1 Department of Biomedical Engineering, Marquette University, Milwaukee 53201-1881; 2 Department of Pulmonary and Critical Care Medicine, 3 Department of Physiology, and 4 Departments of Anesthesiology and Pharmacology/Toxicology, Medical College of Wisconsin, Milwaukee 53226; and 5 Zablocki Veterans Affairs Medical Center, Department of Veterans Affairs, Milwaukee, Wisconsin 53295-1000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hydrogen peroxide generated by monoamine oxidase (MAO)-mediated deamination of biogenic amines has been implicated in cell signaling and oxidative injury. Because the pulmonary endothelium is a site of metabolism of monoamines present in the venous return, this brings into question a role for MAO in hyperoxic lung injury. The objective of this study was to evaluate the O2 dependency of the MAO reaction in the lung. To this end, we measured the pulmonary venous effluent concentrations of the MAO substrate [14C]phenylethylamine and its metabolite [14C]phenylacetic acid after the bolus injection of either phenylethylamine or phenylacetic acid into the pulmonary artery of perfused rabbit lungs over a range of PO2 values from 16 to 518 Torr. The apparent Michaelis constant for O2 was ~18 µM, which is more than an order of magnitude less that measured for purified MAO. The results suggest a minimal influence of high O2 on MAO activity in the normal lung and demonstrate the importance of measuring reaction kinetics in the intact organ.
multiple indicator dilution; mathematical modeling; endothelial cells; pargyline; semicarbazide
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MONOAMINE OXIDASES
(MAOs) purified from various tissues have been found to have rather
high Michaelis constants for O2
(K
The pulmonary endothelium is an important site of metabolism of certain monoamines present in the systemic venous return (15) and is subject to injury during high O2 exposure (13). This brings into question the influence of PO2 on pulmonary endothelial monoamine metabolism and a possible contribution of MAO-generated H2O2 to pulmonary hyperoxic injury. The discrepancy between results from purified enzymes and cell culture indicates that the O2 dependency of MAO activity for cells residing in the intact organ cannot necessarily be predicted from studies on such reduced systems. Thus the objective of this study was to evaluate the O2 dependency of the pulmonary endothelial MAO activity in intact lungs.
To this end, we developed a bolus injection multiple indicator dilution
(MID) method for measuring MAO kinetics in the intact organ where the
factors affecting substrate disposition can be more complex than in
either cell culture or purified enzyme systems (2-6, 10, 22,
38). Thus a key aspect of the approach is the ability to
separate the kinetics of substrate-tissue interactions (e.g., membrane
transport, enzymatic metabolism, nonspecific plasma and tissue protein
interactions) from each other and from the kinematics of organ
perfusion (e.g., perfusion heterogeneity, transit time distribution)
(4, 6, 10, 38). We measured the effect of
PO2 on the pulmonary venous effluent
concentration of the MAO substrate
-[ethyl-1-14C]phenylethylamine
hydrochloride ([14C]PEA) and its
[1-14C]phenylacetic acid ([14C]PAA)
metabolite after the bolus injection of either [14C]PEA
or [14C]PAA into the pulmonary artery of perfused rabbit
lungs. The apparent K
Glossary
B | Site of sequestration of PEA within cells |
CF(t) | 3H2O effluent concentration versus time curve |
Cin(t) = (q/F)hn(t) | Capillary input function |
CR(t) | FITC-dextran (Dex) effluent concentration versus time curve |
CT(t) | Indicator concentration versus time outflow curve for perfusion tubing system without the lung |
hc(t) | Capillary transit time distribution |
hn(t) | Noncapillary (arteries, veins, connecting tubing, and injection system) transit time distribution |
K1 and K2 | PEA and PAA endothelial surface equilibrium dissociation constants, respectively |
k![]() |
Cell sequestration rate constant for PEA |
[H2O][O2]k![]() |
MAO association rate constant |
K![]() |
Michaelis constant for O2 |
kmet = ([H2O][O2]k![]() ![]() |
Measure of the rate of PEA deamination by
pargyline-sensitive MAO, where ![]() |
kmet2 | Measure of the rate of PEA metabolism via semicarbazide-sensitive form of MAO (SSMAO) |
Keq2 and Keq1 | PEA and PAA plasma protein equilibrium dissociation constants, respectively |
Keq3 and Keq4 | PEA and PAA equilibrium dissociation constants for nonspecific intracellular associations, respectively |
kPAA = PS2/![]() |
Measure of PAA egress from the cells where ![]() |
kPEA = PS1/![]() |
Measure of PEA egress from the cells where ![]() |
kseq = k![]() ![]() |
Measure of the PEA sequestration rate within the lung tissue where
![]() |
MAO | Monoamine oxidase |
Pa and Pv | Arterial and venous pressures, respectively |
PAA | Phenylacetic acid |
PAAe(x,t) | (![]() ![]() |
PEA | Phenylethylamine |
PEAB | PEA bound to site of sequestration within cells |
PEAe(x,t) | (![]() ![]() |
PEAPre and PAAPre | PEA and PAA associated with nonspecific intracellular sites, respectively |
PEAPrv and PAAPrv | PEA and PAA bound to plasma protein, respectively |
[PAAc](x,t) | Vascular concentration of PAA at distance x from capillary inlet and time t |
[PAAe](x,t) | Endothelial cell concentration of PAA at distance x from capillary inlet and time t |
[PEAc](x,t) | Vascular concentration of PEA at distance x from capillary inlet and time t |
[PEAe](x,t) | Endothelial cell concentration of PEA at distance x from capillary inlet and time t |
Prv and Pre | Plasma and intracellular proteins, respectively |
PS1 and PS2 | Endothelial influx permeability-surface area products for PEA and PAA, respectively |
q | Mass of the injected indicator |
![]() |
Total organ flow |
Qc | Capillary volume |
Qe | Extravascular volume accessible to PEA and PAA |
Qv | Pulmonary vascular volume |
QW | Extravascular water volume |
Q1 and Q2 | Measures of the magnitude of the rapidly equilibrating nonspecific interactions of PEA and PAA, respectively, with luminal endothelial surface |
[R](x,t) | Vascular concentration of the vascular reference indicator at distance x from the capillary inlet (x = 0) and time t |
t | Time |
W | Average linear flow velocity within Qc |
x | Distance from the capillary inlet (x = 0) |
Z1 and Z2 | Nonspecific PEA and PAA surface binding sites, respectively |
![]() |
EXPERIMENTAL METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolated Rabbit Lung Preparation
As previously described (3, 6), each New Zealand White rabbit [2.68 ± 0.16 (SD) kg; n = 24; New Franken Research Rabbits, New Franken, WI] was given chlorpromazine hydrochloride (25 mg/kg im) followed by pentobarbital sodium (20-25 mg/kg) via an ear vein, heparinized (1200 IU/kg), and exsanguinated via a carotid artery catheter. After cannulation of the pulmonary artery and vein and the trachea, the lungs were removed from the chest and attached to the perfusion system primed with a physiological salt solution containing (in mM) 4.7 KCl, 2.51 CaCl2, 1.19 MgSO4, 2.5 KH2PO4, 118 NaCl, 25 NaHCO3, 5.5 glucose, and 4.5% bovine serum albumin (BSA) (3, 7). The perfusion system included a perfusate reservoir and a MasterFlex roller pump that pumped the perfusate from the reservoir into the pulmonary artery. In the recirculation mode, the perfusate drained from the left atrium back into the reservoir. Pulmonary arterial (Pa) and venous (Pv) pressures referenced to the level of the left atrium were monitored continuously. The lung was ventilated with 5% CO2 and, depending on the experimental condition studied, either 95, 15, or 0% O2 in N2 at 10 breaths/min with end-inspiratory and end-expiratory airway pressures of 7 and 2 cmH2O, respectively. Pulmonary arterial inflow and venous outflow PO2 values were measured with a Radiometer (Copenhagen, Denmark) O2 electrode, and bolus injections were made when the respective PO2 values reached 488 ± 10 (SE) and 548 ± 15 Torr for 95% O2, 107 ± 2 and 105 ± 2 Torr for 15% O2, and 20 ± 2 and 12 ± 2 Torr for 0% O2, with a PCO2 of 36.8 ± 6.0 (SD) Torr and pH of 7.36 ± 0.07 (SD) at 35°C after the change to the respective gas mixtures. For subsequent evaluation of the O2 dependency of MAO activity, the PO2 was taken to be the average of the inflow and outflow PO2 values measured immediately before the injection and after the sample collection. Although for the high and low PO2 values, the system had not completely equilibrated with the ventilating gas at the time the measurements were made, the values did not change significantly over the duration of the data collection period.To produce a bolus injection, a solenoid-operated injection loop (3) was situated in the inflow tubing so that a 1.0-ml bolus could be introduced into the inflow stream without changing the flow or pressure. Just before the injection, the ventilator was stopped at end expiration and the venous outflow was directed into the sample tubes of a modified (3) Gilson Escargot fraction collector. One hundred 2-ml samples were collected at a sampling interval ranging from 0.3 to 2.4 s depending on the flow as described in Experimental Protocols.
After each experiment, the lungs were removed from the perfusion system, and additional bolus injections were made at the various flows studied, with the arterial and venous cannulas connected directly together. The data from these injections were used to obtain the concentration versus time curves [CT(t)] and moments thereof (3, 6, 7) for the passage of the bolus through the tubing from injection to fraction collector in the absence of the lungs at each of the flows studied.
Bolus Composition
The 1.0-ml bolus contained 2.5 mg of fluorescein isothiocyanate-labeled 40,000 molecular weight dextran (FITC-Dex) and 0.1 µCi of either [14C]PEA or [14C]PAA. In 13 of the 24 lungs studied, the injected bolus also included 0.3 µCi of 3H2O. The specific activities for [14C]PEA and [14C]PAA were 50 and 52 mCi/mmol, respectively. The total volume containing the indicators was ~0.1 ml, with the balance composed of perfusate removed from the reservoir just before injection so that the injectate PO2 was approximately equal to that of the arterial inflow.Sample Composition
The concentration of FITC-Dex in the outflow samples was measured spectrophotometrically (494 nm) with a Bausch and Lomb (Rochester, NY) Spectronic 100 spectrophotometer. 3H and 14C were measured by liquid scintillation counting with a Packard Instruments (Downers Grove, IL) model 4530 liquid scintillation spectrometer. For samples collected after [14C]PEA injection, 1.0-ml aliquots were stored atThe identities of the compounds in the venous effluent samples collected after [14C]PEA injection were established with thin-layer chromatography (TLC) on silica gel 60 TLC plates developed in an ethyl acetate-isopropanol-25% NH4OH (50:35:10 vol/vol) mobile phase. With the use of authentic standards, the solute-to-solvent migration ratio values for PEA, PAA, and the intermediary metabolite phenylacetaldehyde were 0.46, 0.26, and 0.92, respectively. TLC analysis of selected peak venous outflow samples collected over the range of conditions studied demonstrated the presence of only [14C]PEA and [14C]PAA. After the identities of the 14C-labeled compounds in the venous effluent were established, [14C]PEA and [14C]PAA in each sample collected were separated with ion-exchange chromatography (43).
Bio-Rex 70 cation-exchange resin (200-400 mesh) was washed and equilibrated to pH 6.0 with 0.05 M sodium phosphate buffer. The resin was packed to a bed height of 1 cm in a plastic Poly-Prep column (Bio-Rad). The lyophilized 1.0-ml samples were redissolved in 2.0 ml of the pH 6.0 buffer before being passed through the columns. This was followed by two 1.0-ml water washes, and the total effluent from each column, which contained mostly [14C]PAA, was collected in a scintillation vial. The [14C]PEA was eluted from each column with two 2.0-ml aliquots of 0.25 M HCl, and the effluent was collected in a separate scintillation vial. The 14C counts in all samples were determined after the addition of 8 ml of Liquiscint (National Diagnostics, Atlanta, GA) on a Packard model 4530 liquid scintillation spectrometer. Recovery of 14C was >95%. The crossovers of PAA into PEA and of PEA into PAA were <0.5 and <5%, respectively, as measured with standards treated as the samples. These percentages include the inherent crossover of the ion-exchange separation procedure and any metabolism that might have occurred in postcollection samples (1). The venous effluent data measured after the bolus injection of [14C]PEA under the various experimental conditions described in Experimental Protocols were corrected for the crossover of PAA into PEA and of PEA into PAA.
Experimental Protocols
The initial experiments were carried out under the various experimental conditions and protocols required to provide the information necessary for the development of the kinetic model and to evaluate the influence of PO2 on the kinetics of the pulmonary disposition of PEA.Flow. One experimental approach for separating the various processes affecting the pulmonary disposition of a given indicator is to vary the flow (2, 3, 6), which, in turn, varies the time the injected indicators are in contact with the pulmonary endothelium. To determine a useful range of flows in this context, a bolus containing FITC-Dex and either [14C]PEA (n = 7 lungs) or [14C]PAA (n = 1 lung) was injected with the flow set at 400, 200, 100, or 50 ml/min at outflow sampling intervals of 0.3, 0.6, 1.2, or 2.4 s, respectively. The [14C]PEA and [14C]PAA bolus injections were carried out in different lungs, and the flow sequence for these and subsequent experiments was randomized. The lungs were ventilated with the high O2 gas mixture to maximize [14C]PEA metabolism. The effect of flow on the vascular volume was minimized by setting the mean pulmonary vascular pressure [(Pa + Pv)/2] at all flows to approximately equal that at 400 ml/min by adjusting the height of the venous outflow (2, 3, 6).
Once it was determined, as indicated in Estimation of Model Parameters, that the effluent concentration versus time data at the two extremes of this flow range provided sufficient information to separately identify the kinetic parameters descriptive of the pulmonary disposition of PEA, two flows, 400 and 50 ml/min, were used in subsequent experiments.MAO inhibition. To evaluate the role of MAO in the pulmonary metabolism of PEA and to provide a positive control for evaluating the ability of the kinetic analysis (see KINETIC MODEL) to distinguish between changes in PEA uptake and metabolism, experiments with the MAO inhibitors pargyline and semicarbazide (21, 43) were carried out by perfusing the lungs with perfusate containing 20 µM pargyline and 1.0 mM semicarbazide for 5 min before the [14C]PEA (n = 4 lungs) or [14C]PAA (n = 1 lung) bolus injections at 400 and 50 ml/min and high PO2. Pargyline (20 µM) and semicarbazide (1.0 mM) were also included in the injected boluses. The pargyline and semicarbazide concentrations were chosen because they had been previously shown to completely inhibit [14C]PEA metabolism by perfused rabbit lungs (21, 43).
Further evaluation of the separate effects of pargyline and semicarbazide on [14C]PEA metabolism was carried out in lungs perfused at 50 ml/min and ventilated with high O2 to provide the maximum window for detecting the effects of MAO inhibition. [14C]PEA was injected before and after the lung was perfused with perfusate containing either pargyline (20 µM) or semicarbazide (1.0 mM). Separate lungs were used for pargyline and semicarbazide, and the concentration of the MAO inhibitor in the injectate was the same as that in the perfusate during the injection-sampling period.Varying PO2. The O2 dependency of [14C]PEA and [14C]PAA pulmonary disposition was measured by injecting boluses containing either [14C]PEA or [14C]PAA at one or more of the O2 levels at 400 and 50 ml/min. During the transition to the low PO2, there was a transient increase in perfusion pressure, reflecting hypoxic vasoconstriction. This constriction had dissipated by the time the low PO2 had been reached as previously described (39).
Additional Experiments
At the 4.5% BSA concentration of the perfusate, 12% of [14C]PEA and 89% of [14C]PAA were albumin bound as measured with centrifugal ultrafiltration as previously described (3, 5, 6). The octanol-water partition coefficients for [14C]PEA and [14C]PAA at pH 7.4 were 0.123 and 0.047, respectively, measured as previously described (3, 5, 6).Drugs and Isotopes
[14C]PEA and [14C]PAA were obtained from American Radiolabeled Chemicals (St. Louis, MO). 3H2O was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). FITC-Dex, pargyline, and semicarbazide hydrochloride were obtained from Sigma (St. Louis, MO). BSA was the Bovuminar standard powder obtained from Intergen (Purchase, NY). The gases were obtained from Praxair (Waukesha, WI). All other chemicals used were of reagent grade. ![]() |
EXPERIMENTAL RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Figure 1 exemplifies the venous
[14C]PEA and [14C]PAA concentration curves
after pulmonary arterial injection of either [14C]PEA or
[14C]PAA over the range of perfusate flows indicated.
Figure 1, top, demonstrates that [14C]PEA is
extensively extracted by the lungs. For instance, at 400 ml/min, which
results in a capillary mean transit time of <1 s (5),
>70% of the injected [14C]PEA was extracted after a
single pass through the pulmonary circulation. The ability of the lungs
to metabolize [14C]PEA is demonstrated by the appearance
of [14C]PAA in the venous effluent after the injection of
[14C]PEA. At 400 ml/min, the amount of
[14C]PAA in the venous effluent was relatively low.
Decreasing the flow increased both [14C]PEA uptake and
the appearance of [14C]PAA. At 50 ml/min, >77% of the
injected [14C]PEA was recovered as [14C]PAA
in the venous effluent compared with ~21% at 400 ml/min as indicated
in Table 1 and Fig. 1.
|
|
The [14C]PAA curves obtained after [14C]PAA injection provide information about the pulmonary disposition of [14C]PAA independent of [14C]PEA metabolism, which is required for subsequent kinetic analysis. It is clear from the data in Fig. 1, bottom, that the rate of [14C]PAA uptake is much slower than that of [14C]PEA.
Treatment with both pargyline and semicarbazide decreased the
[14C]PAA concentration in the venous effluent after
[14C]PEA injection to undetectable levels at both high
and low flows (Fig. 2 and Table 1).
Figure 3C shows that treatment
with only pargyline decreased the fractional recovery of
[14C]PAA in the collected venous effluent samples after
[14C]PEA bolus injection at 50 ml/min by ~ 80%.
On the other hand, any effect of treatment with semicarbazide alone was
undetectable (Fig. 3B).
|
|
The O2 dependency of [14C]PEA metabolism in
the intact lung is exemplified in Fig. 4.
No O2 effect is detectable in the range of
PO2 values from 518 to 106 Torr. However, at a
PO2 of ~16 Torr, [14C]PAA
concentrations in the venous effluent after [14C]PEA
injection were lower (most notably at 50 ml/min) than those at a high
PO2 (Fig. 4).
|
Neither the MAO inhibitors nor PO2 had a significant effect on the [14C]PAA outflow curves after [14C]PAA bolus injection (see Estimation of Model Parameters). Thus only the PAA outflow curves measured after [14C]PAA bolus injection in lungs ventilated with the high PO2 gas mixture are shown (Fig. 1).
The fractions of injected [14C]PEA recovered in the collected venous effluent samples as [14C]PEA or [14C]PAA are given in Table 1 for each of the experimental conditions studied. The fractions of injected [14C]PAA and FITC-Dex recovered were 98.5 ± 2.7 (SD) and 95.2 ± 1.6%, respectively.
The Pa and Pv values at 400 and 50 ml/min under the various
experimental conditions studied are given in Table
2. The pulmonary vascular volume
(Qv) and extravascular water volume (QW)
calculated from the FITC-Dex, CR(t),
3H2O, CF(t), and tubing
CT(t) outflow curves (7) under the
various experimental conditions studied were 8.6 ± 1.3 (SD) and 6.8 ± 1.4 ml, respectively, and were not significantly
affected by experimental condition or flow.
|
![]() |
KINETIC MODEL |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reactions
The metabolism of PEA to PAA is a two-step reaction (21, 43) where the first step involves the oxidative deamination of PEA to the intermediary metabolite phenylacetaldehyde via MAO and the second step involves the oxidation of phenylacetaldehyde to PAA via aldehyde dehydrogenase. The kinetic model developed in the present study for the pulmonary disposition of PEA and PAA assumes the MAO reaction to be the limiting step (28). This assumption is consistent with the TLC results in the present study where only [14C]PEA and [14C]PAA could be detected in venous effluent samples collected after [14C]PEA injection. With this assumption, the metabolism of PEA to PAA can be summarized by the following reaction
![]() |
(a) |
![]() |
![]() |
(b) |
Single-Capillary Element
A single-capillary element of this model is composed of a capillary volume (Qc) and an extravascular volume (Qe) accessible to PEA or PAA. The spatial and temporal variations in the concentrations of the vascular indicator, PEA and PAA within Qc and Qe, are described by the following species balance equations based on the above reactions and the assumption that the O2 concentration was constant during the passage of a bolus
![]() |
(1) |
![]() |
(2) |
![]() |
(3) |
![]() |
![]() |
(4) |
![]() |
(5) |
The identifiable model parameters are kmet
(s1), which is the measure of the rate of PEA deamination
by MAO; kseq (s
1), which is the
measure of the PEA sequestration rate within the lung tissue;
PS1 (ml/s) and PS2
(ml/s), which are the endothelial permeability-surface area products
for PEA and PAA, respectively; kPEA
(s
1) and kPAA (s
1),
which are measures of the respective rate of PEA and PAA egress from
the cells; and the virtual volumes Q1 (ml) and
Q2 (ml), which are measures of the magnitude of the rapidly
equilibrating cell surface interactions of PEA and PAA, respectively.
For PAA injections, Eqs. 2-5 reduce to
Eqs. 4 and 5 with PEAe set to zero,
and the number of identifiable parameters reduces to three, namely
PS2 (ml/s), kPAA
(s
1), and Q2 (ml). The values of
1 and
2 were set at 1.14 and 9.1, respectively, based on the measured PEA-BSA and PAA-BSA binding.
Whole Organ
To construct an organ model from the single-capillary element model, the distribution of pulmonary capillary transit times [hc(t)] needs to be taken into account (4- 6, 10, 38). Previously, Audi et al. (5) estimated that for normal rabbit lungs in this perfusion system, the mean transit time (Estimation of the kinetic model parameters described in Estimation of Model Parameters involved numerically [finite difference method (4)] solving Eqs. 1-5 for the appropriate boundary conditions at each iteration of a Levenberg-Marquardt optimization routine (35). The time step was chosen by successively halving an initial time step until the coefficients of variation between the solutions of Eqs. 1-5 at successive time steps was <2%.
Estimation of Model Parameters
Preliminary investigation of the kinetic model behavior revealed that data from a single bolus injection are not sufficient to robustly estimate all of the identifiable model parameters. Previously, Audi et al. (2, 3, 6) demonstrated the utility of manipulating flow to reduce correlations between model parameters. In the present study, the range of flows studied was chosen as follows. Initial injections at 400 ml/min, which is in the range of rabbit cardiac output [~340 ml/min for a 2.7-kg rabbit (5)], revealed that the outflow concentration curves after [14C]PEA injections are dominated by information about PEA uptake while providing relatively little information on metabolism (Fig. 1A). We progressively reduced the flow until most of the effluent 14C injected as [14C]PEA was in the form of [14C]PAA rather than [14C]PEA. This occurred by 50 ml/min as seen in Fig. 1D. Therefore, the outflow curves measured at 400 and 50 ml/min after [14C]PEA or [14C]PAA injection were used for parameter estimation. The utility of these two flows for reducing correlations between model parameters, particularly [14C]PEA uptake and metabolism, is revealed by the sensitivity functions described in DISCUSSION.The first step in the parameter estimation procedure was to utilize the
[14C]PAA data after [14C]PAA injection to
estimate the PAA model parameters, namely PS2, kPAA, and Q2, independently of PEA
uptake and metabolism to PAA. This was accomplished by simultaneously
fitting the solutions of Eqs. 1, 4, and
5 with the initial conditions [R](x,0) = [PAAc](x,0) = 0 and
PAAe(x,0) = 0 and the boundary conditions
[R](0,t) = Cin(t); [PAAc](0,t) = (1/2)
Cin(t); and
PAAe(0,t) = 0 to the [14C]PAA
concentration versus time data measured after [14C]PAA
bolus injections at 400 and 50 ml/min.
Cin(t) = (q/
)hn(t) is the capillary
input concentration curve (2-6), where
hn(t) is the noncapillary (arteries, veins,
connecting tubing, and the injection system) transit time distribution,
and q and
are the mass of the injected indicator and
total flow through the organ, respectively.
Cin(t) is related to the vascular reference indicator curve CR(t) and the
hc(t) by the convolution relationship CR(t) = Cin(t)*hc(t) as
previously described (2-6). Table
3 shows the estimates of the PAA model
parameters and measures of precision of these estimates, namely the
95% confidence intervals and the correlation matrix (3,
30). To determine whether the values of the PAA model parameters
were affected by any of the experimental conditions, we compared the
model fit obtained using the parameter values estimated from each
individual experiment with the fit obtained using the mean set of PAA
model parameters values estimated from all the PAA experiments given in
Table 3. The F ratios (36) indicated that the
fits to the individual data sets using the mean parameters were not
significantly worse than those using individual parameters estimated
from each data set. Thus it is concluded that any effects of the
different experimental conditions were not detectable, and the kinetic
model parameters descriptive of the pulmonary disposition of PAA were
set to the mean values of 0.27 ml/ml of vascular volume, 0.063 s
1, and 0.50 ml · s
1 · ml
1 of vascular
volume for PS2/Qv,
kPAA, and Q2/Qv,
respectively. The normalization to the Qv measured for each
lung is to accommodate small differences in lung sizes.
|
Knowing the PAA kinetic parameters, the parameters descriptive of PEA
disposition, namely kmet (s1),
kseq (s
1),
PS1 (ml/s), kPEA (1/s),
and Q1 (ml) were estimated by fitting the model to the
[14C]PEA and [14C]PAA data obtained after
[14C]PEA injection. This was accomplished by
simultaneously fitting the solutions of Eqs.
1-5 with the initial conditions
[R](x,0) = [PEAc](x,0) = [PAAc](x,0) = 0 and
PEAe(x,0) = PAAe(x,0) = 0 and the boundary conditions
[R](0,t) = Cin(t);
[PEAc](0,t) = (1/
1)Cin(t); [PAAc](0,t) = 0; and
PEAe(0,t) = PAAe(0,t) = 0 to the [14C]PEA
and [14C]PAA data after [14C]PEA bolus
injections at 400 and 50 ml/min. With this parameter estimation
approach, the effects of MAO inhibitors and PO2
on the pulmonary disposition of PEA were evaluated.
Table 4 shows the estimated values of the
PEA model parameters and measures of precision of these estimates
(3, 30) under the various experimental conditions studied.
The resulting model fit is exemplified in Figs. 2 and 4. The estimated
values of the PEA model parameters are also shown in Table
5, where the extensive parameters
PS1 and Q1 were normalized to the
Qv to account for small differences in lung sizes. With
pargyline and semicarbazide treatment, kmet
became undetectable with little effect on PS1.
The effects on kseq and Q1 were
small but significant. In the range from 518 to 106 Torr,
PO2 had no significant effect on the PEA
kinetic parameters (Table 5). However, at
PO2 = 16 Torr, the estimated value of
kmet was significantly smaller than that
estimated at the higher PO2 levels. The effect
of PO2 = 16 Torr on
kseq was also significant. The apparent
K
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results indicate that the apparent
K
The O2 concentrations used to estimate
K
One aspect of studies on intact organs and cells that
distinguishes them from studies on purified enzymes is that the rate of
entry into the cells needs to be accounted for. In the above model, PEA
uptake is represented by a linear transport mechanism (9)
having a permeability-surface area product
(PS1). Previous investigations (21,
48) have concluded that this mechanism is passive diffusion.
Given the relatively low lipid solubility of PEA, its extensive
pulmonary uptake via passive diffusion is somewhat surprising
(48). The data in Figs. 1-4 and the relative rates of
uptake and metabolism indicate that the intracellular concentration of
PEA during bolus passage became much greater than the vascular
concentration. This would be consistent with a large PEA
tissue-to-perfusate partition coefficient, which in the model would be
a large
3Qe-to-
1Qc ratio.
Alternatively, if an active uptake mechanism were involved, the model
representation would be a larger permeability-surface area product for
PEA uptake (PS1) than for egress, the latter
being lumped with other processes in the group parameter
kPEA. These two effects are not separable with
the data obtained in the present study. However, with additional experimental protocols, the kinetic model and the MID method developed in the present study may be useful for further evaluation of the underlying PEA uptake mechanism.
Another difference between studies with intact organs or cells
and studies with purified enzymes is that in intact organs and cells,
competing processes such as substrate interactions with perfusate
constituents (e.g., plasma proteins) and with any number of cellular
and/or tissue constituents can also affect substrate availability by
altering its partitioning within the cells and between the medium
(perfusate) and the cells. These factors are represented in the model
by Q1 and Q2 for nonspecific cell surface
and/or tissue interactions, 1 and
2 for
perfusate albumin interactions, and
3,
4,
and kseq for intracellular interactions.
The major difference between intact organs and isolated cells is that in the intact organ, access to the cells is generally via the vascular system, which further complicates the evaluation of intracellular functions. For example, changing the flow by itself has a substantial effect on the extracted fraction of PEA (Fig. 1), and at a given flow, the overall extraction fraction is determined by contributions from the capillary pathways with different flows and/or transit times (4, 5, 10) and hence different extraction fractions. This is taken into account in the model by allowing for longitudinal spatial variations in the concentration of the injected indicators within a given capillary element (Eqs. 1-5), by representing the organ by parallel capillary elements with different transit times (4), and by weighing the contributions of these capillary elements according to hc(t) as previously discussed in more detail (4).
Previous studies (21, 43) have concluded that the endothelium is the main site for the MAO responsible for the oxidative deamination of PEA in the lung. Three forms of MAO have been identified in the rabbit lung (20, 21, 42, 43), including MAO-A, MAO-B, and a semicarbazide-sensitive form (SSMAO), with distinct subcellular localizations and substrate and/or inhibitor affinities. MAO-A and MAO-B are both located on the outer mitochondrial membrane (20, 21, 32, 42, 43), but MAO-A has a higher affinity for serotonin and norepinephrine and is selectively inhibited by clorgyline (21, 43), whereas MAO-B has a higher affinity for PEA and is more sensitive to inhibition by pargyline (21, 43). PEA is also a substrate for SSMAO, which is thought to be located on the plasma membrane (11, 17, 21, 43, 50). Roth and Gillis (43) and Gillis and Roth (21) found that treatment with pargyline followed by the addition of semicarbazide reduced PEA metabolism by 70 and ~100%, respectively. This is consistent with the results in the present study where treatment with pargyline alone decreased the fractional recovery of [14C]PAA in the collected venous effluent samples after a [14C]PEA bolus injection at 50 ml/min by ~80% (Fig. 3C) and that treatment with both pargyline and semicarbazide was needed for full inhibition (Fig. 2). However, the lack of effect of semicarbazide alone on PEA metabolism (Fig. 3B) has some interesting implications as indicated below.
In the kinetic model represented by Eqs.
2-5, there is no explicit accommodation for two
types of MAO. To evaluate the possible implications of this
simplification, we modified the kinetic model represented by Eqs.
1-5 to allow for PEA metabolism via both luminal surface (SSMAO) and intracellular MAO (MAO-B). This was accomplished by
substituting Eqs. 2 and 5 with Eqs. 6 and 7
![]() |
(6) |
![]() |
(7) |
The kinetic model represented by Eqs. 2-5 allows for the previously observed PEA sequestration within the cells (21, 43, 47). Table 1 shows that treatment with MAO inhibitors significantly decreased the total amount of 14C recovered in the venous effluent samples collected after a [14C]PEA bolus injection at 50 ml/min (47). This decrease in total recovery reveals competition between the two intracellular processes, namely [14C]PEA metabolism and sequestration. The normal rate of [14C]PEA metabolism (kmet) is faster than that of [14C]PEA sequestration (kseq) as shown in Table 5. Thus, in the absence of MAO inhibitors, most of the [14C]PEA extracted by the pulmonary endothelium was metabolized to [14C]PAA and ultimately returned to the perfusate (Fig. 1C). After treatment with MAO inhibitors, metabolism was no longer competing with sequestration for [14C]PEA, and hence a larger fraction of the PEA taken up was sequestrated and a smaller fraction of the injected 14C was recovered in the venous effluent. This competition between intracellular PEA metabolism and sequestration was not detectable at 400 ml/min (Table 1) because at this flow, less time was available for either [14C]PEA sequestration or metabolism than at the lower flow. Thus a larger fraction of the extracted PEA returned to the perfusate. This observation further demonstrates the utility of varying the flow for revealing competing parallel processes.
To help put this flow dependency (2, 3, 6) in perspective,
the normalized sensitivity function S(t) (6)
obtained for the PEA model parameters estimated from the
[14C]PEA and [14C]PAA data after
[14C]PEA injections at 400 and 50 ml/min are shown in
Fig. 6. For the ith model
parameter
i,
Si(t) =
C(t)/
i, where
C(t) is the calculated PEA or PAA indicator effluent
concentration. The sensitivity function
Si(t) was approximated by the change
in C(t) resulting from a 1% change in
i divided by the change in
i
(6). Multiplying Si(t)
by the value of the parameter estimate,
i,
provides an indication of the relative contribution of the parameter to
the model fit to the data at a given time (6). Comparison
of these normalized sensitivity functions reveals the extent and the
time epoch to which the optimized model parameters make their
contributions to the model fit. The shapes of the sensitivity function
relative to each other reveal how independent the contributions of the individual parameters are to the model fit. For example, at 400 ml/min,
the dominant role of PEA uptake in the model fit is revealed by the
sensitivity function of PS1 (Fig.
6A). The contribution of kmet at 400 ml/min is small relative to that for PS1 (Fig. 6A). At 50 ml/min, on the other hand,
kmet plays a dominant role, whereas the
contribution of PS1 is relatively small (Fig.
6D). Thus fitting the model to the data at both flows
reduces the correlation between model parameters by increasing the
extent of their contributions to the model fit to the data and by
extending and segregating the time epochs over which they make their
contributions.
|
For the above analysis, the data at only the 400 and 50 ml/min flows
were utilized for parameter estimation. However, in several experiments, data were collected at four flows within the same flow
range (Fig. 1). As one measure of the robustness of the above parameter
estimation approach, we determined whether dividing up the flow range
into the four smaller increments would have a significant impact on the
estimated values of the model parameters. To this end, the PEA model
parameters estimated by simultaneously fitting the solution of
Eqs. 1-5 to the [14C]PEA and
[14C]PAA data measured after [14C]PEA
injections at two and four flows were compared. The values of the
kinetic model parameters from two or four flows were not significantly
different (Table 6).
|
The MID method has been used to measure intracellular reactions in organs such as the heart (10) and liver (38). Although widely used in the lungs for measuring tissue composition (4, 7), transcapillary transport (2, 15, 22), and reactions that take place at the luminal endothelial surface (6, 15), its use for studying intracellular metabolism in the lungs has been limited (6, 7). The kinetic model represented by Eqs. 1-5 is similar to the one used by Pang et al. (38) to evaluate the intracellular metabolism of acetaminophen in the intact liver. The kinetic model and experimental protocol developed in the present study establish a basis for utilizing the MID method for evaluating intracellular functions such as MAO activity in the intact lung. The present results with this approach suggest a minimal influence of O2 concentration on the MAO reaction in the normal rabbit lung.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported by National Heart, Lung, and Blood Institute Grant HL-24349 and the Department of Veterans Affairs.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: S. H. Audi, Research Service 151, Zablocki VA Medical Center, 5000 W. National Ave., Milwaukee, WI 53295-1000 (E-mail: audis{at}marquette.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 7 March 2001; accepted in final form 5 June 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Al-Naji, AS,
and
Clarke DE.
Amine oxidase activity in commercial preparations of bovine serum albumin.
Life Sci
32:
635-643,
1983[ISI][Medline].
2.
Audi, SH,
Dawson CA,
Linehan JH,
Krenz GS,
Ahlf SB,
and
Roerig DL.
An interpretation of 14C-urea and 14C-primidone extraction in isolated rabbit lungs.
Ann Biomed Eng
24:
337-351,
1996[ISI][Medline].
3.
Audi, SH,
Dawson CA,
Linehan JH,
Krenz GS,
Ahlf SB,
and
Roerig DL.
Pulmonary disposition of lipophilic amine compounds in the isolated perfused rabbit lung.
J Appl Physiol
84:
516-530,
1998
4.
Audi, SH,
Linehan JH,
Krenz GS,
and
Dawson CA.
Accounting for the heterogeneity of capillary transit times in modeling multiple indicator dilution data.
Ann Biomed Eng
26:
914-930,
1998[ISI][Medline].
5.
Audi, SH,
Linehan JH,
Krenz GS,
Dawson CA,
Ahlf SB,
and
Roerig DL.
Estimation of the pulmonary transport function in isolated rabbit lungs.
J Appl Physiol
78:
1004-1014,
1995
6.
Audi, SH,
Olson LE,
Bongard RD,
Roerig DL,
Schulte ML,
and
Dawson CA.
Toluidine blue O and methylene blue as endothelial redox probes in the intact lung.
Am J Physiol Heart Circ Physiol
278:
H137-H150,
2000
7.
Audi, SH,
Roerig DL,
Ahlf SB,
Lin W,
and
Dawson CA.
Pulmonary inflammation alters the lung disposition of lipophilic amine indicators.
J Appl Physiol
87:
1831-1843,
1999
8.
Bakhle, YS,
and
Vane JR.
Pharmacokinetic function of the pulmonary circulation.
Physiol Rev
54:
1007-1045,
1974
9.
Bakhle, YS,
and
Youdim MBH
Metabolism of phenylethylamine in rat isolated perfused lung: evidence for monoamine oxidase 'type B' in lung.
Br J Pharmacol
56:
125-127,
1976[Abstract].
10.
Bassingthwaighte, JB,
Kroll K,
Schwartz LM,
Raymond GM,
and
King RB.
Strategies for uncovering the kinetics of nucleoside transport and metabolism in capillary endothelial cells.
In: Whole Organ Approaches to Cellular Metabolism, edited by Bassingthwaighte JB,
Goresky CA,
and Linehan JH.. New York: Springer-Verlag, 1998, p. 163-188.
11.
Ben-Harari, RR,
and
Bakhle YS.
Uptake of -phenylethylamine in rat isolated lung.
Biochem Pharmacol
29:
489-494,
1980[ISI][Medline].
12.
Ciccone, CD.
Free-radical toxicity and antioxidant medications in Parkinson's disease.
Phys Ther
78:
313-318,
1998[ISI][Medline].
13.
Clark, JM,
and
Lambertsen CJ.
Pulmonary oxygen toxicity: a review.
Pharmacol Rev
23:
37-133,
1971[ISI][Medline].
14.
Cohen, G,
and
Kesler N.
Monoamine oxidase and mitochondrial respiration.
J Neurochem
73:
2310-2315,
1999[ISI][Medline].
15.
Dawson, CA,
and
Linehan JH.
Biogenic amines. Evaluation of endothelial injury in the human lung.
In: Lung Cell Biology, edited by Massaro D.. New York: Dekker, 1989, vol. 41, chapt. 22, p. 1091-1135. (Lung Biol Health Dis Ser)
16.
Dominguez, J,
Troncoso P,
and
Martinez L.
Monoamine oxidase inhibition prevents ischemia reperfusion damage in rat kidneys.
Transplant Proc
27:
1839-1842,
1995[ISI][Medline].
17.
Enrique-Tarancon, G,
Marti L,
Morin N,
Lizcano JM,
Unzeta M,
Sevilla L,
Camps M,
Palacin M,
Testar X,
Carpene C,
and
Zorzano A.
Role of semicarbazide-sensitive amine oxidase on glucose transport and GLUT4 recruitment to the cell surface in adipose cells.
J Biol Chem
273:
8025-8032,
1998
18.
Fischer, AG,
Schulz AR,
and
Olinger L.
Thyroidal biosynthesis of iodothyronines. II. General characteristics and purification of mitochondrial monoamine oxidase.
Biochim Biophys Acta
159:
460-471,
1968[ISI][Medline].
19.
Fowler, CJ,
and
Callingham BA.
Substrate-selective activation of rat liver mitochondrial monoamine oxidase by oxygen.
Biochem Pharmacol
27:
1995-2000,
1978[ISI][Medline].
20.
Gewitz, MH,
and
Gillis CN.
Uptake and metabolism of biogenic amines in the developing lung.
J Appl Physiol
50:
118-122,
1981
21.
Gillis, CN,
and
Roth JA.
The fate of biogenic monoamine in perfused rabbit lung.
Br J Pharmacol
59:
585-590,
1977[Abstract].
22.
Harris, TR.
The transport of small molecules across the microvascular barrier as a measure of the permeability and functioning exchange area in the normal and acutely injured lung.
In: Whole Organ Approaches to Cellular Metabolism, edited by Bassingthwaighte JB,
Goresky CA,
and Linehan JH.. New York: Springer-Verlag, 1998, p. 495-516.
23.
Hauptmann, N,
Grimsby J,
Shih JC,
and
Cadenas E.
The metabolism of tyramine by monoamine oxidase A/B causes oxidative damage to mitochondrial DNA.
Arch Biochem Biophys
335:
295-304,
1996[ISI][Medline].
24.
Houslay, MD,
and
Tipton KF.
The reaction pathway of membrane-bound rat liver mitochondrial monoamine oxidase.
Biochem J
153:
735-750,
1973.
25.
Husain, M,
Edmondson DE,
and
Singer TP.
Kinetic studies on the catalytic mechanism of liver monoamine oxidase.
Biochemistry
21:
595-600,
1982[ISI][Medline].
26.
Jones, DP.
Benzylamine metabolism at low O2 concentrations.
Biochem Pharmacol
33:
413-417,
1984[ISI][Medline].
27.
Katz, IR,
Wittenberg JB,
and
Wittenberg BA.
Monoamine oxidase, an intracellular probe of oxygen pressure in isolated cardiac myocytes.
J Biol Chem
259:
7504-7509,
1984
28.
Klingman, GI,
and
Klingman JD.
Monoamine oxidase activity in peripheral organs and adrenergic tissues of the rat.
Biochem Pharmacol
15:
77-91,
1966[ISI][Medline].
29.
Kohn, HI.
Tyramine oxidase.
Biochem J
31:
1693-1704,
1937.
30.
Landaw, EM,
and
DiStefano JJ, III.
Multiexponential, multicompartmental, and noncompartmental modeling. II. Data analysis and statistical considerations.
Am J Physiol Regulatory Integrative Comp Physiol
246:
R665-R677,
1984[ISI][Medline].
31.
Lewinsohn, R,
Bohm KH,
Glover V,
and
Sandler M.
A benzylamine oxidase distinct from monoamine oxidase B-widespread distribution in man and rat.
Biochem Pharmacol
27:
1857-1863,
1978[ISI][Medline].
32.
Lizcano, JM,
Tipton KF,
and
Unzeta M.
Purification and characterization of membrane-bound semicarbazide-sensitive amine oxidase (SSAO) from bovine lung.
Biochem J
331:
69-78,
1998[ISI][Medline].
33.
Lizcano, JM,
Tipton KF,
and
Unzeta M.
Time-dependent activities of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes.
Biochem J
351:
789-794,
2000[ISI][Medline].
34.
Malorni, W,
Giammarioli AM,
Matarrese P,
Pietrangeli P,
Agostinelli E,
Ciaccio A,
Grassilli E,
and
Mondovi B.
Protection against apoptosis by monoamine oxidase A inhibitors.
FEBS Lett
426:
155-159,
1998[ISI][Medline].
35.
Marquardt, DW.
An algorithm for least-squares estimation of nonlinear parameters.
J Soc Ind Appl Math
11:
431-441,
1963[ISI].
36.
Motulsky, HJ,
and
Ransnas LA.
Fitting curves to data using nonlinear regression: a practical and nonmathematical review.
FASEB J
1:
365-374,
1987
37.
Oi, S,
Shimada K,
Inamasu M,
and
Yasunobu KT.
Mechanistic studies of beef liver mitochondrial amine oxidase. XVIII. Amine oxidase.
Arch Biochem Biophys
139:
28-37,
1970[ISI][Medline].
38.
Pang, KS,
Barker F,
Simard A,
Schwab AJ,
and
Goresky CA.
Sulfation of acetaminophen by the perfused rat liver: the effect of red blood cell carriage.
Hepatology
22:
267-282,
1995[ISI][Medline].
39.
Peake, MD,
Harabin AL,
Brennan NJ,
and
Sylvester JT.
Steady-state vascular responses to graded hypoxia in isolated lungs in five species.
J Appl Physiol
51:
1214-1219,
1981
40.
Pizzinat, N,
Copin N,
Vindis C,
Parini A,
and
Cambon C.
Reactive oxygen species production by monoamine oxidases in intact cells.
Naunyn Schmiedebergs Arch Pharmacol
359:
428-431,
1999[ISI][Medline].
41.
Raimondi, L,
Banchelli G,
Sgromo L,
Pirisino R,
Ner M,
Parini A,
and
Cambon C.
Hydrogen peroxide generation by monoamine oxidases in rat white adipocytes: role of cAMP production.
Eur J Pharmacol
395:
177-182,
2000[ISI][Medline].
42.
Roth, JA,
and
Gillis CN.
Deamination of -phenylethylamine by monoamine oxidase
inhibition by imipramine.
Biochem Pharmacol
23:
2537-2545,
1974[ISI][Medline].
43.
Roth, JA,
and
Gillis CN.
Multiple forms of amine oxidase in perfused rabbit lung.
J Pharmacol Exp Ther
194:
537-544,
1975[Abstract].
44.
Tipton, KF.
The reaction pathway of pig brain mitochondrial monoamine oxidase.
Eur J Biochem
5:
316-320,
1968[ISI][Medline].
45.
Tipton, KF.
Some properties of monoamine oxidase.
Adv Biochem Psychopharmacol
5:
11-24,
1972[Medline].
46.
Vindis, C,
Seguelas MH,
Bianchi P,
Parini A,
and
Cambon C.
Monoamine oxidase B induces ERK-dependent cell mitogenesis by hydrogen peroxide generation.
Biochem Biophys Res Commun
271:
181-185,
2000[ISI][Medline].
47.
Wu, PH,
and
Boulton AA.
Metabolism, distribution and disappearance of injected -phenylethylamine in the rat.
Can J Biochem
53:
42-50,
1974[ISI].
48.
Youdim, MBH,
Bakhle YS,
and
Ben-Harari RR.
Inactivation of monoamines by the lungs.
Ciba Found Symp
78:
105-128,
1980[Medline].
49.
Youdim, MBH,
and
Woods HF.
The influence of tissue environment on the rates of metabolic processes and the properties of enzymes.
Biochem Pharmacol
24:
317-323,
1975[ISI][Medline].
50.
Yu, PH,
and
Zuo DM.
Oxidative deamination of methylamine by semicarbazide-sensitive amine oxidase leads to cytotoxic damage in endothelial cells. Possible consequences for diabetes.
Diabetes
42:
594-603,
1993[Abstract].
51.
Zhang, J,
and
Piantadosi CA.
Prevention of H2O2 generation by monoamine oxidase protects against CNS O2 toxicity.
J Appl Physiol
71:
1057-1061,
1991
52.
Zychlinski, L,
and
Montgomery MR.
Species differences in lung mitochondrial monoamine oxidase activities.
Comp Biochem Physiol C Pharmacol Toxicol Endocrinol
86:
325-328,
1987[ISI].