Elastase-induced Emphysema In Hamsters Research Articles

Proteasome inhibition improves diaphragm function in an animal model for COPD

Diaphragm muscle weakness in patients with chronic obstructive pulmonary disease (COPD) is associated with increased morbidity and mortality. Recent studies indicate that increased contractile protein degradation by the proteasome contributes to diaphragm weakness in patients with COPD. The aim of the present study was to investigate the effect of proteasome inhibition on diaphragm function and contractile protein concentration in an animal model for COPD. Elastase-induced emphysema in hamsters was used as an animal model for COPD; normal hamsters served as controls. Animals were either treated with the proteasome inhibitor Bortezomib (iv) or its vehicle saline. Nine months after induction of emphysema, specific force-generating capacity of diaphragm bundles was measured. Proteolytic activity of the proteasome was assayed spectrofluorometrically. Protein concentrations of proteasome, myosin, and actin were measured by means of Western blotting. Proteasome activity and concentration were significantly higher in the diaphragm of emphysematous hamsters than in normal hamsters. Bortezomib treatment reduced proteasome activity in the diaphragm of emphysematous and normal hamsters. Specific force-generating capacity and myosin concentration of the diaphragm were reduced by ~25% in emphysematous hamsters compared with normal hamsters. Bortezomib treatment of emphysematous hamsters significantly increased diaphragm-specific force-generating capacity and completely restored myosin concentration. Actin concentration was not affected by emphysema, nor by bortezomib treatment. We conclude that treatment with a proteasome inhibitor improves contractile function of the diaphragm in emphysematous hamsters through restoration of myosin concentration. These findings implicate that the proteasome is a potential target of pharmacological intervention on diaphragm weakness in COPD.

A Trail of Research Revisted

The author describes how his interest in the nutritional value of soybeans led to a trail of research that had many ramifications. In an attempt to account for the poor nutritional value of raw soybeans and the beneficial effect of heat treatment, a protein displaying hemagglutinating activity and capable of inhibiting the growth of rats was isolated and characterized. This protein subsequently proved to be an example of a class of proteins that were later referred to as "lectins". Lectins were also isolated from kidney beans and shown to be even more toxic than the soybean lectin. Concurrent studies with the soybean trypsin inhibitors revealed that these proteins inhibited growth by stimulating the secretory activity of the pancreas and could in the long term cause acinar cell adenoma of the pancreas. Acute experiments with human subjects showed that the human pancreas also responded to the stimulatory effects of the so-called Bowman-Birk soybean inhibitor. The studies on soybean trypsin inhibitors were expanded to include a protease inhibitor present in blood, alpha-1-antitrypsin, a deficiency of which leads to emphysema in humans. The mechanism whereby this protein inhibits leucocyte elastase was investigated. On the basis of these results the intratracheal administration of a synthetic peptide inhibitor of elastase attached to albumin microspheres was found to prevent elastase-induced emphysema in hamsters.

Pulmonary vascular reactivity and hemodynamic changes in elastase-induced emphysema in hamsters

Changes in pulmonary hemodynamics and vascular reactivity in emphysematous hamsters were studied in an isolated lung preparation perfused at constant flow with blood and 3% dextran. Hamsters were treated with intratracheal porcine pancreatic elastase at 70 days of age, and experimental studies were conducted at 1, 3, and 8 mo after treatment. Baseline pulmonary arterial pressure in elastase-treated lungs was increased compared with saline-treated control lungs 1 mo after treatment, but this increase did not progress at 3 and 8 mo. Increases in pulmonary arterial pressure in elastase-treated lungs were temporally correlated with the morphological development of emphysema and right ventricular hypertrophy; both of these were evident at 1 mo after treatment and showed little change thereafter. Pressor responses to hypoxia and angiotensin II were not different between elastase-treated and control lungs at 1 and 3 mo. At 8 mo, however, pressor responses in emphysematous lungs to 0% O2 (but not to angiotensin II) were significantly increased. This was the result of a lack of the normal age-related fall in the hypoxic pressor response. Our results suggest that the right ventricular hypertrophy found in these emphysematous animals results from a chronically increased pulmonary vascular resistance. Furthermore, increases in pulmonary vascular resistance in the early development of emphysema are likely a result of the loss of vascular beds and supporting connective tissue.

Differential Modification of Elastase-Induced Emphysema in Hamsters by Trypsin

The administration of trypsin 24 h before, admixed with, or 24 h after administration of an emphysema-inducing dose of porcine pancreatic elastase (PPE) to hamsters resulted in significantly enhanced destruction of lung tissue as evidenced by mean linear intercept values 4 weeks post PPE. The coadministration of trypsin with a nonemphysema-inducing dose of PPE resulted in a significant destructive lung lesion. Administration of trypsin 24 h before or admixed with human leukocyte elastase (HLE) resulted in a lesion that was significantly reduced relative to that produced by administration of HLE alone. When trypsin was administered 24 h after HLE, no effect on the lesion was observed. In vitro, coincubation of trypsin with PPE resulted in a slight enhancement of rate of hydrolysis of elastin, while coincubation of trypsin with HLE resulted in a significant reduction of the rate of hydrolysis. These results suggest that interaction with other proteases may offer an additional physiological control mechanism to prevent inappropriate tissue destruction.

In VivoandIn VitroLung Reactivity in Elastase-induced Emphysema in Hamsters

Although patients with chronic obstructive lung disease often show airway hyperresponsiveness to constrictor challenge, the mechanisms underlying this hyperreactivity are unknown. In this study, we tested whether the elastase-induced hamster model of emphysema and bronchial secretory cell metaplasia shows a similar hyperreactivity. Four weeks after intratracheal administration of 0.2 mg/100 g body weight porcine pancreatic elastase, the animals were anesthetized and ventilated with a constant tidal volume of 5 ml/kg. Changes in airway pressure (Paw) were monitored before and after intravenous challenge with 0.7 mg/kg acetylcholine (ACh). There was a significant decrease in baseline Paw from 5.7 +/- 0.6 cm H2O in control animals (n = 6) to 4.0 +/- 0.6 cm H2O in emphysematous animals (n = 6). The peak Paw response to the intravenous challenge, normalized to the baseline Paw, was 2.9 +/- 0.4 in control animals, but it was significantly increased in the emphysematous animals to 4.4 +/- 1.1. Trachea, bronchi, and parenchyma from these lungs were challenged with cumulative dose of ACh and KCl. Sensitivity of the trachea and bronchi to ACh challenge, assessed as the log ED50, did not show differences between the two groups. However, the emphysematous parenchyma showed greater sensitivity to ACh compared with the control parenchyma. Trachea and bronchi from emphysematous animals showed significantly decreased maximal contractility to challenge with both ACh and KCl. In contrast, the emphysematous parenchyma showed significantly greater maximal contractility. These findings were independent of the baseline passive tension. This increased responsiveness may relate to increased passive distensibility of the emphysematous parenchyma.

Amelioration of human neutrophil elastase-induced emphysema in hamsters by pretreatment with an oligopeptide chloromethyl ketone

Human neutrophils are a likely source of elastase in the pathogenesis of human pulmonary emphysema. A study was undertaken to determine whether emphysema, induced in hamsters by intratracheal treatment with human neutrophil elastase (HNE), could be ameliorated by intratracheal instillation of succinyl-alanyl-alanyl-prolyl-valine-chloromethyl ketone (CMK). One mg of CMK was given to hamsters 1 h before 300 or 360 micrograms HNE or 1 h or 4 h after 360 micrograms HNE. The animals were studied eight weeks after treatment. The CMK given 4 h after HNE did not ameliorate the emphysema. The CMK given 1 h before HNE, ameliorated the development of emphysema but not bronchial secretory cell metaplasia. A molar ratio of instilled CMK to HNE of 128 was required for 50% in vivo effectiveness in ameliorating emphysema. Clearance studies indicated that 6.9% of the instilled CMK could be lavaged from the lungs 1 h after instillation. Therefore, an 8.9 to 1 molar ratio of lavageable CMK to HNE, at the time of HNE instillation, resulted in 50% protection. Using an in vitro assay with 3H-elastin as substrate, a 3 to 1 molar ratio of CMK to HNE was required to inhibit 50% of the elastolytic activity; 14% of the activity remained with an 18 to 1 molar ratio of CMK to HNE. Study of the in vivo effectiveness of anti-elastases, given as pretreatment in ameliorating HNE-induced emphysema and secretory cell metaplasia, is a reasonable bioassay, which may be used as a step in evaluating such agents for possible use in the prevention of human disease.

Characterization of bronchoalveolar lavage cells and macrophage-derived chemoattractant activity in pancreatic elastase-induced emphysema in hamsters.

Bronchoalveolar lavage (BAL) was performed 30 days after endotracheal saline (C) or pancreatic elastase (E) administration to hamsters. E animals had twice as many cells as C animals (7.04 +/- 0.07 vs. 6.69 +/- 0.05 log10 cells, p less than 0.0001, paired t-test) and greater numbers of polymorphonuclear neutrophils (PMN) (6.61 +/- 0.05 vs. 5.27 +/- 0.04 log10 cells/animal, p less than 0.0001, t-test). On flow cytometric analysis and cell sorting, BAL cells from C animals showed a homogeneous population of cells with high light scatter and laser-excited intrinsic autofluorescence that were predominantly (greater than 99%) pulmonary alveolar macrophages (PAM). BAL cells from E animals had 80% PAM with characteristics similar to those of C animals; the remaining 20% of cells were smaller with lower forward and 90 degrees light scatter and four- to fivefold lower autofluorescence. These were found to be predominantly PMN on cytological examination of a sorted fraction. Fc receptors were expressed by greater than 95% of BAL cells from both C and E animals. To explain the altered cell population in E animals, PAM from both E and C animals were cultured in vitro, and the supernatants were assayed for neutrophil chemoattractant activity (NCA). In vitro PAM from E animals produced greater NCA than control PAM after 48 h in culture when stimulated with aggregated immunoglobulin or serum activates zymosan (p less than or equal to 0.003, ANOVA). Analyses of BAL supernatants 24 h after elastase administration indicated the presence of a heat-stable chemoattractant for PMN; such chemotactic activity, however, was not detectable 1 week or 4 weeks after the administration of elastase.

The prevention of elastase-induced emphysema in hamsters by the intratracheal administration of a synthetic elastase inhibitor bound to albumin microspheres.

The peptide, succinyl-alanyl-alanyl-prolyl-valine chloromethylketone (SPCK), a synthetic inhibitor of elastase, was covalently attached to human albumin microspheres (HAM) and administered intratracheally to hamsters 15 min and 8 h prior to the instillation of porcine pancreatic elastase. Pressure-volume relationships and histologic examination of excised lungs after 4 wk showed complete protection from emphysema when SPCK-HAM was administered either 15 min or 8 h before elastase exposure. Concurrent experiments with free SPCK showed that protection was achieved only if elastase was administered within 15 min after the instillation of SPCK. Extending this period to 8 h not only led to a failure of free SPCK to prevent emphysema but actually resulted in more extensive air-space enlargement. The prolonged effectiveness conferred by the attachment of SPCK to a biodegradable carrier should reduce the frequency with which it would have to be administered for the therapeutic intervention of emphysema and should minimize any toxic side reactions.

Cigarette smoke inhalation potentiates elastase-induced emphysema in hamsters.

Cigarette smoke is shown, in this study, to potentiate emphysema induced by intratracheal administration of elastase, whereas elastase alone caused minimal disease. Elastase-treated hamsters with prior or ongoing smoke exposure uniformly had severe emphysema with decreases in lung elastic recoil and increases in mean linear intercept (Lm). In contrast, hamsters receiving elastase alone, cigarette smoke alone, or postelastase cigarette smoke developed no or barely detectable histopathologic evidence of emphysema. Lungs from these animals had normal elastic recoil and Lm. These data support the idea that cigarette smoke exposure may unmask or amplify protease-induced emphysema caused by acute lung injury.

The effect of lung growth on the evolution of elastase-induced emphysema in the hamster

The effect of lung growth on the evolution of elastase-induced emphysema in hamsters was investigated using morphometric methods. Young (21–22 days) rapidly growing hamsters received intratracheally a single instillation of porcine pancreatic elastase (0.02 mg, the maximal tolerated dose). Adult hamsters (approximately 75 days old) received the same dose of elastase, per unit of body weight, as the young hamsters. Control young and adult animals were given saline by the same route. Animals from all groups were sacrificed at 1 and 50 days after the insult. The young animals, during this period, underwent a process of rapid somatic growth (body weight, body length) and of growth of the lungs (lung weight, lung volume). The increase in lung volume was accompanied by alveolar proliferation as assessed indirectly by measurement of the mean linear intercept and of the internal surface area and directly by alveolar counting. In the adult animals both the somatic and lung changes were of a much lower order of magnitude than in the young animals. Elastase administration resulted in an increase of the mean linear intercept and a decrease of the internal surface area at day 1 both in the young and in the adult hamsters. The severity of these changes was markedly increased by day 50 in the adult but not in the young animals. These results indicate that in rapidly growing hamsters, contrary to what happens in the adult animal, the severity of the elastase-induced emphysema does not progress with time. Alveolar multiplication may, in part or in whole, be responsible for this effect.

Effect of chloromethyl ketone on the progression of elastase-induced emphysema in hamsters.

Intratracheal administration of an elastase inhibitor (Succinyl alanyl alanyl prolyl valine chloromethyl ketone) into hamsters 20 days after induction of emphysema by porcine pancreatic elastase did not affect the lesion seen at 120 days. This study suggests that the progressive increase in lung volume between 20 and 120 days after emphysema induction was not due to a residue of active elastase in the lungs.

Role of alpha-macroglobulin-elastase complexes in the pathogenesis of elastase-induced emphysema in hamsters.

Radiolabeled, enzymatically active or chloromethyl ketone-inactivated porcine pancreatic elastase was endotracheally instilled into hamsters. Gel filtration of the bronchopulmonary lavage fluid revealed two major radioactive fractions: one, eluting at 780,000 daltons, corresponding to an alpha-macroglobulin-pancreatic elastase complex, and another, at 68,000 daltons, corresponding to an alpha-1-protease inhibitor-pancreatic elastase complex. Elastolytic activity was recovered in the bronchopulmonary lavage fluid up to 4 d after elastase instillation and was associated with the alpha-macroglobulin-pancreatic elastase complex. Small amounts of this complex were recovered 14 d after instillation. When less than 1% (1.5--1.7 micrograms) of the usual dose of elastase was instilled into hamsters, the major radioactive complex was alpha-1-protease inhibitor-pancreatic elastase complex, and little or no elastolytic activity was found in the lavage fluid. In contrast to the instillation of 220 micrograms of elastase, no disease or hemorrhagic reaction was detected with this low dose, and without hemorrhage only insignificant amounts of alpha-macroglobulin-pancreatic elastase complexes were recovered from the lungs. To study the interaction of circulating antiproteases with elastase, hamster plasma was allowed to interact directly with the radiolabeled elastase; alpha-macroglobulin bound much more of the elastase than alpha-1-protease inhibitor, confirming the findings in the lung lavage experiments. The hamster's susceptibility to pancreatic elastase-induced emphysema may depend on the preferential binding of elastase to alpha-macroglobulin, which protects the elastolytic potential, rather than to alpha-1-protease inhibitor, which inactivates elastase. We speculate that if even a fraction of the residual radioactivity found in the hamster lungs as long as 144 d after instillation of elastase represents enzymatically active alpha-macroglobulin-pancreatic elastase complex, this could serve as a source of persistent elastolytic activity, which might explain the progressive nature of the pulmonary lesion.

The effects of small doses of oligopeptide elastase inhibitors on elastase-induced emphysema in hamsters: a dose-response study.

Our previous studies have indicated that the synthetic elastase inhibitor N-acetyl-L-alanyl-L-alanyl-L-prolyl-L-alanyl chloromethylketone (AAPACK) administered intraperitoneally in divided doses totaling 19 mg, prior to and after a single intratracheal injection of elastase, substantially inhibited the development of experimental emphysema. The present studies evaluated the effects of 1.1, 4.1 and 8.0 mg AAPACK administered in divided doses 10 min prior to and 10, 30, and 50 min after a single intratracheal dose of elastase. The development of emphysema was essentially eliminated by 4.1 and 8.0 mg of AAPACK and markedly diminished with 1.1 mg AAPACK. The AAPACK was excreted rapidly in the urine at 30 to 60 min after its administration. Elastase inhibitory capacity (EIC) in urine was elevated to 14 times that in serum at the corresponding collection times. The plasma elastase inhibitory capacity was only slightly increased after administration of AAPACK and was not a sensitive indicator of elastase inhibition in the plasma.

The moderation of elastase-induced emphysema in the hamster by intratracheal pretreatment or post-treatment with succinyl alanyl alanyl prolyl valine chloromethyl ketone.

The capacity of the synthetic elastase inhibitor, succinyl alanyl alanyl prolyl valine chloromethyl ketone (CMK), to moderate elastase-induced emphysema in hamsters was examined using morphometric and physiologic measurements. When 0.5 mg of CMK in saline was injected intratracheally 1 h before the intratracheal injection of 0.1 mg of porcine pancreatic elastase, the hamsters did not develop emphysema. When CMK was injected intratracheally 1 h after elastase, the severity of emphysema was reduced by approximately 50% compared with control animals receiving saline 1 h after elastase. The CMK was ineffective when administered intratracheally 4 h after elastase.

5 -Hydroxytryptamine Uptake by Lungs of Hamsters with Pulmonary Emphysema

Elastase-induced emphysema in hamsters was used as a model to determine whether this type of chronic lung injury affected the metabolic function of pulmonary endothelium. 5-Hydroxytryptamine (5-HT) uptake was studied in isolated perfused lungs of hamsters from a control group and from those with emphysema of various severity. After measuring 5-HT uptake, we determined the mean linear intercept and internal surface area for each lung. The concentration of 5-HT used in the perfusate ranged from the level in the resting hamster to supraphysiologic levels. The results reveal that elastase-induced emphysema does not affect 5-HT uptake by the isolated perfused hamster lung despite significant loss of alveolar surface area.

Development of a Canine Model of Experimental Emphysema by Intratracheal Instillation of Elastase

216 22nd ASPEN LUNG CONFERENCE CHEST, 77: 2, FEBRUARY, 1980 SUPPLEMENT by alveolar macrophages. Am Rev Respir Dis 117:65, 1978 18 Carp H, Janoff A: Possible mechanisms of emphysema in smokers. In vitro suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants. Am Rev Respir Dis 118:617-21, 1978 19 Powers JC, Carroll DL, Tuhy P: Synthetic active sitedirected inhibitors of elastolytic proteases. Ann NY Acad SC! 256:420-25 20 Stone PJ, Lucey EC, Calore JD, et al: The moderation of elastase-induced emphysema in hamsters by post-treatment with a chloromethyl ketone elastase inhibitor. Am Rev Respir Dis 119:384, 1979

The effect of physical exercise on elastase-induced emphysema in hamsters.

The effect of physical exercise on the evolution of elastase-induced emphysema was investigated with morphometric methods in hamsters. The animals were given an intratracheal injection of 0.1 mg of pancreatic elastase and were divided into rest and exercise groups. Exercise was done in motor-driven activity wheels for 4 h/day, 5 days/wk. Controls consisting of animals given a saline injection followed the same schedule. One wk after the insult, both elastase-treated groups (rest and exercise) showed significant emphysematous changes, as assessed by an increase of the mean linear intercept and a decrease of the internal surface area of the lungs. By the fifth wk, emphysema had progressed significantly (approximately 20%) in both elastase groups. However, at neither point was there a significant difference between the rest and exercise groups. At 35 days, ultrastructural morphometry of the heart revealed a significant decrease in the percentage of mitochondria and an increase in the percentage of contractil...

Internal surface area and number of respiratory air spaces in elastase-induced emphysema in hamsters.

A stereologic study of the characteristics of the respiratory air spaces was carried out in 16 hamsters with emphysema induced by 0.2 mg of porcine pancreatic elastase per 100 g of body weight given endotracheally; 8 untreated hamsters served as control animals. The total number of alveoli was decreased from a mean ± SE of 5.10 ± 0.31 × 106 in the normal animals to 2.18 ± 0.27 × 106 in the emphysematous animals. The average ± SE mean linear intercept was 55 ± 1 × 10−4 cm for the normal group and 105 ± 4 × 10−4 cm for the emphysematous group. The mean ± SE internal surface area calculated from the mean linear intercept was 3,830 ± 125 cm2 for the normal group and 2,794 ± 85 cm2 for the emphysematous group; the comparable values calculated by the air space density method were 6,221 ± 183 and 4,281 ± 127 cm2, respectively. The mean internal surface area of the emphysematous lungs was 73 per cent of normal by the mean linear intercept method and 67 per cent of normal by the air space density method; the inter...

A one-year study of the evolution of elastase-induced emphysema in hamsters.

ARTICLESA one-year study of the evolution of elastase-induced emphysema in hamstersG. L. Snider, and C. B. SherterG. L. Snider, and C. B. SherterPublished Online:01 Oct 1977https://doi.org/10.1152/jappl.1977.43.4.721MoreSectionsPDF (3 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByElastase-coupled beads as a tool for characterizing localized alveolar tissue destruction associated with the onset of emphysemaJ. M. Craig, A. L. Scott, and W. Mitzner1 June 2013 | Journal of Applied Physiology, Vol. 114, No. 11Differences in acute lung response to elastase instillation in two rodent species may determine differences in severity of emphysema developmentAndrea Vecchiola, Juan Francisco de la Llera, Rodrigo Ramírez, Pablo Olmos, Cristobal I. Herrera, and Gisella Borzone1 July 2011 | American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Vol. 301, No. 1Differences in lung glutathione metabolism may account for rodent susceptibility in elastase-induced emphysema developmentGisella R. Borzone, Leonel F. Liberona, Andrea P. Bustamante, Claudia G. Saez, Pablo R. Olmos, Andrea Vecchiola, Andrea Villagrán, Carolina Serrano, and Tatiana P. Reyes1 April 2009 | American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Vol. 296, No. 4Rat and hamster species differences in susceptibility to elastase-induced pulmonary emphysema relate to differences in elastase inhibitory capacityGisella Borzone, Leonel Liberona, Pablo Olmos, Claudia Sáez, Manuel Meneses, Tatiana Reyes, Rodrigo Moreno, and Carmen Lisboa1 September 2007 | American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Vol. 293, No. 3Adenovirus-Mediated Transfer and Overexpression of Heme Oxygenase 1 cDNA in Lungs Attenuates Elastase-Induced Pulmonary Emphysema in MiceHuman Gene Therapy, Vol. 16, No. 3Cortical bone dynamics, strength, and densitometry after induction of emphysema in hamstersJill E. Shea, Scott C. Miller , David C. Poole, and John P. Mattson1 August 2003 | Journal of Applied Physiology, Vol. 95, No. 2Distribution of Lung Density and Mass in Patients With Emphysema as Assessed by Quantitative Analysis of CTChest, Vol. 118, No. 6Effects of emphysema and training on glutathione oxidation in the hamster diaphragmLeo M. A. Heunks, Aalt Bast, Cees L. A. van Herwaarden, Guido R. M. M. Haenen, and P. N. Richard Dekhuijzen1 June 2000 | Journal of Applied Physiology, Vol. 88, No. 6Effect of Lung Volume Reduction Surgery on Bony Thorax Configuration in Severe COPDChest, Vol. 116, No. 1Pulmonary emphysema decreases hamster skeletal muscle oxidative enzyme capacityJohn P. Mattson, and David C. Poole1 July 1998 | Journal of Applied Physiology, Vol. 85, No. 1Effects of emphysema on diaphragm blood flow during exerciseWilliam L. Sexton, and David C. Poole1 March 1998 | Journal of Applied Physiology, Vol. 84, No. 3Effect of pulmonary emphysema on diaphragm capillary geometryDavid C. Poole, and Odile Mathieu-Costello1 February 1997 | Journal of Applied Physiology, Vol. 82, No. 2Biological Effects of Inhaled 239PuO2 in Rats with Pre-existing Pulmonary Emphysema2 July 2016 | Human & Experimental Toxicology, Vol. 9, No. 5Strain specific respiratory air space enlargement in aged ratsExperimental Gerontology, Vol. 25, No. 6Early effects of intratracheal instillation of elastase on mortality, respiratory function, and pulmonary morphometry of F-344 ratsToxicology and Applied Pharmacology, Vol. 64, No. 3 More from this issue > Volume 43Issue 4October 1977Pages 721-729 Copyright & PermissionsCopyright © 1977 the American Physiological Societyhttps://doi.org/10.1152/jappl.1977.43.4.721PubMed908690History Published online 1 October 1977 Published in print 1 October 1977 Metrics