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Anesth Analg 2000;90:1445-1449
© 2000 International Anesthesia Research Society

GENERAL ARTICLES

Halothane Does Not Decrease Amiloride-Sensitive Alveolar Fluid Clearance in Rabbits

Vance G. Nielsen, MD*, Manuel S. Baird, MS*, Brian T. Geary, BS*, and Sadis Matalon, PhD{dagger}

Departments of *Anesthesiology and {dagger}Physiology and Biophysics, The University of Alabama at Birmingham, Birmingham, Alabama


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Halothane decreases alveolar fluid clearance (AFC), a function required for efficient gas exchange in the rat. Further, halothane decreases amiloride-sensitive Na+ transport in rat alveolar type II cells, a process responsible for a significant portion of AFC. We tested the hypothesis that halothane would decrease amiloride-sensitive AFC in rabbits. Rabbits anesthetized with 1.8% halothane had 5% albumin in 0.9% NaCl instilled into the right lung with (n = 11) or without (n = 11) 1 mM amiloride present in the instillate. Similarly, animals anesthetized with IV fentanyl and droperidol were administered 5% albumin solution with (n = 11) or without (n = 11) amiloride. At 90 min after instillation, alveolar fluid samples were obtained, and AFC was determined by changes in fluid protein concentration. Rabbits anesthetized with halothane or fentanyl and droperidol in the absence of amiloride had similar AFC values (35% ± 12% and 35% ± 7%, respectively, mean ± SD). Rabbits anesthetized with halothane or fentanyl and droperidol in the presence of amiloride had similar AFC values (20% ± 10% and 16% ± 12%, respectively) that were significantly less than the groups not administered amiloride (P < 0.01). Unlike the rat, the ability of the rabbit to clear fluid from the alveolar space through amiloride-sensitive pathways is not decreased by halothane anesthesia.

Implications: Unlike the rat, the ability of the rabbit to clear fluid from the alveolar space through amiloride-sensitive pathways is not decreased by halothane anesthesia.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The beneficial and adverse effects of volatile anesthetics on critical lung functions have recently been the focus of several investigators (1–6). Desflurane (1) and halothane (2) increase alveolar-capillary membrane permeability in vivo after hepatoenteric ischemia-reperfusion and acid aspiration, respectively. However, isoflurane administration decreases ischemia-reperfusion injury in isolated, perfused lungs (3). Halothane decreases surfactant biosynthesis (4) and vectorial Na+ transport (5) in alveolar type II cells in vitro. Of further interest, halothane anesthesia decreases alveolar fluid clearance (AFC) in the rat (6). Taken as a whole, these studies suggest that volatile anesthetics may alter vital lung functions under normal and pathologic conditions.

Alveolar fluid clearance is of particular clinical interest because an impairment of AFC in patients with acute lung injury or acute respiratory distress syndrome is associated with increased morbidity and mortality (7,8). Iso-osmolar clearance of alveolar fluid occurs via active alveolar epithelial Na+ transport through apical Na+ channels and basolateral Na+, K+ adenosinetriphosphatase (ATPase) activity (9). Water and Cl- passively follow Na+, affecting fluid clearance from the alveolar space. A significant fraction of AFC occurs via amiloride-sensitive Na+-dependent pathways in rabbits (10–12), rats (13), and humans (14). Given that AFC in the rat is decreased by both halothane and isoflurane in vivo (6) and that halothane decreases amiloride-sensitive Na+ absorption in alveolar type II cells in vitro (5), it is possible that volatile anesthetics could decrease the amiloride-sensitive AFC in vivo. Consequently, perhaps species other than the rat have total and amiloride-sensitive AFC decreased by volatile anesthetics. As the rabbit has been extensively used to investigate determinants of AFC during normal and pathological states (10–12,15,16), we proposed to determine whether the amiloride-sensitive portion of AFC in the rabbit would be decreased by halothane anesthesia as compared with IV fentanyl and droperidol anesthesia.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The study was approved by our animal review committee. Male New Zealand white rabbits (1.8–2.8 kg) were anesthetized with 10 mg/kg ketamine IV via a marginal ear vein. Anesthesia was maintained with either 60 µg · kg-1 · h-1 fentanyl IV and 3.0 mg · kg-1 · h-1 droperidol IV (n = 22) or 1.3 minimum alveolar anesthetic concentration halothane (1.8% inhaled, n = 22) (17). Halothane administration (inspired concentration) was monitored with an anesthetic-specific monitor (8100; BCI International, Waukesha, WI). After tracheotomy, a 3.5-mm inner diameter endotracheal tube was placed into the trachea. Rabbits were ventilated (fraction of inspired oxygen, 0.98–1.0, 98%–100%) with a ventilator (Harvard Apparatus, Southnatick, MA). Tidal volume and ventilation rate were adjusted to yield a peak inspiratory pressure between 10 and 15 mm Hg measured from within the endotracheal tube and a PaCO2 of 32–45 mm Hg. To insure relaxed chest wall muscle tone 0.3 mg · kg-1 · h-1 pancuronium bromide IV was administered. Arterial pressure was monitored by placement of a 22-gauge central ear artery catheter. All pressures were recorded on a polygraph (7D; Grass Instruments, Quincy, MA). All rabbits received a maintenance infusion of lactated Ringer’s solution at 20 mL · kg-1 · h-1, and esophageal temperatures were maintained at 38°-39°C with a heating pad. A 15-min equilibration period followed completion of the surgical preparation. The pHa, PaCO2, and PaO2 were determined after 15 min of equilibration and every 30 min thereafter, throughout the experimental period at 37°C.

After equilibration and 60 min of ventilation, a modified 4F Fogarty catheter was placed into the trachea next to the endotracheal tube. The Fogarty catheter was modified by removal of the balloon tip and placement of a yellow suture bootie that was punctured on its distal end. Placement of this catheter approximately 9–10 cm into the trachea resulted in an air-tight intubation of the right caudal lobe of the lung that was confirmed postmortem. A 4-mL/kg bolus of 5% fatty acid-free bovine serum albumin (BSA) dissolved in 0.9% NaCl was instilled into the right caudal lobe (over 2 min) of animals anesthetized with fentanyl and droperidol (n = 11) or halothane (n = 11). A similar bolus of BSA solution containing 1 mM amiloride was administered to the other rabbits anesthetized with fentanyl and droperidol (n = 11) or halothane (n = 11). The dead space in the modified Fogarty catheter was cleared by injection of 600 µL of 100% O2. The fluid in the right caudal lobe was subsequently withdrawn in 1-mL increments 90 min after instillation with the last 500 µL of fluid collected for analysis. The 500-µL samples were centrifuged at 1,000g for 5 min to pellet cells and debris, and the protein concentration was determined by a modification of a spectrophotometric method (18).

Expressed as percent of total instilled volume (excluding the volume of albumin), AFC was calculated from the following relationship, as described previously (11,13): Go


where the variables Ci and Ct are, respectively, the protein concentrations at time zero and 90 min. Time zero was considered to be the end of the instillation. Lastly, the concentration of amiloride in alveolar samples was determined by measuring the fluorescence intensity at an excitation of 360 nm and emission of 415 nm (19). All rabbits were killed with 1 mL of a saturated KCl solution after the 90-min alveolar sample was obtained.

To determine whether halothane directly decreases amiloride in 5% BSA in 0.9% NaCl, samples containing 1 mM amiloride had 1.8% halothane in 98% O2 (n = 6) or 100% O2 (n = 6) were bubbled through the solution for 1 min. The solutions were subsequently incubated in a 39°C water bath in air-tight sealed glass tubes for 90 min. The concentration of amiloride was then determined as previously mentioned (19).

All variables were expressed as mean ± SD. Analyses of the effects of the anesthetic administered and the presence of amiloride on AFC were conducted by using one-way analysis of variance (ANOVA). Analyses of the effects of the anesthetic administered and the presence of amiloride on hemodynamic and arterial blood gas variables were conducted by using repeated measures ANOVA. Post hoc analysis was conducted with the Tukey test after data analysis with ANOVA. Analysis of the anesthetic administered in vivo on alveolar amiloride concentration and in vitro on amiloride concentration was conducted with the Student’s t-tests. An {alpha} error of 0.05 was considered significant for one-way ANOVA and t-test analyses. A Bonferroni correction of the {alpha} error to <0.0125 was used during repeated ANOVA analyses in comparing the following combinations: 1) fentanyl and droperidol versus halothane; 2) fentanyl and droperidol with amiloride versus halothane with amiloride; 3) fentanyl and droperidol with amiloride versus fentanyl and droperidol; and 4) halothane versus halothane with amiloride.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
There was no significant difference in AFC between the group administered fentanyl and droperidol anesthesia and the group administered halothane anesthesia (Figure 1). Alveolar fluid clearance was significantly decreased by the administration of amiloride concomitant with either fentanyl and droperidol or halothane anesthesia. However, there was no significant difference in AFC between the two groups administered amiloride. Halothane anesthesia was associated with a significantly decreased alveolar amiloride concentration (0.48 ± 0.27 mM) compared with fentanyl and droperidol anesthesia (0.72 ± 0.20 mM). In vitro, samples exposed to 1.8% halothane in oxygen had amiloride concentrations (0.89 ± 0.01 mM) not significantly different from samples exposed to oxygen alone (0.85 ± 0.05 mM).



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  		Figure 1. Alveolar fluid clearance in rabbits anesthetized with fentanyl and droperidol or 1.3 minimum alveolar anesthetic concentration halothane in the presence or absence of amiloride. The anesthetic administered did not affect alveolar fluid clearance; however, amiloride administration significantly decreased alveolar fluid clearance. *P < 0.05 fentanyl and droperidol versus fentanyl and droperidol with amiloride, {dagger}P < 0.05 halothane versus halothane with amiloride.

 
With regard to hemodynamic variables, halothane anesthesia was associated with a significantly lower mean arterial pressure (MAP) and higher heart rate (HR) than fentanyl and droperidol anesthesia (Table 1). Compared with 15-min equilibration, values of MAP and HR were significantly lower after 60 min of ventilation and 90 min after instillation of 5% BSA solutions into the right lung during halothane administration. Amiloride administration did not affect MAP or HR. Peak inspiratory pressure increased significantly in all groups after placement of the modified Fogarty catheter and instillation of 5% BSA solutions compared with the 15-min equilibration and 60-min ventilation time points (Table 1).


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  		Table 1. Mean Arterial Pressure, Heart Rate and Peak Inspiratory Pressure Data
 
Arterial blood gas analyses demonstrated small, significant differences in pHa values at 15-min equilibration and 90 min after alveolar instillation of 5% BSA solution between the fentanyl and droperidol with amiloride and halothane with amiloride groups (Table 2). PaO2 was significantly lower 90 min after alveolar instillation of 5% BSA solution compared with all other time points in all groups.


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  		Table 2. Arterial Blood Gas Data
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Unlike the rat (6), the rabbit does not have AFC significantly decreased by the administration of halothane. Of further interest, the current study is the first to report that halothane does not affect the amiloride-sensitive fraction of AFC in vivo. The rabbit clears 5% BSA solutions instilled into the lung at a rate of approximately 20% to 25% per hour during fentanyl and droperidol anesthesia (12) and after more than five hours of halothane anesthesia (0.8% inhaled) (15,16). An AFC value of approximately 23% is observed in the rat during halothane anesthesia (2% inhaled for two hours), whereas an AFC value of 30% is observed concomitantly with pentobarbital anesthesia (6). Both Na+ absorption and Na+, K+ ATPase activity have been shown to decrease in the presence of 1%, 2%, or 4% halothane in vitro in rat alveolar type II cells (5). However, the mechanism by which halothane decreased both Na+ absorption and Na+, K+ ATPase activity could not be determined (5). This apparent species difference with regard to the effects of volatile anesthetics on AFC serves as an impetus for future investigation.

Another important finding was that halothane administration resulted in a significantly decreased alveolar amiloride concentration (approximately 0.5 mM) compared with animals anesthetized with fentanyl and droperidol (approximately 0.7 mM). Our in vitro experiments demonstrate that halothane does not directly decrease the amiloride concentration in 5% BSA in 0.9% NaCl. Amiloride is actively transported from the airways of sheep (20), so perhaps halothane may up-regulate this process in rabbits. The alveolar amiloride concentration observed after fentanyl and droperidol anesthesia was similar to that reported by us in rabbits anesthetized with 1% isoflurane (11) and in rabbits anesthetized with fentanyl and droperidol (12). Indeed, if the alveolar amiloride concentration was <0.5 mM in rabbits administered isoflurane, there was no discernible inhibition of AFC (11). Consequently, halothane-anesthetized rabbits had alveolar amiloride concentrations associated with ineffective inhibition of amiloride-sensitive AFC pathways that nevertheless inhibited AFC to a similar extent as that observed in rabbits administered fentanyl and droperidol. One explanation for this phenomenon is that, although halothane may increase alveolar amiloride clearance, halothane may paradoxically decrease AFC by synergistically interacting with the amiloride present to inhibit amiloride-sensitive AFC pathways. However, the tacit assumption of this hypothesis is that the alveolar amiloride concentration is the primary determinant of amiloride-mediated inhibition of AFC, and the pharmacokinetics and pharmacodynamics of amiloride dissolved in 5% BSA in the alveolar space have not been extensively investigated. Overall, depending on the anesthetic administered, differing alveolar amiloride concentrations may be associated with similar inhibition of the amiloride-sensitive fraction of AFC.

Our previous investigations of AFC in the rabbit reported that the amiloride-sensitive fraction of AFC two hours after instillation of BSA solution varied between 67% and 75% of total AFC (11,12). The current study reports an amiloride-sensitive fraction of approximately 50%, 90 minutes after instillation of 5% BSA solution. The only methodological difference between the present and previous (11,12) studies is that the rabbits were subjected to one hour of mechanical ventilation before placement of the modified Fogarty catheter and instillation of 5% BSA solution. In our previous investigations, the catheter was placed concomitantly with the endotracheal tube, removing the instilled lobe from the barotrauma associated with mechanical ventilation. Consequently, it may be possible that mechanical ventilation may adversely impact on amiloride-mediated inhibition of AFC.

In conclusion, halothane anesthesia, at clinically relevant concentrations, neither decreased total AFC nor decreased the amiloride-sensitive fraction of AFC in the rabbit as compared with fentanyl and droperidol anesthesia. Although we observed rabbits for a short time (2.5 hours), similar, total AFC values have been observed after several hours by other investigators by using rabbits anesthetized with halothane (18,19). The species difference (rat versus rabbit) in AFC in response to exposure to volatile anesthetics serve as a rational basis to determine how human AFC is affected by inhaled anesthetics. This issue is of great clinical interest as volatile anesthetics are routinely administered to patients with both normal and compromised pulmonary function.


   Acknowledgments
 
Supported, in part, by the American Heart Association (Southeast Affiliate, 9850201V), the National Institutes of Health Grants HL31197 and HL51173, and the Department of Anesthesiology, The University of Alabama at Birmingham, Birmingham, AL.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication February 25, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2000 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press