.8 to 2.1 pmol · cm
2 · s
1. No signals
were evident in cells that were not treated with IFN-
(data not
shown).
|
With the self-referencing probe, NO fluxes were also measured from the
surface of small aggregates of macrophages (see Fig. 3C for
representative example). In aggregates, NO flux measurements ranged
from 4 to 10 pmol · cm2 · s
1. Greater
fluxes of NO were measured from macrophage aggregates compared with
those in individual cells, which most likely represents the
contribution of several surrounding cells to the flux measurements. Figure 3D illustrates recordings from a representative
aggregate that displayed an initial NO flux of ~5
pmol · cm
2 · s
1. As
described in individual cells, in aggregates, the NO signal remained
stable until the addition of Hb to the cultures and returned to initial
levels when the Hb-containing medium was replaced with fresh medium. In
five measurements from macrophages in distinct aggregates, NO flux was
reduced by ~94% by Hb (6.4 ± 0.3 to 0.43 ± 0.43 pmol · cm
2 · s
1). NO fluxes
were also dependent on L-arginine. Only very small NO
fluxes were measured in cells maintained in Hanks' balanced salt
solution without L-arginine. The addition of
L-arginine to the culture medium (5 mM final concentration)
caused a marked increase in NO flux (Fig.
4A). Figure 4,
inset, shows the increase in NO flux with
L-arginine from five separate aggregates that had been
deprived of this amino acid for 4 h.
|
Our experiments measuring nitrate and nitrite levels indicated that the addition of L-NAME to the cultures readily inhibited production of NO. However, it was unclear just how rapidly this inhibitory effect could be observed (Fig. 2). With aggregates of macrophages, L-NAME (1 mM) was readily found to inhibit NO fluxes (Fig. 4B). Inhibition was ~96% within 4 min after addition of the inhibitor. When L-NAME was removed from the cultures, the NO flux returned to ~80% of initial levels within 2 h (Fig. 4B, inset).
We next characterized the spatial extent of the signal for NO from
macrophages to estimate its diffusion field from a cellular source. For
these studies, measurements were made from macrophages in cell
aggregates to approximate the distribution of activated macrophages in
tissues because larger signals could be obtained. Figure
5A shows the spatial profile
of NO flux for three different aggregates. Measurements were made by
placing the self-referencing probe at various distances from a
macrophage in an aggregate and then monitoring the differential signal
at each of these locations. The initial size of the signal and the
distance to which it extended from the cell appeared to depend on the
number of cells within the aggregate. Thus the NO signal from the
smallest aggregate, which contained ~15-20 cells, could be
detected at distances of up to ~100 µm (10 cell diameters), whereas
in the largest aggregate, which contained ~50-75 cells, the
signal could be detected at distances of up to ~400-500 µm
(40-50 cell diameters), although the contribution from the cells
in the remote vicinity may not be discounted. Our observations closely
resemble those measured from an artificial source as described in Fig.
1C.
|
Cellular sulfhydryls are thought to be an important sink for NO or its
oxidation products and may limit the extent of NO-mediated injury
(8). Figure 5B shows recordings from an
aggregate in the presence of increasing concentrations of reduced
glutathione (GSH). Surprisingly, only minimal changes were observed in
either the peak value of NO flux or the extent of its spatial
distribution. Similar results were noted with oxidized glutathione
(GSSG) and with a 1:1 mixture (1 and 5 mM, respectively) of GSSG and
GSH (data not shown). In separate experiments, the initial signal for
five aggregates was determined to be 6.2 ± 0.3 (SE)
pmol · cm2 · s
1 before the
addition of 5 mM GSH and 6.5 ± 0.5 pmol · cm
2 · s
1 after its addition.
In contrast, BSA produced an immediate decline in both the peak value
and the spatial extent of the NO signal (Fig. 5C). The decline depended on the concentration of BSA (20-60 µg/ml) and was reversed when BSA-containing medium was replaced with fresh medium.
In separate experiments, we determined that the initial signal for five
aggregates was 6.9 ± 0.9 pmol · cm2 · s
1 before the
addition of 60 µg/ml of BSA and 4.1 ± 0.4 pmol · cm
2 · s
1 after its
addition, a 41% decline in the signal. Similarly, a marked reduction
in signal was observed after the addition of phosphatidylcholine-containing liposomes to the culture medium (Fig.
5D). Again, the reduction correlated with the concentration of the added liposomes. These effects were independent of the charge on
the liposomes because generally similar effects were observed with
neutral and positively and negatively charged liposomes. Thus in four
measurements from macrophages in separate aggregates on a culture dish
with 1 mg/ml of liposomes, NO fluxes were reduced by ~53% for
neutral liposomes (5.8 ± 0.5 to 2.7 ± 0.2 pmol · cm
2 · s
1), 61% for
negatively charged liposomes (6.2 ± 0.4 to 2.4 ± 0.3 pmol · cm
2 · s
1), and 70%
for positively charged liposomes (5.6 ± 0.2 to 1.7 ± 0.2 pmol · cm
2 · s
1). In these
experiments, the same cell aggregates were used to measure the effects
of each type of liposome. Between experiments, the liposome-containing
medium was replaced by fresh medium and initial signals returned to
control levels. During experiments with either BSA or lipids,
significant reductions in the NO signals measured adjacent to the cells
were observed. Presumably, this reflects interactions between NO and
these moieties that occur between the cell surface and the position of
the NO probe. The BSA and lipids may also alter the diffusion
coefficient of NO or its solubility characteristics. These hypotheses
are strengthened by our observations that the NO signals returned to
levels similar to those initially measured after the removal of these substances.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our experiments demonstrated that NO microelectrodes used in a
self-referencing format were capable of real-time detection of NO
fluxes from individual macrophages and small aggregates of macrophages,
potentially reflecting cellular distributions likely to be found in
vivo. The NO signals were abolished by the NO scavenger Hb and the NOS
inhibitor L-NAME, effects that were readily reversible.
Moreover, the addition of the NOS substrate L-arginine to
cultures deprived of this crucial amino acid resulted in a marked
increase in the signal detected by the self-referencing electrode.
Fluxes of 0.8-2.1
pmol · cm2 · s
1 were
recorded from individual IFN-
-stimulated macrophages, whereas fluxes
of 5-10
pmol · cm
2 · s
1 were
recorded from aggregates of macrophages. Of particular interest was our
ability to use the NO sensor to obtain information on the spatial
distribution of NO released by macrophages. As expected, when NO flux
was measured from macrophage aggregates, there was a direct
relationship between the number of cells comprising the aggregate and
the initial size of the NO signal as well as the distance from the
aggregate at which a signal could be detected. Thus for larger cell
aggregates (50-75 cells), initial NO flux reached 9-10
pmol · cm
2 · s
1, and the
signal for NO could be detected at distances of up to 400-500
µm, whereas for smaller aggregates (15-20 cells), the signal
reached ~5
pmol · cm
2 · s
1 and could
be detected at distances of up to ~100 µm. It should be noted that
a linear relationship between the number of cells analyzed and the
initial NO flux measurements was not observed. Although the microsensor
was placed adjacent to an individual cell in an aggregate, the net
signal recorded was a result of a combination of the NO fluxes from
several cells near the probe (2, 13). Thus it is likely
that different numbers of cells contributed to the signals in each
aggregate, and in the larger aggregates, more cells appeared to be in
the vicinity of the probe. In macrophages, this effect may be important
in generating sufficient quantities of NO for tumor cytostasis or
nonspecific host defense (21). In this regard, clustering
of macrophages in tissues after inflammation, tumor cell invasion, or
xenobiotic-induced tissue injury has been reported to be important in
mediating cytotoxicity (16, 17). It should be noted,
however, that the contribution of multiple cells to a signal measured
from a microsensor is a limiting factor in attempting to measure NO
production in individual cells in tissues (22).
A limited field of diffusion of NO in biological systems was originally proposed based on the short half-life of individual NO molecules (13). However, current models predict a larger field based on the rapid rate of NO diffusion (2, 13). In the present studies, we report that the spatial distribution of NO released from aggregates of macrophages more closely correlates with this latter model. Our data are consistent with the rapid diffusion rate of NO and suggest that once released from cells, significant amounts may migrate to distances in excess of 40-50 cell diameters. Thus, based on our experimental findings, it is apparent that substantial quantities of NO may act at a distance from the macrophage. Indeed, this may be a requirement for effective host defense against pathogens (1). However, it should be noted that during inflammation in sensitive tissues such as those of the central nervous system or the myocardium, the ability of NO to diffuse from cells into surrounding tissues may lead to significant inadvertent damage and tissue injury (4, 10, 21, 30). The pattern of diffusional spread of NO released by macrophages may also be important in cells expressing calcium-dependent isoforms of NOS (endothelial NOS and neuronal NOS). As previously suggested (2, 13), the distances over which NO can act may be sizable and will be in proportion to both the amount generated and the duration of NO released from these cells. For example, in the central nervous system, diffusional spread of NO by neurons can potentially affect a large number of nearby neurons in three-dimensional space (32). In this regard, using the self-referencing microsensor, we found that NO flux could be detected at distances >500 µm from aggregates of electrochemically stimulated neuronal NOS-containing neuroectodermal-derived tumor cells (31; unpublished observations).
Although diffusion of NO is rapid, within tissues, numerous components may interact with NO, limiting its biologically effective radius. Likely molecules include sulfhydryl moieties, cellular antioxidants, multivalent metal centers, proteins, and lipids (10, 21). The formation and decay of sulfhydryl-derived S-nitrosothiols such as S-nitroso-GSH and S-nitrososerum albumin have been postulated to play important roles in the storage and/or transport of NO (1, 26, 28, 29). GSH, a ubiquitous thiol-containing antioxidant, is a powerful cellular reducing agent believed to provide protection from an array of reactive species (23). Interestingly, we observed that the diffusion field of NO generated by macrophages was not altered by either GSSG or GSH. Earlier studies (1, 26) indicated that NO does not react directly with GSH to form S-nitroso-GSH but may generate GSH disulfide and nitroxyl anion. Based on our data, the latter reaction does not appear to be a significant factor limiting the diffusion of NO outside the cells. It should be noted, however, that S-nitroso-GSH may still be formed by the cells via the reaction of GSH with oxidized NO. Thus as suggested previously (23), it appears that GSH may exert its protective effects indirectly, perhaps by maintaining the reduced state of critical cellular sulfhydryls.
Significant S-nitrosoalbumin is found in human serum (28), and this protein may be an important sink for NO. The present studies demonstrate that albumin, when added directly to the culture medium, significantly reduced NO fluxes recorded from macrophages. This was presumably due to the reaction of NO with sulfhydryl residues in albumin close to the cell surface. Albumin is known to contain a cysteine residue (Cys34) with an unusually low negative log of the acidic dissociation constant, which may facilitate the reaction with NO (28). However, at the present time, we cannot exclude the possibility that albumin either nonspecifically sequesters NO (see below) or contains reactive ligands, including transition metals, which may catalyze its oxidation (3). The fact that the extracellular environment contains an array of proteins with the potential to react with and/or sequester NO may be a critical factor controlling the diffusional spread of NO. This may be especially important in wounding and inflammation during which plasma leakage of albumin may occur and large amounts of NO are synthesized (15).
NO is highly soluble in hydrophobic environments, and thus it may be sequestered in lipid-containing cellular structures such as plasma membranes and mitochondria as well as in hydrophobic pockets found within numerous proteins including albumin (3). We found that various artificial membranes prepared as liposomes also reduced peak signals and the diffusional spread of NO from the macrophages. The lipid-favored equilibria and the rate at which NO partitions into micelles imply that macrophage-derived NO may accumulate within cellular lipids and preferentially interact with target molecules in lipid environments. In this regard, Liu et al. (20) have shown that soy phospholipid vesicles, detergent micelles, and isolated membranes from rat hepatocytes readily accelerate the disappearance of NO from aqueous solutions. These authors demonstrated that the reaction of NO with O2 is rapid in a hydrophobic environment and suggest that this may be an important site for the formation of NO-derived reactive species as well as for the interaction of NO with lipid-soluble antioxidants. Within many tissues, lipid moieties or lipid coatings of tissue structures such as in ingrowths of subepicardial fat in the ventricles or lipid-rich myelin sheaths may act as conduits, specifically delivering high concentrations of lipid-soluble agents such as NO to sensitive targets. This may be important in regulating normal physiological functioning. However, pathological lipid accumulations and an excess of NO may lead either to damage of lipid-containing structures or to the delivery of NO to distal sites, potentially causing toxicity.
In summary, our results demonstrate that the self-referencing microsensor can be used to estimate the diffusional spread of NO from macrophages and, potentially, other cell types including neuronal cells and that materials commonly found in the extracellular environment, e.g., albumin and lipids, can readily modify this function. These results provide further support for the idea that NO has the potential to act at significant distances from its point of origin. It should be pointed out that a variety of additional components including Hb contained in red blood cells or metal binding serum proteins may also limit diffusion of NO in vivo (19), and these remain to be investigated.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank an anonymous reviewer for constructive guidance toward the completion of this investigation.
![]() |
FOOTNOTES |
---|
This research was funded in part by National Institute of Environmental Health Sciences (NIEHS) Grants P01-ES-06817 (to J. D. Laskin and D. E. Heck), NIEHS Grant ES-03647 (to J. D. Laskin), NIEHS Center of Excellence Grant ES-05022, National Eye Institute Grant E4-09411 (to R. P. Malchow), and Marine Biological Laboratory Grant P41RR01395 to P. J. S. Smith.
Present address of D. M. Porterfield: Dept. of Biological Sciences, Univ. of Missouri-Rolla, 105 Schrenk Hall, 1870 Miner Cir., Rolla, MO 65409.
Address for reprint requests and other correspondence: D. E. Heck, 170 Frelinghuysen Rd., Rutgers Univ., Piscataway, NJ 08854 (E-mail: heck{at}eohsi.rutgers.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 July 2000; accepted in final form 10 May 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Broillet, MC.
S-nitrosylation of proteins.
Cell Mol Life Sci
55:
1036-1042,
1999[ISI][Medline].
2.
Cahill, PS,
and
Wightman RM.
Simultaneous amperometric measurement of ascorbate and catecholamine secretion from individual bovine adrenal medullary cells.
Anal Chem
67:
2599-2605,
1995[ISI][Medline].
3.
Carter, DC,
and
Ho JX.
Structure of serum albumin.
Adv Protein Chem
45:
153-203,
1994[ISI][Medline].
4.
Chabrier, PE,
Demerle-Pallardy C,
and
Auguet M.
Nitric oxide synthases: targets for therapeutic strategies in neurological diseases.
Cell Mol Life Sci
55:
1029-1035,
1999[ISI][Medline].
5.
Faaland, CA,
Mermelstein FH,
Hayashi J,
and
Laskin JD.
Rapid uptake of tyrphostin into A431 human epidermoid cells is followed by delayed inhibition of epidermal growth factor (EGF)-stimulated EGF receptor tyrosine kinase activity.
Mol Cell Biol
11:
2697-2703,
1991[ISI][Medline].
6.
Friedemann, MN,
Robinson SW,
and
Gerhardt GA.
o-Phenylenediamine-modified carbon fiber electrodes for the detection of nitric oxide.
Anal Chem
68:
2621-2628,
1996[ISI][Medline].
7.
Gardner, CR,
Heck DE,
Yang CS,
Thomas PE,
Zhang XJ,
DeGeorge GL,
Laskin JD,
and
Laskin DL.
Role of nitric oxide in acetaminophen-induced hepatotoxicity in the rat.
Hepatology
27:
748-754,
1998[ISI][Medline].
8.
Griffith, OW,
and
Mulcahy RT.
The enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase.
Adv Enzymol Relat Areas Mol Biol
73:
209-267,
1999[ISI][Medline].
9.
Heck, DE,
Laskin DL,
Gardner CR,
and
Laskin JD.
Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing.
J Biol Chem
267:
21277-21280,
1992
10.
Hobbs, AJ,
Higgs A,
and
Moncada S.
Inhibition of nitric oxide synthase as a potential therapeutic target.
Annu Rev Pharmacol Toxicol
39:
191-220,
1999[ISI][Medline].
11.
Jansen, A,
Drazen J,
Osborne JA,
Brown R,
Loscalzo J,
and
Stamler JS.
The relaxant properties in guinea pig airways of S-nitrosothiols.
J Pharmacol Exp Ther
261:
154-160,
1992[Abstract].
12.
Lancaster, JR, Jr.
Simulation of the diffusion and reaction of endogenously produced nitric oxide.
Proc Natl Acad Sci USA
91:
8137-8141,
1994[Abstract].
13.
Lancaster, JR, Jr.
A tutorial on the diffusibility and reactivity of free nitric oxide.
Nitric Oxide
1:
18-30,
1997[ISI][Medline].
14.
Lander, HM,
Sehajpal P,
Levine DM,
and
Novogrodsky A.
Activation of human peripheral blood mononuclear cells by nitric oxide-generating compounds.
J Immunol
150:
1509-1516,
1993
15.
Laskin, DL,
Heck DE,
and
Laskin JD.
Role of inflammatory cytokines and nitric oxide in hepatic and pulmonary toxicity.
Toxicol Lett
102:
289-293,
1998.
16.
Laskin, DL,
and
Pendino KJ.
Macrophages and inflammatory mediators in tissue injury.
Annu Rev Pharmacol Toxicol
35:
655-677,
1995[ISI][Medline].
17.
Laskin, DL,
Pilaro AM,
and
Ji S.
Potential role of activated macrophages in acetaminophen hepatotoxicity. II. Mechanism of macrophage accumulation and activation.
Toxicol Appl Pharmacol
86:
216-226,
1986[ISI][Medline].
18.
Lide, DR.
Handbook of Chemistry and Physics (71st ed.). Boca Raton, FL: CRC, 1991, p. 6-151.
19.
Liu, X,
Miller MJ,
Joshi MS,
Sadowska-Krowicka H,
Clark DA,
and
Lancaster JR, Jr.
Diffusion-limited reaction of free nitric oxide with erythrocytes.
J Biol Chem
273:
18709-18713,
1998
20.
Liu, X,
Miller MJS,
Joshi MS,
Thomas DD,
and
Lancaster JR, Jr.
Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes.
Proc Natl Acad Sci USA
95:
2175-2179,
1998
21.
MacMicking, J,
Xie QW,
and
Nathan C.
Nitric oxide and macrophage function.
Annu Rev Immunol
15:
323-350,
1997[ISI][Medline].
22.
Malinski, T,
and
Taha Z.
Nitric oxide release measured in situ by a porphyrinic-based microsensor.
Nature
358:
676-678,
1992[ISI][Medline].
23.
Meister, A.
Glutathione biosynthesis and its inhibition.
Methods Enzymol
252:
26-30,
1995[ISI][Medline].
24.
Raschke, WC,
Baird S,
Ralph P,
and
Nakoinz I.
Functional macrophage cell lines transformed by Abelson leukemia virus.
Cell
15:
261-267,
1978[ISI][Medline].
25.
Shaffer, JE,
Han BJ,
Chern WH,
and
Lee FW.
Lack of tolerance to a 24-h infusion of S-nitroso-N-acetylpenicillamine (SNAP) in conscious rabbits.
J Pharmacol Exp Ther
260:
286-293,
1992[Abstract].
26.
Singh, RJ,
Hogg N,
Joseph J,
and
Kalyanaraman B.
Mechanism of nitric oxide release from S-nitrosothiols.
J Biol Chem
271:
18596-18603,
1996
27.
Smith, PJ,
Hammer K,
Porterfield DM,
Sanger RH,
and
Trimarchi JR.
Self-referencing, noninvasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux.
Microsc Res Tech
46:
398-417,
1999[ISI][Medline].
28.
Stamler, JS,
Jaraki O,
Osborne J,
Simon DI,
Keaney J,
Vita J,
Singel D,
Valeri CR,
and
Loscalzo J.
Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.
Proc Natl Acad Sci USA
89:
7674-7677,
1992[Abstract].
29.
Stamler, JS,
Simon DI,
Jaraki O,
Osborne JA,
Francis S,
Mullins M,
Singel D,
and
Loscalzo J.
S-nitrosylation of tissue-type plasminogen activator confers vasodilatory and antiplatelet properties on the enzyme.
Proc Natl Acad Sci USA
89:
8087-8091,
1992[Abstract].
30.
Wang, D,
Yang XP,
Liu YH,
Carretero OA,
and
LaPointe MC.
Reduction of myocardial infarct size by inhibition of inducible nitric oxide synthase.
Am J Hypertens
12:
174-182,
1999[ISI][Medline].
31.
Wolff, DJ,
and
Lubeskie A.
Aminoguanidine is an isoform-selective, mechanism-based inactivator of nitric oxide synthase.
Arch Biochem Biophys
316:
290-301,
1995[ISI][Medline].
32.
Wood, J,
and
Garthwaite J.
Models of the diffusional spread of nitric oxide: implications for neural nitric oxide signalling and its pharmacological properties.
Neuropharmacology
33:
1235-1244,
1999.