The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.
- surfactant protein B
- surfactant protein C
pulmonary surfactant is a complex mixture of proteins and phospholipids synthesized and secreted by alveolar type II cells. Alveolar surfactant is metabolized and removed from the lung by reuptake of inactive surfactant by both alveolar macrophages and alveolar type II cells. At least four proteins are known to exist in endogenous surfactant, surfactant protein A (SP-A), SP-B, SP-C, and SP-D. Of these surfactant-specific proteins, SP-B and SP-C, two small hydrophobic proteins, have been demonstrated to play a role in the formation of a stable surfactant monolayer. SP-B especially has been shown to be essential for normal surfactant function, lowering surface tension (6, 23), as the absence of SP-B at birth leads to death caused by respiratory insufficiency (26, 30), and conditional knockout of SP-B in adult animals leads to respiratory failure (17). In addition, previous studies have demonstrated altered concentrations of SP-B in bronchoalveolar lavage fluids of patients suffering acute respiratory distress syndrome (10, 11).
SP-C also enhances surface-active properties of surfactant (6, 19, 23, 29, 32), although absence of SP-C at birth is not lethal like SP-B, but experiments performed in specific SP-C knockout mice demonstrated that despite the fact that surfactant pool sizes and lung morphology were similar in wild-type and SP-C-knockout mice, the absence of SP-C leads to a decreased stability of the surfactant at low volumes (9). Another function of SP-C is increasing the resistance of surfactant against inactivation by plasma proteins (31).
Today, exogenous surfactant is used in clinical practice in neonates, whereas the use of exogenous surfactant in adults is under consideration (16). These exogenous surfactant preparations commonly contain SP-B and/or SP-C since these proteins play an important role in lowering surface tension. Although the beneficial effects of addition of the proteins on surface tension-lowering activity will not be a point of discussion, these effects on the uptake of surfactant have to be clarified. Uptake of surfactant is an important step in the recycling of surfactant, which is known to be crucial in the maintenance of surfactant homeostasis.
Although both these hydrophobic surfactant proteins have been demonstrated to participate in the reuptake of inactive surfactant (14, 24) by alveolar type II cells, these studies were done in vitro. Recently, we were able to show the influence of the local environment on the uptake of surfactant by alveolar cells (22), indicating the need for studying surfactant metabolism, especially uptake, not only in vitro but also in vivo.
Therefore, we examined the effect of SP-B and SP-C on the uptake of lipids by both alveolar type II cells and alveolar macrophages in vivo in ventilated rats as well as in vitro using fluorescently labeled liposomes, flow cytometry, and confocal laser scanning microscopy.
MATERIALS AND METHODS
This study was approved by the Institutional Animal Committee at Erasmus University, Rotterdam, The Netherlands.
Both SP-B and SP-C were purified from lipid extracts of porcine lung lavage to homogeneity by Sephadex LH-60 chromatography (Pharmacia, Uppsala, Sweden) (20). The concentrations of SP-B and SP-C were determined using a fluorescamine protein assay using BSA as standard (4). Surfactant-like liposomes were prepared by mixing the following lipids: dipalmitoyl phosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidylethanolamine labeled with rhodamine in the head group (rhodamine DHPE; Molecular Probes, Leiden, The Netherlands) and cholesterol in a weight ratio of 55:21:8:2:6:8 and the indicated concentrations of surfactant proteins. The lipids and proteins were dissolved in chloroform/methanol/0.1 M HCl (1:1:0.05 by vol). Subsequently, this mixture was dried under a stream of nitrogen. The lipids were purchased from Sigma, Zwijndrecht, The Netherlands, unless stated otherwise. The liposomes were either suspended in PBS or in solution 2 (140 mM NaCl, 5 mM KCl, 2.5 mM phosphate buffer, 10 mM HEPES, 6 mM glucose, 2.0 mM CaCl2, and 1.3 mM MgSO4) at a concentration of 1 mg of lipids/ml using glass pearls and vortexing, resulting in large multilamellar liposomes. Immediately before use, the liposome suspension was sonicated for 2 min on ice using an ultrasonic disintegrator (Branson Sonifier 250) to prepare small unilamellar liposomes (22). The size of the liposomes was determined by dynamic light scattering at 25°C with a Malvern 4700 system using a 25-mW He-Ne laser (NEC, Tokyo, Japan) and automeasure version 3.2 software (Malvern). As a measure of particle size distribution of the dispersion, the system reports a polydispersity index. This index ranges from 0.0 for a monodisperse and up to 1.0 for an entirely polydisperse dispersion. After ultrasonification, the liposomes size ranged from 140 to 165 nm, and the polydispersity index ranged from 0.2 to 0.35.
Intratracheal instillation of fluorescent liposomes.
The studies were performed with male Sprague-Dawley rats (IFFA Credo) with a body weight of 318 ± 16 g. After induction of anesthesia with a mixture of nitrous oxide (66%), oxygen (33%), and isoflurane (1–2%), a sterile polyethylene catheter (0.8 mm outer diameter) was inserted into one of the carotid arteries. The animals were then tracheotomized, and a sterile metal cannula was inserted into the trachea.
After these surgical procedures, gaseous anesthesia was ended and replaced with an intraperitoneal injection of pentobarbital sodium (60 mg/ml Nembutal; Algin, Maassluis, The Netherlands) at a dose of 30 mg/kg body wt every hour.
Muscle relaxation was induced and maintained by an hourly intramuscular injection of pancuronium bromide (2 mg/kg Pavulon; Organon Technika, Boxtel, The Netherlands). The animals were then mechanically ventilated with a Servo ventilator 900C (Siemens-Elema, Solna, Sweden) set to pressure control mode using a frequency of 30 breaths/min, an inspiratory/expiratory ratio of 1:2, a positive end-expiratory pressure (PEEP) of 2 cmH2O, a peak inspiratory pressure (PIP) of 12 cmH2O, and FiO2 was set to 1.
Before instillation of the labeled liposomes, PEEP was increased to 6 cmH2O and PIP was increased to 26 cmH2O. After disconnection from the ventilator, the liposomes were administered intratracheally at the indicated dosage. The suspension of liposomes (1 mg of lipids/ml unless stated otherwise) was administered as a bolus of 3 ml/kg followed by a bolus of air (12 ml/kg) directly into the endothracheal tube via a syringe, and the animals were immediately reconnected to the ventilator. Thirty minutes after instillation of the liposomes, PEEP was reduced to 2 cmH2O and PIP to 12 cmH2O.
Arterial blood gas values were measured with conventional methods (ABL 555; Radiometer, Copenhagen, Denmark) at the start of ventilation, immediately after instillation of the liposomes, and every 30 min thereafter. One hour after ventilation, the animals were killed by exsanguination via the abdominal aorta, and the alveolar cells were isolated to determine the cell-associated fluorescence. Untreated animals were killed immediately after anesthesia, and their isolated alveolar type II cells and alveolar macrophages were used to correct for autofluorescence.
Isolation of alveolar type II cells and alveolar macrophages.
Before isolation of the cells, the thorax was opened, and the blood cells were removed from the lungs by perfusing the pulmonary artery with warmed saline (37°C) supplemented with 20 IE heparin per milliliter (Leo Pharma; Weesp, The Netherlands). The lungs were removed from the thoracic cavity en bloc and lavaged with 10 ml of solution 1 (140 mM NaCl, 5 mM KCl, 2.5 mM Na2HPO4, 2 H2O, 10 mM HEPES, 6 mM glucose, and 0.2 mM EGTA, pH 7.40) at 37°C. This procedure was repeated four times. The lung lavages were pooled per animal and centrifuged (100 g, 10 min, 4°C). The cellular pellet, i.e., alveolar macrophages, were suspended in solution 2 to a concentration of 2 × 106 cells/ml and stored on ice until further use. The alveolar type II cells were isolated according to Dobbs et al. (7) using enzymatic digestion as previously described (22). Alveolar type II cells were suspended in solution 2 at a concentration of 2 × 106 cells/ml and stored on ice until further use. Alveolar macrophages were identified by using monoclonal antibodies specific for rat macrophages (ED9), and alveolar type II cells were identified using an alkaline phosphatase assay as described by Edelson et al. (8). The average yield of alveolar type II cells was 16 × 106 with a purity of 80 ± 5% and 5 × 106 for alveolar macrophages with a purity of 92 ± 5% per rat.
In vitro uptake of SP-B- and SP-C-containing liposomes.
Alveolar cells of untreated animals were isolated as described above and suspended in solution 2 to a concentration of 2 × 106 cells/ml. A total of 3 × 105 cells were incubated with the indicated concentration of the specified liposomes at 37°C (final volume 500 μl) in a shaking water bath. After 1 h, the incubation was terminated by addition of 2 ml of ice-cold PBS (4°C). The cell suspension was centrifuged at 100 g for 10 min at 4°C. The supernatant was removed, and the cells were suspended in 2 ml of ice-cold PBS and centrifuged again. This wash procedure was repeated twice. Finally, the pellet was resuspended in 200 μl of cold PBS, and cell-associated fluorescence was determined as described below.
Cell-associated fluorescence of the alveolar type II cells and alveolar macrophages as a measure for internalized liposomes was determined using flow cytometry (FACSCalibur; Becton Dickinson). The cell-associated fluorescence of 15,000 cells was determined. Alveolar macrophages and alveolar type II cells derived from control animals were used in each experiment to determine the autofluorescence of the cells. Subsequently, the mean cell-associated fluorescence was determined only for those cells that had a higher fluorescence than that caused by autofluorescence (gated cells).
Localization of cell-associated fluorescence.
To localize the cell-associated fluorescence, intracellular or extracellular confocal laser micrographs were obtained using a confocal microscope (LSM510NLO, Carl Zeiss Jena). Images were created with plan apochromat 63 × 1.4 numerical aperture objectives and photomultiplier tubes, and emission filter was set at 500–550 nm (rhodamine, excitation 543-nm He-Ne laser). Images of alveolar cells were serially sectioned pinhole at 1 airy unit with an interval of 0.5 μm to distinguish cell membrane-associated fluorescence from the intracellular fluorescence.
Differences between the different groups that received liposomes with a different composition were analyzed using an ANOVA followed by Tukey’s post hoc test. The unpaired t-test was used for analysis of the effect of calcium. Blood gas values were analyzed using a repeated measurement ANOVA.
Differences were considered statistically significant at a P < 0.05. Values are expressed as means ± SD.
Effect of SP-B and SP-C on uptake in vivo.
Although SP-B and SP-C have been shown to influence the uptake by alveolar cells (13, 15, 25), most studies were performed in vitro (13, 25), and due to the methods used, no comparison between in vivo and in vitro results can easily be made. Therefore, in the current study, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes containing SP-B and/or SP-C. Because it has been described that divalent cations affect the function of these surfactant proteins, additional groups were included that were instilled with liposomes suspended in solution 2 that include calcium and magnesium.
The inclusion of SP-B and/or SP-C in the liposome does not influence the uptake by alveolar cells when these liposomes are suspended in PBS (Fig. 1, A and C). However, suspending the liposomes in solution 2 leads to a significant increase in uptake by alveolar macrophages of liposomes with SP-C or both SP-B and SP-C incorporated (Fig. 1C).
The use of flow cytometry to measure the uptake of these fluorescently labeled liposomes also provides the opportunity to measure the percentage of cells involved in the uptake. Hence, the current experiments demonstrate that the incorporation of SP-B and/or SP-C in the liposome does not affect the number of alveolar cells involved in the uptake (Fig. 1, B and D), although suspending the liposomes in solution 2 results in a significant increase in the percentage of alveolar type II cells involved in the uptake of SP-C-containing liposomes and the amount of alveolar macrophages taking up liposomes containing both SP-B and SP-C (Fig. 1, B and D).
Effect of increasing SP-B:lipid ratio on the uptake in vivo.
The absence of any effect with the inclusion of SP-B in the liposomes raised the question of whether or not the amount of SP-B was sufficient. Therefore, the SP-B:lipid ratio in the liposomes was varied. Higher SP-B:lipid ratios, however, did not affect the amount of liposomes taken up by individual alveolar type II cells or the number of alveolar type II cells involved in the uptake (Fig. 2, A and B).
In addition, increasing the SP-B:lipid ratio did not have an effect on the uptake of the liposomes by alveolar macrophages, since cell-associated fluorescence, as a measure for the amount of liposomes internalized, did not differ significantly from standard liposomes (Fig. 2C). The number of alveolar macrophages taking up the labeled liposomes tends to decrease with higher SP-B:lipid ratios, although this decrease is not significant (Fig. 2D).
Resuspending the liposomes in solution 2 did not have any effect on the uptake by either alveolar type II cells or by alveolar macrophages (Fig. 2).
Effect of SP-C:lipid ratio on the uptake in vivo.
To determine an optimal SP-C:lipid ratio for internalization of these liposomes by alveolar cells, liposomes with different SP-C:lipid ratios were instilled. SP-C has a maximal effect on the uptake by alveolar type II cells at a SP-C:lipid ratio of 2:100 (Fig. 3A). Increasing the SP-C:lipid ratio did not affect the number of alveolar type II cells involved in the uptake (Fig. 3C).
Interestingly, uptake of liposomes by alveolar macrophages is unchanged with increasing SP-C:lipid ratios when these liposomes are resuspended in PBS. However, when the liposomes are resuspended in solution 2, significant increases in uptake are demonstrated when SP-C is incorporated in the liposome, with a maximal uptake at a SP-C:lipid ratio of 2:100 (Fig. 3C). However, the number of alveolar macrophages involved in the uptake remains unaffected by the incorporation of SP-C in the liposome or resuspension of the liposomes in solution 2 (Fig. 3D).
Effect of SP-B- or SP-C-containing liposomes on gas exchange.
The instillation of the fluorescently labeled liposomes did not have any effect on gas exchange as determined by arterial blood gas values (Table 1). There was also no effect on arterial oxygenation by the incorporation of the studied surfactant proteins or suspending the liposomes in solution 2.
In vitro effects on the uptake of liposomes with SP-B or SP-C incorporated.
A possible explanation for the absence of any effects of the inclusion of SP-B in the liposomes on the uptake might be that in the healthy lung, the amount of SP-B is sufficient or optimal, and additional SP-B, added by instillation of liposomes with SP-B incorporated, does not affect uptake. To study the effects of the inclusion of SP-B in the liposome in the absence of any natural SP-B, alveolar cells of untreated animals were isolated, and 3 × 105 cells were incubated with different concentrations of liposomes in the presence of solution 2. These liposomes had SP-B incorporated in a SP-B-lipid ratio of 1:100. Uptake of these liposomes was unchanged compared with liposomes without SP-B. Neither uptake per cell (Fig. 4, A and C) nor the number of cells involved in the uptake (Fig. 4, B and D) was affected by the incorporation of SP-B in the liposomes.
For SP-C, liposomes with incorporated SP-C in a protein/lipid ratio of 1:100 were added at different concentrations to isolated alveolar cells and incubated for 1 h. At the highest-used liposome concentration (50 μg/ml), inclusion of SP-C results in an approximately fourfold increase in uptake by alveolar type II cells (Fig. 5A). In addition, the incorporation of SP-C in the liposomes not only increased uptake of these liposomes by alveolar type II cells but also induced a significantly higher uptake of these liposomes by alveolar macrophages.
Furthermore, the incorporation of SP-C does not affect the number of cells involved in the uptake at an SP-C:lipid ratio of 1:100 (Fig. 5, B and D).
Localization of cell-associated fluorescence.
To ascertain that the cell-associated fluorescence was located within the cell rather than binding to the outer membrane of the cell, confocal laser scanning microscopy was used. One hour after intratracheal instillation of the liposomes containing SP-B or SP-C in mechanically ventilated rats, the alveolar cells were isolated and confocal scans were made. These confocal scans, through the middle of the cell, show a punctuate fluorescence throughout the cell limited to its circumference with the exception of its nucleus, demonstrating the intracellular presence of the fluorescent liposomes. By scanning the cells at different levels, each 1 μm apart, we ensured that the used confocal scan was indeed through the middle of the cell, and, in addition, light microscopy of the same cell at the same setting demonstrated the limitation of the cell-associated fluorescence to the cytoplasm (see Fig. 6).
Effect of SP-B:lipid ratio on the uptake in vitro.
Although in the current study, no effect of inclusion of SP-B in the liposomes on the uptake by alveolar cells was observed, Rice et al. (24) have demonstrated a significant increase in uptake of liposomes by alveolar type II cells with SP-B incorporated. However, in their study, higher protein/lipid ratios were used. Therefore, using our method of fluorescently labeled liposomes, isolated alveolar cells of untreated animals were incubated with 50-μg/ml liposomes that had an increasing amount of SP-B included, varying from 1:100 to 8:100. Increasing the SP-B:lipid ratio results in a significantly enhanced uptake of liposomes by alveolar type II cells at an SP-B:lipid ratio of 8:100 (Fig. 7A). In addition, the inclusion of SP-B in the liposome at this particular ratio resulted in an increased number of alveolar type II cells involved in the uptake (Fig. 7B).
The effect of an increased SP-B:lipid ratio on the uptake of liposomes by alveolar macrophages demonstrated a similar significant increase at a SP-B:lipid ratio of 8:100. An increased ratio, however, did not influence the number of alveolar macrophages involved in the uptake of these liposomes.
In the current study, we investigated the effects of both SP-B and SP-C on the uptake of surfactant-like liposomes in vivo and in vitro.
As demonstrated in this study, SP-B does not influence the uptake of liposomes in vivo, not even at a SP-B:lipid ratio of 4:100. However, these liposomes were suspended in PBS before instillation in the trachea of the rats. Because the intra-alveolar fluid phase in rats is estimated to be ∼200 μl, and the liposomes instilled were suspended in 1 ml of PBS, this might lead to dilution of the intra-alveolar pool of solutes in the lung, including calcium.
Previous studies have demonstrated a crucial role of calcium on the biophysical function of SP-B as well in interaction with PG or formation of tubular myelin (12, 35). Therefore, to ensure more physiological circumstances, the liposomes were resuspended in solution 2 containing calcium, magnesium, potassium, and glucose in similar concentrations as the endogenous pool. However, despite resuspending the liposomes in solution 2, the incorporation of SP-B in the liposomes did not affect the uptake of lipids by alveolar cells.
Another explanation might be that the physiological amount of SP-B present in vivo is the optimal concentration for the SP-B-mediated lipid uptake by alveolar cells. In other words, raising the SP-B concentration by using SP-B-incorporated liposomes does not further influence the uptake. Therefore, the uptake of liposomes by isolated alveolar cells was studied in vitro using liposomes with and without incorporated SP-B. These experiments clearly demonstrate that at the physiological SP-B:lipid ratio of 1:100, no effect on the uptake of liposomes by alveolar type II cells was observed compared with the uptake of liposomes devoid of SP-B. However, Rice et al. (24) demonstrated a significant increase in uptake of liposomes by alveolar type II cells in vitro due to the presence of SP-B, although at higher SP-B:lipid ratios. To determine whether these results could be repeated and extended to alveolar macrophages using our method, we isolated alveolar cells and incubated these cells with a fixed concentration of lipids while increasing SP-B:lipid ratio up to 8:100. At the highest ratio, a significant increase in uptake was observed, indicating that SP-B is indeed able to increase uptake, in accordance with the results of the previous studies (14, 24).
Uptake of liposomes is suggested to take place via a receptor-mediated pathway (18, 21, 27, 28), although not exclusively, that is, where these “phospholipid” receptors tend to have a higher affinity for negatively charged lipids compared with neutrally charged lipids (21). However, the increase in uptake due to the incorporation of SP-B, although in a high protein:lipid ratio (8:100), seems unlikely to be caused by the presence of a specific SP-B receptor. The relatively high concentrations of SP-B necessary to induce an effect on the uptake make a specific receptor physiologically unlikely or suggest a receptor with a very low affinity. In summary, the SP-B-containing liposomes are internalized by the phospholipid receptors and not by additional SP-B receptors on the alveolar cells.
The increment in uptake caused by SP-B at the high protein- to-lipid ratio could also be explained by changes in lipid conformation of the liposomes because SP-B is able to change lipid conformation of surfactant (5).
In vivo alveolar type II cells internalize more liposomes when SP-C is incorporated in the liposomes at a protein-to-lipid ratio of 2:100. At other protein-to-lipid ratios, no effect of SP-C was detected even when the liposomes were suspended in solution 2. In contrast, significant stimulation of the uptake of liposomes containing SP-C by alveolar macrophages was observed at various lipid-to-protein ratios when these liposomes were suspended in solution 2.
To study the influence of possible additional effects of “environmental” factors in vivo, the uptake of liposomes with SP-C incorporated (SP-C:lipid ratio of 1:100) was studied in vitro. At this ratio, SP-C enhanced the uptake; at a lipid concentration of 50 μg/ml, the uptake of SP-C-containing liposomes was approximately six times greater by both alveolar macrophages and alveolar type II cells. These results suggest that in vivo, the uptake of SP-C-containing liposomes by alveolar cells may be suppressed by some unknown factor(s).
How may the incorporation of SP-C in liposomes enhance the uptake by alveolar cells? Rice et al. (24) and Horowitz et al. (14) have demonstrated previously that SP-C does enhance the uptake of lipids in a nonsaturable way. In addition, SP-C is known to associate very rapidly with lung tissue and alveolar macrophages (2, 3). This increased association, even more rapidly than DPPC, might be a possible explanation for an increased uptake of liposomes containing SP-C. However, other factors, such as the conformational changes observed in liposomes after incorporation of SP-C, may also affect the binding and uptake of these liposomes by alveolar cells, as was suggested by Rice et al. (24).
In summary, this study indicates that SP-B has no effect on the uptake of liposomes in vivo by alveolar cells. However, SP-C was shown to enhance the uptake of liposomes in vivo by alveolar cells, an effect that could be further increased by suspending the liposomes in a buffer that contained calcium, magnesium, and other solutes necessary for normal alveolar function. It is probable that other factors and not only these solutes may effect the uptake of the “SP-C-mediated uptake” of liposomes. We were able to demonstrate in vitro in the presence of physiological concentrations of calcium and magnesium that the SP-C-mediated uptake of liposomes is much higher by alveolar cells than in vivo, suggesting that in vivo the uptake appears to be downregulated. The precise mechanism of this possible downregulation of the uptake is not known but may include cell-to-cell contact and unknown soluble factors.
The results of the current study, absence of effects of SP-B and increased uptake due to SP-C, might be useful in clinical practice. Today, more and more exogenous surfactant preparations contain SP-B and SP-C, as these proteins have been demonstrated to increase efficiency of the surfactant (1).
To date it is widely accepted that small “inactive” surfactant aggregates are removed from the alveolar space by alveolar cells (33, 34). Exogenous surfactant is surface active and consists mainly of large “active” surfactant aggregates. However, after various breathing cycles, the exogenous surfactant will become small, inactive surfactant aggregates and need to be recycled. The presence of SP-B in these aggregates will probably not influence the uptake of these liposomes by alveolar type II cells, the first step in the recyling. On the other hand, inclusion of SP-C in exogenous surfactant in either the absence or presence of calcium and other solutes may enhance the recycling of the surfactant by increasing the uptake of surfactant by the alveolar cells. Therefore, variations in the SP-B concentrations of exogenous surfactant will probably have little influence on the recyling of surfactant, although variations in the SP-C concentrations may stimulate the recyling. However, it is still unknown which situation is most beneficial for the therapy of respiratory failure, an enhanced recycling or not.
This study was financially supported by LEO Pharmaceutical Products, Ballerup, Denmark.
The authors thank A. Visscher for technical assistance and Laraine Visser-Isles for English language editing.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 the American Physiological Society