| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Physiology, St. George's Hospital Medical School, London SW17 ORE, United Kingdom
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Wells, U. M., S. Duneclift, and J. G. Widdicombe.
H2O2
increases sheep tracheal blood flow, permeability, and vascular response to luminal capsaicin. J. Appl.
Physiol. 82(2): 621-631, 1997.
Exogenous hydrogen
peroxide
(H2O2)
causes airway epithelial damage in vitro. We have studied the effects
of luminal
H2O2
in the sheep trachea in vivo on tracheal permeability to
low-molecular-weight hydrophilic (technetium-99m-labeled
diethylenetriamine pentaacetic acid;
99mTc-DTPA) and lipophilic
([14C]antipyrine;
[14C]AP) tracers and
on the tracheal vascular response to luminal capsaicin, which
stimulates afferent nerve endings. A tracheal artery was perfused, and
tracheal venous blood was collected. H2O2
exposure (10 mM) reduced tracheal potential difference
(
42.0 ± 6.4 mV) to zero. It increased arterial and
venous flows (56.7 ± 6.1 and 57.3 ± 10.0%,
respectively; n = 5, P < 0.01, paired t-test) but not tracheal lymph flow
(unstimulated flow 5.0 ± 1.2 µl · min
1 · cm1,
n = 4). During
H2O2
exposure, permeability to 99mTc-DTPA increased from
2.6 to 89.7 × 107 cm/s
(n = 5, P < 0.05), whereas permeability to
[14C]AP (3,312.6 × 107 cm/s,
n = 4) was not altered significantly
(2,565 × 107
cm/s). Luminal capsaicin (10 µM) increased tracheal blood flow (10.1 ± 4.1%, n = 5)
and decreased venous 99mTc-DTPA
concentration (19.7 ± 4.0, P < 0.01), and these effects were
significantly greater after epithelial damage (28.1 ± 6.0 and
45.7 ± 4.3%, respectively,
P < 0.05, unpaired
t-test). Thus H2O2
increases the penetration of a hydrophilic tracer into tracheal blood
and lymph but has less effect on a lipophilic tracer. It also enhances
the effects of luminal capsaicin on blood flow and tracer uptake.
tracheal lymph; hydrogen peroxide; vascular hyperresponsiveness; potential difference
INTRODUCTION EPITHELIAL DAMAGE MAY BE a feature of asthmatic airways
(3, 18, 20), although not all studies support this view (21). Epithelial damage induced experimentally greatly increases the permeability of a low-molecular-weight hydrophilic tracer,
technetium-99m-labeled diethylenetriamine pentaacetic acid
(99mTc-DTPA) in the sheep trachea
in vivo (30). This suggests that uptake of inhaled hydrophilic
respiratory drugs may be increased in airways with substantial damage.
Less is known about the effects of epithelial damage on the
permeability of lipophilic molecules (15, 32). Furthermore, although
airway injury increases sheep tracheal lymph flow (1), nothing is known
about the penetration of low-molecular-weight tracers from tracheal
lumen to tracheal lymph and whether this is increased by agents that
cause epithelial damage.
Epithelial damage may have other consequences that could alter the
uptake of hydrophilic molecules from the airway lumen. For example, it
is not known whether agents that cause epithelial damage also damage
the sensory nerve endings found in and near the tracheal epithelium or
whether their responsiveness is enhanced. Changes in the resulting
degree of vasodilatation would alter tracer uptake because venous
99mTc-DTPA concentration is
negatively related to tracheal blood flow (14, 29).
One endogenous agent that causes epithelial damage, at least when
applied exogenously in vitro, is hydrogen peroxide
(H2O2) (16, 19).
H2O2
is produced by inflammatory cells; these are present in large numbers
in inflamed airways so that high concentrations of
H2O2
may be produced locally (17).
H2O2
is converted to the hydroxyl radical, which is highly reactive and
causes tissue damage (9). It is scavenged by catalase and glutathione,
two enzymes present in the airway and in epithelial lining fluid in the
lower airway (7-9).
The effects of
H2O2
on airway permeability have not been studied in vivo, but in vitro
epithelial damage was associated with only a threefold change (19) or
no change at all (16) in the flux of low-molecular-weight hydrophilic
molecules. However, in vitro studies may not reflect uptake in vivo
because of the lack of a functional vasculature. Changes in
99mTc-DTPA permeability in vivo
(30) appear to be much greater than those produced by the corresponding
agent in vitro (Z. Hanafi, U. M. Wells and J. G. Widdicombe,
unpublished observations).
We have assessed in sheep the short-term effects (up to 2 h) of 10-min
exposure of the tracheal lumen to 10 mM
H2O2
on the tracheal epithelium [by histology and by measurement of
electrical potential difference (PD) across the airway wall] on
tracheal blood and lymph flow and on the tracheal permeability of two
low-molecular-weight molecules, one hydrophilic
(99mTc-DTPA, 492 Da) and one
lipophilic
{[14C]antipyrine
(14C-AP), 200 Da}. In
addition, we have investigated the effect of stimulating sensory nerve
endings by using luminal
8-methyl-N-vanillyl-6-nonenamide (capsaicin) on tracheal blood flow and venous
99mTc-DTPA concentration before
and after
H2O2
exposure.
METHODS Twenty-four sheep were used: five were used for the
concentration-response study, ten to test the effects of 10 µM
capsaicin in the presence or absence of epithelial damage due to
H2O2
exposure, five to test the effects of
H2O2
on [14C]antipyrine
permeability, and four to investigate the effects of
H2O2
on lymph flow and radiolabel uptake.
Experimental Preparation
A tracheal artery from the left carotid artery was isolated, and the
left carotid artery cranial to this was ligated (Fig. 1). The occluded segment of the left
carotid artery was bypassed from the right carotid artery by using a
plastic Y-tube connection. Tracheal arterial flow was measured by using
an electromagnetic flow probe inserted between two catheters in the
left carotid artery proximal to the isolated artery and connected to a
square-wave electromagnetic flowmeter (Carolina Medical Electronics).
The mean flow for each 5-min period was calculated. A tracheal vein on
the left side that drained the perfused region of the trachea was
catheterized (3 FG, Portex), and venous blood was collected for 5-min
periods continuously throughout the experiment. The distribution of
perfused tracheal circulation was tested by close-arterial injection of
Evans blue dye diluted in 0.15 M NaCl.
The cervical trachea was isolated by inserting two balloon-cuffed tracheostomy tubes into the trachea, one below the larynx and the other just above the low cervical tracheostomy supplying airflow to the lungs. The enclosed segment was usually 15-20 cm long and included the area perfused by the isolated tracheal artery. Before each experiment, Krebs-Henseleit solution (KH) was flushed through the lumen two or three times.
Cannulation of Efferent Tracheal Lymphatics
In four experiments, the deep caudal cervical lymph node was exposed (2) and 0.1 ml Evans blue (5 mg/ml) was injected into the node to identify the efferent lymphatics. All visible efferent lymphatics were ligated, and a single vessel was cannulated retrogradely by using medical-grade tubing (0.025-cm internal diameter, SF Medical, Watford, UK). The end of the tubing was positioned over a 2-ml Bijou tube containing 20 µl heparin (25,000 U/ml), and lymph was collected for 15-min intervals. In these experiments, the tracheal lumen was isolated as described above, and a tracheal vein was cannulated. However, arterial flow was not measured. In addition, all visible efferent lymphatics running close to the carotid artery (at the level of the most caudal part of the trachea) were ligated to reduce lymph flow from the head and neck.Protocols
99mTc-DTPA experiments. The tracheal lumen was filled with KH containing 99mTc-DTPA. This was replaced, by using a constant volume, at 15-min intervals. In each set of experiments, the lumen was exposed to the capsaicin vehicle in the period preceding each stimulus with capsaicin. The capsaicin and vehicle stock solutions (see Drugs) and H2O2 were diluted in KH containing 99mTc-DTPA. In five sheep, the tracheal lumen was exposed to 10 µM capsaicin during the fourth 15-min period and to 100 µM capsaicin during the eighth period. This protocol was carried out to check that 10 µM capsaicin had a clear but submaximal effect on tracheal blood flow. The duration of the protocol was 2.5 h. In ten additional sheep, the lumen was exposed to 10 µM capsaicin during the fourth and tenth periods. In five of these experiments, the lumen was also exposed to 10 mM H2O2 for 10 min during the sixth period. The duration of the protocol was 3 h. In one of the experiments without H2O2, venous flow could not be measured. [14C]AP experiments. Five experiments were carried out to test whether H2O2 exposure altered the baseline permeability of [14C]AP. Capsaicin was not given in these experiments. The tracheal lumen was filled with KH containing [14C]AP. This remained in the lumen until the end of the fifth 15-min period, when it was replaced by 10 mM H2O2 (in KH containing [14C]AP) for 10 min. This was replaced again by KH containing only [14C]AP, which remained in the lumen until the end of the experiment. The duration of the protocol was 3 h. In all experiments using 99mTc-DTPA or [14C]AP, femoral arterial blood samples were taken at 30-min intervals before H2O2 administration and every 15 min thereafter to monitor the background levels of radioactivity. Lymph experiments. In four sheep in which an efferent tracheal lymphatic had been cannulated, the effects of 10 mM luminal H2O2 on lymph flow and tracer uptake were studied. KH containing both 99mTc-DTPA and [14C]AP was washed into the tracheal lumen. After 75-90 min, it was removed and replaced by KH containing 10 mM H2O2 plus both radiolabels. This was removed after 10 min and replaced by KH containing both radiolabels. This KH remained in the lumen until the end of the experiment at 3 or 3.25 h. Samples of luminal fluid were taken midway through each 15-min period (see Measurement of [14C]AP Output), and the concentration of both radiolabels in each sample was measured. Venous blood and lymph samples were collected continuously for 15-min periods throughout the experiment. Arterial flow was not measured.Measurement of PD
The electrical PD across the tracheal wall was measured by using two calomel reference electrodes. These were filled with 3.8 M KCl and placed in separate beakers of the same solution. Electrical contact was made with the preparation by using two agar bridges constructed from polyethylene tubing (0.5-mm internal diameter) filled with 3.8 M KCl in 2.5% (wt/vol) agar solution. The end of one agar bridge was inserted into the lumen of the isolated segment of trachea via the lower cannula; the end of the other agar bridge was placed in a pool of saline touching the external tracheal wall. Output from the two electrodes was via a high input-impedance buffer amplifier (>109 M
) and displayed on a
digital voltmeter.
Histology
Sections of the trachea were taken at the end of three of the experiments in which the lumen was exposed to 10 µM capsaicin before and after treatment with 10 mM H2O2 and from two control experiments (2 exposures to 10 µM capsaicin/experiment). These were compared with sections from earlier experiments in which the tracheal lumen was exposed only to KH for 3 h. All sections were taken immediately postmortem and were fixed in 20% formal saline for standard paraffin processing. They were stained by using a routine hematoxylin and eosin stain. Photomicrographs were obtained by using a Zeiss photomicroscope and Ilford Pan F 50 black-and-white film.Measurement of 99mTc-DTPA Output
Each venous blood sample was weighed to give venous flow, and the 99mTc-DTPA concentration (counts · min1 · ml1)
in each sample was measured by using a gamma counter (Beckman Gamma
5500). Corrections were made for decay. The
99mTc-DTPA concentration was
multiplied by venous flow to obtain the total
99mTc-DTPA output
(counts/min2) in the
catheterized vein for each 5-min period.
Measurement of [14C]AP Output
The mean [14C]AP concentration in KH when the solution was initially placed in the tracheal lumen was 449,303 ± 34,808 counts · min1 · ml1
(n = 5). Because of the high
permeability of
[14C]AP, the luminal
counts drop by ~50% in 90 min (13). A luminal sample was taken
midway through each 15-min period by withdrawing the luminal solution
in a syringe and then immediately returning all but 0.15 ml to the
lumen. The
[14C]AP concentration
in a 0.1-ml sample was then measured by 
-scintillation counting
(Beckman LC 6000IC).
All arterial and venous samples were centrifuged (10,000 revolutions/min for 10 min). The hematocrit of selected samples was measured; because this varied little throughout the experiment, a mean
value was used for all samples. The plasma concentration of
[14C]AP was measured
by -scintillation counting. The ratio of
[14C]AP in plasma to
cellular fraction was found to be 1:1.1 in an earlier study (13), and
this value was used here for calculation of the concentration of
[14C]AP in the
cellular fraction.
Analysis of Tracheal Lymph Samples
Lymph samples collected over 15-min periods were weighed, and results were expressed as flow (µl/min). Protein concentrations were measured by using a clinical refractometer (model 5711-5020, Schuco International, London, UK). The 99mTc-DTPA and [14C]AP concentrations in 100-µl aliquots were measured as described above. The total outputs of protein, 99mTc-DTPA, and [14C]AP in each sample were calculated by mutiplying the concentrations by the sample volume.Analysis of Results
PD results were expressed in millivolts. Tracheal arterial flow, venous flow, venous 99mTc-DTPA concentration, and venous 99mTc-DTPA output during each 5 min of the 15-min test period and posttest periods were expressed as mean (±SE) percentage changes from the last 5 min of the pretest controls. The statistical significance of changes was tested by using Student's two-tailed paired t-tests by comparison with the values of the final 5 min of the 15-min pretest controls. The unpaired t-test was used to test whether the effects of 100 µM capsaicin were significantly different from the effects of 10 µM capsaicin and whether the effects of 10 µM capsaicin were significantly greater after epithelial damage.The baseline venous 99mTc-DTPA concentration remains fairly constant during the experiment because the low permeability of 99mTc-DTPA means that the luminal concentration does not alter significantly. However, venous [14C]AP concentration decreases rapidly with time because the high permeability of [14C]AP leads to a rapid reduction in its luminal concentration and, hence, in the concentration gradient across the airway wall. For this reason, and because the luminal [14C]AP solution was replaced with fresh stock solution after H2O2 exposure, the [14C]AP results are presented only as a permeability coefficient (see below), which takes into account the concentration gradient between lumen and venous blood, and not as venous concentration and output.
Data from consecutive 15-min tracheal lymph samples were pooled to give results for each 30-min period (except for the 15-min period during which H2O2 was instilled into the tracheal lumen for 10 min). Results from the first 15-min period were not used in this analysis. The effects of H2O2 on lymph flow and tracer concentrations were assessed by comparison with the preceding control period by using the paired test.
Permeability coefficients. The permeability coefficients for 99mTc-DTPA (PDTPA) and [14C]AP (PAP) were calculated during test periods and also during the control period immediately preceding the first test period in each experiment. The permeability coefficient is calculated as follows: P =(d
/dt)/(S · 
C),
where d/dt is the
output of 99mTc-DTPA or
[14C]AP (in
counts/min2), C is the
concentration gradient of
99mTc-DTPA from tracheal lumen to
venous blood
(counts · min1 · ml1),
and S is that part of the surface area
of the isolated trachea that is perfused.
For 99mTc-DTPA, C was
calculated as the luminal concentration at the start of each 15-min
period (because it did not change significantly during 15 min) minus
the mean venous concentration. For
[14C]AP, C was
calculated as the luminal concentration halfway through each 15-min
period (because the luminal concentration drops significantly during 15 min) minus the mean venous concentration.
With the use of Evans blue injections, the perfused surface area
(S in equation above) was shown in an
earlier study, which used similar variety and weights of sheep, to be
30 cm2 (14). This
value, which is slightly smaller than the total surface area of the
tracheal segment (14), has been used here. Because only part of the
arterial blood is collected in the cannulated vein, it is assumed that
total tracer uptake is the measured output times the ratio of arterial
blood inflow to venous outflow (14). The mean (±SE) permeability
coefficients for each stimulus were compared by using the Student's
t-test.
Drugs
Evans blue dye was obtained from Aldrich Chemical; heparin sodium from CP Pharmaceuticals; gallamine triethiodide and pentobarbital sodium (Sagatal) from May and Baker; H2O2 (AnalaR) from BDH Laboratory Supplies; and capsaicin (98% purity) from Sigma Chemical. A stock solution of 10 mM capsaicin in a vehicle (70% ethanol-30% saline) was freshly made for each experiment. This was diluted with KH containing 99mTc-DTPA to give a 100 or 10 µM capsaicin solution as required. The vehicle was diluted in a similar way so that the vehicle for 10 µM capsaicin contained 0.7 µl ethanol/ml KH.RESULTS
Baseline Blood Pressure
Mean systemic arterial pressure in all experiments involving capsaicin was 109 ± 6 mmHg (n = 15) at the beginning of the experiment and 93 ± 4 mmHg (n = 15) at the end of the experiment. This change was significantly different (P < 0.01). There were no significant differences in these values between the concentration-response, control, or H2O2 experiments.Effects of 10 mM H2O2
PD. Mean baseline PD was42.0 ± 6.4 mV at the start of the
experiment (lumen negative). On exposure of the tracheal lumen to H2O2,
PD rapidly became zero (P < 0.01, n = 5). In one of five experiments, PD
became more negative for the first 7 min of exposure (maximum change
was 20% of baseline) before very rapidly reversing to become very
close to zero. No significant recovery of PD occurred subsequently.
Tracheal blood flow.
H2O2
also produced rapid and large changes in arterial and venous flows
(Fig. 2). Arterial flow increased within 15 s of washing the
H2O2
into the lumen. Flow did not return to baseline until 15-20 min
after removal of the
H2O2.
,
Period of exposure to
H2O2.
Results were normalized to last 5 min of preceding control.
Significantly different vs. last 5 min preceding control:
* P < 0.05;
** P < 0.01 (paired
t-test).
99mTc-DTPA and [14C]AP permeability. H2O2 greatly increased venous 99mTc-DTPA concentration (Fig. 3A) and venous 99mTc-DTPA output (Fig. 3B). PDTPA is shown in Table 1. PDTPA reached a peak in the control after H2O2 exposure and decreased thereafter. In the final control, permeability was
68.7 ± 28.1 × 107 cm/s, ~50% lower
than immediately after
H2O2
exposure. In separate experiments,
H2O2
did not alter PAP
significantly (Table 1).
|
|||||||||||||||||||||||||||
2%) (Table 2) and
increased slightly during the experiment. The baseline lymph-to-luminal
concentration ratio for
[14C]AP was 100-fold
higher than that for 99mTc-DTPA.
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
), which represents 15-min exposure period].
Significantly different vs. control preceding H2O2:
* P < 0.05;
** P < 0.01 (paired
t-test).
Capsaicin Experiments: Baseline Tracheal Blood Flow
Baseline tracheal arterial and venous flows before capsaicin administration are shown in Table 3. There were no significant differences in baseline flows between control periods or between the H2O2 and control experiments.
|
||||||||||||||||||||||||||||||||||||||||
Capsaicin Concentration-Response Study
PD was unaffected by capsaicin (10 µM:39.1 ± 1.3 mV, 100 µM: 37.1 ± 2.4 mV compared with controls of 39.0 ± 2.3 and 39.9 ± 3.3 mV, respectively). The capsaicin
vehicle had no significant effect on blood flow or on PD (data not
shown).
Both 10 and 100 µM luminal capsaicin significantly increased arterial
and venous flows (Fig. 5). Capsaicin (100 µM) had a significantly greater effect than 10 µM capsaicin
(P < 0.05).
P < 0.05 (unpaired t-test).
Effect of H2O2 Exposure on Vascular Response to 10 µM Capsaicin
Capsaicin (10 µM) produced significantly larger increases in tracheal arterial and venous flows after exposure of the tracheal lumen to H2O2 (Fig. 6, A and C). Similar changes were not seen in control experiments in which the lumen was not exposed to H2O2 (Fig. 6, B and D).
P < 0.05 (unpaired t-test).
Capsaicin (10 µM) also produced a significantly greater percent
decrease in venous 99mTc-DTPA
concentration after
H2O2
exposure than before (Fig. 7). However,
baseline 99mTc-DTPA concentration
was decreasing markedly at this point in the experiment (Fig. 3).
P < 0.05;
P < 0.01 (unpaired
t-test).
Luminal capsaicin administration did not alter systemic arterial pressure. In the experiments with H2O2, mean arterial pressure during the first (104 ± 8 mmHg, n = 5) and second (93 ± 12 mmHg, n = 5) capsaicin administrations were not significantly different from the values in the 5 min preceding capsaicin treatment (104 ± 8 and 88 ± 9 mmHg, respectively).
Histology
In three tracheas taken at the end of the experiments in which both 10 µM capsaicin and H2O2 were used, the epithelial layer was not eroded, but large intercellular spaces were present toward the base of the epithelium (Fig. 8). Cilia were visible along the epithelial surface, and goblet cells were present. The upper one-half of epithelium in one trachea stained intensely pink. Relatively few subepithelial inflammatory cells were present. In two tracheas exposed only to 10 µM capsaicin (twice), and in two tracheas exposed only to KH for 3 h, a normal well-ordered epithelium was seen.
DISCUSSION
Exposure of the sheep tracheal lumen to H2O2 in vivo caused epithelial damage and greatly increased the penetration of a low-molecular-weight hydrophilic tracer (99mTc-DTPA) but not of a lipophilic tracer ([14C]AP) into tracheal venous blood and lymph. H2O2 also increased tracheal blood flow and enhanced the vascular response to luminal capsaicin.
PDTPA and PAP
Luminal H2O2 (10 mM) caused tracheal epithelial damage, as shown by the abnormal appearance of epithelial cells, the enlarged intracellular spaces in the epithelium, and the large decrease in PD across the tracheal wall. It also increased PDTPA 53-fold. Large increases in PDTPA also occur after sloughing of tracheal epithelial cells induced by Triton X-100 (30). H2O2 was first shown to increase paracellular permeability in Madin-Darby canine kidney cells (31). Our results contrast with evidence from in vitro studies on airways, in which 10 mM H2O2 had little effect on the gross structure of the epithelium, whereas 100 mM H2O2 caused epithelial denudation (16, 19). This was associated with only a threefold increase in terbutaline flux across the guinea pig trachea (19) and no change in histamine permeability in human bronchi (16). Whether such high levels of H2O2 are produced within the airway wall in vivo during inflammation, when large numbers of inflammatory cells may be present, is not known; however, macrophages can produce H2O2 at a concentration of 1-100 mM (17).The increase in PDTPA is probably due to a change in the integrity of tight junctions and perhaps also to cellular damage. H2O2 alters the cell cytoskeleton (31), and cytoskeleton-destabilizing drugs increase the penetration of 99mTc-DTPA or 111In-labeled DTPA across the rat trachea (4). PDTPA usually peaked 15-30 min after H2O2 exposure, then steadily decreased to ~50% of the peak by the end of the experiment. Transmural PD showed little recovery. Similar changes in PDTPA and PD also occurred in experiments in which Triton X-100 was used (30). Whether this reflects epithelial repair, cellular repair, or some other change that reduces 99mTc-DTPA flux, for example edema of the airway wall, is not known.
In contrast, epithelial damage did not increase PAP. This is in agreement with an earlier study showing that platelet-activating factor, which may open epithelial tight junctions, increased PDTPA but not PAP in the ferret trachea in vitro (15). Similarly, denudation of the guinea pig trachea in vitro only doubled the flux of theophylline (lipophilic, molecular mass 180 Da) (32).
The decrease in PAP during and immediately after H2O2 exposure (Table 1) is probably due to a decrease in venous [14C]AP concentration secondary to the H2O2-induced increase in blood flow. A negative relationship between blood flow and the venous tracer concentration has been established for both 99mTc-DTPA (14, 30) and [14C]AP (Z. Hanafi, D. Corfield, and J. G. Widdicombe, unpublished observations). A similar drop in venous 99mTc-DTPA concentration did not occur, presumably because any flow-related decrease would be overridden by the increased 99mTc-DTPA flux resulting from epithelial damage.
Tracheal Lymph
Baseline tracheal lymph flow was 5 µl · min1 · cm1.
This is higher than a previous measurement of 1.3 µl · min1 · cm1
in conscious sheep (2). Although we tried to eliminate the contribution
of lymph from the head and neck by ligation of lymphatics near the
larynx, we cannot be certain that all lymph from this region was
excluded from our samples. In addition, some lymph from areas such as
the esophagus and the respiratory muscles may drain via the deep caudal
cervical lymph node. Because the lymph concentration of the tracers was
only 1-3% of the tracheal venous concentration, this also
suggests that much of the lymph was not tracheally derived and limits
the conclusions that can be drawn. Nevertheless, the presence of the
radiolabeled tracers in the lymph samples and the rapid and large
increase in lymph 99mTc-DTPA
concentration after
H2O2
exposure suggest that some tracheal lymph was collected.
The lack of effect of H2O2 on lymph flow may therefore be due to the presence of "nontracheal" lymph, masking an increase in tracheal lymph flow. Alternative explanations are that luminal H2O2 does not cause edema or that edema formation is late in onset. An increase in tracheal lymph flow has been detected 4-24 h after tracheal injury with cotton smoke (1), but earlier measurements were not made.
The penetration of luminally applied low-molecular-weight tracers into tracheal lymph has not previously been demonstrated. As would be expected, the qualitative changes in tracer lymph-to-luminal concentration ratio mirror the venous-to-luminal concentration ratio. Thus epithelial damage increases the 99mTc-DTPA lymph-to-luminal concentration ratio but has much less effect on [14C]AP.
H2O2 and Tracheal Blood Flow
H2O2 produced a large and very rapid increase in tracheal blood flow. The mechanism has not been established. Acute ozone exposure also increases sheep bronchial blood flow (26). However, this was a slow onset effect at the concentrations used. The H2O2-induced vasodilatation may be due to release of prostaglandins or leukotrienes because H2O2 can stimulate arachidonic acid metabolism via both the cyclooxygenase and lipoxygenase pathways (5, 10, 25). However, nonprostanoids could be involved. For example, H2O2 produces a relaxation of rabbit airway smooth muscle that is partly due to an epithelium-dependent release of NO (11). Infusion of H2O2 into the pulmonary vasculature usually causes a thromboxane-mediated vasoconstriction (12, 25, 27). However, in precontracted pulmonary vessels an endothelium-independent relaxation due to a nonprostanoid occurs (6). H2O2 also relaxes dog coronary artery in vitro (23). This effect is partly endothelium dependent and is partly due to a direct action on the smooth muscle.Capsaicin
Capsaicin stimulates tracheal sensory nerves, particularly C fibers. Local intra-arterial injection of capsaicin into the dog tracheal vasculature causes a rapid vasoconstriction followed by a longer lasting vasodilation (24). In our study, luminal capsaicin caused only a vasodilation. Both central reflexes and local axon reflexes may be initiated by capsaicin, but no change in systemic blood pressure was detected, suggesting that local axon reflexes may be the primary route for the vasodilation.H2O2 exposure caused a tracheal vascular hyperresponsiveness to luminal capsaicin. A similar effect occurs with luminal histamine after tracheal epithelial damage induced by Triton X-100 (29). In contrast to histamine, the increased response to capsaicin is probably not due to an increase in its permeability because capsaicin, like [14C]AP, is very lipophilic (22). Further experiments would be needed to establish the mechanism of the hyperresponsiveness. There are at least four possible explanations; these are not mutually exclusive. First, epithelial damage may increase the exposure of sensory nerve endings. Second, H2O2 exposure may cause loss of neutral endopeptidase and therefore enhance the effects of neuropeptides released from sensory nerve endings. Third, H2O2 may in some way increase the sensitivity of sensory nerve endings to capsaicin itself. Finally, H2O2 might have a direct action on blood vessels that alters their responsiveness.
After reaching a peak during the first 5 min of capsaicin stimulation, arterial and venous blood flows decreased (Fig. 6). This occurred both in the presence and absence of epithelial damage, and the reason is unknown. Two possible explanations are that neuropeptide release decreases on maintained stimulation of sensory nerves, and, second, that the capsaicin concentration at sensory nerve endings decreased during the stimulation period (because, being lipophilic, the luminal concentration of capsaicin may drop markedly during 15 min).
Luminal capsaicin decreased venous 99mTc-DTPA concentration (Fig. 7). This may be a direct consequence of the increase in blood flow (see above). However, because venous 99mTc-DTPA output (99mTc-DTPA concentration × venous flow) was also decreased by capsaicin, vasodilation may be accompanied by other factors, for example, a redistribution of blood flow away from subepithelial capillaries, that influence the relationship between blood flow and venous 99mTc-DTPA concentration.
Capsaicin apparently produced a significantly greater reduction in venous 99mTc-DTPA concentration after H2O2 exposure. This is true when the results are expressed as percent changes; however, the absolute changes in venous 99mTc-DTPA concentration are even greater because the baseline PDTPA was increased initally by 50-fold by H2O2 exposure (Fig. 7). At least part of this apparent effect of capsaicin is due to the decreasing baseline venous 99mTc-DTPA concentration (Fig. 7), so firm conclusions cannot be drawn. Nevertheless, a similar effect occurred with luminal histamine in an earlier study, in which the baseline venous 99mTc-DTPA concentration after epithelial damage was more stable (30). In that study, the effect may have been due to the enhanced blood flow response, or removal of the epithelium may have altered the relationship between venous 99mTc-DTPA concentration and blood flow. It is conceivable that capsaicin could produce a similar effect. For example, if epithelial damage increased stimulation of sensory nerve endings by capsaicin, then enhanced edema formation would increase the diffusion distance for 99mTc-DTPA from lumen to capillary.
In summary, the epithelial damage produced by exposure of the tracheal lumen to H2O2 is associated with an increase in the penetration of 99mTc-DTPA into tracheal venous blood and lymph, whereas the PAP is not increased. H2O2 increased tracheal blood flow and also increased the vascular response to luminal capsaicin. These results suggest that the uptake of lipophilic drugs taken by inhalation will not be increased in the presence of epithelial damage, whereas the uptake of hydrophilic drugs will be greatly increased. Despite this, the effects of lipophilic drugs that stimulate C fibers in the airway wall may be enhanced by epithelial damage.
ACKNOWLEDGEMENTS
The authors thank Dr. D. Traber and L. Traber, the Shriners' Burn Institute, University of Texas Medical School, Galveston, TX, for teaching the technique for cannulating tracheal lymphatics, and Dr. Cathy Corbishley, Department of Histopathology, St. George's Hospital Medical School, for processing and photographing histological sections.
FOOTNOTES
Address for reprint requests: U. Wells, Dept. of Physiology, St. George's Hospital Medical School, London SW17 ORE, UK.
Received 15 April 1996; accepted in final form 24 September 1996.
REFERENCES
| 1. |
Barrow, R. E.,
S. E. Morris,
J. O. Basadre,
and
D. N. Herndon.
Selective permeability changes in the lungs and airways of sheep after toxic smoke inhalation.
J. Appl. Physiol.
68:
2165-2170,
1990.
|
| 2. |
Barrow, R. E.,
S. E. Morris,
H. A. Linares,
and
D. N. Herndon.
Tracheal venous blood and lymph collection: a model to study airway injury in sheep.
J. Appl. Physiol.
70:
1645-1649,
1991.
|
| 3. | Beasley, R., W. R. Roche, J. A. Roberts, and S. T. Holgate. Cellular events in mild asthma and after bronchial provocation. Am. Rev. Respir. Dis. 139: 806-817, 1989. [Medline] |
| 4. | Bhalla, D. K., S. M. Lavan, and T. T. Crocker. Airway permeability in rats exposed to ozone or treated with cytoskeleton-destabilising drugs. Exp. Lung Res. 14: 501-525, 1988. [Medline] |
| 5. | Burghuber, O. C., R. J. Strife, J. Zirrolli, P. M. Henson, J. E. Henson, M. M. Mathias, J. T. Reeves, R. C. Murphy, and N. F. Voelkel. Leukotriene inhibitors attenuate rat lung injury induced by hydrogen peroxide. Am. Rev. Respir. Dis. 131: 778-785, 1985. [Medline] |
| 6. | Burke-Wolin, T., P. D. Cherry, and G. H. Gurtner. Tone-dependent effects of peroxides in rabbit pulmonary artery and isolated perfused lung (Abstract). FASEB J. 4: A573, 1990. |
| 7. | Cantin, A. M., G. A. Fells, R. C. Hubbard, and R. G. Crystal. Antioxidant macromolecules in the epithelial lining fluid of the normal human lower respiratory tract. J. Clin. Invest. 86: 962-971, 1990. |
| 8. |
Cantin, A. M.,
S. L. North,
R. C. Hubbard,
and
R. G. Crystal.
Normal alveolar epithelial lining fluid contains high levels of glutathione.
J. Appl. Physiol.
63:
152-157,
1987.
|
| 9. |
Cheeseman, K. H.,
and
T. F. Slater.
An introduction to free radical biochemistry.
Br. Med. Bull.
49:
481-493,
1993.
|
| 10. |
Gao, Y.,
and
P. Vanhoutte.
Products of cyclooxygenase mediate the responses of the guinea-pig trachea to hydrogen peroxide.
J. Appl. Physiol.
74:
2105-2111,
1993.
|
| 11. |
Gupta, J. B.,
and
K. Prasad.
Mechanism of H2O2-induced modulation of airway smooth muscle.
Am. J. Physiol.
263:
L714-L722,
1992.
|
| 12. |
Gurtner, G. H.,
A. Knoblauch,
P. Smith,
H. Sies,
and
N. F. Adkinson.
Oxidant- and lipid-induced pulmonary vasoconstriction mediated by arachidonic acid metabolites.
J. Appl. Physiol.
55:
949-954,
1983.
|
| 13. | Hanafi, Z. The Control of Drug Clearance From the Tracheal Lumen of Experimental Animals (PhD thesis). London: University of London, 1993. |
| 14. |
Hanafi, Z.,
D. R. Corfield,
S. E. Webber,
and
J. G. Widdicombe.
Tracheal blood flow and luminal clearance of 99mTc-DTPA in sheep.
J. Appl. Physiol.
73:
1273-1281,
1992.
|
| 15. |
Hanafi, Z.,
S. E. Webber,
and
J. G. Widdicombe.
Permeability of ferret trachea in vitro to 99mTc-DTPA and [14C]antipyrine.
J. Appl. Physiol.
77:
1263-1273,
1994.
|
| 16. | Hulsmann, A. R., H. R. Raatgeep, J. C. den Hollander, T. Stijnen, P. R. Saxena, K. F. Kerrebijn, and J. C. de Jongste. Oxidative epithelial damage produces hyperresponsiveness of human peripheral airways. Am. J. Respir. Crit. Care Med. 149: 519-525, 1994. [Abstract] |
| 17. | Iyer, G. Y. N., D. M. F. Islam, and J. H. Quastel. Biochemical aspects of phagocytosis. Nature Lond. 192: 535-541, 1961. |
| 18. | Jeffery, P. K., A. J. Wardlaw, F. C. Nelson, J. V. Collins, and A. B. Kay. Bronchial biopsies in asthma-an ultrastructural, quantitative study and correlation with hyperreactivity. Am. Rev. Respir. Dis. 140: 1745-1753, 1989. [Medline] |
| 19. | Jeppsson, A. B., F. Sundler, A. Luts, B. Waldeck, and E. Widmark. Hydrogen peroxide-induced tracheal epithelial damage increases terbutaline transport in guinea-pig tracheal wall: implications for drug delivery. Pulm. Pharmacol. 4: 73-79, 1991. [Medline] |
| 20. | Laitinen, L. A., M. Heino, A. Laitinen, T. Kava, and T. Haahtela. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am. Rev. Respir. Dis. 131: 599-606, 1985. [Medline] |
| 21. | Lozewicz, S., C. Wells, E. Gomez, H. Ferguson, P. Richman, J. Devalia, and R. J. Davies. Morphological integrity of the bronchial epithelium in mild asthma. Thorax 45: 12-15, 1990. [Abstract] |
| 22. | Merck Index (12th ed.). Whitehouse Station, NJ: Merck & Co., 1996. |
| 23. | Rubanyi, G. M., and P. M. Vanhoutte. Oxygen-derived free radicals, endothelium and responsiveness of vascular smooth muscle. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H815-H821, 1986. |
| 24. | Salonen, R. O., S. E. Webber, and J. G. Widdicombe. Effects of neuropeptides and capsaicin on the tracheal vasculature in vivo. Br. J. Pharmacol. 95: 1262-1270, 1988. [Medline] |
| 25. | Seeger, W., N. Suttorp, F. Schmidt, and H. Neuhof. The glutathione redox cycle as a defense system against hydrogen peroxide induced prostanoid formation and vasoconstriction in rabbit lungs. Am. Rev. Respir. Dis. 133: 1029-1036, 1986. [Medline] |
| 26. | Schelegle, E. S., R. A. Gunther, G. H. Parsons, S. R. Colbert, M. A. A. Youssef, and C. E. Cross. Acute ozone exposure increases bronchial blood flow in conscious sheep. Respir. Physiol. 82: 325-336, 1990. [Medline] |
| 27. | Tate, R. M., K. M. VanBenthuysen, D. M. Shasby, I. F. McMurtry, and J. E. Repine. Oxygen-radical-mediated permeability, edema and vasoconstriction in isolated perfused rabbit lungs. Am. Rev. Respir. Dis. 126: 802-806, 1982. [Medline] |
| 28. |
Wells, U. M.,
Z. Hanafi,
and
J. G. Widdicombe.
Osmolality alters tracheal blood flow, tracer uptake and ion and water fluxes in sheep in vivo.
J. Appl. Physiol.
77:
2400-2407,
1994.
|
| 29. | Wells, U. M., Z. Hanafi, and J. G. Widdicombe. Tracheal epithelial damage alters the effects of luminal histamine on blood flow and tracer flux in anaesthetised sheep. Eur. Respir. J. 9: 78-85, 1996. [Abstract] |
| 30. |
Wells, U. M.,
A. J. Woods,
Z. Hanafi,
and
J. G. Widdicombe.
Epithelial damage alters the effects of tracheal osmolality on water and tracer fluxes in sheep in vivo.
J. Appl. Physiol.
78:
1921-1930,
1995.
|
| 31. | Welsh, M. J., D. M. Shasby, and R. M. Husted. Oxidants increase paracellular permeability in a cultured epithelial cell line. J. Clin. Invest. 76: 1155-1168, 1985. |
| 32. |
Yang, J.,
W. Mitzner,
and
C. Hirshman.
Role of the epithelium in airway smooth muscle responses to relaxant agonists.
J. Appl. Physiol.
71:
1434-1440,
1991.
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
