Proteins and lipids define the diffusional field of nitric oxide

D. Marshall Porterfield1, Jeffrey D. Laskin2, Sung-Kwon Jung1, Robert Paul Malchow3, Blase Billack2, Peter J. S. Smith1, and Diane E. Heck2

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

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 · cm-2 · 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN RESPONSE TO AN ARRAY of inflammatory cytokines, most notably, interferon (IFN)-gamma , 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
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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-gamma (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-alpha -phosphatidylcholine, dicetyl phosphate, and sterylamine in a molar ratio of 7:2:1. Positively charged liposomes were prepared from egg yolk L-alpha -phosphatidylcholine, sterylamine, and cholesterol in a molar ratio of 7:2:1. Neutral liposomes were prepared from L-alpha -phosphatidylcholine,beta -oleoyl-gamma -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.


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Fig. 1.   Characteristics and calibration of the nitric oxide (NO)-selective probe. A: schematic illustration of the probe used to measure NO flux. A 5-µm carbon fiber was inserted into a glass capillary tube and coated with Nafion and o-phenylenediamine to impart selectivity for NO. B: calibration of electrode output for different concentrations of NO when used in the static, nonoscillating mode. I, current. , Probe 1; , probe 2. Note linear dependence of the signal on NO concentration and the similarity between the 2 probes tested. C: NO concentration as a function of distance from a source pipette containing 10.0 mM NO donor S-nitroso-N-acetylpenicillamine in 0.5% agar. Electrode was held steady at a variety of measured distances from the source pipette. open circle , Output of the electrode; , modeled gradient based on the assumption that NO spherically diffuses from a sphere source (radius 5 µm) of constant NO production and disappears at a half-life of 1 min. D: flux measurements in an artificial NO gradient. Data points are flux values obtained at varying locations throughout the diffusion field with a stationary electrode (black-lozenge ) or an electrode oscillating as a square wave over 10 µm at a rate of 0.3 Hz (diamond ). All measurements were performed in HEPES solution at 37°C.

To impart selectivity for the oxidation of NO, the carbon fibers were treated with Nafion and OPD as first described by Friedemann et al. (6). Nafion is a polysulfonated Teflon that carries an intrinsic negative charge that repels electrochemically active anions (e.g., nitrate, nitrite, and ascorbate). OPD is an electrochemically active material thought to impart selectivity to NO by size exclusion of noncharged interferents such as electrochemically active catecholamines. The carbon fiber was first coated with Nafion (5% in aliphatic alcohols; Aldrich) and dried at 110°C for 5-10 min. After two additional Nafion coatings, the probe was plated with a 5 mM OPD plating solution containing 0.1 mM ascorbic acid in 100 mM PBS (pH 7.4). The OPD, prepared fresh for each use, was plated on the probe at a constant +0.9-V potential until a stable current with desirable noise characteristics was obtained. The modified carbon fiber electrodes had final tip diameters of 7-8 µm and were operated at +0.90 V vs. a Ag-AgCl return electrode that completed the circuit in solution via a 3 M KCl-5% agar bridge.

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
C(<IT>r</IT>)<IT>=</IT>C<SUB>o</SUB><IT>R</IT><SUB>o</SUB><IT>/r</IT> (1)
where r is the distance from the center of the spherical source, C is concentration, and Co is the concentration at Ro. Approximating that NO molecules disappear with a first-order rate constant of k, the fraction of NO molecules that remains after a given period of time is given by
<FR><NU>N<SUB>t</SUB></NU><DE>N<SUB>0</SUB></DE></FR>=exp(−<IT>kt</IT>) (2)
where N0 is the number of NO molecules at time 0 and Nt is the number of NO molecules after a period of time t. The travel distance of NO in time t is described by the Einstein equation as
r−R<SUB>o</SUB><IT>=</IT>(2<IT>Dt</IT>)<SUP>½</SUP> (3)
where r - Ro is the distance from a spherical source and D is the diffusion coefficient of NO (3.3 × 10-5 cm2/s at 37°C) (18). Incorporating Eq. 3 into Eq. 2 gives
<FR><NU>N<SUB>t</SUB></NU><DE>N<SUB>0</SUB></DE></FR>=exp[−(<IT>r−R</IT><SUB>o</SUB>)<SUP>2</SUP><IT>k/</IT>2<IT>D</IT>] (4)
Combining Eq. 4 with Eq. 1 gives
C(<IT>r</IT>)<IT>=</IT>(C<SUB>o</SUB><IT>R</IT><SUB>o</SUB><IT>/r</IT>) exp[−(<IT>r−R</IT><SUB>o</SUB>)<SUP>2</SUP><IT>k/</IT>2<IT>D</IT>] (5)
The first-order rate constant k is related to the half-life as t1/2 = (ln 2)/k. Assuming that t1/2 in aqueous medium under air is 1 min, k would be 0.012 s. Based on Eq. 5, we modeled a NO gradient from a sphere source with a radius of 5 µm (Fig. 1C). As shown in Fig. 1C, the measured values fit the modeled values. The modeled gradient allows an estimation of the strength of 10.0 mM SNAP. The approximate concentration of NO in SNAP was 17 µM.

To demonstrate the effectiveness of the self-referencing technique, the NO electrode was oscillated over a distance of 10 µm at a speed of 0.3 Hz. Flux values for NO are given by the Fick equation
J=−<IT>D </IT><FR><NU><IT>&Dgr;</IT>C</NU><DE><IT>&Dgr;x</IT></DE></FR> (6)
where J is the flux rate in moles per centimeter squared per second, Delta C is the concentration difference in moles between the two points of measurement, and Delta x is the distance of measurement in centimeters. The flux values (Fig. 1D) before compensation were 0.85 times the values measured in static mode (Fig. 1D). This disparity was a result of system response time and is constant between electrodes and thus can be compensated for. As shown in Fig. 1D, in static mode, it is, in practice, difficult to derive fluxes <15 pmol · cm-2 · s-1. On the other hand, in self-referencing mode, fluxes as low as 0.4 pmol · cm-2 · s-1 can be measured directly.

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.


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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-gamma (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-gamma 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).


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Fig. 2.   Expression of NO synthase in RAW 264.7 cells. A: NO production, measured by the accumulation of nitrite and nitrate in the culture medium, was quantified spectrophotometrically with the Griess reaction as indicated in MATERIALS AND METHODS. Note that interferon (IFN)-gamma (100 U/ml) stimulated NO production in cells (curve at top), whereas NO was not produced in control cells or in cells treated with N-nitro-L-arginine methyl ester (L-NAME; 1 mM) or L-NAME plus IFN-gamma (overlapping curves at bottom). B: Western blot showing inducible NO synthase (NOS2) in cultures treated with IFN-gamma (lane 3) and IFN-gamma plus L-NAME (lane 4) but not in control cultures (lane 1) or cultures treated with L-NAME alone (lane 2). C-F: immunofluorescence detection of NOS2 in control cells without (C) and with (E) L-NAME and in IFN-gamma -treated macrophages without (D) and with (F) L-NAME. Note the intense labeling of the cytoplasm, characteristic of NOS2 distribution in these cells. For Western blotting and immunofluorescence, cells were treated with IFN-gamma and/or L-NAME for 24 h.

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-gamma -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-gamma (data not shown).


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Fig. 3.   NO signals recorded from individual macrophages. A: phase-contrast photomicrograph of an isolated macrophage with a self-referencing electrode placed directly over the cell. Bright image in middle, tip of the electrode; blurred image at far right, body of the probe. Bar, 5 µm. B: recording from individual macrophage. At time 0, the electrode was placed within 1 µm of the cell surface, and a differential signal of ~1 pmol · cm-2 · s-1 was detected. During the background time, the electrode was raised ~1 mm above the cell. At 580 s into the recording, Hb (5 mg/ml final concentration) was added to the dish with a handheld micropipette (+Hb). Note that the signal was abolished and indistinguishable from the signal at background location. At 1,000 s, medium in the dish was replaced with fresh medium to remove the Hb (-Hb). C: phase-contrast photomicrograph of a cell aggregate with a self-referencing electrode placed directly over a cell. Bar, 10 µm. D: recording from a cell aggregate. At time 0, the electrode was placed within 1 µm of the surface of the cells, and a differential signal of ~4.2 pmol · cm-2 · s-1 was detected. Note that Hb reversibly inhibited the signal.

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 · cm-2 · 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.


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Fig. 4.   Effects of L-arginine and L-NAME on signals recorded from small cell aggregates. A: L-arginine stimulated NO flux. Cells were cultured for 4 h in arginine-free modified Hanks' balanced salt solution. At indicated time (arrow), L-arginine (1 mM final concentration) was added to the culture medium. Note that the addition of L-arginine to the culture medium resulted in a marked increase in the size of the signal. Inset, time-averaged values for NO flux from 5 separate cell aggregates (A-E) deprived of L-arginine (solid bars) and 5 min after addition of arginine (open bars). Values are means ± SD. B: L-NAME blocks NO flux. Note rapid decline of the signal after the addition of 1 mM L-NAME (arrow) to the cultures. Inset, responses from 5 cell aggregates (A-E) before (solid bars), during (open bars), and after (hatched bars) a 2-h recovery from the addition of 1 mM L-NAME. Values are means ± SD of time-averaged signals obtained under each condition; time of averaging varied from 1 to several minutes within each treatment group.

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.


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Fig. 5.   Spatial profiles delineating the extent of the NO signal. Data are time-averaged values of varying durations at different distances from each of the aggregates. A: recordings obtained from 3 separate cell aggregates. Smallest aggregate (group 3) was estimated to contain 15-20 cells; midsize aggregate (group 2), 35-40 cells; and the largest aggregate (group 1), 50-75 cells. Self-referencing electrode was oscillated at various distances from the membrane of the cells to determine the extent of NO flux. Values are means ± SD of signal strength of each recording. B: lack of effect of GSH on the spatial extent of NO flux. Recordings were made from an aggregate in the absence and presence of increasing concentrations of GSH. Note that there was no significant difference in either the peak amount or spatial spread of the signal in the presence of 1 and 5 mM GSH. C: decline in peak response and diffusional spread of NO signal after addition of BSA. D: reduction in peak value and diffusional spread of NO flux after the addition of positively charged liposomes.

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 · cm-2 · 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 · cm-2 · 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.


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REFERENCES

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 · cm-2 · s-1 were recorded from individual IFN-gamma -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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