Kinetics of NO, air space absorption in isolated rat lungs EDWARD
M. POSTLETHWAIT,
Pulmonary Research Laboratories, Galveston, Texas 77555-0876
SHANNON Department
POSTLETHWAIT,EDWARDM., SHANN~ND.LANGFORD,AND AKHIL BIDANI. Kinetics of NO, air space absorption in isolated rat lungs. J. Appl. Physiol. 73(5): 1939-1945, 1992.-We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO, uptake displays reaction-mediated characteristics (J. Appl. Physiol. 68: 594-603, 1990). In vitro kinetic studies of pulmonary epithelial lining fluid (ELF) demonstrated that NO, interfacial transfer into ELF exhibits firstorder kinetics with respect to NO,, attains [N&]-dependent rate saturation, and is aqueous substrate dependent (J. Appl. Physiol. 71: 1502-1510,199l). We have extended these observations by evaluating the kinetics of NO, gas phase disappearance in isolated ventilating rat lungs. Transient exposures (23/lung at 25°C) employed rebreathing (NO,-air) from a noncompliant continuously stirred closed chamber. We observed that 1) NO, uptake rate is independent of exposure period, 2) NO, gas phase disappearance exhibited first-order kinetics {initial rate (r”) saturation occurred when [NOJ > 11 ppm}, 3) the mean effective rate constant (/Z*) for NO, gas phase disappearance ([NO,] s 11 ppm, tidal volume = 2.3 ml, functional residual capacity = 4 ml, ventilation frequency = 50/min) was 83 t 5 ml/min, 4) with [NO,1 5 11 ppm, k* and r” were proportional to tidal volume, and 5) NO, fractional uptakes were constant across [NO,] (~11 ppm) and tidal volumes but exceeded quasisteady-state observations. Preliminary data indicate that this divergence may be related to the inspired Pco,. These results suggest that NO, reactive uptake within rebreathing isolated lungs follows first-order kinetics and displays initial rate saturation, similar to isolated ELF. Differences observed between transient (rebreathing) and steady-state studies suggest an effect of inspired [NO,] on the distribution of air space NO2 and the epithelial surfaces participating in NO, reactive uptake. reactive gases; absorption kinetics; pulmonary dosimetry; rebreathing; nitrogen dioxide toxicology; isolated lungs
WE PREVIOUSLY presented evidence that reactive uptake constitutes a primary mode for absorption of inhaled nitrogen dioxide (NO,) from the pulmonary air space gas phase (5). Those in vitro exposures (rat lungs) were conducted under quasi-steady-state conditions where the inspired [NO,] ([NO,],), tidal volume (VT), functional residual capacity (FRC), and frequency (f) were experimentally fixed and pulmonary absorption was determined from the difference between [NO,li and expired [NO,]. Under similar in vitro quasi-steady-state exposures, we also observed that freshly isolated rat pulmonary epithelial lining fluid (ELF) displayed NO, absorption characteristics similar to those of the intact lung (6). In an effort to characterize the kinetics of NO, reactive absorp0161-7567/92 $2.00 Copyright
D. LANGFORD,
of Internal
Medicine,
AND
AKHIL
University
BIDANI
of Texas Medical Branch,
tion, we initially utilized transient exposure conditions to investigate the kinetics of NO, interfacial transfer to isolated ELF (8). The results demonstrated that the rate of NO, uptake by ELF displayed first-order kinetics with respect to NO, and was aqueous substrate dependent,. However, at initial gas phase [NOJ > 11 ppm, we observed a change in reaction order that produced saturation of the initial gas phase disappearance rate. Additional results and data from a corresponding study (‘7) demonstrated that NO, reactive uptake by biological systems appears to be localized near the gas-liquid interface, suggesting that little or no NO, diffuses unreacted through an overlying aqueous reactant layer. Surface-localized reaction-mediated absorption of an inhaled pollutant gas has important toxicological implications both in terms of site and extent of local dosimetry and in mechanisms of toxicity. Presumably, cytotoxicity must be initiated by the reaction product(s) that is formed as a consequence of NO, absorption. In addition, this mode of interfacial transfer would explain why acute NO, uptake in the lung is apparently independent of physical solubility, diffusion, and blood flow (5, S), because the epithelial surface layer functions as a reaction sink where NO, per se is rapidly removed from the system. Despite our observations of the determinants that govern NO, gas phase removal by model biological systems, whether these same governing characteristics held true within the intact ventilating lung was unclear. Because ventilation induces non-steady-state conditions at the pulmonary gas-liquid interfaces, it is implicit that interfacial interactions are ultimately characterized by kinetic descriptions to improve our ability to predict reactive gas dosimetry. Consequently, we have developed a nonsteady-state (transient) exposure isolated rat lung preparation that permits direct kinetic evaluations of NO, reactive uptake within the lung. The results suggest that, similar to freshly harvested ELF, pulmonary NO, uptake follows first-order kinetics with respect to NO,. In addition, the initial rate of NO, absorption is proportional to the VT and was also observed to saturate near [NO,], similar to that for ELF. This experimental system provides the methodology to determine true kinetic parameters of reactive gas uptake by a ventilating lung. METHODS Animals
gen-free
and lung isolation. The lungs from viral anti-
male Sprague-Dawley
0 1992 the American Physiological
Society
rats (Harlan
Sprague1939
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
1940
PULMONARY
ABSORPTION
KINETICS
OF NO,
SIGNAL TQ RECORDER 1
SAMPLE
PORT
/ L-------w \
TO VACUUM
VENTILATOR
FIG. 1. Schematic representation of transient exposure isolated lung apparatus. Dashed arrows, direction of gas flow used to control functional residual capacity and tidal volumes (VT). Upper vessel containing belt-driven gas phase stirrer is designated the “exposure flask.” Differential pressure transducers are labeled to indicate obtained measurements. Ptp, transpulmonary pressure.
Dawley, Houston, TX), SO-275 g body wt, were used for
driven by ventilator-induced
all experimental
tion of gas (artificial
preparations.
Animals were allowed free
access to food and water until induction of anesthesia with pentobarbital sodium (70 mg/kg ip). Depth of anesthesia was determined by foot pinch. The surgical procedure for lung isolation was unchanged from previous experimental protocols and has been described in detail elsewhere (5). Briefly, the trachea was cannulated, and positive support ventilation was started at the time of pneumothorax. The heart and lungs were exposed, and the pulmonary artery was cannulated via a trocar inserted through the right ventricle. The left atrium was removed, and the lungs were perfused free of blood by use of 50 ml of Krebs bicarbonate buffer containing 8.33 mM glucose and bovine serum albumin (5 g/100 ml, pH 7.4). The remaining heart tissue was resected, and the lungs were removed en bloc. Transient exposure (rebreathing) apparatus and procedure. Figure 1 is a schematic representation of the rebreathing isolated lung preparation. All components were constructed of glass, Teflon, or stainless steel. The lungs were suspended in an artificial thorax by the tracheal cannula, which extended through a Teflon-covered rubber stopper. End-expiratory pleural pressure (i.e., lung flask internal pressure) was controlled by positivepressure introduction and vacuum withdrawal of humidified 95% air-5% CO,. Flows were regulated and monitored by systems of high-accuracy needle valves and rotameters. Respiratory movements were controlled by cyclic changes in pleural pressure (subatmospheric)
withdrawal
and reintroduc-
thorax).
For NO, exposure, the tracheal cannula stopper was inserted into the bottom of the exposure flask (4,025 ml). The gas phase was continuously stirred. With a sample port slightly open to prevent pressurization, NO, (550 ppm in NJ was injected into the flask. The ports were closed, and the gas phase was allowed to mix for 1 min. Subsequently, a gas phase sample (1.5 ml) was collected via a glass syringe and a permanent stainless steel sampling needle and considered as the time 0 [NO,] (W0,1,)(J* At the t ime of gas phase sample collection, another aperature was vented slightly open to limit sampling-induced pressure changes within the exposure flask. The lungs were allowed to rebreathe the NO,-air for ~15 min, with gas phase samples collected approximately every 3 min {([NO&,). Then the tracheal cannula stopper was disconnected and the exposure flask flushed with room air while the lungs continued to ventilate room air. The 15-min exposure protocol was repeated twice more (total of 3Aung) for a total isolated ventilation time of
M. POSTLETHWAIT,
Pulmonary Research Laboratories, Galveston, Texas 77555-0876
SHANNON Department
POSTLETHWAIT,EDWARDM., SHANN~ND.LANGFORD,AND AKHIL BIDANI. Kinetics of NO, air space absorption in isolated rat lungs. J. Appl. Physiol. 73(5): 1939-1945, 1992.-We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO, uptake displays reaction-mediated characteristics (J. Appl. Physiol. 68: 594-603, 1990). In vitro kinetic studies of pulmonary epithelial lining fluid (ELF) demonstrated that NO, interfacial transfer into ELF exhibits firstorder kinetics with respect to NO,, attains [N&]-dependent rate saturation, and is aqueous substrate dependent (J. Appl. Physiol. 71: 1502-1510,199l). We have extended these observations by evaluating the kinetics of NO, gas phase disappearance in isolated ventilating rat lungs. Transient exposures (23/lung at 25°C) employed rebreathing (NO,-air) from a noncompliant continuously stirred closed chamber. We observed that 1) NO, uptake rate is independent of exposure period, 2) NO, gas phase disappearance exhibited first-order kinetics {initial rate (r”) saturation occurred when [NOJ > 11 ppm}, 3) the mean effective rate constant (/Z*) for NO, gas phase disappearance ([NO,] s 11 ppm, tidal volume = 2.3 ml, functional residual capacity = 4 ml, ventilation frequency = 50/min) was 83 t 5 ml/min, 4) with [NO,1 5 11 ppm, k* and r” were proportional to tidal volume, and 5) NO, fractional uptakes were constant across [NO,] (~11 ppm) and tidal volumes but exceeded quasisteady-state observations. Preliminary data indicate that this divergence may be related to the inspired Pco,. These results suggest that NO, reactive uptake within rebreathing isolated lungs follows first-order kinetics and displays initial rate saturation, similar to isolated ELF. Differences observed between transient (rebreathing) and steady-state studies suggest an effect of inspired [NO,] on the distribution of air space NO2 and the epithelial surfaces participating in NO, reactive uptake. reactive gases; absorption kinetics; pulmonary dosimetry; rebreathing; nitrogen dioxide toxicology; isolated lungs
WE PREVIOUSLY presented evidence that reactive uptake constitutes a primary mode for absorption of inhaled nitrogen dioxide (NO,) from the pulmonary air space gas phase (5). Those in vitro exposures (rat lungs) were conducted under quasi-steady-state conditions where the inspired [NO,] ([NO,],), tidal volume (VT), functional residual capacity (FRC), and frequency (f) were experimentally fixed and pulmonary absorption was determined from the difference between [NO,li and expired [NO,]. Under similar in vitro quasi-steady-state exposures, we also observed that freshly isolated rat pulmonary epithelial lining fluid (ELF) displayed NO, absorption characteristics similar to those of the intact lung (6). In an effort to characterize the kinetics of NO, reactive absorp0161-7567/92 $2.00 Copyright
D. LANGFORD,
of Internal
Medicine,
AND
AKHIL
University
BIDANI
of Texas Medical Branch,
tion, we initially utilized transient exposure conditions to investigate the kinetics of NO, interfacial transfer to isolated ELF (8). The results demonstrated that the rate of NO, uptake by ELF displayed first-order kinetics with respect to NO, and was aqueous substrate dependent,. However, at initial gas phase [NOJ > 11 ppm, we observed a change in reaction order that produced saturation of the initial gas phase disappearance rate. Additional results and data from a corresponding study (‘7) demonstrated that NO, reactive uptake by biological systems appears to be localized near the gas-liquid interface, suggesting that little or no NO, diffuses unreacted through an overlying aqueous reactant layer. Surface-localized reaction-mediated absorption of an inhaled pollutant gas has important toxicological implications both in terms of site and extent of local dosimetry and in mechanisms of toxicity. Presumably, cytotoxicity must be initiated by the reaction product(s) that is formed as a consequence of NO, absorption. In addition, this mode of interfacial transfer would explain why acute NO, uptake in the lung is apparently independent of physical solubility, diffusion, and blood flow (5, S), because the epithelial surface layer functions as a reaction sink where NO, per se is rapidly removed from the system. Despite our observations of the determinants that govern NO, gas phase removal by model biological systems, whether these same governing characteristics held true within the intact ventilating lung was unclear. Because ventilation induces non-steady-state conditions at the pulmonary gas-liquid interfaces, it is implicit that interfacial interactions are ultimately characterized by kinetic descriptions to improve our ability to predict reactive gas dosimetry. Consequently, we have developed a nonsteady-state (transient) exposure isolated rat lung preparation that permits direct kinetic evaluations of NO, reactive uptake within the lung. The results suggest that, similar to freshly harvested ELF, pulmonary NO, uptake follows first-order kinetics with respect to NO,. In addition, the initial rate of NO, absorption is proportional to the VT and was also observed to saturate near [NO,], similar to that for ELF. This experimental system provides the methodology to determine true kinetic parameters of reactive gas uptake by a ventilating lung. METHODS Animals
gen-free
and lung isolation. The lungs from viral anti-
male Sprague-Dawley
0 1992 the American Physiological
Society
rats (Harlan
Sprague1939
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
1940
PULMONARY
ABSORPTION
KINETICS
OF NO,
SIGNAL TQ RECORDER 1
SAMPLE
PORT
/ L-------w \
TO VACUUM
VENTILATOR
FIG. 1. Schematic representation of transient exposure isolated lung apparatus. Dashed arrows, direction of gas flow used to control functional residual capacity and tidal volumes (VT). Upper vessel containing belt-driven gas phase stirrer is designated the “exposure flask.” Differential pressure transducers are labeled to indicate obtained measurements. Ptp, transpulmonary pressure.
Dawley, Houston, TX), SO-275 g body wt, were used for
driven by ventilator-induced
all experimental
tion of gas (artificial
preparations.
Animals were allowed free
access to food and water until induction of anesthesia with pentobarbital sodium (70 mg/kg ip). Depth of anesthesia was determined by foot pinch. The surgical procedure for lung isolation was unchanged from previous experimental protocols and has been described in detail elsewhere (5). Briefly, the trachea was cannulated, and positive support ventilation was started at the time of pneumothorax. The heart and lungs were exposed, and the pulmonary artery was cannulated via a trocar inserted through the right ventricle. The left atrium was removed, and the lungs were perfused free of blood by use of 50 ml of Krebs bicarbonate buffer containing 8.33 mM glucose and bovine serum albumin (5 g/100 ml, pH 7.4). The remaining heart tissue was resected, and the lungs were removed en bloc. Transient exposure (rebreathing) apparatus and procedure. Figure 1 is a schematic representation of the rebreathing isolated lung preparation. All components were constructed of glass, Teflon, or stainless steel. The lungs were suspended in an artificial thorax by the tracheal cannula, which extended through a Teflon-covered rubber stopper. End-expiratory pleural pressure (i.e., lung flask internal pressure) was controlled by positivepressure introduction and vacuum withdrawal of humidified 95% air-5% CO,. Flows were regulated and monitored by systems of high-accuracy needle valves and rotameters. Respiratory movements were controlled by cyclic changes in pleural pressure (subatmospheric)
withdrawal
and reintroduc-
thorax).
For NO, exposure, the tracheal cannula stopper was inserted into the bottom of the exposure flask (4,025 ml). The gas phase was continuously stirred. With a sample port slightly open to prevent pressurization, NO, (550 ppm in NJ was injected into the flask. The ports were closed, and the gas phase was allowed to mix for 1 min. Subsequently, a gas phase sample (1.5 ml) was collected via a glass syringe and a permanent stainless steel sampling needle and considered as the time 0 [NO,] (W0,1,)(J* At the t ime of gas phase sample collection, another aperature was vented slightly open to limit sampling-induced pressure changes within the exposure flask. The lungs were allowed to rebreathe the NO,-air for ~15 min, with gas phase samples collected approximately every 3 min {([NO&,). Then the tracheal cannula stopper was disconnected and the exposure flask flushed with room air while the lungs continued to ventilate room air. The 15-min exposure protocol was repeated twice more (total of 3Aung) for a total isolated ventilation time of
