Pulmonary and renal pressure-flow relationships: what should be taught?

This article is from a symposium presented at the annual meeting of the Human Anatomy and Physiology Society (HAPS) on June 11, 2000. The presentation was funded under the auspices of a National Science Foundation Course, Curriculum, and Laboratory Improvement Program entitled "Development of Active Learning Materials for Physiology and Functional Anatomy: A Cooperative HAPS-APS Initiative." This symposium was part of the first module to be developed on "gradients and conductances: what flows where and why?" This presentation was designed to model the usefulness of the general model of gradients and conductances in the physiology and pathophysiology of the respiratory and renal systems. Thirteen different examples of pressure-flow-resistance and concentration-flux relationships are introduced; several ideas for active-learning activities and simple figures appropriate for undergraduate physiology classes are included. The symposium assumes that undergraduate students have already learned about diffusion, osmosis, and the basic principles of cardiovascular physiology. The presentation was designed to follow a symposium entitled: "Cardiovascular pressure-flow relationships: what should be taught?"

Structural abnormalities underlying alveolar hypoventilation and fluid imbalance in the dystrophic hamster lung.

Bio 14.6 dystrophic hamsters exhibit alveolar hypoventilation and increased lung hydration. This study evaluated whether age- and genotype-related morphometric differences in lungs exist and correlate with the development of lung pathophysiology. Morphometry was used to characterize lungs of young (Y) and mature (M) control (C) and dystrophic (D) hamsters. With age, both C and D had increased barrier surface area [S(a-b,p)] and morphometric diffusing capacity index [mdci], and decreased harmonic thickness. In C but not D, mean capillary diameter [d(c)] and parenchymal volume density [V(v)(p,L)] increased with age, whereas barrier arithmetic thickness decreased. Chord length increased with age, whereas the ratio of parenchymal surface area to airspace volume [S/V] and the intersection density of the air-blood interface [I(v)(a-b,s)] decreased in D but not C. At both ages, lung volume relative to body mass was greater in D than C. With that exception, no genotype differences were found in young hamsters. Mature D displayed lower V(v)(p,L), S/V, d(c), I(v)(a-b,s), S(a-b,p), and mdci than mature C. Independent of age, chord length was greater but arithmetic thickness, airspace surface density, frequency of type II cells, and lamellar body area and volume density were lower in D than C. We conclude: 1) lung volume relative to body growth was greater in dystrophics than controls; 2) parenchymal remodeling was delayed or abnormal in dystrophics; 3) lower diffusing capacity in mature dystrophics may effect alveolar hypoventilation; 4) lower tissue volume, surface area, and the type II cell abnormalities in dystrophics could reduce sodium and water transport leading to greater lung hydration.

Protein kinase and phosphatase activity in the lungs of normoxic versus hyperoxic rats.

Studies were designed to examine aspects of phosphorylation and dephosphorylation in rat lung cells in response to hyperoxic exposure. Protein kinase and phosphatase activities were measured in preparations of lungs from normoxic rats, hyperoxia-exposed rats (95% O2 for 60h), and rats recovering in room air for 1 and 3 days. Protein kinase C (PKC) activity immediately postexposure was significantly lower than in normoxic controls (normoxia 127.1 +/- 13 vs. hyperoxia 101.5 +/- 6 pmol/min mg-1) and continued to decline during the recovery period (85.3 +/- 4 and 78.2 +/- 6 pmol/min mg-1 at 1 and 3 days recovery, respectively). The PKC activity did not translocate from cytoplasm to the membranes. In contrast, PKA activity did not change in response to hyperoxia exposure or recovery. Protein phosphatase activity was decreased significantly by hyperoxia exposure (normoxia 30.7 +/- 3 vs. hyperoxia 21.9 +/- 1 pmol/min microgram-1) but returned to normoxic control levels by 1 and 3 days (24.1 +/- and 31.5 +/- 1 pmol/min microgram -1, respectively). Protein phosphatase activity was inhibited by okadaic acid (Ki = 1 nM) and calyculin A (Ki = 0.61 pM), indicating a type 2A protein phosphatase. Enzyme activities in cultured type II alveolar cells paralleled those observed in whole lung preparations. Decreased enzyme activities in the lung may be located to the development of acute lung injury during hyperoxic exposure.

Active 22Na+ transport by the intact lung during early postnatal life.

The lung relies upon epithelial active transport of Na+ to aid in the clearance of fluid from its air spaces. Because it is unknown whether the rate of active Na+ transport by the distal lung epithelium varies during early postnatal age, we performed studies in young guinea pigs (7 and 30 days after birth). We used a single pass isolated perfused lung model in which a Krebs Ringer bicarbonate solution containing 22Na+, [14C]sucrose, and FITC-dextran was placed into the air spaces of the lungs, and apparent permeability-surface area (PS) products were calculated after determining the changes in lung weight and the concentrations of the isotopes in the vascular effluent. The PS product for 22Na+, but not [14C]sucrose, decreased significantly at both ages when amiloride was infused (final concentration of 10(-4) M). Amiloride also decreased the rate of fluid clearance, as assessed by changes in organ weight, at both ages. Although the absolute rate of amiloride-sensitive 22Na+ transport increased with age, morphometric measurement of the alveolar region demonstrated that the rate of amiloride-sensitive 22Na+ transport per unit alveolar surface area was similar. These data indicate that although the guinea pig lung undergoes significant growth shortly after birth, the rate of amiloride-sensitive active Na+ transport per unit surface area remains constant. Since a component of weight loss was insensitive to amiloride, these in vivo studies suggest that the amiloride-insensitive Na+ transport pathways previously identified in cultured lung epithelium exist in the intact lung.

Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs.

We investigated effects of acute hyperoxia on solute transport from air space to vascular space in isolated rat lungs. Air spaces were filled with Krebs-Ringer bicarbonate solution containing fluorescein isothiocyanate-labeled dextran (FD-20; mol wt 20,000) and either 22Na+ and [14C]sucrose, or D-[14C]glucose and L-[3H]glucose. Apparent permeability-surface area products for tracers over time (up to 120 min) were calculated for isolated perfused lungs from control rats (room air) and rats exposed to > 95% O2 for 48 or 60 h immediately postexposure. After O2 exposures, mean fluxes for [14C]sucrose and FD-20 were significantly higher than in room-air control lungs. However, amiloride-sensitive Na+ and active D-glucose fluxes were unchanged after hyperoxic exposure. Therefore, it is unlikely that decreases in net solute transport in this lung-injury model contributed to pulmonary edema resulting from O2 toxicity. Increased net solute transport shown to help resolve pulmonary edema after acute hyperoxic exposure must therefore begin during the recovery period. In summary, our data show increases in passive solute fluxes but no changes in active solute fluxes immediately after acute hyperoxic lung injury.

Sodium transport and fluid balance in lungs from normal and dystrophic hamsters.

Gravimetric and sodium transport characteristics of lungs from BIO 14.6 (dystrophic) hamsters were compared with those of lungs from golden Syrian (normal) hamsters at 30 and 150 days of age. Isolated perfused lungs were used to determine lung permeability and fluid balance differences between normal and dystrophic animals at both ages. Apparent permeability-surface area products for air space-to-vascular space sodium, sucrose, and fluorescein isothiocyanate-labeled dextran fluxes were compared in the four groups of hamsters. Morphometric analysis of fixed lungs of representative hamsters from each group was also performed. Dystrophic hamsters exhibited higher lung wet-to-dry weight ratios than normal hamsters at both ages. Lungs from dystrophic hamsters were less sensitive to inhibition of sodium transport by amiloride than lungs from age-matched normal hamsters. Dystrophic hamster lungs had higher absolute permeabilities of the passively transported solutes, lower permeability values for sodium, and only one-half of the amiloride-sensitive sodium transport of lungs from age-matched normal hamsters. Differences in lung fluid balance between dystrophic and normal hamsters may be related to differences in sodium clearance.

Differences in sodium and D-glucose transport between hamster and rat lungs.

The purpose of this study was to characterize phloridzin- and amiloride-sensitive transport across blood-gas barrier of hamster and rat lungs. Air spaces of isolated perfused lungs were instilled with a solution containing 22Na or L-[3H]glucose, D-[14C]glucose, and fluorescein isothiocyanate-labeled dextran. Apparent permeability-surface area products (PS) were calculated. Phloridzin (Na(+)-dependent D-glucose transport inhibitor) had no effect on D-glucose or sodium transport out of air spaces in hamster lungs. In contrast, in rat lungs, phloridzin decreased PS for D-glucose by 89% and that for Na by 28%. Trapping of 14CO2 in vascular samples was measured to estimate metabolism. Unlabeled air space D-glucose increased appearance of perfused D-[14C]glucose in air spaces of rat lungs. We conclude that Na(+)-dependent D-glucose transport is important for D-glucose uptake in rat lungs but not in hamster lungs. In hamster lungs, amiloride (Na+ transport inhibitor) also decreased PS for sodium, but drugs known to stimulate sodium transport in rat lungs had no effect. Thus, species differences in active transport processes exist in the distal air spaces of mammalian lungs.

Evidence for regulation of sodium transport from airspace to vascular space by cAMP.

Evidence has been accumulating that regulation of the rate of solute and fluid removal from the alveolar spaces may play an important role in the prevention and/or resolution of alveolar pulmonary edema. In this study, the isolated perfused rat lung was used to investigate the effects of an adenosine 3',5'-cyclic monophosphate (cAMP) analogue or a phosphodiesterase inhibitor on active sodium transport from airspace to vascular space. Three tracers were instilled into the airways of isolated Krebs-Ringer bicarbonate solution (KRB)-perfused rat lungs. The appearance of tracers in the single-pass perfusate was measured, and the apparent permeability-surface area products (PS) were calculated for each tracer at each sample time based on Fick's first law of diffusion. After steady-state PS values had been reached, a cAMP analogue or phosphodiesterase inhibitor was added to the perfusate. Both agents caused significant increases in the PS for 22Na. In another group of experiments, a cAMP analogue was added to the perfusate, followed by the subsequent addition of a sodium transport inhibitor and the resultant large decrease in the PS for 22Na. These data are consistent with the regulation of active sodium transport across the intact mammalian alveolar epithelium by a cAMP-mediated process leading to removal of sodium from the alveolar spaces, with anions and water following passively.

Recent advances in characterizing clearance of water from lungs.

The investigation of pulmonary fluid balance has been hampered by the complexity of the factors involved. New techniques using cultured monolayers of alveolar epithelial cells have improved the study of the transport properties of the lung. Recent studies using isolated lung preparations and isolated alveolar epithelial cells give additional insight into the mechanisms and pathophysiology of alveolar pulmonary edema. There may be potential for new therapies of clinical pulmonary edema in humans.

Characteristics and regulation of active transport in lungs from young and aged mammals.

From the data presented here, it appears that the ability to stimulate solute (and water) clearance from the alveolar spaces may decrease with aging. This implies that the management of elderly patients with respiratory diseases or complications may necessitate a different approach than that useful in younger patients. Many of the active transport mechanisms well-characterized in other epithelia now seem to be important for maintaining the alveolar air spaces in their normally "dry" condition in the lungs of fetal and young, sexually mature mammals. In situations in which excess fluid is present in the air spaces of the lung, it is likely that the normal regulation of these active transport processes may play a major processes may play a major role in alveolar fluid clearance. Various exogenous agents may be helpful in stimulating the active solute and water reabsorption from air space to vascular space in both fetal and young sexually mature mammals. Further studies on the effects of aging on the characteristics and regulation of lung fluid balance are still needed.

Evidence for active sodium transport across alveolar epithelium of isolated rat lung.

We have previously presented evidence that cultured alveolar epithelial cell monolayers actively transport sodium from medium to substratum, a process that can be inhibited by sodium transport blockers and stimulated by beta-agonists. In this study, the isolated perfused rat lung was utilized in order to investigate the presence of active sodium transport by intact adult mammalian alveolar epithelium. Radioactive tracers (22Na and [14C]sucrose) were instilled into the airways of isolated Ringer-perfused rat lungs whose weight was continuously monitored. The appearance of isotopes in the recirculated perfusate was measured, and fluxes and apparent permeability-surface area products were determined. A pharmacological agent (amiloride, ouabain, or terbutaline) was added to the perfusate during each experiment after a suitable control period. Amiloride and ouabain resulted in decreased 22Na fluxes and a faster rate of lung weight gain. Terbutaline resulted in increased 22Na flux and a more rapid rate of lung weight loss. [14C]sucrose fluxes were unchanged by the presence of these pharmacological agents. These data are most consistent with the presence of a regulable active component of sodium transport across intact mammalian alveolar epithelium that leads to removal of sodium from the alveolar space, with anions and water following passively. Regulation of the rate of sodium and fluid removal from the alveolar space may play an important role in the prevention and/or resolution of alveolar pulmonary edema.

Effects of terbutaline on sodium transport in isolated perfused rat lung.

We have previously presented evidence that cultured alveolar epithelial cell monolayers actively transport sodium from medium to substratum, and that this process can be stimulated by beta-agonists. In this study the isolated perfused rat lung was utilized to investigate sodium transport across intact mammalian alveolar epithelium. Radioisotopic tracer(s) (22Na and/or [14C]sucrose) were instilled into the airways of isolated Ringer-perfused rat lungs. The appearance of isotope(s) in the recirculated perfusate was measured and a permeability-surface area product was calculated. Pharmacological agent(s) (terbutaline and/or propranolol) were present in the instillate or were added to the perfusate during the experiments. Terbutaline alone, whether in the instillate or perfusate, caused a significant increase in 22Na flux. This increase was prevented by the presence of propranolol. [14C]sucrose fluxes were unaffected by the presence of terbutaline. These data are consistent with the presence of an active component of sodium transport across intact mammalian alveolar epithelium that leads to removal of sodium from the alveolar space.

Sodium-amino acid cotransport by type II alveolar epithelial cells.

Type II alveolar epithelial cell monolayers have been shown to actively transport sodium (Na+). Coupling to amino acid uptake could be an important mechanism for Na+ entry into these cells. This study demonstrates the presence of such a coupled cotransport mechanism in the plasma membrane of isolated type II cells by use of the nonmetabolizable amino acid analogue alpha-methylaminoisobutyric acid (MeAIB). Transport of MeAIB in 137 mM Na+ is saturable, with the uptake constant (Vmax) equaling 13.9 pmol X mg prot-1 X s-1 and the Michaelis-Menten constant (Km) equaling 0.13 mM. In the presence of Na+, MeAIB is accumulated against a concentration gradient. MeAIB uptake in the absence of Na+ is linear with MeAIB concentration, as expected for simple diffusion. The Hill coefficient for Na+-MeAIB cotransport is 1.11, suggesting a 1:1 stoichiometry. Proline inhibits Na+-MeAIB cotransport, with Ki equaling 0.5 mM. These findings suggest that Na+-amino acid cotransport may be an important pathway for Na+ (and/or amino acid) uptake into type II alveolar epithelial cells.

Regulation of transport across pulmonary alveolar epithelial cell monolayers.

Domes are formed in large numbers by primary cultured monolayers of type II alveolar epithelial cells from rat lungs. These fluid-filled structures are formed by active transport of solute from medium to substratum, with water following passively. In the present study, we used dome-forming monolayers to study the regulation of alveolar epithelial transport processes by determining the effects on dome formation of adenosine 3',5'-cyclic monophosphate (cAMP) analogues, phosphodiesterase inhibitors, neurotransmitters, and vasopressin (antidiuretic hormone, ADH). The cAMP analogues (dibutyryl cAMP and 8-bromo-cAMP) and phosphodiesterase inhibitors (theophylline, papaverine, and isobutylmethylxanthine) caused large increases in dome formation by 24 h. ADH and beta-adrenergic agonists (epinephrine, terbutaline, and isoproterenol) also caused significant increases in dome density. The beta-agonist response was completely eliminated in the presence of the beta-blocker propranolol. Dibutyryl guanosine 3',5'-cyclic monophosphate and acetylcholine (cholinergic agonist) had no effect on dome formation, whereas the alpha-adrenergic agonist methoxamine caused a small but significant decrease in dome formation. These findings suggest that the active solute flux resulting in dome formation by type II alveolar epithelial cell monolayers is increased by substances expected to elevate intracellular cAMP (or analogue) concentrations. An attractive speculation having major implications for lung fluid balance is that transepithelial fluxes can be modulated by endogenous, and perhaps exogenous, chemical agents in adult mammalian alveolar epithelium in vivo.

Evidence for active Na+ transport by cultured monolayers of pulmonary alveolar epithelial cells.

Primary cultured type II alveolar epithelial cells grown to confluence on nonporous surfaces form many small fluid-filled hemicysts or domes. These domes are generally thought to result from active transport of solutes from the medium above the cell monolayer to the substratum, with water following passively. We have investigated the characteristics of active transport by primary cultured monolayers of type II alveolar epithelial cells from rat lungs. Changes in dome density were measured after exposure to metabolic inhibitors, Na+ or Cl- transport inhibitors, and low-Na+ or low-Cl- culture media. Metabolic and Na+ transport inhibitors, and low-Na+ medium, lead to disappearance of domes, whereas Cl- transport inhibitors and low-Cl- medium seem to have no effect on dome density. These results suggest the presence of a Na+-dependent active transepithelial transport process across the monolayer, which is responsible for the formation of domes. This finding implies that absorption of fluid by mammalian alveolar epithelium in vivo may be important in the maintenance of normal lung fluid balance.

Dome formation in primary cultured monolayers of alveolar epithelial cells.

We have observed the formation of domes by type II alveolar epithelial cells harvested from rat lungs. The cells were harvested using elastase and grew to confluence in 3-4 days after plating on plastic. Numerous domes were observed in the monolayers 4-18 days after plating, with peak dome density occurring at days 6-9. When trypsin was used instead of elastase as the harvesting enzyme, many fewer domes were formed by the monolayers, with peak dome density observed at day 5 and no domes seen after 8 days. The life span of an individual dome was about 3-4 h. The presence of domes indicates an intact active transport function of the cells in the monolayer, which may represent an important mechanism for the maintenance of fluid-free air spaces and normal alveolar fluid balance in mammalian lungs in vivo.

Alveolar epithelium permeability to small solutes: developmental changes.

To determine whether alveolar epithelium permeability to small lipid-insoluble solutes changes during development we measured transport across the blood-gas barrier in isolated Ringer-perfused lungs from prenatal, 1-day-old, 4-wk-old, and adult rabbits. Radioactive test molecules, one of which was always sucrose, were dissolved in Ringer solution and instilled into the trachea of degassed lungs. Samples taken from recirculating perfusate were used to calculate permeability-surface area (PS) products. Results were expressed as the ratio (PS)/(PS)sucrose, and as absolute permeability. Lungs from 4-wk-old rabbits were studied most thoroughly; the (PS)/(PS) sucrose ratios obtained are urea 4.0, erythritol 1.3, mannitol 0.98, L-glucose 1.4, and D-glucose 5.6. These and other data imply that the most lipid-insoluble molecules (erythritol, mannitol, L-glucose, and sucrose) are transported by a nonselective bulk process. Urea transport is primarily through lipid membranes; D-glucose seems to involve a special process. Sucrose and L-glucose permeability decreased during development, but their relative permeabilities did not change. Small lipid-insoluble solutes apparently do not cross the alveolar epithelium through small water-filled pores, and their permeability decreases as the animal matures.