Cellular acid-base status has been found to exert selective actions on the effector functions of ... more Cellular acid-base status has been found to exert selective actions on the effector functions of activated macrophages (mφ). We examined the effects of extracellular pH (pH o ) on the production of tumour necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in resident alveolar mφ. Cells were obtained by bronchoalveolar lavage of rabbits, activated in vitro with LPS, and cultured at pH o 5.5, 6.5 or 7.4 for up to 18 h. The relative abundance of TNF-α mRNA peaked at " 2 h. The peak transcript abundance was increased at lower pH o values. This finding probably reflected pre-transcription/transcription effects of pH, in as much as the stability of TNF-α mRNA induced with phorbol ester was unaffected by the experimental pH o values. TNFα secretion by LPS-treated mφ decreased at lower pH o values. The TNF-α content of mφconditioned media decreased progressively with decrements in pH o . The reduced TNF-α secretion at pH o 5.5 was accompanied by an increase in the cytosolic TNF-α content (compared with that at pH o 7.4), indicating that pH o altered TNF-α secretion due, in part, to the intracellular retention of synthesized cytokine (i.e. a post-translation effect). The data show that pH o has multiple effects (pre-transcription/transcription and post-translation) on TNF-α production induced by LPS in resident alveolar mφ. These results suggest that the role of alveolar mφ in inflammatory responses is modulated by pH o , which may be important in tumours/ abscesses and sites of infection where the external milieu is acidic.
Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 5... more Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 594-603, 1990 and J. Appl: Physiol. 69: 523-531, 1990), have suggested that the steady-state absorption of inhaled NO2 is mediated by chemical reaction(s) between NO2 and ELF solute reactants. To characterize the kinetics of NO2 absorption into aqueous biological substrates across a gas-liquid interface, we utilized a closed system of known geometry and initial gas phase [NO2] [([NO2]g)0] to expose ELF (as bronchoalveolar lavage; BAL) and a biochemical model system (glutathione, GSH). Assessments of NO2 reactive uptake, into both GSH and ELF, indicated first-order NO2 kinetics [([NO2]g)0 less than or equal to 10.5 ppm] with effective rate constants of (kNO2)GSH = 4.8 and (kNO2)BAL = 2.9 ml.min-1.cm-2 (stirred). Above 10.5 ppm (1 mM GSH), zero-order kinetics were observed. Both (kNO2)GSH and (kNO2)BAL showed aqueous reactant dependence. The reaction order with respect to GSH and BAL was 0.47 and 0.64, respectively. We found no effect of interfacial surface area or bulk phase volume on kNO2. In unstirred systems, significant interfacial resistance was observed and was related to reactant concentration. These results indicate that NO2 reactive uptake follows first-order kinetics with respect to NO2 ([NO2]g less than or equal to 10.5 ppm) and displays aqueous substrate dependence. Furthermore the site of reactive absorption appears to be limited to near the aqueous surface interface. Unstirred conditions confine interfacial mass transfer kinetics in a dose-dependent manner. These phenomenological coefficients may provide the basis for direct extrapolation to environmentally relevant exposure concentrations.
The role of plasmalemmal V-type H+ translocating ATPase (V-ATPase) in regulation of intracellular... more The role of plasmalemmal V-type H+ translocating ATPase (V-ATPase) in regulation of intracellular pH (pHi) is unclear in monocytes. This study examined the plasmalemmal V-ATPase and Na+/H+ exchanger (NHE) in U937 human monocytes. The fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pHi and the kinetics of pHi recovery from cytosolic acid-loads (NH4Cl prepulse). Bafilomycin A1 and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were used to delineate the activities of the H+-pump and NHE, respectively. Baseline pHi was approximately 7.13 at an extracellular pH (pHo) of 7.4 and fell progressively at lower pHo values. EIPA had no effect on baseline pHi at pHo 7.4, but caused a sustained decrement in pHi at pHo 6.0-7.0. Bafilomycin A1 had biphasic effects on baseline pHi at pHo 6.5-7.4; pHi declined approximately 0.1 units over the course of several minutes and then recovered. At pHo 6.0, bafilomycin A1 caused a sustained decrement in baseline pHi. Recovery from the bafilomycin-induced acidosis at pHo 6.5-7.4 was prevented by EIPA. Similarly, pHi recovery from NH4Cl prepulse acid-loads (pHo 7.4) was sensitive to both EIPA and bafilomycin A1. During this recovery process, Na+/H+ exchange (EIPA-sensitive component of apparent H+ efflux) was the predominant mechanism for H+ extrusion at acid-loaded pHi values < 7.0. At acid-loaded pHi values > or = 7.0, the V-ATPase (bafilomycin-sensitive component) and NHE contributed almost equally to H+ extrusion. The data provide the first evidence that plasmalemmal V-ATPase participates in pHi regulation in U937 cells. The H+-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocytes.
The present study determined the effects of cotton smoke inhalation on the functioning of alveola... more The present study determined the effects of cotton smoke inhalation on the functioning of alveolar macrophages (mphi). Smoke inhalation led to dose-dependent impairment of respiratory gas exchange by 48 h postexposure and pulmonary edema by 96 h. Maximal effects were observed in animals ventilated with 54 breaths of cotton smoke (3-min exposure, 18 breaths/min). Macrophages were obtained at 48 h postexposure by bronchoalveolar lavage of rabbits subjected to 54 breaths of smoke or room air (control). Phagocytosis of opsonized bacteria and adherence to solid substratum were reduced in smoke-exposed mphi. Smoke inhalation primed mphi for release of tumor necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS). Smoke-exposed mphi were also primed for TNF-alpha release induced by phorbol myristate acetate, which suggests that the priming event occurred downstream of protein kinase C activation in the signal transduction pathway. Further, smoke exposure attenuated the inhibitory effects of phosphodiesterase inhibitors on LPS-induced TNF-alpha release. Thus, the priming event may be mediated through cAMP and/or protein kinase A. The data indicate that cotton smoke inhalation suppresses the antimicrobial activities of alveolar mphi and can lead to excessive mphi production of TNF-alpha. These mphi effects would be expected to contribute to the pathophysiological abnormalities associated with smoke inhalation injury.
The present studies determined the effects of extracellular pH (pH o ) on the production of tumou... more The present studies determined the effects of extracellular pH (pH o ) on the production of tumour necrosis factor-α (TNF-α) in the macrophage-like cell lines RAW 246.7 and J774 A.1. The cells were activated with lipopolysaccharide (LPS) at pH o 5.5, 6.5 or 7.4. TNF-α gene transcription was monitored by Northern blot analysis. Synthesis of the cytokine was monitored by ELISA measurements of the TNF-α content of cell-conditioned media (extracellularly released TNF-α) and cell lysates (cytosolic TNF-α). The magnitude of the TNF-α response differed markedly between the two cell lines. RAW cells were more responsive to LPS than were J774 cells. However, the effects of pH o on TNF-α production were similar in the two cell lines. TNF-α gene transcription was insensitive to experimental pH o . The pH o had no effect on the abundance of TNF-α mRNA at 2, 4 or 18 h. Nonetheless, synthesis of TNF-α was affected significantly by pH o . The TNF-α contents of cell-conditioned medium and cell lysate at 18 h were reduced progressively at lower pH o values. The data indicate that pH o alters TNF-α production in RAW and J774 cells at a post-transcriptional level. These findings suggest that pH o influences the phenotypic responses of macrophages to activating stimuli and modifies the role that macrophages play in inflammatory and immune actions.
American Journal of Physiology Lung Cellular and Molecular Physiology, Aug 1, 1995
Three different mechanisms interact to control the cytosolic pH (pHi) of alveolar macrophages (M ... more Three different mechanisms interact to control the cytosolic pH (pHi) of alveolar macrophages (M phi), namely, plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase), Na+/H+ exchange, and Na(+)-independent HCO3-/Cl- exchange. To investigate the activity of plasmalemmal V-ATPase in alveolar M phi, we developed a nonlinear mathematical model of pHi regulation that incorporates the biophysical determinants of pHi and the fluxes of individual acid-base equivalents. The model was used to analyze the acid-base responses of rabbit alveolar M phi to a weak acid (propionic acid) under conditions that favored V-ATPase-mediated effects (presence of 1 mM amiloride and nominal absence of CO2). The pHi was measured using the fluorescent probe, 2',7'-biscarboxethyl-5,6-carboxyfluorescein. M phi exposure to propionic acid caused a rapid fall in pHi. Recovery of pHi after acid loading varied directly with the magnitude of the acid load. Mathematical analyses showed that pHi recovery was hindered by persistent influx of propionic acid driven, in turn, by transporter-mediated H+ extrusion and propionate efflux. Eventually, a new steady state was established in which propionate and H+ were cycled out of the M phi and propionic acid was recycled into the cell. As a consequence, model predictions of the rate of V-ATPase-mediated H+ efflux were almost twice that estimated from experimental determinations of the initial rate of pHi recovery.
Lung carbonic anhydrase (CA) participates directly in plasma CO2-HCO3(-)-H+ reactions. To charact... more Lung carbonic anhydrase (CA) participates directly in plasma CO2-HCO3(-)-H+ reactions. To characterize pulmonary CA activity in situ, CO2 excretion and capillary pH equilibration were examined in isolated saline-perfused rat lungs. Isolated lungs were perfused at 25, 30, and 37 degrees C with solutions containing various concentrations of HCO3- and a CA inhibitor, acetazolamide (ACTZ). Total CO2 excretion was partitioned into those fractions attributable to dissolved CO2, uncatalyzed HCO3- dehydration, and catalyzed HCO3- dehydration. Approximately 60% of the total CO2 excretion at each temperature was attributable to CA-catalyzed HCO3- dehydration. Inhibition of pulmonary CA diminished CO2 excretion and produced significant postcapillary perfusate pH disequilibria, the magnitude and time course of which were dependent on temperature and the extent of CA inhibition. The half time for pH equilibration increased from approximately 5 s at 37 degrees C to 14 s at 25 degrees C. For the HCO3- dehydration reaction, pulmonary CA in situ displayed an apparent inhibition constant for ACTZ of 0.9-2.2 microM, a Michaelis-Menten constant of 90 mM, a maximal reaction velocity of 9 mM/s, and an apparent activation energy of 3.0 kcal/mol.
We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO2 uptake di... more We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO2 uptake displays reaction-mediated characteristics (J. Appl. Physiol. 68: 594-603, 1990). In vitro kinetic studies of pulmonary epithelial lining fluid (ELF) demonstrated that NO2 interfacial transfer into ELF exhibits first-order kinetics with respect to NO2, attains [NO2]-dependent rate saturation, and is aqueous substrate dependent (J. Appl. Physiol. 71: 1502-1510, 1991). We have extended these observations by evaluating the kinetics of NO2 gas phase disappearance in isolated ventilating rat lungs. Transient exposures (2-3/lung at 25 degrees C) employed rebreathing (NO2-air) from a non-compliant continuously stirred closed chamber. We observed that 1) NO2 uptake rate is independent of exposure period, 2) NO2 gas phase disappearance exhibited first-order kinetics [initial rate (r*) saturation occurred when [NO2] > 11 ppm], 3) the mean effective rate constant (k*) for NO2 gas phase disappearance ([NO2] < or = 11 ppm, tidal volume = 2.3 ml, functional residual capacity = 4 ml, ventilation frequency = 50/min) was 83 +/- 5 ml/min, 4) with [NO2] < or = 11 ppm, k* and r* were proportional to tidal volume, and 5) NO2 fractional uptakes were constant across [NO2] (< or = 11 ppm) and tidal volumes but exceeded quasi-steady-state observations. Preliminary data indicate that this divergence may be related to the inspired PCO2. These results suggest that NO2 reactive uptake within rebreathing isolated lungs follows first-order kinetics and displays initial rate saturation, similar to isolated ELF.(ABSTRACT TRUNCATED AT 250 WORDS)
Alveolar macrophages (m) participate in inflammatory and immune responses in acidic microenvironm... more Alveolar macrophages (m) participate in inflammatory and immune responses in acidic microenvironments such as the interstitial fluids of tumors and abscesses. Two plasmalemmal H + extruders interact to control the acid-base status of alveolar m, namely a V-type H + pump (V-ATPase) and a Na + /H + exchanger. The present study examined the effects of extracellular pH (pH o ) and H + transport inhibitors on tumor necrosis factor-␣ (TNF-␣) release induced by endotoxin (lipopolysaccharide) in rabbit alveolar m. The amount and activity of TNF-␣ in mconditioned media were determined by enzyme-linked immunosorbent assay and L929 fibroblast bioassay, respectively. TNF-␣ release was suppressed progressively at lower pH o values (Յ7.0). Also, bafilomycin A 1 (a specific inhibitor of V-ATPases) significantly reduced the amount and activity of TNF-␣ in mconditioned media (pH o 7.4). However, bafilomycin caused a significant increase in the nonspecific cytotoxicity (i.e. bioactivity insensitive to TNF-␣ antibody) of mconditioned media. The effects of bafilomycin specifically on TNF-␣ release followed a time course similar to that of acidic pH o , suggesting that both treatments acted on similar events in the lipopolysaccharide signal transduction pathway. Amiloride (an inhibitor of Na + transporters including the Na + /H + exchanger) also suppressed TNF-␣ release but displayed a time course of action different from the acidic pH o or bafilomycin.
The first intravascular oxygenator and carbon dioxide (CO2) removal device (IVOX), conceived by M... more The first intravascular oxygenator and carbon dioxide (CO2) removal device (IVOX), conceived by Mortensen, was capable of removing 30% of CO2 production of an adult at normocapnia with a measurable reduction in ventilator requirements. Through studies of mathematical modeling, an ex vivo venovenous bypass circuit to model the human vena cava, animal models of severe smoke inhalation injury, and patients with acute respiratory failure, the practice of permissive hypercapnia has been established to enhance CO2 removal by IVOX. By allowing the blood pCO2 to rise gradually, the CO2 excretion by IVOX can be linearly increased in a 1:1 relationship. Experimental and clinical studies have shown that CO2 removal by IVOX can increase from 30 to 40 ml/min at a normal blood pCO2 to 80 to 90 ml/min at a pCO2 of 90 mm Hg. In addition, IVOX with permissive hypercapnia allows a significant reduction in minute ventilation and peak airway pressure. Active blood mixing to decrease the boundary layer resistance in the blood can significantly improve O2 transfer by up to 49% and CO2 removal by up to 35%. Design changes can also improve the performance of IVOX. Increased surface area with more fibers and enhanced mixing by increased fiber crimping in new prototypes of IVOX significantly increased CO2 removal. Other groups have used alternative designs to address the limited performance of intravascular gas exchange devices. With improved design and patient management, clinically meaningful gas exchange and reduction in mechanical ventilatory support may be achieved during treatment of severe respiratory failure.
Inhibition of carbonic anhydrase (CA) activity (activity in red blood cells and activity availabl... more Inhibition of carbonic anhydrase (CA) activity (activity in red blood cells and activity available on capillary endothelium) results in decrements in CO2 excretion (VCO2) and plasma-erythrocyte CO2-HCO(-3)-H+ disequilibrium as blood travels around the circulation. To investigate the kinetics of changes in blood PCO2 and pH during progressive CA inhibition, we used our previously detailed mathematical model of capillary gas exchange to analyze experimental data of VCO2 and blood-gas/pH parameters obtained from anesthetized, paralyzed, and mechanically ventilated dogs after treatment with acetazolamide (Actz, 0-100 mg/kg i.v.). Arterial and mixed venous blood samples were collected via indwelling femoral and pulmonary arterial catheters, respectively. Cardiac output was measured by thermodilution. End-tidal PCO2, as a measure of alveolar PCO2, was obtained from continuous records of airway PCO2 above the carina. Experimental results were analyzed with the aid of a mathematical model of lung and tissue-gas exchange. Progressive CA inhibition was associated with stepwise increments in the equilibrated mixed venous-alveolar PCO2 gradient (9, 19, and 26 Torr at 5, 20, and 100 mg/kg Actz, respectively). The maximum decrements in VCO2 were 10, 24, and 26% with 5, 20, and 100 mg/kg Actz, respectively, without full recovery of VCO2 at 1 h postinfusion. Equilibrated arterial PCO2 overestimated alveolar PCO2, and tissue PCO2 was underestimated by the measured equilibrated mixed venous blood PCO2. Mathematical model computations predicted hysteresis loops of the instantaneous CO2-HCO(-3)-H+ relationship and in vivo blood PCO2-pH relationship due to the finite reaction times for CO2-HCO(-3)-H+ reactions. The shape of the hysteresis loops was affected by the extent of Actz inhibition of CA in red blood cells and plasma.
Arteriovenous carbon dioxide removal (AVCO 2 R) as an alternative treatment for acute respiratory... more Arteriovenous carbon dioxide removal (AVCO 2 R) as an alternative treatment for acute respiratory distress syndrome uses a low resistance gas exchanger in a simple arteriovenous shunt to achieve total CO 2 removal and allow lung rest. We have previously shown in our clinically relevant LD 40 ovine model of smoke/burn induced acute respiratory distress syndrome that AVCO 2 R allows significant decreases in respiratory rate, tidal volume, peak airway pressure, and FiO 2 , as compared with standard mechanical ventilation. In addition, we have shown in a prospective randomized outcomes study that AVCO 2 R increases ventilator free days, decreases ventilator dependent days, and significantly improves survival. The purpose of this study is to further define the limits of AVCO 2 R through hemodynamic augmentation and evaluation of peak end expiratory pressure (PEEP). Administration of an alpha agonist (phenylephrine) and a beta agonist (isoproterenol) increased mean arterial pressure (MAP) and cardiac output (CO), respectively. MAP increases ranged from 2.4% to 94.4% and CO increases ranged from 33% to 146%. Phenylephrine caused elevations in MAP (2.4 -94.4%) and AVCO 2 R flow (9 -67%), and CO never decreased more than 10%. Isoproterenol administration increased CO (33-146%), decreased MAP (9 -54%), and decreased AVCO 2 R flow (11-42%). In a second group, PEEP was increased stepwise from 0 (baseline) to 20 cm H 2 O. Increasing PEEP did not result in significant hemodynamic changes (<10% change from baseline PEEP) for MAP, CO, or AVCO 2 R flow. In conclusion, alpha agonist administration increased AVCO 2 R blood flow, whereas beta agonist administration decreased MAP and AVCO 2 R blood flow, despite CO elevation. Various levels of PEEP are well tolerated and thus allow a range of options during AVCO 2 R.
Jama the Journal of the American Medical Association, Sep 28, 1994
To evaluate the potential efficacy of pressure limitation with permissive hypercapnia in the trea... more To evaluate the potential efficacy of pressure limitation with permissive hypercapnia in the treatment of acute respiratory failure/adult respiratory distress syndrome on the basis of current theories of ventilator-induced lung injury, potential complications of systemic hypercarbia, and available human outcome studies. Articles were identified through MEDLINE, reference citations of published data, and consultation with authorities in their respective fields. Animal model experimentation and human clinical trials were selected on the basis of whether they addressed the questions of pressure limitation with or without hypercapnia, the pathophysiologic effects of hypercapnia, or the concept of ventilator-induced parenchymal lung injury. Frequently cited references were preferentially included. Data were analyzed with particular emphasis on obtaining the following variables from the clinical studies: peak inspiratory pressures, tidal volumes, minute ventilation, and PCO2. Quantitative aspects of respiratory physiology were used to analyze the theoretical effects of permissive hypercapnia on ventilatory requirements in normal and injured lungs. Extensive animal model data support the hypothesis that ventilator-driven alveolar overdistention can induce significant parenchymal lung injury. The heterogeneous nature of lung injury in adult respiratory distress syndrome, with its small physiologic lung volume, may render the lung susceptible to this type of injury through the use of conventional tidal volumes (10 to 15 mL/kg). Permissive hypercapnia is an approach whereby alveolar overdistention is minimized through either pressure or volume limitation, and the potential deleterious consequences of respiratory acidosis are accepted. Uncontrolled human trials of explicit or implicit permissive hypercapnia have demonstrated improved survival in comparison with models of predictive mortality. Avoidance of alveolar overdistention through pressure or volume limitation has significant support based on animal models and computer simulation. Deleterious effects of the associated hypercarbia in severe lung injury do not appear to be a significant limiting factor in preliminary human clinical trials. Although current uncontrolled studies suggest benefit, controlled trials are urgently needed to confirm these findings before adoption of the treatment can be endorsed.
American Journal of Physiology Heart and Circulatory Physiology, Dec 1, 2001
A human cardiopulmonary system model applied to the analysis of the Valsalva maneuver. Am J Physi... more A human cardiopulmonary system model applied to the analysis of the Valsalva maneuver. Am J Physiol Heart Circ Physiol 281: H2661-H2679, 2001.-Previous models combining the human cardiovascular and pulmonary systems have not addressed their strong dynamic interaction. They are primarily cardiovascular or pulmonary in their orientation and do not permit a full exploration of how the combined cardiopulmonary system responds to large amplitude forcing (e.g., by the Valsalva maneuver). To address this issue, we developed a new model that represents the important components of the cardiopulmonary system and their coupled interaction. Included in the model are descriptions of atrial and ventricular mechanics, hemodynamics of the systemic and pulmonic circulations, baroreflex control of arterial pressure, airway and lung mechanics, and gas transport at the alveolar-capillary membrane. Parameters of this combined model were adjusted to fit nominal data, yielding accurate and realistic pressure, volume, and flow waveforms. With the same set of parameters, the nominal model predicted the hemodynamic responses to the markedly increased intrathoracic (pleural) pressures during the Valsalva maneuver. In summary, this model accurately represents the cardiopulmonary system and can explain how the heart, lung, and autonomic tone interact during the Valsalva maneuver. It is likely that with further refinement it could describe various physiological states and help investigators to better understand the biophysics of cardiopulmonary disease. cardiopulmonary modeling; ventricular interaction; closedloop hemodynamics; baroreflex control; airway mechanics; gas exchange THE DIAGNOSIS AND TREATMENT of cardiopulmonary disease may be improved by using mathematical models of the cardiovascular and pulmonary systems. With this in mind, we developed a model of the cardiopulmonary system of the normal human subject that not only represents the system accurately but also predicts its response to a variety of commonly used diagnostic procedures. To our knowledge, this is the first example of a truly integrative model of the cardiopulmonary system.
... New Technique for Performing Bronchoalveolar Lavage in Sheep. Savage, Clare MD*; Zwischenberg... more ... New Technique for Performing Bronchoalveolar Lavage in Sheep. Savage, Clare MD*; Zwischenberger, Joseph BMD*; Deyo, Donald JDVM; Heming, Thomas A. Ph.D.; Alpard, Scott K.*; Bidani, Akhil MD, Ph.D.. ... The FFB (Olympus URF-P, San Jose, CA)-70 cm in length ...
Cellular acid-base status has been found to exert selective actions on the effector functions of ... more Cellular acid-base status has been found to exert selective actions on the effector functions of activated macrophages (mφ). We examined the effects of extracellular pH (pH o ) on the production of tumour necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in resident alveolar mφ. Cells were obtained by bronchoalveolar lavage of rabbits, activated in vitro with LPS, and cultured at pH o 5.5, 6.5 or 7.4 for up to 18 h. The relative abundance of TNF-α mRNA peaked at " 2 h. The peak transcript abundance was increased at lower pH o values. This finding probably reflected pre-transcription/transcription effects of pH, in as much as the stability of TNF-α mRNA induced with phorbol ester was unaffected by the experimental pH o values. TNFα secretion by LPS-treated mφ decreased at lower pH o values. The TNF-α content of mφconditioned media decreased progressively with decrements in pH o . The reduced TNF-α secretion at pH o 5.5 was accompanied by an increase in the cytosolic TNF-α content (compared with that at pH o 7.4), indicating that pH o altered TNF-α secretion due, in part, to the intracellular retention of synthesized cytokine (i.e. a post-translation effect). The data show that pH o has multiple effects (pre-transcription/transcription and post-translation) on TNF-α production induced by LPS in resident alveolar mφ. These results suggest that the role of alveolar mφ in inflammatory responses is modulated by pH o , which may be important in tumours/ abscesses and sites of infection where the external milieu is acidic.
Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 5... more Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 594-603, 1990 and J. Appl: Physiol. 69: 523-531, 1990), have suggested that the steady-state absorption of inhaled NO2 is mediated by chemical reaction(s) between NO2 and ELF solute reactants. To characterize the kinetics of NO2 absorption into aqueous biological substrates across a gas-liquid interface, we utilized a closed system of known geometry and initial gas phase [NO2] [([NO2]g)0] to expose ELF (as bronchoalveolar lavage; BAL) and a biochemical model system (glutathione, GSH). Assessments of NO2 reactive uptake, into both GSH and ELF, indicated first-order NO2 kinetics [([NO2]g)0 less than or equal to 10.5 ppm] with effective rate constants of (kNO2)GSH = 4.8 and (kNO2)BAL = 2.9 ml.min-1.cm-2 (stirred). Above 10.5 ppm (1 mM GSH), zero-order kinetics were observed. Both (kNO2)GSH and (kNO2)BAL showed aqueous reactant dependence. The reaction order with respect to GSH and BAL was 0.47 and 0.64, respectively. We found no effect of interfacial surface area or bulk phase volume on kNO2. In unstirred systems, significant interfacial resistance was observed and was related to reactant concentration. These results indicate that NO2 reactive uptake follows first-order kinetics with respect to NO2 ([NO2]g less than or equal to 10.5 ppm) and displays aqueous substrate dependence. Furthermore the site of reactive absorption appears to be limited to near the aqueous surface interface. Unstirred conditions confine interfacial mass transfer kinetics in a dose-dependent manner. These phenomenological coefficients may provide the basis for direct extrapolation to environmentally relevant exposure concentrations.
The role of plasmalemmal V-type H+ translocating ATPase (V-ATPase) in regulation of intracellular... more The role of plasmalemmal V-type H+ translocating ATPase (V-ATPase) in regulation of intracellular pH (pHi) is unclear in monocytes. This study examined the plasmalemmal V-ATPase and Na+/H+ exchanger (NHE) in U937 human monocytes. The fluorescent probe 2&amp;amp;amp;amp;amp;amp;amp;amp;#39;,7&amp;amp;amp;amp;amp;amp;amp;amp;#39;-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pHi and the kinetics of pHi recovery from cytosolic acid-loads (NH4Cl prepulse). Bafilomycin A1 and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were used to delineate the activities of the H+-pump and NHE, respectively. Baseline pHi was approximately 7.13 at an extracellular pH (pHo) of 7.4 and fell progressively at lower pHo values. EIPA had no effect on baseline pHi at pHo 7.4, but caused a sustained decrement in pHi at pHo 6.0-7.0. Bafilomycin A1 had biphasic effects on baseline pHi at pHo 6.5-7.4; pHi declined approximately 0.1 units over the course of several minutes and then recovered. At pHo 6.0, bafilomycin A1 caused a sustained decrement in baseline pHi. Recovery from the bafilomycin-induced acidosis at pHo 6.5-7.4 was prevented by EIPA. Similarly, pHi recovery from NH4Cl prepulse acid-loads (pHo 7.4) was sensitive to both EIPA and bafilomycin A1. During this recovery process, Na+/H+ exchange (EIPA-sensitive component of apparent H+ efflux) was the predominant mechanism for H+ extrusion at acid-loaded pHi values &amp;amp;amp;amp;amp;amp;amp;amp;lt; 7.0. At acid-loaded pHi values &amp;amp;amp;amp;amp;amp;amp;amp;gt; or = 7.0, the V-ATPase (bafilomycin-sensitive component) and NHE contributed almost equally to H+ extrusion. The data provide the first evidence that plasmalemmal V-ATPase participates in pHi regulation in U937 cells. The H+-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocytes.
The present study determined the effects of cotton smoke inhalation on the functioning of alveola... more The present study determined the effects of cotton smoke inhalation on the functioning of alveolar macrophages (mphi). Smoke inhalation led to dose-dependent impairment of respiratory gas exchange by 48 h postexposure and pulmonary edema by 96 h. Maximal effects were observed in animals ventilated with 54 breaths of cotton smoke (3-min exposure, 18 breaths/min). Macrophages were obtained at 48 h postexposure by bronchoalveolar lavage of rabbits subjected to 54 breaths of smoke or room air (control). Phagocytosis of opsonized bacteria and adherence to solid substratum were reduced in smoke-exposed mphi. Smoke inhalation primed mphi for release of tumor necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS). Smoke-exposed mphi were also primed for TNF-alpha release induced by phorbol myristate acetate, which suggests that the priming event occurred downstream of protein kinase C activation in the signal transduction pathway. Further, smoke exposure attenuated the inhibitory effects of phosphodiesterase inhibitors on LPS-induced TNF-alpha release. Thus, the priming event may be mediated through cAMP and/or protein kinase A. The data indicate that cotton smoke inhalation suppresses the antimicrobial activities of alveolar mphi and can lead to excessive mphi production of TNF-alpha. These mphi effects would be expected to contribute to the pathophysiological abnormalities associated with smoke inhalation injury.
The present studies determined the effects of extracellular pH (pH o ) on the production of tumou... more The present studies determined the effects of extracellular pH (pH o ) on the production of tumour necrosis factor-α (TNF-α) in the macrophage-like cell lines RAW 246.7 and J774 A.1. The cells were activated with lipopolysaccharide (LPS) at pH o 5.5, 6.5 or 7.4. TNF-α gene transcription was monitored by Northern blot analysis. Synthesis of the cytokine was monitored by ELISA measurements of the TNF-α content of cell-conditioned media (extracellularly released TNF-α) and cell lysates (cytosolic TNF-α). The magnitude of the TNF-α response differed markedly between the two cell lines. RAW cells were more responsive to LPS than were J774 cells. However, the effects of pH o on TNF-α production were similar in the two cell lines. TNF-α gene transcription was insensitive to experimental pH o . The pH o had no effect on the abundance of TNF-α mRNA at 2, 4 or 18 h. Nonetheless, synthesis of TNF-α was affected significantly by pH o . The TNF-α contents of cell-conditioned medium and cell lysate at 18 h were reduced progressively at lower pH o values. The data indicate that pH o alters TNF-α production in RAW and J774 cells at a post-transcriptional level. These findings suggest that pH o influences the phenotypic responses of macrophages to activating stimuli and modifies the role that macrophages play in inflammatory and immune actions.
American Journal of Physiology Lung Cellular and Molecular Physiology, Aug 1, 1995
Three different mechanisms interact to control the cytosolic pH (pHi) of alveolar macrophages (M ... more Three different mechanisms interact to control the cytosolic pH (pHi) of alveolar macrophages (M phi), namely, plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase), Na+/H+ exchange, and Na(+)-independent HCO3-/Cl- exchange. To investigate the activity of plasmalemmal V-ATPase in alveolar M phi, we developed a nonlinear mathematical model of pHi regulation that incorporates the biophysical determinants of pHi and the fluxes of individual acid-base equivalents. The model was used to analyze the acid-base responses of rabbit alveolar M phi to a weak acid (propionic acid) under conditions that favored V-ATPase-mediated effects (presence of 1 mM amiloride and nominal absence of CO2). The pHi was measured using the fluorescent probe, 2&#39;,7&#39;-biscarboxethyl-5,6-carboxyfluorescein. M phi exposure to propionic acid caused a rapid fall in pHi. Recovery of pHi after acid loading varied directly with the magnitude of the acid load. Mathematical analyses showed that pHi recovery was hindered by persistent influx of propionic acid driven, in turn, by transporter-mediated H+ extrusion and propionate efflux. Eventually, a new steady state was established in which propionate and H+ were cycled out of the M phi and propionic acid was recycled into the cell. As a consequence, model predictions of the rate of V-ATPase-mediated H+ efflux were almost twice that estimated from experimental determinations of the initial rate of pHi recovery.
Lung carbonic anhydrase (CA) participates directly in plasma CO2-HCO3(-)-H+ reactions. To charact... more Lung carbonic anhydrase (CA) participates directly in plasma CO2-HCO3(-)-H+ reactions. To characterize pulmonary CA activity in situ, CO2 excretion and capillary pH equilibration were examined in isolated saline-perfused rat lungs. Isolated lungs were perfused at 25, 30, and 37 degrees C with solutions containing various concentrations of HCO3- and a CA inhibitor, acetazolamide (ACTZ). Total CO2 excretion was partitioned into those fractions attributable to dissolved CO2, uncatalyzed HCO3- dehydration, and catalyzed HCO3- dehydration. Approximately 60% of the total CO2 excretion at each temperature was attributable to CA-catalyzed HCO3- dehydration. Inhibition of pulmonary CA diminished CO2 excretion and produced significant postcapillary perfusate pH disequilibria, the magnitude and time course of which were dependent on temperature and the extent of CA inhibition. The half time for pH equilibration increased from approximately 5 s at 37 degrees C to 14 s at 25 degrees C. For the HCO3- dehydration reaction, pulmonary CA in situ displayed an apparent inhibition constant for ACTZ of 0.9-2.2 microM, a Michaelis-Menten constant of 90 mM, a maximal reaction velocity of 9 mM/s, and an apparent activation energy of 3.0 kcal/mol.
We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO2 uptake di... more We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO2 uptake displays reaction-mediated characteristics (J. Appl. Physiol. 68: 594-603, 1990). In vitro kinetic studies of pulmonary epithelial lining fluid (ELF) demonstrated that NO2 interfacial transfer into ELF exhibits first-order kinetics with respect to NO2, attains [NO2]-dependent rate saturation, and is aqueous substrate dependent (J. Appl. Physiol. 71: 1502-1510, 1991). We have extended these observations by evaluating the kinetics of NO2 gas phase disappearance in isolated ventilating rat lungs. Transient exposures (2-3/lung at 25 degrees C) employed rebreathing (NO2-air) from a non-compliant continuously stirred closed chamber. We observed that 1) NO2 uptake rate is independent of exposure period, 2) NO2 gas phase disappearance exhibited first-order kinetics [initial rate (r*) saturation occurred when [NO2] &gt; 11 ppm], 3) the mean effective rate constant (k*) for NO2 gas phase disappearance ([NO2] &lt; or = 11 ppm, tidal volume = 2.3 ml, functional residual capacity = 4 ml, ventilation frequency = 50/min) was 83 +/- 5 ml/min, 4) with [NO2] &lt; or = 11 ppm, k* and r* were proportional to tidal volume, and 5) NO2 fractional uptakes were constant across [NO2] (&lt; or = 11 ppm) and tidal volumes but exceeded quasi-steady-state observations. Preliminary data indicate that this divergence may be related to the inspired PCO2. These results suggest that NO2 reactive uptake within rebreathing isolated lungs follows first-order kinetics and displays initial rate saturation, similar to isolated ELF.(ABSTRACT TRUNCATED AT 250 WORDS)
Alveolar macrophages (m) participate in inflammatory and immune responses in acidic microenvironm... more Alveolar macrophages (m) participate in inflammatory and immune responses in acidic microenvironments such as the interstitial fluids of tumors and abscesses. Two plasmalemmal H + extruders interact to control the acid-base status of alveolar m, namely a V-type H + pump (V-ATPase) and a Na + /H + exchanger. The present study examined the effects of extracellular pH (pH o ) and H + transport inhibitors on tumor necrosis factor-␣ (TNF-␣) release induced by endotoxin (lipopolysaccharide) in rabbit alveolar m. The amount and activity of TNF-␣ in mconditioned media were determined by enzyme-linked immunosorbent assay and L929 fibroblast bioassay, respectively. TNF-␣ release was suppressed progressively at lower pH o values (Յ7.0). Also, bafilomycin A 1 (a specific inhibitor of V-ATPases) significantly reduced the amount and activity of TNF-␣ in mconditioned media (pH o 7.4). However, bafilomycin caused a significant increase in the nonspecific cytotoxicity (i.e. bioactivity insensitive to TNF-␣ antibody) of mconditioned media. The effects of bafilomycin specifically on TNF-␣ release followed a time course similar to that of acidic pH o , suggesting that both treatments acted on similar events in the lipopolysaccharide signal transduction pathway. Amiloride (an inhibitor of Na + transporters including the Na + /H + exchanger) also suppressed TNF-␣ release but displayed a time course of action different from the acidic pH o or bafilomycin.
The first intravascular oxygenator and carbon dioxide (CO2) removal device (IVOX), conceived by M... more The first intravascular oxygenator and carbon dioxide (CO2) removal device (IVOX), conceived by Mortensen, was capable of removing 30% of CO2 production of an adult at normocapnia with a measurable reduction in ventilator requirements. Through studies of mathematical modeling, an ex vivo venovenous bypass circuit to model the human vena cava, animal models of severe smoke inhalation injury, and patients with acute respiratory failure, the practice of permissive hypercapnia has been established to enhance CO2 removal by IVOX. By allowing the blood pCO2 to rise gradually, the CO2 excretion by IVOX can be linearly increased in a 1:1 relationship. Experimental and clinical studies have shown that CO2 removal by IVOX can increase from 30 to 40 ml/min at a normal blood pCO2 to 80 to 90 ml/min at a pCO2 of 90 mm Hg. In addition, IVOX with permissive hypercapnia allows a significant reduction in minute ventilation and peak airway pressure. Active blood mixing to decrease the boundary layer resistance in the blood can significantly improve O2 transfer by up to 49% and CO2 removal by up to 35%. Design changes can also improve the performance of IVOX. Increased surface area with more fibers and enhanced mixing by increased fiber crimping in new prototypes of IVOX significantly increased CO2 removal. Other groups have used alternative designs to address the limited performance of intravascular gas exchange devices. With improved design and patient management, clinically meaningful gas exchange and reduction in mechanical ventilatory support may be achieved during treatment of severe respiratory failure.
Inhibition of carbonic anhydrase (CA) activity (activity in red blood cells and activity availabl... more Inhibition of carbonic anhydrase (CA) activity (activity in red blood cells and activity available on capillary endothelium) results in decrements in CO2 excretion (VCO2) and plasma-erythrocyte CO2-HCO(-3)-H+ disequilibrium as blood travels around the circulation. To investigate the kinetics of changes in blood PCO2 and pH during progressive CA inhibition, we used our previously detailed mathematical model of capillary gas exchange to analyze experimental data of VCO2 and blood-gas/pH parameters obtained from anesthetized, paralyzed, and mechanically ventilated dogs after treatment with acetazolamide (Actz, 0-100 mg/kg i.v.). Arterial and mixed venous blood samples were collected via indwelling femoral and pulmonary arterial catheters, respectively. Cardiac output was measured by thermodilution. End-tidal PCO2, as a measure of alveolar PCO2, was obtained from continuous records of airway PCO2 above the carina. Experimental results were analyzed with the aid of a mathematical model of lung and tissue-gas exchange. Progressive CA inhibition was associated with stepwise increments in the equilibrated mixed venous-alveolar PCO2 gradient (9, 19, and 26 Torr at 5, 20, and 100 mg/kg Actz, respectively). The maximum decrements in VCO2 were 10, 24, and 26% with 5, 20, and 100 mg/kg Actz, respectively, without full recovery of VCO2 at 1 h postinfusion. Equilibrated arterial PCO2 overestimated alveolar PCO2, and tissue PCO2 was underestimated by the measured equilibrated mixed venous blood PCO2. Mathematical model computations predicted hysteresis loops of the instantaneous CO2-HCO(-3)-H+ relationship and in vivo blood PCO2-pH relationship due to the finite reaction times for CO2-HCO(-3)-H+ reactions. The shape of the hysteresis loops was affected by the extent of Actz inhibition of CA in red blood cells and plasma.
Arteriovenous carbon dioxide removal (AVCO 2 R) as an alternative treatment for acute respiratory... more Arteriovenous carbon dioxide removal (AVCO 2 R) as an alternative treatment for acute respiratory distress syndrome uses a low resistance gas exchanger in a simple arteriovenous shunt to achieve total CO 2 removal and allow lung rest. We have previously shown in our clinically relevant LD 40 ovine model of smoke/burn induced acute respiratory distress syndrome that AVCO 2 R allows significant decreases in respiratory rate, tidal volume, peak airway pressure, and FiO 2 , as compared with standard mechanical ventilation. In addition, we have shown in a prospective randomized outcomes study that AVCO 2 R increases ventilator free days, decreases ventilator dependent days, and significantly improves survival. The purpose of this study is to further define the limits of AVCO 2 R through hemodynamic augmentation and evaluation of peak end expiratory pressure (PEEP). Administration of an alpha agonist (phenylephrine) and a beta agonist (isoproterenol) increased mean arterial pressure (MAP) and cardiac output (CO), respectively. MAP increases ranged from 2.4% to 94.4% and CO increases ranged from 33% to 146%. Phenylephrine caused elevations in MAP (2.4 -94.4%) and AVCO 2 R flow (9 -67%), and CO never decreased more than 10%. Isoproterenol administration increased CO (33-146%), decreased MAP (9 -54%), and decreased AVCO 2 R flow (11-42%). In a second group, PEEP was increased stepwise from 0 (baseline) to 20 cm H 2 O. Increasing PEEP did not result in significant hemodynamic changes (<10% change from baseline PEEP) for MAP, CO, or AVCO 2 R flow. In conclusion, alpha agonist administration increased AVCO 2 R blood flow, whereas beta agonist administration decreased MAP and AVCO 2 R blood flow, despite CO elevation. Various levels of PEEP are well tolerated and thus allow a range of options during AVCO 2 R.
Jama the Journal of the American Medical Association, Sep 28, 1994
To evaluate the potential efficacy of pressure limitation with permissive hypercapnia in the trea... more To evaluate the potential efficacy of pressure limitation with permissive hypercapnia in the treatment of acute respiratory failure/adult respiratory distress syndrome on the basis of current theories of ventilator-induced lung injury, potential complications of systemic hypercarbia, and available human outcome studies. Articles were identified through MEDLINE, reference citations of published data, and consultation with authorities in their respective fields. Animal model experimentation and human clinical trials were selected on the basis of whether they addressed the questions of pressure limitation with or without hypercapnia, the pathophysiologic effects of hypercapnia, or the concept of ventilator-induced parenchymal lung injury. Frequently cited references were preferentially included. Data were analyzed with particular emphasis on obtaining the following variables from the clinical studies: peak inspiratory pressures, tidal volumes, minute ventilation, and PCO2. Quantitative aspects of respiratory physiology were used to analyze the theoretical effects of permissive hypercapnia on ventilatory requirements in normal and injured lungs. Extensive animal model data support the hypothesis that ventilator-driven alveolar overdistention can induce significant parenchymal lung injury. The heterogeneous nature of lung injury in adult respiratory distress syndrome, with its small physiologic lung volume, may render the lung susceptible to this type of injury through the use of conventional tidal volumes (10 to 15 mL/kg). Permissive hypercapnia is an approach whereby alveolar overdistention is minimized through either pressure or volume limitation, and the potential deleterious consequences of respiratory acidosis are accepted. Uncontrolled human trials of explicit or implicit permissive hypercapnia have demonstrated improved survival in comparison with models of predictive mortality. Avoidance of alveolar overdistention through pressure or volume limitation has significant support based on animal models and computer simulation. Deleterious effects of the associated hypercarbia in severe lung injury do not appear to be a significant limiting factor in preliminary human clinical trials. Although current uncontrolled studies suggest benefit, controlled trials are urgently needed to confirm these findings before adoption of the treatment can be endorsed.
American Journal of Physiology Heart and Circulatory Physiology, Dec 1, 2001
A human cardiopulmonary system model applied to the analysis of the Valsalva maneuver. Am J Physi... more A human cardiopulmonary system model applied to the analysis of the Valsalva maneuver. Am J Physiol Heart Circ Physiol 281: H2661-H2679, 2001.-Previous models combining the human cardiovascular and pulmonary systems have not addressed their strong dynamic interaction. They are primarily cardiovascular or pulmonary in their orientation and do not permit a full exploration of how the combined cardiopulmonary system responds to large amplitude forcing (e.g., by the Valsalva maneuver). To address this issue, we developed a new model that represents the important components of the cardiopulmonary system and their coupled interaction. Included in the model are descriptions of atrial and ventricular mechanics, hemodynamics of the systemic and pulmonic circulations, baroreflex control of arterial pressure, airway and lung mechanics, and gas transport at the alveolar-capillary membrane. Parameters of this combined model were adjusted to fit nominal data, yielding accurate and realistic pressure, volume, and flow waveforms. With the same set of parameters, the nominal model predicted the hemodynamic responses to the markedly increased intrathoracic (pleural) pressures during the Valsalva maneuver. In summary, this model accurately represents the cardiopulmonary system and can explain how the heart, lung, and autonomic tone interact during the Valsalva maneuver. It is likely that with further refinement it could describe various physiological states and help investigators to better understand the biophysics of cardiopulmonary disease. cardiopulmonary modeling; ventricular interaction; closedloop hemodynamics; baroreflex control; airway mechanics; gas exchange THE DIAGNOSIS AND TREATMENT of cardiopulmonary disease may be improved by using mathematical models of the cardiovascular and pulmonary systems. With this in mind, we developed a model of the cardiopulmonary system of the normal human subject that not only represents the system accurately but also predicts its response to a variety of commonly used diagnostic procedures. To our knowledge, this is the first example of a truly integrative model of the cardiopulmonary system.
... New Technique for Performing Bronchoalveolar Lavage in Sheep. Savage, Clare MD*; Zwischenberg... more ... New Technique for Performing Bronchoalveolar Lavage in Sheep. Savage, Clare MD*; Zwischenberger, Joseph BMD*; Deyo, Donald JDVM; Heming, Thomas A. Ph.D.; Alpard, Scott K.*; Bidani, Akhil MD, Ph.D.. ... The FFB (Olympus URF-P, San Jose, CA)-70 cm in length ...
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