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Department of Physiology and Neuroscience, Lund University, S-223 62 Lund, Sweden
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The hypothesis that neuroepithelial endocrine (NEE) cells control spontaneous tone in isolated guinea pig tracheal preparations was examined. Epithelium-denuded preparations were unable to develop a normal oscillating tone in 12% oxygen (corresponding to systemic arterial oxygen levels) and, instead, developed a strong, smooth tone, similar to the "classic" tone in 94% oxygen. Inhibition of the hydrogen peroxide-producing NADPH oxidase in the NEE cells by 20 µM diphenyleneiodonium chloride transformed, in intact preparations in 94% oxygen, the tone from a strong, smooth type to an oscillating tone of considerably less force. Similar experiments in denuded preparations showed no change of tone and no oscillations. After pretreatment with the catalase inhibitor 3-amino-1,2,4-triazole (1 mM), addition of 2 mM hydrogen peroxide to intact preparations displaying the oscillating tone caused a transformation to a strong, smooth type. These findings support the hypothesis that the spontaneous tone in this preparation is largely controlled by the oxygen-sensing NEE cells. For the first time, previous findings on isolated cells can be linked to effects in intact tissue preparations. The results also suggest that the regulation by the NEE cells involves the release of powerful relaxing and contracting factors from the epithelium.
epithelium denudation; hydrogen peroxide; diphenyleneiodonium chloride; oscillating spontaneous tone; epithelium-derived relaxing factor
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IN A PREVIOUS STUDY (20), we described that the spontaneous tone in guinea pig in vitro tracheal preparations is highly dependent on the oxygen concentration in the superfusing solution. In 94% oxygen, the preparations display the "classic" type of relatively strong, irregular spontaneous tone. In 12% oxygen, corresponding to systemic arterial oxygen concentration, the preparations instead develop a highly stable tone with repetitive bursts of oscillations, which we have called a complex spontaneous tone.
During these examinations, we speculated that much of the oxygen-induced effects are due to an altered activity in the neuroepithelial endocrine (NEE) cells found throughout the airways (16, 17). These cells may be solitary or organized into innervated clusters, often referred to as neuroepithelial bodies. It has been suggested that also solitary NEE cells may be innervated (18), although this issue has not yet been fully clarified. The neuroendocrine cells have in several studies been found to be oxygen sensitive, and the oxygen-sensing mechanism has been proposed to comprise an NADPH oxidase that produces the oxygen free radical product H2O2 and a closely associated H2O2-sensitive K+ channel (25, 27, 28). A high oxygen concentration could in theory lead to an increase in the formation of H2O2, causing an activation of the K+ channels and a hyperpolarization of the neuroendocrine cells, thereby reducing the release of transmitters from them.
To further investigate whether and how the NEE cells participate in the generation of the spontaneous tracheal tone, we performed various experiments interfering with the NEE cell functions. One method was removal of the NEE cells by epithelium denudation. As an alternative procedure, changes in the concentration of H2O2, assumed to play a key role in the NEE cells' signal transduction, were achieved by pharmacological means.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparations
Male Dunkin Hartley guinea pigs (400-700 g, Harlan Winkelmann, Borchen, Germany) were anesthetized by thiopental (sodium salt) intraperitoneally (25 mg/kg) and killed by inhalation of nitrogen gas. Because there have been reports that acute hypoxia can deplete NEE cell transmitters (12), it was questioned whether the nitrogen exposure could influence the experimental behavior of the preparations. Therefore, three animals were, after thiopental treatment, killed by either a blow to the head or bleeding. Preparations from these animals developed an oscillating tone with regular complexes that appeared identical to the tone seen in nitrogen-exposed animals, which probably means that the standard method of killing in this study most likely does not result in persistent hypoxic artifacts. The lungs were quickly removed and placed in a 30-ml dissection chamber continuously perfused with 5 ml/min of a physiological saline solution (PSS; for composition see Solutions) at room temperature. The trachea was dissected free, and preparations with six cartilaginous rings each were obtained. The preparations were cut open along a line opposite the muscular portion of the trachea. Some preparations were gently rubbed with a cotton swab to remove the epithelium (see Histological Examinations). Such preparations will, henceforth, be referred to as denuded preparations, whereas preparation not subject to this treatment will be called intact preparations. One cut end of the preparation was tied to a small steel hook connected to a force transducer while the other end of the preparation was attached to a fixed hook. During the mounting procedure, great care was taken to avoid touching the epithelium in intact preparations. The preparations were then allowed to adjust in the experimental chamber, as described in Initiation of experiments.Experimental Chamber
The experimental chamber, with a volume of 5 ml, was continuously perfused with solutions at a rate of 3 ml/min. The inflow pipe of the chamber was, to prevent the appearance of unstirred layers, positioned near the preparations so that the solutions flowed directly toward them. The fast access of the solutions to the preparations was apparent from the fact that many tested substances gave a clear contraction within as soon as 15-30 s after the solutions had entered the chamber. The temperature was kept at 38.0 ± 0.1°C by means of an electronically controlled Peltier element. The pH in the experimental chamber was monitored continuously by a small pH electrode (model MI-710 combination pH electrode, Microelectrodes, Bedford, NH). The oxygen concentration in the chamber was measured by a small oxygen electrode (model MI-730 oxygen electrode, Microelectrodes). Mechanical measurements were performed by means of two separate force transducers (model AME 801, SensoNor, Horten, Norway) for simultaneous registration on two parallel preparations. Each force transducer was connected to a micrometer screw, with an accuracy of 10 µm. For signal recording, use was made of a CED Spike2 data-acquisition system (Cambridge Electronic Design, Cambridge, UK).Solutions
The PSS contained the following (in mM): 117 NaCl, 4.87 KCl, 1.20 MgSO4, 21.4 NaHCO3, 1.20 CaCl2, and 5.29 glucose. The standard PSS was bubbled with 12% oxygen (corresponding to a systemic arterial oxygen pressure of 95 Torr) and 6% carbon dioxide in nitrogen, giving a pH of 7.40 ± 0.05 and 12 ± 2% O2 in the experimental chamber. Because it was concluded in the end of this study that the NEE cells control the oxygen-induced changes of the spontaneous tone, the question was raised whether it perhaps would be more appropriate to use a slightly higher oxygen concentration to reflect the situation in the tracheal lumen. Theoretically, the correct amount would probably be an average of the inspired humidified air with an oxygen pressure of 149 Torr and the expired air with an oxygen pressure of 120 Torr, which is 135 Torr, corresponding to 18% oxygen. However, experiments showed that a change from 12 to 18% oxygen caused small effects. One preparation exhibited a more irregular complex tone in 18% oxygen, whereas four other preparations did not display any clear change of the complex tone. Therefore, 12% oxygen can perhaps be considered a little better for experiments in which it is desirable to have a spontaneous tone that is as regular as possible. In some experiments, where indicated, 25, 40, 60, and 94% oxygen with 6% carbon dioxide in nitrogen gas was used instead. Also, for the experiments with differing carbon dioxide concentrations, the PSS was bubbled with 12% oxygen and 4% carbon dioxide, or with 12% oxygen and 8% carbon dioxide, in nitrogen gas. The pH was, during these experiments, maintained at 7.40 by titration with HCl or NaHCO3. During experiments with differing pH, the correct pH was obtained by addition of either HCl or NaHCO3. Capsaicin and indomethacin were prepared daily as stock solutions dissolved in ethanol. Control experiments with vehicle in equivalent concentrations had in a previous study (20) been found to be without effect on the complex spontaneous tone. Diphenylene-iodonium chloride (DPI) was dissolved in DMSO as stock solution. All other chemicals were dissolved in water and, as stock solutions, stored in a freezer until used. All chemicals were purchased from Sigma Chemical (St. Louis, MO).Experimental Procedures
Initiation of experiments. The preparations were, after being mounted in the experimental chamber, allowed to adjust with a very low passive tone for 1.5 h. After the adjustment period, the preparations were stretched so that the cut ends of the cartilage were positioned ~3 mm apart. Preparations with intact epithelium were allowed to adjust for 1-3 h after stretch until they had developed a regular complex tone, at which time the experiments begun. Epithelium-denuded preparations were normally allowed to adjust 15-30 min after stretch before the experiments were started. Some epithelium-denuded preparations were used for two different experiments, separated by 30 min of rest in control conditions. In no case were more that two preparations from the same animal used for a specific experiment.
Tests of various agents on the spontaneous tone. After development of a steady-state control tone for ~30 min, the effects of various agents on the tone were tested by applying them (a complete exchange of solution in the chamber was achieved within 5-6 min) and letting them act long enough for the tone to assume new steady-state properties for ~15 min. The tone observed during these 15 min was then estimated as time average for further statistical evaluation. In long-term tests of denuded preparations, an average of the 3-h period after stretch was used for calculations.
Histological Examinations
Morphological evaluation of preparations.
Tracheal preparations were, after completion of the experiments,
immersed in buffered 4% paraformaldehyde (pH 7.2), dehydrated, and
embedded in paraffin. Four sections of 6 µm (separated by at least
400 µm) from each animal were stained with hematoxylin and
erythrosin, for assessment of general airway morphology and to examine
the extent of remaining airway epithelium and damage of subepithelial
tissue. Intact preparations exhibited a normal respiratory epithelium
with eosinophils (Fig.
1A),
which are normally present in the guinea pig tracheal mucosa (4, 5).
Rupture of the reticular layer of the airway epithelial basement
membrane was used as a measure of subepithelial tissue damage. To
visualize the reticular layer of the basement membrane (as depicted in
Fig. 1B), Nomarski optics was used.
The reticular layer forms a continuous linear structure just beneath
the airway epithelium and is readily detectable in the light
microscope. Disruption of this "line" was used as a measure of
subepithelial tissue damage. The percentage of basement membrane
covered with airway epithelium and the percentage of damaged airway
epithelial basement membrane in each section were calculated by using a
computerized image-analysis system. All slides were coded and examined
in a blinded fashion. Tracheal segments with >10% remaining airway
epithelium, or >10% damaged airway epithelial basement membrane,
were excluded from the study (9 of 46 preparations).
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Immunohistochemical visualization of NEE cells.
Tracheal specimens (n = 5) were
immersed overnight in Stefanini's fixative (2% paraformaldehyde and
0.2% picric acid in 0.1 M phosphate buffer, pH 7.2), rinsed repeatedly
in buffer (Tyrode buffer supplemented with 10% sucrose), frozen in
mounting medium (Tissue-Tek, Miles, Elkhart, IN), and stored at
80°C until sectioning. Antibodies against protein gene
product 9.5 (PGP 9.5; polyclonal rabbit anti-human PGP 9.5 antibody,
Ultraclone, Wellow, UK), secretory protein-1 (SP-1)/chromogranin A
(polyclonal rabbit anti-bovine SP-1/chromogranin antibody, INCSTAR,
Stillwater, MN), and neuron-specific enolase (monoclonal mouse
anti-human neuron- specific enolase antibody, Zymed Laboratories, San
Francisco, CA) were used for immunohistochemical demonstration of NEE
cells in cryostat (10 µm) sections (22, 23). The sections were
exposed to the primary antiserum overnight in 4°C in a moist
chamber. The site of the antigen-antibody reaction was revealed by
application of fluorescein isothiocyanate-conjugated swine anti-rabbit
(DAKO, Glostrup, Denmark) or goat anti-mouse (Jackson Immunoresearch
Laboratories, West Grove, PA) Ig secondary antibodies for 1 h at room temperature.
Statistics
Measurement values are given as means ± SE. Tests of statistical significance were performed by using a two-tailed version of the Mann-Whitney U-test.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of Epithelium Denudation on Spontaneous Tone
In this study, a total of 37 denuded preparations was used. None of these displayed a complex spontaneous tone in 12% oxygen, but eight of the preparations spontaneously exhibited weak oscillations during short periods of time.To clarify the long-term behavior of denuded preparations, examinations
with exposure to standard PSS for 4 h were performed. All preparations
developed some tone during the initial adjustment period. After
stretch, they exhibited a relatively strong, irregular (so-called
"smooth") tone with an average force of 0.56 ± 0.14 mN
(n = 7), which is about five times
larger than the tone found in intact preparations with oscillations and
complexes [0.10 ± 0.01 mN; n = 19; (19)]. This difference in average tone between intact and
denuded preparations proved to be highly significant (P < 0.001). An
example of spontaneous tone in denuded and intact preparations observed
for 4 h is seen in Fig. 2.
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Pharmacologically, the tone of denuded preparations behaved as the
classic tone of intact preparations in a high oxygen concentration (cf.
Ref. 19) in that it could be suppressed completely both by 1 h of
treatment with 10 µM indomethacin and, after a transient increase in
force, by 10 µM capsaicin. Typical recordings of such effects are
shown in Fig. 3.
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Pharmacological Manipulations of the NEE Cells in Intact Preparations
Because the change of the spontaneous tone in denuded preparations could be due to a lack of oxygen-sensitive NEE cells, experiments were performed with substances that are expected to modulate the release of transmitters from these cells.Effects of DPI.
To test the hypothesis that the classic tone in intact preparations is
caused by an increased production of
H2O2
by the NEE cells, the
H2O2-producing
NADPH oxidase was blocked by DPI (3, 25) after the tone had been
transformed to the strong, smooth tone by exposure to 94% oxygen. As
seen in Fig. 4A, this
resulted within 30 min in a decline of average tone by >80% (Table
1) and in the development of oscillations.
The oscillations did, however, not group into complexes during the
following 1 h, which is similar to what is found in preparations
exposed to 12% oxygen after some time in 94% oxygen (20). The
DPI-induced effects were only slowly reversible, and subsequent
exposure to drug-free solutions during 30 min led to some increase in
average tone and to less-frequent oscillations. However, the strong,
nonoscillating tone was not displayed during this
time.
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-nitro-L-arginine methyl ester
(10 µM) and
N-monomethyl-L-arginine (200 µM) given to preparations displaying a complex tone, no effect could
be detected. Thus NO appears not to be involved in the
regulation of the oscillating tone.
Effects of H2O2. To further clarify the role of H2O2 in the generation of spontaneous tone, preparations in 12% oxygen were exposed to H2O2 in concentrations up to 10 mM. This caused only weak effects on the spontaneous tone. However, because H2O2 normally is rapidly degraded by catalase, we also examined H2O2 in preparations pretreated with 1 mM of the catalase inhibitor 3-amino-1,2,4-triazole (11). Thirty minutes of exposure to this substance alone did not affect the spontaneous tone at all. Addition of 2 mM H2O2 in preparations pretreated with 3-amino-1,2,4-triazole caused a transformation of tone from a weak, oscillating complex type to a much stronger smooth tone, as shown in Fig. 4B1 and further accounted for in Table 1. A restitution of an oscillating tone of low average force took place on withdrawal of H2O2, thus showing the reversible effect by H2O2.
In other experiments, preparations were exposed to a combination of indomethacin and H2O2 after a 1-h exposure to H2O2. These preparations lost all tone within 1 h of indomethacin treatment (Fig. 4B2), thus showing a clear resemblance between the H2O2-induced tone and the classic spontaneous tone. In a previous study (20), it was concluded that the tracheal tone is determined by prostaglandin-dependent (strong, nonoscillating, force-generating) mechanisms and prostaglandin-independent (oscillating force-generating) mechanisms. To explore the effects of H2O2 on the prostaglandin-independent part of the spontaneous tone, preparations were exposed to 2 mM H2O2 after 30 min pretreatment with 1 mM 3-amino-1,2,4-triazole and 10 µM indomethacin, as seen in Fig. 4C. This resulted in a complete elimination of the oscillations and a reduction of the average tone by 92 ± 6% (P < 0.05; n = 4). In a few preparations, the treatment with H2O2 was discontinued by addition of drug-free PSS. This led to a reappearance of an oscillating tone, thus showing once more the reversibility of the effects by H2O2.Pharmacological Control Experiments on Epithelium-Denuded Preparations
For a control of whether the drug effects in the preceding section were due to nonepithelial mechanisms, both DPI and H2O2 were applied to denuded preparations. For DPI (20 µM) it was found that, in 94% oxygen, 1 h of exposure to the drug had no significant effect on the average level of spontaneous tone (Table 1) and, also, that DPI was unable to elicit oscillations.Application of
H2O2
for 30 min in standard PSS resulted in preparations with functioning
prostaglandin synthesis, an increase in smooth tone, as shown in Fig.
5. The increase was 352 ± 96% (Table 1) and not statistically different from that produced by
H2O2
in intact preparations. This increase of force appears to be caused by
a stimulation of the prostaglandin synthesis, because
H2O2
did not cause any contraction in preparations pretreated with 10 µM
indomethacin, as is also illustrated in Fig. 5.
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Effects of Varying Oxygen Concentrations on the Spontaneous Tone
Against the background of the hypothesis of the oxygen effect on the generation of tone being mediated by NEE cells, it seemed appropriate to determine more precisely the oxygen concentration at which the tone is transformed from an oscillating to a smooth type. For this purpose, six intact preparations were exposed to 12, 25, 40, 60, and 94% oxygen for 30 min each. Three preparations lost the oscillating tone completely at 25% oxygen, one at 40%, and two at 60%. Thus, on an average, the tone was transformed to the smooth type at ~40% oxygen. An example of this is shown in Fig. 6A.
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Because there is evidence that the transition from oscillating to smooth tone implies prostaglandin-independent as well as prostaglandin-dependent mechanisms, it was decided to repeat these experiments on six additional preparations after 30-min pretreatment with 10 µM indomethacin. It was found that a complete abolition of the complex tone took place at 25% oxygen in two preparations and at 40% in four preparations. Thus, on average, 40% oxygen completely removed the complexes and oscillations also in preparations without prostaglandin synthesis. A typical recording of this experiment is seen in Fig. 6B.
As a control, 10 denuded preparations were also exposed to varying oxygen concentrations. A change in the oxygen concentration from 12 to 94% produced a decrease in tone in seven and an increase in tone in three preparations after 30 min of exposure. On average, preparations in 94% oxygen had a tone that was 74 ± 17% [P = 0.14 (not significant); n = 10] of control tone in 12% oxygen. Thus the epithelium appears vital for oxygen-induced changes of spontaneous tone in this preparation.
Effects of Carbon Dioxide and pH on the Spontaneous Tone
Pulmonary NEE cells have been compared with cells in the carotid bodies (13) that are sensitive to carbon dioxide and pH, as well as to oxygen (6). To see whether carbon dioxide and pH are also affecting tracheal NEE cells and, thereby, the tracheal tone, intact and denuded preparations were exposed for 30 min to varying amounts of carbon dioxide and pH. The results of the experiments are compiled in Table 1, and some typical recordings are shown in Fig. 7. It appears that, in intact preparations, both a decrease to 4% carbon dioxide and an increase to 8% carbon dioxide at a constant pH of 7.40 gave rise to some increase in tone by evoking more continuous oscillations with few relaxations. In other experiments, a quite significant increase in tone arose in response to an increased pH to 7.80 at constant carbon dioxide. Very similar effects to changes in carbon dioxide and pH were, however, also observed in denuded preparations, and, therefore, it does not appear likely that pulmonary NEE cells are mainly responsible for the effects of changing pH and carbon dioxide on the spontaneous tracheal tone.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Principal Findings and Conclusions
This paper deals with the functional role of NEE cells in the generation of spontaneous tone in isolated tracheal preparations. The principal findings are as follows: 1) epithelium-denuded preparations were unable to develop an oscillating tone with complexes and instead in many respects exhibited the classic type of relatively strong, nonoscillating spontaneous tone; 2) inhibition of NADPH-induced production of H2O2 in the NEE cells by DPI caused in intact preparations in 94% oxygen a transformation of the strong, smooth tone to a weaker tone with oscillations; and 3) exposure to H2O2 in the presence of catalase inhibition by 3-amino-1,2,4-triazole caused a transformation from a complex type of tone to a strong, smooth tone in 12% oxygen. Taken together, these findings 1) support the notion that the NEE cells in the epithelium are able to detect the ambient oxygen concentration via a H2O2-producing NADPH oxidase and a closely associated H2O2-activated K+ channel and 2) suggest that the pulmonary NEE cells release factors that are essential for the normal oscillating spontaneous tone. Thus we were able to show, for the first time, that NEE cells have a crucial role in controlling the spontaneous tone in isolated guinea pig tracheal preparations.Agents Affecting NEE Cell Function
In Fig. 8, a schematic drawing of the proposed control of spontaneous tone by NEE cell-influencing drugs used in this study is shown. This figure is examined in detail below.
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DPI. DPI caused a transformation of a high, smooth tone to a weak, oscillating type. It appears that this substance reduced the average tone only by affecting the NEE cell function, and not by altering the prostaglandin synthesis, because no reduction of spontaneous tone was seen when it was given to denuded preparations, which in other experiments had been found to relax completely when exposed to indomethacin. We also found that the NO synthesis was without influence on the complex tone, which makes it unlikely that the studied effects are caused by inhibition of NO production.
The DPI concentration used in this study (20 µM) can perhaps by considered as fairly high, because it has been described that concentrations of 3-10 µM might give nonselective ion-channel inhibition in isolated pulmonary artery smooth muscle cells (26). We obtained the concentration by administration of gradually higher amounts; 5 µM caused only small effects, 10 µM transformed the tone to an oscillating type, whereas 20 µM also reduced the spontaneous force development to near the average control force before exposure to 94% oxygen. DPI in that concentration did probably not cause any unspecific effects, because exposure of denuded preparations to DPI did not significantly change the spontaneous tone. One explanation of the discrepancy for unspecific DPI effects might be that we used intact tissue, in which the NEE cells are embedded in the epithelium and in many cases only reach the lumen by a tiny process, instead of dissociated cells, which are exposed to the solution on all sides. Also, in our study we used tracheal muscle rather than pulmonary artery muscle.High oxygen concentration. A high oxygen concentration does not appear to give rise to an increase of spontaneous tone by stimulating the prostaglandin synthesis, because DPI, which probably acts only by activating the NEE cell function, was able to return average tone to around control level in 12% oxygen (cf. Fig. 4A).
A high oxygen concentration gave rise to a strong, smooth tone only if the prostaglandin synthesis was intact. This might appear paradoxical if oxygen is presumed not to stimulate the prostaglandin synthesis. However, an explanation could be that normally, contractile prostaglandins are synthesized in the preparation independently of the NEE cells. The contractile effects by the prostaglandins (Fig. 8, arrow vii) are, however, normally suppressed by powerful relaxing factor(s) from the epithelium. When a high oxygen concentration is administered, the NEE cells cease to release their relaxing factor(s), which unmasks the contractile effects by the prostaglandins.H2O2. H2O2 transformed the complex tone to the strong, smooth type of tone. In addition, it completely abolished the oscillations when given to preparations pretreated with indomethacin. Therefore, it appears that H2O2 inhibits the activity of the NEE cells, similar to the effects by a high oxygen concentration. However, H2O2 also caused a contraction in denuded preparation that was completely abolished by indomethacin treatment. Therefore, H2O2 apparently stimulates the prostaglandin synthesis as well, a mechanism previously shown in other preparations (e.g., Refs. 9, 10).
Because H2O2 clearly has a stimulating effect on the prostaglandin synthesis, one might wonder whether the other effect by H2O2, abolition of the oscillations, also in some way is caused by an increase of prostaglandins. However, previous examinations (20) showed that addition of a prostaglandin (PGF2
) to the bath increased
the size of the complexes, rather than eliminating them. Furthermore,
H2O2
abolished all oscillations and complexes also in preparations with
inhibited prostaglandin synthesis (cf. Fig.
4C), which clearly demonstrates that
this effect is prostaglandin independent.
Factors Controlling Spontaneous Airway Tone
Relaxing factor(s). Preparations without epithelium developed a much stronger spontaneous tone than did intact preparations. In addition, activation of the NEE cells (by inhibition of the H2O2 production) by DPI distinctly lowered the average tone in intact, but not denuded, preparations by >80%. These findings indicate that the NEE cells release powerful relaxing factor(s).
The existence of a relaxing factor could explain the similarities between the classic high-oxygen-concentration type of tone and the tone in denuded preparations in 12% oxygen. This explanation would imply that the classic, nonoscillating tone can be accomplished by a reduction of the release of relaxing factor(s) from the NEE cells in two different ways. One is a high oxygen concentration, which by production of H2O2 reduces the activity of the NEE cells, leading to a reduced release of epithelial factors, and the other is denudation, which, of course, completely eliminates these factors. It may be speculated that the relaxing factor(s) in this study may include paracrine as well as neuronal mechanisms. That the relaxing factor(s) responsible for reduction of tone in preparations with a complex tone might involve neuronal mechanisms could be indicated by previous findings (20) showing that preparations exposed to the local anesthetic lidocaine (1 mM), in concentrations that did not affect neurokinin A-induced contractions, transformed the tone from a complex type to a stronger, nonoscillating type of tone. EPITHELIUM-DERIVED RELAXING FACTOR (EPDRF). Several studies have suggested the existence of an EpDRF. This concept has been based mainly on observations that epithelium removal shifts the concentration-contraction curves for many agonists to the left, without affecting the maximal response (21), and on findings with bioassay experiments in which solutions passing through an epithelium-intact preparation could relax precontracted vascular preparations (24). The nature of the EpDRF has been extensively speculated on but appears not to be NO (14). The relationship between the EpDRF and the relaxing factor in this study is unclear. It is, however, interesting to note that the concept of EpDRF has been challenged on the basis of that it only can be demonstrated in coaxial bioassays in low-oxygen-concentration environment (8). However, as is evident from our study, the NEE cells are only active and ready to release a relaxing factor in low (that is physiological)-oxygen concentrations. Therefore, it is not unlikely that the EpDRF and the relaxing factor studied in this paper are identical.Contracting factor(s). A high oxygen concentration, when given to indomethacin-pretreated preparations, completely abolished the oscillating tone (cf. Fig. 6B). The spontaneous tone in indomethacin-treated preparations is probably regulated only by NEE cell transmitters (and C-fiber transmitters). The hyperoxia-induced suppression of tone in indomethacin-treated preparations is therefore caused by a reduced release of contracting factors from the NEE cells, considering that hyperoxia is believed to reduce the activity in these cells.
Possible NEE Cell Transmitters Influencing the Spontaneous Tone
Several bioactive substances have been demonstrated in the NEE cells (1, 17). 5-HT (serotonin) appears ubiquitously in NEE cells from various animals. Because 5-HT is a potent bronchoconstrictor, it is not impossible that this substance might be (one of) the contracting factor(s) from the NEE cells. Calcitonin gene-related peptide (CGRP) has also been demonstrated in these cells. The effect of CGRP on airway smooth muscle is controversial, and it has been described both to relax precontracted airway preparations (2) and to moderately contract tracheal preparations (15). Perhaps CGRP is involved in the NEE cell-induced relaxation. Furthermore, calcitonin, enkephalin, somatostatin, substance P, peptide YY, and other bioactive substances have been described in the NEE cells. The importance of these substances in control of the spontaneous airway tone remains to be resolved.Arguments Supporting the Notion That the Oscillating Tone is not Caused by Hypoxia
An additional important result of this study is that it clearly supports our view that the oscillating tone is physiologically normal and is not caused by airway smooth muscle hypoxia. The evidence for this is threefold. 1) Denuded preparations develop a nonoscillating strong tone despite being exposed to 12% oxygen. It appears unlikely that removal of the 50-µm-thick epithelium would dramatically improve the oxygen concentration in the preparation, because the diffusion length would only be marginally shorter. 2) It is possible to pharmacologically control the type of spontaneous tone independently of the oxygen concentration. Thus intact preparations in 12% oxygen exhibit the classic type of tone if exposed to H2O2, and preparations in 94% oxygen exhibit an oscillating type of tone if the synthesis of H2O2 is abolished. 3) This study shows that the NEE cells are important for detecting the oxygen concentration. The epithelium is in contact with the surrounding solution and directly detects its oxygen concentration. Therefore, within limits, the important factor is the oxygen concentration in the epithelium and not in the center of the preparation.| |
ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. Frank Sundler for valuable viewpoints concerning the histological methods used in this paper.
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FOOTNOTES |
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This study was supported by the Swedish Medical Research Council (project no. 2082), Anders Otto Swärds Stiftelse, Vårdalstiftelsen, and the Swedish Heart and Lung Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests: S. Skogvall, Dept. of Physiology and Neuroscience, Lund Univ., Sölvegatan 19, S-223 62 Lund, Sweden (E-mail: staffan.skogvall{at}mphy.lu.se).
Received 28 July 1998; accepted in final form 13 November 1998.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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