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Am J Physiol Regul Integr Comp Physiol 277: R147-R153, 1999;
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Vol. 277, Issue 1, R147-R153, July 1999

Differential regulation of functional responses by beta -adrenergic receptor subtypes in brown adipocytes

Archana Chaudhry and James G. Granneman

Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences, Wayne State University, School of Medicine, Detroit, Michigan 48201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Brown adipose tissue contains both beta 1- and beta 3-adrenergic receptors (beta -ARs), and whereas both receptor subtypes can activate adenylyl cyclase, recent studies suggest that these subtypes have different pharmacological properties and may serve different signaling functions. In this study, primary brown adipocyte cultures were used to determine the role of beta -AR subtypes in mediating lipolysis and uncoupling protein-1 (UCP1) gene expression, elicited by the physiological neurohormone norepinephrine (NE). NE increased both lipolysis and UCP1 mRNA levels in brown adipocyte cultures; the beta 1-receptor-selective antagonist CGP-20712A strongly antagonized the increase in UCP1 gene expression but had little effect on lipolysis. The beta 3-receptor-selective agonist CL-316243 (CL) also increased lipolysis and UCP1 mRNA levels, yet CL was more potent in stimulating lipolysis than UCP1 gene expression. NE also increased the phosphorylation of cAMP response element-binding protein (CREB) and perilipin (PL), both of which are protein kinase A substrates that are differentially targeted to the nucleus and lipid droplets, respectively. beta 1-receptor blockade inhibited NE-stimulated phosphorylation of CREB but not PL. The results suggest that beta -AR subtypes regulate different physiological responses stimulated by NE in brown adipocyte cultures in part by differentially transducing signals to subcellular compartments.

protein kinase A; adenosine 3',5'-cyclic monophosphate response element-binding protein; perilipin; lipid droplet; beta receptors; uncoupling protein; lipolysis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MAIN FUNCTION OF BROWN adipose tissue (BAT) is thermogenesis in response to sympathetic stimulation, and defects in BAT thermogenesis have been linked to obesity (28). The release of norepinephrine (NE) by sympathetic nerves and the subsequent stimulation of adenylyl cyclase via beta -adrenergic receptors (beta -ARs) are central to the control of BAT function (14). beta -ARs mediate many functional responses such as lipolysis, induction of uncoupling protein-1 (UCP1) gene expression, and cellular proliferation (3, 7, 8). BAT coexpresses beta 1- and beta 3-receptors (16, 20), and whereas the physiological relevance of these receptor subtypes remains to be determined, differences in coupling efficiency, desensitization pattern, and coupling to G proteins suggest that they might regulate different NE-stimulated functional responses in BAT (11, 16-18, 21). For example, previous studies have reported that beta 1- receptors regulate cellular proliferation (7) and beta 3- receptors regulate lipolysis (30). There has been no systematic investigation examining the simultaneous coupling of the beta -AR subtypes to NE-stimulated functional responses of BAT. In the present study, we have used primary brown adipocyte cultures to examine the contribution of beta 1- and beta 3-receptor subtypes to lipolysis and UCP1 gene expression elicited by NE. These results indicate that NE stimulates UCP1 induction mainly via beta 1-receptors and lipolysis mainly through activation of beta 3-receptors. Furthermore, this differential regulation of metabolic and genetic responses is paralleled by the ability of these receptors to phosphorylate proteins that are compartmentalized to the lipid droplet and nucleus of brown adipocytes.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue culture. Brown adipocyte progenitors were isolated from interscapular BAT from 19- to 23-day-old male Sprague-Dawley rats (Hilltop Lab Animals, Scottdale, PA) as previously described (29) but without a hyposmotic shock. Isolated precursor cells were seeded at a density of 2,000 cells/mm2 in DMEM supplemented with 10% neonatal calf serum, 4 nM insulin, 10 mM HEPES, and 50 IU/ml penicillin, 50 µg/ml streptomycin, and 25 µg/ml sodium ascorbate and grown at 37°C in an atmosphere of 10% CO2 in air. The medium was changed 1 day after plating, and after 3 days cells were grown in serum-free chemically defined medium (24) of the following composition: 50% DMEM, 50% Ham's F-12 medium, 16 µM biotin, 18 µM pantothenic acid, 5 mM glutamine, 16 mM glucose, 15 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, 100 µM ascorbate, 10 µg/ml transferrin, 510 nM insulin, and 200 nM 3,3',5-triiodo-L-thyronine. Progenitors differentiated 6-7 days after seeding.

Drug treatment. Differentiated brown adipocyte cultures were used 6-7 days after plating. Cultures were exposed to different concentrations of NE or CL-316243 (CL) in the presence or absence of various antagonists. For measurements involving lipolysis and UCP1 gene expression, cells were incubated with drugs for 4 h, and an aliquot of the medium was taken for measurement of glycerol released. The adipocytes were then washed once with phosphate-buffered saline, and total RNA was extracted as described previously (19). In experiments in which drug-stimulated phosphorylation of cAMP response element-binding protein (CREB) and perilipin (PL) was examined, cells were exposed to agonists for various times, the medium was aspirated, and the cells were lysed by addition of PAGE buffer. Proteins were determined using bicinchoninic acid protein assay reagent.

Preparation of subcellular fractions. Differentiated adipocytes were untreated or exposed to 100 nM NE. After 30 min, cells were washed once with phosphate-buffered saline and then homogenized in 25 mM HEPES containing 2 mM MgCl2, 1 mM EDTA, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 50 µg/ml Nalpha -tosyl-Lys-chloromethyl ketone, 100 µg/ml Nalpha -tosyl-Phe-chloromethyl ketone, and 240 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride. The homogenate was centrifuged at 12,000 g for 10 min. The resulting fat cake and nuclear pellet were subjected to SDS-PAGE as previously described.

Quantification of UCP1 mRNA. mRNA was measured by ribonuclease protection assay. The BamH I-Hind III fragment of rat UCP1 (6) was subcloned into PGEM 7Z vector (Promega) and used for preparing the [32P]cRNA probe. Tissue mRNA (5-15 µg) was coprecipitated with 3 × 104 counts/min [32P]cRNA probe, and samples were resuspended in 30 µl hybridization buffer containing 75% formamide, 400 mM NaCl, and 1 mM EDTA. Samples were resuspended in 30 µl hybridization buffer containing 75% formamide, 400 mM NaCl, 1 mM EDTA, and 40 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.4, and hybridized at 55°C for 12-18 h. Samples were diluted in 10 vol 300 mM NaCl, 5 mM EDTA, and 10 mM Tris, pH 7.5, and 300 U T1 ribonuclease were added to each sample. Digestions were stopped after a 60-min incubation at 37°C, and samples were precipitated in ethanol. The [32P]RNA probes that were protected from RNase digestion were electrophoretically resolved on a denaturing polyacrylamide gel containing 8 M urea. The gels were dried and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) for 18-72 h. The resulting autoradiograms were scanned in transmission mode with a Microtek Scanmaker E6 (Redondo Beach, CA) using Adobe Photoshop (San Jose, CA) and quantitated with Scion Image (Fredrick, MD).

Immunochemical detection of phosphoproteins. Cell lysates were denatured by the addition of 2-mercaptoethanol and boiling. The cell lysate (20-30 µg) was resolved by SDS-PAGE (12% polyacrylamide), transferred to nitrocellulose, and then blocked with either milk (for phospho-CREB) or BSA (for phosphoserine). The blots were then probed with either an antibody against phospho-CREB (1:1,000) or phosphoserine (1:2,000). Binding of the primary antibody to phosphoproteins was visualized by enhanced chemiluminescence (Pierce, Rockford, IL). Immunoreactivity was quantitated as described previously for the protection experiments.

Measurement of lipolysis. Brown adipocyte cultures were incubated for 4 h with various drugs, and lipolysis was monitored as the amount of glycerol released into the medium. Glycerol was measured by a coupled enzymatic assay as described previously (13).

Materials. Materials for nuclease protection were obtained from sources previously described (18). Collagenase was obtained from Worthington (Freehold, NJ). Bicinchoninic acid protein assay reagent and Supersignal chemiluminescent substrate kit were obtained from Pierce. CL was provided by American Cyanamid (Pearl River, NY). NE bitartrate was from Sigma Chemical (St. Louis, MO). ICI-118551 was a gift of Imperial Chemical Industries (Macclesfield, UK). CGP-20712A was provided by Ciba-Geigy (Summit, NJ). Rabbit polyclonal antibody to phospho-CREB was obtained from New England Biolabs (Beverly, MA). Rabbit polyclonal antibody to phosphoserine was obtained from Zymed (South San Francisco, CA).

Data analysis. Dose-response curves for agonists were fitted with nonlinear regression analysis for sigmoidal curves using GraphPad Prism software (San Diego, CA).

Differences between means were analyzed with Student's t-test. Differences between dose-response curves were analyzed with a two-way analysis of variance using repeated measures (GraphPad Prism).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Differential regulation of functional responses by beta -AR subtypes. In most experiments, we examined lipolysis (glycerol release) and UCP1 mRNA levels in 6- to 7-day-old primary brown adipocyte cultures after 4 h of exposure to NE. Glycerol release elicited by NE was linear over this time period (Fig. 1). Glycerol release and UCP1 mRNA levels were measured simultaneously in the same cultures. NE stimulated substantial glycerol release with a mean potency of 9.7 nM (Fig. 2A). Coincubation with the beta 1-receptor-selective antagonist CGP-20712A at a concentration that saturates beta 1- receptors (1 µM) did not affect NE-mediated lipolysis. NE also increased UCP1 mRNA levels with a mean potency of 2.4 nM (Fig. 2B). Unlike lipolysis, CGP-20712A strongly antagonized the increase in UCP1 gene expression, causing an eighteen-fold shift in the dose-response curve for UCP1 gene expression (P < 0.05). To rule out participation of beta 2- and alpha 1-adrenergic receptors in NE-mediated lipolysis and UCP1 gene expression, we investigated the effects of prazosin (Fig. 3, A and B) and ICI-118551 (Fig. 3, C and D), which are alpha 1- and beta 2-selective antagonists, respectively. Prazosin and ICI-118551 failed to significantly antagonize lipolysis or UCP1 gene expression elicited by NE (Fig. 3).


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  		Fig. 1.   Norepinephrine (NE)-stimulated lipolysis in brown adipocytes. Differentiated brown adipose tissue (BAT) cultures were incubated with 1 µM NE and 1 aliquot of medium was withdrawn at various times for glycerol determination. Results are shown from a representative experiment.



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  		Fig. 2.   Differential effects of CGP-20712A on NE-stimulated lipolysis and uncoupling protein-1 (UCP1) gene expression in brown adipocytes. Differentiated BAT cultures were incubated for 4 h with different concentrations of NE in absence and presence of CGP-20712A (1 µM). At end of incubation period, medium was taken for glycerol determination and the cells were used for determination of UCP1 mRNA levels as described in METHODS. Effect of CGP-20712A on dose-response curves was analyzed using 2-way analysis with repeated measures. A: NE-stimulated glycerol release is expressed as percent of maximal glycerol release (2.8 µmol/culture). NE dose-response curves for lipolysis in absence and presence of CGP-20712A were not significantly different from each other. B: NE dose-response curves for UCP1 gene expression in presence of CGP-20712A were significantly different from control curve (P < 0.05). Inset shows autoradiogram of nuclease protection assay of UCP1. Values are means ± SE; n = 6.



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  		Fig. 3.   Effect of prazosin and ICI-118 on NE-stimulated lipolysis (A and C) and UCP1 gene expression (B and D) in brown adipocytes. Differentiated BAT cultures were incubated for 4 h with different concentrations of NE in absence and presence of either prazosin (1 µM) or ICI-118551 (0.1 µM). Glycerol and UCP1 mRNA levels were determined in same cultures. Values are means ± SE; n = 3 or 4.

NE stimulates both beta 1- and beta 3-receptors, and the failure of CGP-20712A to antagonize NE-stimulated lipolysis, but not UCP1 gene expression, suggests that beta 3-receptors stimulate lipolysis more effectively than UCP gene expression. To test this hypothesis, we examined the effects CL, a highly selective beta 3-receptor agonist (4, 21). CL stimulated both lipolysis and UCP1 gene expression in differentiated brown adipocytes (Fig. 4), and the maximal response induced by CL was comparable to that induced by a maximal dose of NE (data not shown). CL was eight times more potent at stimulating lipolysis (EC50 0.17 nM) than it was in inducing UCP1 (EC50 1.43 nM; P < 0.05).


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  		Fig. 4.   Effects of CL-316243 (CL) on lipolysis and UCP1 mRNA levels. Differentiated BAT cultures were incubated for 4 h with different concentrations of CL. Glycerol and UCP mRNA levels were determined in same cultures. Concentration-response curves for CL-stimulated lipolysis and UCP1 induction were significantly different by 2-way analysis of variance (P < 0.05). Values are means ± SE; n = 6.

Phosphorylation of protein kinase A substrates by beta -ARs in brown adipocytes. The differential control of lipolysis and gene expression by the beta -AR subtypes suggests that the signals generated by the two receptor subtypes might be targeted to different subcellular domains and thus would be expected to phosphorylate distinct protein kinase A (PKA) substrates. Therefore, we examined NE-mediated phosphorylation of two proteins, CREB and PL, which are differentially compartmentalized in the nucleus and lipid droplet of adipocytes, respectively (22, 23, 34). Antibodies to phospho-CREB and phosphoserine were used to examine phosphorylation of CREB and PL, respectively. Brown adipocyte cultures were exposed for 30 min to NE and the subcellular distribution of phosphoproteins was examined in the nuclear pellet and lipid droplet, respectively. As expected, NE treatment caused the appearance of phospho-CREB in the nuclear pellet but not the lipid droplet (Fig. 5A). PL is the major PKA substrate in adipocytes where it is tightly bound to neutral lipids (22). NE treatment resulted in the strong phosphorylation of 63- to 64-kDa protein corresponding to PL (22) in the lipid droplet fraction but not nuclear pellet (Fig. 5B). Both phospho-CREB and phospho-PL were also detectable in total lysates obtained from NE-treated cells (Fig. 5, C and D), which were routinely used in subsequent experiments.


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  		Fig. 5.   NE-induced phosphorylation of cAMP response element-binding protein (CREB) and perilipin (PL). A and B: differentiated BAT cultures were treated with 100 nM NE for 30 min. Nuclear extracts and lipid droplets were prepared and subjected to SDS-gel electrophoresis as described in METHODS. Immunoblots were probed with either anti-phospho-CREB (A) or anti-phosphoserine (B). C and D: differentiated brown adipocytes were incubated for 10 min with 100 nM NE and immunoblot analysis of cell lysates was performed with anti-phospho-CREB (C) or anti-phosphoserine (D) antibodies.

To examine whether beta -AR subtypes differentially regulate the phosphorylation of compartmentalized PKA substrates, the effects of beta 1-receptor blockade on NE-induced phosphorylation of PL and CREB were examined in the same cultures. Cultures were examined after 30 min of stimulation at a time when CREB and PL are maximally phosphorylated. As shown in Fig 6, beta 1-receptor blockade with CGP-20712A strongly antagonized NE-induced phosphorylation of CREB but not PL.


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  		Fig. 6.   Effect of CGP-20712A (CGP-207) on NE-induced phosphorylation of CREB and PL. Differentiated BAT cultures were incubated for 30 min with 100 nM NE in presence or absence of 1 µM CGP-20712A. Cell lysates were probed with either anti-phospho-CREB or anti-phosphoserine antibodies. Results are expressed as percent of NE-induced CREB or PL phosphorylation in absence of CGP-20712A. Values are means ± SE; n = 3. * Significantly different from control samples incubated with NE in the absence of CGP-20712A (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

beta 1- and beta 3-receptors are coexpressed in brown adipocytes where they both stimulate cAMP production. Biochemical analysis of beta 1- and beta 3-receptors in transfected cells has pointed to differences in agonist affinity, coupling efficiency, and desensitization pattern, suggesting that these beta -AR subtypes might serve distinct, yet overlapping functions in adipocytes (17). Despite these suggestions, little work has been done to examine the coupling of the subtypes to different responses in cells that normally express these receptors. We therefore examined whether the beta -AR subtypes differentially contribute to the control of lipolysis and UCP1 induction in brown adipocytes. Differentiated adipocytes were used because beta 3-receptors, UCP1, and agonist-stimulated lipolytic responses are restricted to differentiated adipocytes (9, 24, 25).

The results indicate that beta 1-receptors participate more in NE-mediated UCP1 gene expression, whereas beta 3-receptors participate more in NE-mediated lipolysis. The differential control of lipolysis and UCP1 gene expression by the beta -AR subtypes was particularly evident at low, physiological concentrations of NE. At higher concentrations, NE stimulated UCP1 gene expression via both beta 1- and beta 3-receptors. The finding that the beta 3-agonist CL stimulates lipolysis more potently than UCP1 gene expression, also indicates that beta 3- receptors are better coupled to lipolysis. In other words, fewer beta 3-receptors need to be occupied to stimulate lipolysis versus UCP induction. That is not to say that beta 3-receptors are incapable of inducing UCP1 gene expression because maximal doses of CL stimulated UCP1 gene expression to the same degree as did NE. Indeed, beta 3-agonists stimulate UCP1 gene expression both in vitro and in vivo (8, 9, 15, 24, 29, 31, 32), but the potencies of these compounds in stimulating lipolysis and UCP1 gene expression have never been simultaneously compared in the same experiments.

The differential involvement of beta 1- and beta 3-receptors in NE-mediated responses in brown adipocytes appears to involve differential phosphorylation of compartmentalized PKA substrates. Thus phosphorylation of CREB was highly dependent on beta 1-AR activation whereas phosphorylation of PL was not. How beta -AR signals are differentially transduced to PKA substrates is not clear. It is possible that beta -AR subtypes are targeted to different plasma membrane domains. In this context, the carboxyl tail of the beta 1-, but not beta 3-, receptor contains a PDZ domain-binding motif that could mediate subcellular targeting (26). It is also possible that beta -AR subtypes are not compartmentalized, but that the signaling molecules with which they differentially interact are targeted to different subcellular compartments. cAMP generation involves multiple G proteins and adenylyl cyclase subtypes. For example beta 3-, but not beta 1-, receptors interact with Gi in white and brown adipocytes (11; unpublished results). A greater abundance of Gi near the nucleus versus the lipid droplet, might inhibit beta 3-receptor-mediated gene expression but not lipolysis. In this regard, recent studies have shown that beta 1- coexist with beta 2-receptors in cardiac myocytes but elicit qualitatively different cellular responses (35), possibly because beta 2- but not beta 1-receptors couple to a pertussis-sensitive G protein (36).

The findings discussed indicate that beta -AR signaling is functionally compartmentalized in brown adipocytes. Past evidence also indicates that G protein-mediated signaling and generation of cAMP are functionally compartmentalized in both white and brown adipose tissue. For example, catecholamine activation of lipolysis in both brown and white adipocytes requires far lower levels of cAMP than does activation by forskolin, suggesting that adrenergic receptors more effectively target cAMP generation to activation of lipolysis (2, 27). Also, whereas serotonin and catecholamines each increase cAMP levels in epididymal adipocytes, serotonin increases only phosphorylase activity and not lipolysis, whereas catecholamines increase both (23). These data further support the concept that cAMP generated by different receptors might be targeted to different subcellular compartments and PKA substrates in adipose tissue.

Perspectives

At first glance it is unclear why adipocytes express both beta 1- and beta 3-receptors given that either subtype stimulates adenylyl cyclase. Recent studies indicate that these receptor subtypes have different pharmacological sensitivities to endogenous catecholamines and differentially couple to G proteins and adenylyl cyclase subtypes (17, 18). Also, acute agonist exposure of adipocytes causes rapid desensitization of beta 1- but not beta 3-receptors (16). It has been suggested that beta 1-receptors could be important in mediating acute effects of low level sympathetic stimulation, whereas beta 3-receptors could be involved in mediating the chronic effects of somewhat higher sympathetic stimulation (17). CREB phosphorylation, which activates transcription of various genes (1), is a transient response, whereas PL phosphorylation is more sustained, as is lipolysis (data not shown). Therefore, beta 1-receptors are well suited to mediating transient CREB phosphorylation because these receptors are rapidly desensitized. In contrast, beta 3- receptors are more suited to mediating chronic responses such as lipolysis because these receptors do not undergo rapid desensitization. beta 3-receptors should also mediate NE-stimulated thermogenesis, which is triggered by free fatty acids resulting from lipolysis (3).

It remains to be determined whether beta 1- and beta 3- receptors differentially regulate catecholamine-stimulated BAT responses in vivo. The differential activation of functional responses by beta -AR subtypes could play a role in cold- and diet-induced thermogenesis. Both conditions increased sympathetic stimulation of BAT, resulting in the induction of both metabolic and genetic responses (14, 18, 28). beta 1-receptors probably control transient NE-mediated responses such as cellular proliferation and UCP1 gene expression, which increase the overall thermogenic capacity of BAT. beta 3-receptors are more likely to be involved in sustained NE-stimulated metabolic responses such as lipolysis and thermogenesis. On the other hand, the participation of beta -AR subtypes in BAT functional responses might not mirror that seen in brown adipocyte cultures and might depend on the expression of the various components of the beta -AR-PKA system. As stated previously, recent studies suggest that beta 1- and beta 3-receptors differentially couple to G proteins and adenylyl cyclase subtypes (17, 18). The coupling of beta 1- and beta 2-receptors to BAT adenylyl cyclase changes during periods of cold stress, and these alterations are associated with changes in the expression of beta -ARs, Gsalpha isoforms, GIalpha , and adenylyl cyclase subtypes (10, 12, 18). Thus it is likely that the relative contribution of beta -AR subtypes to specific responses will vary with the physiological state of the tissue.

Like BAT, beta 3-receptors control catecholamine-stimulated lipolysis in white adipose tissue (WAT) (5). Also, beta 3-, but not beta 1-, receptors couple to GI in this tissue (11). The participation of beta 1- and beta 3-receptors in WAT functional responses has not been systematically investigated despite evidence that suggests that PKA signaling is compartmentalized in this tissue. For example, catecholamine activation of lipolysis in white adipocytes requires lower levels of cAMP than does forskolin (2). Isoproterenol and serotonin, both of which elevate cAMP, produce different physiological responses in WAT (23). Insulin inhibits catecholamine-stimulated lipolysis but not glycogenolysis in adipocytes even though both responses are dependent on cAMP (33). Therefore, it is likely, that beta -AR subtypes control different catecholamine-stimulated functional responses in WAT as well.

In conclusion, our results show that NE stimulates functional responses in brown adipocytes via activation of both beta 1- and beta 3-receptors. beta 1-receptors are more involved in regulating UCP1 gene expression whereas beta 3-receptors are more involved in regulating lipolysis.


    ACKNOWLEDGEMENTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37006.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Chaudhry, Dept. of Cell Biology, Parke-Davis Pharmaceutical Res. Div., Warner-Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105 (E-mail: Archana.Chaudhry{at}wl.com).

Received 17 December 1998; accepted in final form 12 March 1999.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Compar Physiol 277(1):R147-R153
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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