A water channel of the nematode C. elegans and its implications for channel selectivity of MIP proteins

Michio Kuwahara1, Kenichi Ishibashi1, Yong Gu1, Yoshio Terada1, Yuji Kohara2, Fumiaki Marumo1, and Sei Sasaki1

1 Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519; and 2 Gene Network Laboratory, National Institute of Genetics, Mishima 441-8540, Japan

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

A genome project focusing on the nematode Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein superfamily. We functionally characterized one of these cDNAs named C01G6.1. Injection of C01G6.1 cRNA increased the osmotic water permeability (Pf) of Xenopus oocytes 11-fold and the urea permeability 4.5-fold but failed to increase the glycerol permeability. It has been speculated that the MIP family may be separated into two large subfamilies based on the presence or absence of two segments of extra amino acid residues (~15 amino acids) at the second and third extracellular loops. Because C01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with those of AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that of wild-type AQP-CE1, although the values of Pf and urea permeability were decreased by 39-74% and 28-65%, respectively. These results suggest that the two segments of extra amino acid residues may not contribute to channel selectivity or formation of the route for small solutes.

aquaporin; glycerol facilitator; water permeability; glycerol permeability; urea permeability; major intrinsic protein; Caenorhabditis elegans

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

WATER MOVEMENT across the plasma cell membrane is a fundamental process for the maintenance of the intracellular environment. Aquaporins (AQPs) are a family of integral membrane proteins that transport water. AQPs are members of the major intrinsic protein (MIP) superfamily and are found throughout nature (reviewed in Refs. 7 and 18). In Escherichia coli, there are two MIP family proteins, namely, glycerol facilitator (GlpF) (15) and water channel (AQPZ) (1). Yeast (Saccharomyces cerevisiae) has four MIP proteins in its genome. Plants have many MIP family proteins; for example, there are >23 MIP family proteins in Arabidopsis thaliana (21). Ten AQPs (AQP0-AQP9) have been identified so far in mammals, and these are widely distributed in water-transporting epithelia and endothelia of a variety of tissues (2, 4-6, 9, 14). In lower animals, functional water channels have been cloned in insects (12).

The hydropathy analysis of AQP/MIP family proteins predicts six transmembrane regions with the NH2 terminus and COOH terminus localized in the cytosol (Fig. 1). It has been suggested that AQP/MIP proteins may be divided into two groups, depending on the presence or absence of longer amino acid sequences at the second and third extracellular loops (7). In this study, we call these segment I and segment II (Figs. 1B and 2). Previous functional expression studies have demonstrated that AQPs lacking these segments (Fig. 1A, e.g., AQP1, AQP2, AQP4, and AQP5) are all water selective and exclude solutes (3, 7, 9). On the other hand, AQPs having these segments (Fig. 1B, e.g., AQP3, AQP7, and AQP9) are permeable to small molecules such as glycerol and urea in addition to water (2, 4, 5, 15). Another MIP protein that also has these segments, GlpF, permeates glycerol but not water (11, 15). Thus it was previously speculated that segments I and II participate in the formation of the transport pathway for small solutes (2) or in the alteration of channel selectivity (7).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Membrane topology of two types of major intrinsic protein (MIP) family proteins (A and B). In one type (B), extra amino acid residues (~15 amino acids) are present at the second and third extracellular loops. These extra segments are designated segment I and segment II in this study. In the other type (A), these segments are absent. Both types possess 6 presumed transmembrane segments, 5 connecting loops, and 2 conserved Asn-Pro-Ala (NPA) motifs.


View larger version (85K):
[in this window]
[in a new window]
 
Fig. 2.   Amino acid sequence of AQP-CE1 (C01G6.1) aligned with human AQP3, glycerol facilitator protein (GlpF), human AQP1, and human AQP2. Gaps (dashes) are inserted to maximize the matching. White letters in black boxes denote conserved amino acid residues.

Genome projects focusing on Caenorhabditis elegans have revealed the presence of eight cDNAs encoding MIP proteins (7, 18). Of these, segments I and II are present in C01G6.1, C35A5.1, F32A5.5, and M02F4.8 and are absent in CEH09F14 and F40F9.9. K02G10.7 possesses only segment I, whereas C32C4.2 posseses only segment II. However, their functions have not yet been examined. In this study, we compared the channel function of the C01G6.1 gene product with those of AQP3 and GlpF. Moreover, we performed chimeric studies of segments I and II to determine their physiological roles.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Preparation of cDNA. The cDNA library was constructed from mRNA of total embryo of the wild-type nematode C. elegans directionally into the EcoR I/Xho I site of Lambda ZAPII vector (Y. Kohara, unpublished observations). The random sequencing of the cDNA clones identified 14 clones encoding an MIP family protein. One clone was proved to be a cDNA reported as C01G6.1 in GenBank (accession no. Z35595). The cDNA insert for C01G6.1 (containing the open reading frame and untranslated sequences) was a blunt-end ligand inserted into the Bgl II site of a pSP64T-derived Bluescript vector containing 5'- and 3'-untranslated sequences of the beta -globin gene of Xenopus (pXBG-ev1; a generous gift from Dr. Peter Agre).

In vitro cRNA synthesis and site-directed mutagenesis. Capped RNA transcripts for C01G6.1, human AQP3, and GlpF were synthesized in vitro with T3 RNA polymerase after a digestion with Not I to linearize the plasmids. Mutants of C01G6.1 were made with the PCR technique using C01G6.1 cDNA as a template (10, 11). A fragment between the Hind III and Xba I sites was replaced by a PCR fragment coding the mutants. Amino acids of segment I (DGGVRTVG) and segment II (SIFYGGAVFTK) in C01G6.1 were replaced by those in human AQP3 (ADNQLFVS and ALAGWGSAVFTTGQH, respectively) or GlpF (EQTHHIVR and WLAGWGNVAFTGGRDIP, respectively) using the mutation primers listed in Table 1. After the mutations were confirmed by a DNA sequencer (Applied Biosystems 373A), these mutant cRNAs were constructed.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Oligonucleotide primers for mutagenesis of AQP-CE1

Water, glycerol, and urea permeability of oocytes. Oocytes at stages V-VI were obtained from Xenopus laevis. Each oocyte was injected with 40 nl of water, 5 ng of wild-type or mutated C01G6.1 cRNA, human AQP3 cRNA, or GlpF cRNA and was incubated for 48 h at 20°C in Barth's buffer. The osmotic water permeability (Pf) of the oocytes was measured at 20°C from the time course of osmotic cell swelling as previously described (10). After the initial incubation, the oocytes were transferred from 200 mosM Barth's buffer to 70 mosM buffer and were imaged on a charge-coupled device camera connected to an area analyzer (Hamamatsu Photonics C3160). Serial images were stored at 0.5-s intervals in a computer. Pf was calculated from the initial 15-s response of cell swelling. To examine the effect of mercury on oocyte Pf, the oocytes were incubated in Barth's buffer containing 0.3 mM HgCl2 for 5 min before the assay. Pf was also measured at 4°C and 30°C to obtain the activation energy (Ea) from an Arrhenius plot.

The glycerol permeability (Pgly) and urea permeability (Purea) were measured from the initial rate of glycerol and urea uptake into the oocytes, respectively (8, 11). Oocytes were incubated for 5 min at 20°C in Barth's buffer containing [U-14C]glycerol (Amersham) or [14C]urea (Amersham). After the incubation, the oocytes were rapidly rinsed three times in ice-cold Barth's buffer. The individual oocytes were lysed in 0.2 ml of 10% SDS and then were subjected to liquid scintillation counting.

Immunoblot analysis. Plasma membrane fractions of oocytes were obtained as previously described (19). The samples were heated at 70°C for 10 min and then separated by SDS-PAGE. Oocyte plasma membranes from 20 oocytes were applied in each lane. The samples were transferred to Immobilon-P filters (Millipore) using a semidry system. The filters were incubated for 1 h with an affinity-purified antibody against 15 COOH-terminal amino acids of the C01G6.1 gene product. The filters were further incubated for 1 h with 125I-labeled protein A solution and then examined by autoradiography.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The amino acid sequence of C01G6.1 (AQP-CE1) is shown in Fig. 2. The open reading frame encodes a protein of 290 amino acids with a relative molecular mass calculated at 31.4 kDa. Sequence alignment clearly shows that, just as in the case of AQP3 and GlpF, C01G6.1 also has two segments of extra amino acid sequences (segments I and II) that are not present in AQP1 and AQP2. No potential N-linked glycosylation sites are present in the sequences. There is one potential phosphorylation site by cAMP-dependent protein kinase (residue Ser-179) and two potential phosphorylation sites by protein kinase C (residues Ser-69 and Thr-280). A search of the protein database revealed the highest amino acid sequence identity with AQP3 (44%) and a lesser identity with other MIP proteins, i.e., E. coli GlpF (38%), AQPZ (36%), AQP1 (34%), AQP2 (36%), AQP4 (32%), AQP5 (34%), and AQP-gamma tonal intrinsic protein (36%).

We expressed C01G6.1, AQP3, and GlpF in Xenopus oocytes and compared their functions. Pf of water-injected (control) oocytes was 17 ± 2 × 10-4 (SE) cm/s (Fig. 3A). Injection with C01G6.1 cRNA increased Pf 10.9-fold, indicating that C01G6.1 is a water channel (designated AQP-CE1). Similar Pf was observed in AQP3-expressing oocytes. The Pf of AQP-CE1-expressing oocytes was not inhibited by incubation with 0.3 mM HgCl2, suggesting that AQP-CE1 is a mercury-insensitive water channel. Pf of oocytes injected with GlpF cRNA was not different from control Pf. To examine the temperature dependency of Pf, the Pf was measured at 4°C and 30°C (n = 10 each). The calculated Ea from the Arrhenius equation of Pf was 3.9 kcal/mol, a value in the range expected for a water channel (<6 kcal/mol).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3.   Function of AQP-CE1 (C01G6.1), AQP3, and GlpF expressed in Xenopus oocytes. A: osmotic water permeability (Pf). B: glycerol permeability (Pgly). C: urea permeability (Purea). Oocytes were injected with 40 nl of water (control) or 5 ng of cRNAs of AQP-CE1, human AQP3, and GlpF. Pf was calculated from the time course of osmotic cell swelling of the oocytes. Pgly and Purea were measured from the initial 5-min uptake of glycerol and urea into oocytes, respectively. Where indicated, the oocytes were preincubated with 0.3 mM HgCl2 (+Hg). Each bar represents means ± SE of 14-21 measurements.

Next, we measured Pgly and Purea in oocytes. Pgly was not stimulated after injection of AQP-CE1 cRNA, indicating that AQP-CE1 excludes glycerol (Fig. 3A). By contrast, Pgly was increased 4.8- and 6.7-fold after injection with human AQP3 cRNA and GlpF cRNA, respectively, a finding consistent with previous reports (8, 11, 13, 15). Purea of AQP-CE1-expressing oocytes was 4.7 times higher than that of control, indicating that AQP-CE1 has a urea permeability (Fig. 3B). The Purea of AQP-CE1 was not inhibited by 0.3 mM HgCl2, as observed with Pf. The Purea of AQP3 was as high as that of AQP-CE1, whereas Purea of GlpF was similar to that of control.

To examine the role of segments I and II (Figs. 1 and 2), these segments of AQP3 and GlpF were substituted for the corresponding segments of AQP-CE1. As summarized in Fig. 4A, Pf of segment I mutants [replacement of AQP-CE1 segment I with that of AQP3 segment I (AQP3 I) or GlpF segment I (GlpF I)] was decreased by ~66-70% but was ~2.7-3.2 times higher than control Pf. Segment II mutants (AQP3 II and GlpF II) also had water channel function, although Pf was lowered by ~39-42%. In double mutants of segments I and II (AQP3 I/II and GlpF I/II), ~67-74% decreases of Pf were observed, but Pf was still ~2.4-3.1 times higher than control Pf. All these mutants failed to gain a glycerol permeability (Fig. 4B). As shown in Fig. 4C, Purea of segment I, II, and I/II mutants was ~2.3-6.0 times higher than control Purea, indicating that all these mutants were permeable to urea. The immunoblot of oocyte membrane detected bands at 27 kDa in wild-type and mutated AQP-CE1s (Fig. 5). The band intensities were similar, and no glycosylated bands were detected.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Pf, Pgly, and Purea of wild-type and mutated AQP-CE1s expressed in oocytes. Segments I and II of AQP-CE1 were replaced by those of human AQP3 (AQP3 I and AQP3 II, respectively) and/or those of GlpF (GlpF I and GlpF II, respectively). Each bar represents means ± SE of 12-20 measurements.


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 5.   Immunoblot of oocyte plasma membrane fractions probed with an affinity-purified antibody against AQP-CE1. Oocytes were injected with water (control) or cRNA of wild-type or mutated AQP-CE1s. Segments I and II of AQP-CE1 were replaced by those of human AQP3 (AQP3 I and AQP3 II, respectively) and/or those of GlpF (GlpF I and GlpF II, respectively). Samples of the plasma membrane fraction from 20 equivalent oocytes were loaded in each lane.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

We characterized for the first time the function of a C. elegans MIP protein encoded by a cDNA named C01G6.1. Because the protein showed a water permeability when expressed in Xenopus oocytes (Fig. 3), we named it AQP-CE1. AQP-CE1 has a urea permeability in addition to its water channel function, but it does not have a glycerol permeability. AQP-CE1 is highly homologous to mammalian AQP3 (44%; Fig. 2). The phylogenetic comparison among MIP family members demonstrated that AQP3 is most related to bacterial GlpF and that AQP3 and GlpF have developed in a different branch from other AQP/MIP proteins (7, 18). This group is also characterized by the presence of segments I and II. Based on its sequence homology and the presence of segments I and II, AQP-CE1 seems to belong to this branch.

In human AQP3, we previously observed that the Tyr-to-Cys mutation at residue 212 (the position equivalent to the mercury-sensitive cysteine in AQP1 and AQP2) increased the inhibitory effect of mercury on Pf and Pgly in parallel and that the Tyr-to-Trp mutation at residue 212 decreased both Pf and Pgly to control levels (15). In addition, Ea for Pgly was 4.5 kcal/mol. Thus we concluded that water and small solutes may share a common pore in AQP3 and that the location of the pore site may be similar in AQP1, AQP2, and AQP3. In contrast, Echevarria et al. (2) previously observed no mercurial inhibition on Pgly and a high Ea for Pgly and Purea (>12 kcal/mol) in AQP3, raising the hypothesis that segments I and II contribute to a formation of the transport pathway for small solutes that is independent of the pore for water transport. We replaced segments I and II of AQP-CE1 with those of AQP3 and GlpF. If the above speculation were correct, the mutants of AQP3 I, AQP3 II, and/or AQP3 I/II could be expected to gain glycerol permeability, and the mutants of GlpF I, GlpF II, and/or GlpF I/II could be expected to lose urea permeability and gain glycerol permeability. Our results indicated that the basic characteristics of these mutants were not different from those of wild-type AQP-CE1, namely, the oocytes expressing these mutants possessed water and urea permeability but no glycerol permeability (Fig. 4). Thus segments I and II may not be directly related to the channel selectivity or to formation of the route for small solutes. However, the decreases in Pf (~39-74%) and Purea (~28-65%) were evident in these mutants, suggesting that segments I and II may play roles in the activity of the channel.

The present study and previous studies have revealed different patterns of water and solute selectivity in AQP/MIP proteins. For example, AQP1 and AQP2 are water selective and exclude solutes (3, 9); GlpF is glycerol selective and excludes water and urea (Fig. 3 and Ref. 15); AQP3 and AQP7 are permeable to water, glycerol, and urea (4, 8, 11); and AQP-CE1 and AQP9 are permeable to water and urea but exclude glycerol (Fig. 3 and Ref. 5). If we assume that water and solutes pass through the same pore, then we can safely conclude that the physical pore size alone is not the determinant of the selectivity of the pore. Recently, the three-dimensional structure of AQP1 has been partially explored (16, 20). These studies suggest that six alpha -helices form a central pore and that the first intracellular loop and third extracellular loop [both contain Asn-Pro-Ala (NPA) motifs] bend into the lipid bilayer to the center of this pore. Strictly conserved NPA motifs seem to be indispensable for the formation of the pore. If this model for AQP1 were found to be applicable to other AQP/MIP proteins, it would be tempting to speculate that amino acid sequences surrounding NPA motifs define the selectivity of the channel. This possibility should be examined in future.

The physiological significance of water channels has not been determined in nematodes. In situ hybridization of C. elegans embryo demonstrated transient expression of AQP-CE mRNA (17). The hybridization signal was first detected at the internal cells of midgastrulation. The cells of C lineage, which later develop to epidermis, seemed to express AQP-CE1 mRNA. The cells expressing AQP-CE1 mRNA increased progressively until the comma stage, eventually covering the whole embryo. At the 1.5-fold stage, the expression at the tail portion started to disappear while the cells at the head portion continued to express AQP-CE1 mRNA. The mRNA expression decreased gradually from the twofold stage and disappeared before hatching. The specific role of AQP-CE1 at this relatively narrow period in C. elegans embryogenesis remains to be clarified. The functional deletion of AQP-CE1 by induction of the mutation may shed light on its physiological significance.

    ACKNOWLEDGEMENTS

This work was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture, Japan, and by a grant from The Salt Science Research Foundation.

    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: M. Kuwahara, Second Dept. of Internal Medicine, Tokyo Medical and Dental Univ., Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.

Received 11 May 1998; accepted in final form 12 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Calamita, G., W. R. Bishai, G. M. Preston, W. B. Guggino, and P. Agre. Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli. J. Biol. Chem. 270: 29063-29066, 1995[Abstract/Free Full Text].

2.   Echevarria, M., E. E. Windhager, and G. Frindt. Selectivity of the renal collecting duct water channel aquaporin-3. J. Biol. Chem. 271: 25079-25082, 1996[Abstract/Free Full Text].

3.   Fushimi, K., S. Sasaki, T. Yamamoto, M. Hayashi, T. Furukawa, S. Uchida, M. Kuwahara, K. Ishibashi, M. Kawasaki, I. Kihara, and F. Marumo. Functional characterization and cell immunolocalization of AQP-CD water channel in kidney collecting duct. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F573-F582, 1994[Abstract/Free Full Text].

4.   Ishibashi, K., M. Kuwahara, Y. Gu, Y. Kageyama, A. Tohsaka, F. Suzuki, F. Marumo, and S. Sasaki. Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea. J. Biol. Chem. 272: 20782-20786, 1997[Abstract/Free Full Text].

5.   Ishibashi, K., M. Kuwahara, Y. Gu, F. Marumo, and S. Sasaki. Cloning and functional expression of a new aquaporin abundantly expressed in the peripheral leukocytes permeable to water and urea, but not to glycerol. Biochem. Biophys. Res. Commun. 244: 268-272, 1998[Medline].

6.   Ishibashi, K., M. Kuwahara, Y. Kageyama, A. Tohsaka, F. Marumo, and S. Sasaki. Cloning and functional expression of a second new aquaporin abundantly expressed in testis. Biochem. Biophys. Res. Commun. 237: 714-718, 1997[Medline].

7.   Ishibashi, K., and S. Sasaki. The dichotomy of MIP family suggests the origin of water channels. News Physiol. Sci. 13: 137-142, 1998.[Abstract/Free Full Text]

8.   Ishibashi, K., S. Sasaki, K. Fushimi, S. Uchida, M. Kuwahara, H. Saito, T. Furukawa, K. Nakajima, Y. Yamaguchi, T. Gojobori, and F. Marumo. Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells. Proc. Natl. Acad. Sci. USA 91: 6269-6273, 1994[Abstract].

9.   King, L. S., and P. Agre. Pathophysiology of the aquaporin water channels. Annu. Rev. Physiol. 58: 619-648, 1996[Medline].

10.   Kuwahara, M., K. Fushimi, Y. Terada, L. Bai, F. Marumo, and S. Sasaki. cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes. J. Biol. Chem. 270: 10384-10387, 1995[Abstract/Free Full Text].

11.   Kuwahara, M., Y. Gu, K. Ishibashi, F. Marumo, and S. Sasaki. Mercury-sensitive residues and pore site in AQP3 water channel. Biochemistry 36: 13973-13978, 1997[Medline].

12.   Le Caherec, F., S. Deschamps, C. Delamarche, I. Pellerin, G. Bonnec, M.-T. Guillam, D. Thomas, J. Gouranton, and J.-F. Hubert. Molecular cloning and characterization of an insect aquaporin: functional comparison with aquaporin 1. Eur. J. Biochem. 241: 707-715, 1996[Abstract].

13.   Ma, T., A. Frigeri, H. Hasegawa, and A. S. Verkman. Cloning of a water channel homolog expressed in brain meningeal cells and kidney collecting duct that functions as a stilbene-sensitive glycerol transporter. J. Biol. Chem. 269: 21845-21849, 1994[Abstract/Free Full Text].

14.   Ma, T., B. Yang, W.-L. Kuo, and A. S. Verkman. cDNA cloning and gene structure of a new water channel expressed exclusively in human kidney: evidence for a gene cluster of aquaporins at chromosome locus 12q13. Genomics 35: 543-550, 1996[Medline].

15.   Maurel, C., J. Reizer, J. I. Schroeder, M. J. Chrispeels, and M. H. Saier, Jr. Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. J. Biol. Chem. 269: 11869-11872, 1994[Abstract/Free Full Text].

16.   Mitra, A. K., A. N. Van Hoek, M. C. Wiener, A. S. Verkman, and M. Yaeger. The CHIP28 water channel visualized in ice by electron cryo-crystallography. Nature Struct. Biol. 2: 726-729, 1995[Medline].

17.  NEXTDB. The nematode expression pattern database. http://watson.genes.nig.ac.jp:8080/db/index.html.

18.   Park, J. H., and M. H. Saier, Jr. Phylogenetic characterization of the MIP family of transmembrane channel proteins. J. Membr. Biol. 153: 171-180, 1996[Medline].

19.   Wall, D. A., and S. Patel. Isolation of plasma membrane complexes from Xenopus oocytes. J. Membr. Biol. 107: 189-201, 1989[Medline].

20.   Waltz, T., B. L. Smith, P. Agre, and A. Engel. The three-dimensional structure of human erythrocyte aquaporin CHIP. EMBO J. 13: 2985-2993, 1994[Abstract].

21.   Weig, A., C. Deswarte, and M. J. Chrispeels. The major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group. Plant Physiol. 114: 1347-1357, 1997[Abstract/Free Full Text].


Am J Physiol Cell Physiol 275(6):C1459-C1464
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society