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Molecular and functional evidence for a Na+–HCO3 −-cotransporter in sheep ruminal epithelium

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Abstract

The present study aimed to identify the HCO3 −-dependent mechanisms contributing to the homeostasis of the intracellular pH (pHi) in ruminal epithelial cells of sheep. Therefore, pHi was measured spectrofluorometrically in primary cultured ruminal epithelial cells loaded with the pH-sensitive fluorescent dye, 2′,7′-bis(carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester. Switching from a HEPES-buffered to a CO2/HCO3 −-buffered solution caused a rapid intracellular acidification followed by a counter-regulation towards alkaline levels. The counter-regulation was totally dependent upon extracellular Na+, but independent of intracellular Cl−. Adding 30 μM EIPA to the solutions did not affect the pHi counter-regulation following the acidification. Presence of 500 M H2DIDS inhibited the counter-regulation of pHi by 67%. These results pointed to a Na+–HCO3 −-cotransporter (NBC) as the main pHi regulatory mechanism in the presence of HCO3 −. Existence of an NBC in both cultured ruminal epithelial cells and intact ruminal epithelium was verified by reverse transcription polymerase chain reaction (RT-PCR) studies. RT-PCR yielded a band of the expected molecular size of 333 bp in both cultured cells and intact epithelium. The mRNA sequences were identical and shared a homology of 62% with human kidney NBC (Genebank accession number AF007216), of 66% with rat kidney NBC (AF004017) and of 65% with mouse duodenal NBC (AF141934).

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Abbreviations

BCECF/AM :

2′,7′-bis(carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester

EIPA :

5-(N-ethyl-N-isopropyl)-amiloride

FCS :

fetal calf serum

H 2 DIDS :

DIDS4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid

DPBS :

Dulbecco's phosphate-buffered saline

HEPES :

N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid

M-199 :

Medium 199

MCT :

monocarboxylate transporter

NBC :

Na+–HCO3 − -cotransporter

NCBE :

Na+-dependent Cl-/HCO3 −-exchanger

NHE :

Na+/H+ exchanger

NMDG + :

N-methyl-D-glucamine

PCR :

polymerase chain reaction

pH i :

intracellular pH

RT-PCR:

reverse transcription polymerase chain reaction

SCFA :

short-chain fatty acids

References

  • Amlal H, Burnham CE, Soleimani M (1999) Characterization of Na+/HCO3 − cotransporter isoform NBC3. Am J Physiol 276:F903–F913

    CAS  PubMed  Google Scholar 

  • Ash RW, Dobson A (1963) The effect of absorption on the acidity of rumen contents. J Physiol 169:39–61

    CAS  Google Scholar 

  • Bergmann EN (1990) Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol Rev 70:567–590

    PubMed  Google Scholar 

  • Boron WF, Boulpaep EL (1983) Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3 − transport. J Gen Physiol 81:53–94

    CAS  PubMed  Google Scholar 

  • Brune T, Fetzer S, Backus KH, Deitmer JW (1994) Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes. Pflugers Arch 429:64–71

    CAS  PubMed  Google Scholar 

  • Burnham CE, Amlal H, Wang Z, Shull GE, Soleimani M (1997) Cloning and functional expression of a human kidney Na+:HCO3 − cotransporter. J Biol Chem 272:19111–19114

    Article  CAS  PubMed  Google Scholar 

  • Choi I, Aalkjer C, Boulpaep EL, Boron WF (2000) An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel. Nature 405:571–575

    Article  CAS  PubMed  Google Scholar 

  • Gäbel G, Bestmann M, Martens H (1991) Influences of diet, short-chain fatty acids, lactate and chloride on bicarbonate movement across the reticulo-rumen wall of sheep. Zentralbl Vetreinarmed A 38:523–529

    Google Scholar 

  • Gálfi P, Neogrády S, Kutas F (1981) Culture of epithelial cells from bovine ruminal mucosa. Vet Res Commun 4:295–300

    PubMed  Google Scholar 

  • Gálfi P, Gäbel G, Martens H (1993) Influence of extracellular matrix components on growth and differentiation of ruminal epithelial cells in primary culture. Res Vet Sci 54:102–109

    PubMed  Google Scholar 

  • Grichtchenko II, Choi I, Zhong X, Bray-Ward P, Russel JM, Boron WF (2001) Cloning, characterization and chromosomal mapping of a human electroneutral Na+-driven Cl-HCO3 exchanger. J Biol Chem 276:8358–8367

    Article  CAS  PubMed  Google Scholar 

  • Heyer M, Muller-Berger S, Romero MF, Boron WF, Fromter E (1999) Stoichiometry of the rat kidney Na+-HCO3− cotransporter expressed in Xenopus laevis oocytes. Pflugers Arch 438:322–329

    Article  CAS  PubMed  Google Scholar 

  • Ilundáin A (1991) Intracellular pH regulation in intestinal and renal epithelial cells. Comp Biochem Physiol A 101:413–424

    Google Scholar 

  • Ishiguro H, Steward MC, Lindsay AR Case RM (1996) Accumulation of intracellular HCO3 − by Na(+)-HCO3 − cotransport in interlobular ducts from guinea-pig pancreas. J Physiol 495:169–178

    CAS  PubMed  Google Scholar 

  • Jensen LJ, Schmitt BM, Berger UV, Nsumu NN, Boron WF, Hediger MA, Brown D, Breton S (1999) Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis. Biol Reprod 60:573–579

    CAS  PubMed  Google Scholar 

  • Ko Yp, lang HJ, Loh SH, Chu KC, Wu ML (1999) Cl-dependent and Cl-independent Na+/HCO3 − acid extrusion in cultured rat cerebellar astrocytes. Chin J Physiol 42:237–248

    CAS  PubMed  Google Scholar 

  • Kramer T, Michelberger T, Gürtler H, Gäbel G (1996) Absorption of short-chain fatty acids across ruminal epithelium of sheep. J Comp Physiol B 166:262–269

    CAS  PubMed  Google Scholar 

  • Kristensen NB, Danfaer A, Agergaard N (1996) Diurnal patterns of ruminal concentrations and portal appearance rates of short-chain fatty acids in sheep fed a hay or a concentrate/straw diet in two meals daily. Acta Agric Scand Sect A, Anim Sci 46:227–238

    Google Scholar 

  • Little PJ, Neylon CB, Farelly CA, Weissberg PL, Cragoe EJ Jr, Bobik A (1995) Intracellular pH in vascular smooth muscle: regulation by sodium-hydrogene exchange and multiple sodium dependent HCO3 − mechanisms. Cardiovasc Res 29:239–246

    Article  CAS  PubMed  Google Scholar 

  • Müller F, Aschenbach JR, Gabel G (2000) Role of Na+/H+ exchange and HCO3 − transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen. J Comp Physiol B 170:337–343

    Article  PubMed  Google Scholar 

  • Müller F, Huber K, Pfannkuche H, Aschenbach JR, Breves G, Gäbel G (2002) Transport of ketone bodies and lactate in the sheep ruminal epithelium by monocarboxylate transporter 1. Am J Physiol Gastrointest Liver Physiol 283:G1139–G1146

    PubMed  Google Scholar 

  • Noël J, Pouysségur J (1995) Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. Am J Physiol 268:C283–C296

    CAS  Google Scholar 

  • Peral MJ, Calonge ML, Ilundain AA (1995) Na+-HCO3(−) cotransporter and intracellular pH regulation in chicken enterocytes. Pflugers Arch 430:612–616

    CAS  PubMed  Google Scholar 

  • Planelles G, Thomas SR, Anagnostopoulos T (1993) Change of apparent stoichiometry of proximal-tubule Na(+)-HCO3 − cotransport upon experimental reversal of its orientation. Proc Natl Acad Sci USA 90:7406–7410

    CAS  PubMed  Google Scholar 

  • Praetorius J, Hager H, Nielsen S, Aalkjaer C, Friis UG, Ainsworth MA, Johansen T (2001) Molecular and functional evidence for electrogenic and electroneutral Na(+)-HCO(3)(−) cotransporters in murine duodenum. Am J Physiol Gastrointest Liver Physiol 280: G332–G343

    CAS  PubMed  Google Scholar 

  • Pushkin A, Abdulaze N, Lee I, Newman D, Hwang J, Kurtz I (1999) Cloning, tissue distribution, genomic organization, and functional characterization of NBC3, a new member of the sodium bicarbonate cotransporter family. J Biol Chem 273:16569–16575

    Article  Google Scholar 

  • Romero MF, Boron WF (1999) Electrogenic Na+/HCO3 − cotransporters: cloning and physiology. Ann Rev Physiol 61:699–732

    Article  CAS  Google Scholar 

  • Rossmann H, Bachmann O, Vieillard-Baron D, Gregor M, Seidler U (1999) Na+/HCO3 − cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal and mucous cells. Gastroenterology 116:1389–1398

    CAS  PubMed  Google Scholar 

  • Sassani P, Pushkin A, Gross E, Gomer A, Abdulaze N, Dukkipati R, Carpentino G, Kurtz I (2002) Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter. Am J Physiol 282:C408–C416

    CAS  Google Scholar 

  • Schmitt BM, Biemesderfer D, Romero MF, Boulpaep EL, Boron WF (1999) Immunolocalization of the electrogenic Na+-HCO3 − cotransporter in mammalian and amphibian kidney. Am J Physiol 276: F27–F38

    CAS  PubMed  Google Scholar 

  • Sehested J, Diernaes L, Moller PD, Skadhauge E (1996) Transport of sodium across the isolated bovine ruminal epithelium: interaction with short-chain fatty acids, chloride and bicarbonate. Exp Physiol 81:79–94

    CAS  PubMed  Google Scholar 

  • Seidler U, Bachmann O, Jacob P, Christiani S, Blumenstein I, Rossmann H (2001) Na(+)/HCO(3)(−) cotransport in normal and cystic fibrosis intestine. JOP 2:247–256

    CAS  PubMed  Google Scholar 

  • Soleimani M, Burnham CE (2001) Na+:HCO3 − Cotransporters (NBC): cloning and characterization. J Membr Biol 183:71–84

    Article  CAS  PubMed  Google Scholar 

  • Teleky B, Hamilton G, Cosentini E, Bischof G, Riegler M, Koperna T, Feil W, Schiessel R, Wenzl E (1994) Intracellular pH regulation of human colonic crypt cells. Pflugers Arch 426:267–275

    CAS  PubMed  Google Scholar 

  • Thevenod F, Roussa E, Schmitt BM, Romero MF (1999) Cloning and immunolocalization of a rat pancreatic Na(+) bicarbonate cotransporter. Biochem Biophys Res Commun 264:291–298

    Article  CAS  PubMed  Google Scholar 

  • Thomas JA, Buchsbaum RN, Zimniak A, Racker E (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210–2218

    CAS  PubMed  Google Scholar 

  • Tobey NA, Reddy SP, Khalbuss WE, Silvers SM, Cragoe EJ Jr, Orlando RC (1993) Na(+)-dependent and -independent Cl−/HCO3 − exchangers in cultured rabbit esophageal epithelial cells. Gastroenterology 104:185–195

    CAS  PubMed  Google Scholar 

  • Vaneckova I, Vylitova-Pletichova M, Beskid S, Zicha J, Pacha J (2001) Intracellular pH regulation in colonocytes of rat proximal colon. Biochim Biophys Acta 1536:103–115

    CAS  PubMed  Google Scholar 

  • Virkki LV, Wilson DA, Vaughan-Jones R, Boron W (2002) Functional characterisation of human NBC4 as an electrogenic Na+-HCO3 − cotransporter (NBCe2). Am J Physiol 282: C1278–C1289

    CAS  Google Scholar 

  • Walter A, Gutknecht J (1986) Permeability of small nonelectrolytes through lipid bilayer membranes. J Membr Biol 90:207–217

    CAS  PubMed  Google Scholar 

  • Wang CZ, Yano H, Nagashima K, Seino S (2000) The Na+-driven Cl−/HCO3 −-exchanger. Cloning, tissue distribution, and functional characterization. J Biol Chem 275:35486–35490

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to Ines Urbansky for expert technical assistance. The experiments described in this report comply with the current legislation covering the protection of animals.

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Authors and Affiliations

  1. Veterinär-Physiologisches Institut, Universität Leipzig, An den Tierkliniken7, 04103, Leipzig, Germany

    K. Huhn, F. Müller, K. U. Honscha, H. Pfannkuche & G. Gäbel

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  1. K. Huhn
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  2. F. Müller
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  3. K. U. Honscha
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  4. H. Pfannkuche
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  5. G. Gäbel
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Corresponding author

Correspondence to H. Pfannkuche.

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Communicated by G. Heldmaier

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Huhn, K., Müller, F., Honscha, K.U. et al. Molecular and functional evidence for a Na+–HCO3 −-cotransporter in sheep ruminal epithelium. J Comp Physiol B 173, 277–284 (2003). https://doi.org/10.1007/s00360-003-0333-0

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