HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chew, S.B. C. Articles by Wong, P.Y.D. Search for Related Content PubMed PubMed Citation Articles by Chew, S.B. C. Articles by Wong, P.Y.D. Agricola Articles by Chew, S.B. C. Articles by Wong, P.Y.D.

Polarized Distribution of NHE1 and NHE2 in the Rat Epididymis

^a Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China ^b Gastroenterology Division, Department of Medicine, The John Hopkins University, School of Medicine, Baltimore, Maryland 21205-2195

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Previous studies from our laboratory have provided evidence that the rat epididymis utilizes the Na⁺/H⁺ exchanger to transport acid and base. The present study was undertaken to use immunohistochemistry for investigating the localization (apical versus basolateral) and distribution of NHE1 and NHE2 proteins along intact rat epididymis. Both proteins were found to be exclusively localized within the epithelium. Immunoreactivity for NHE1 was detected on the basolateral surface, whereas NHE2 immunoreactivity was detected on the apical side of the epithelium. Interestingly, NHE1 was found along the entire length of the epididymal tubule whereas NHE2 was absent in the initial segment but present in the caput, corpus, and cauda regions. These results, when interpreted along with those of previous functional studies, may suggest that the apical NHE2 is involved in Na⁺ reabsorption and the basolateral NHE1 in HCO₃^- secretion in the rat epididymis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Na⁺/H⁺ exchangers are expressed by eukaryotic cells, in which they have been shown to take part in the homeostatic control of intracellular pH, cell volume, and cell division. In epithelial tissues such as the gut and the kidney tubules, they also mediate transepithelial transport of salt and water. At least 6 Na⁺/H⁺ exchanger isoforms have now been cloned. Among them, NHE1, NHE2, and NHE3 are best characterized. NHE1 is ubiquitously expressed and is the basolateral isoform in the renal and intestinal epithelial cells. Functionally, it is responsible for pH and volume homeostasis. In contrast, expression of NHE3 is restricted to epithelial tissues such as the intestine, kidney, and testis. In intestinal and renal epithelial cells, NHE3 is located in the apical membrane, where it is thought to be responsible for Na⁺ reabsorption. Although NHE2 is also expressed in epithelial tissues, its cellular localization can be either apical or basolateral, depending on the cell type. For example, in intestinal epithelial cells, NHE2 is apically located [1] and together with NHE3 is involved in Na⁺ reabsorption under basal conditions [2]. In the medullary thick ascending limb of the kidney, both NHE2 and NHE3 are coexpressed on the apical membrane, where they are believed to participate in Na⁺ reabsorption [3]. However, in the inner segment of inner medullary collecting duct (IMCD3#) cells of the kidney, NHE2 is present in the basolateral membrane, where it is believed to participate in intracellular pH and cell volume regulation [4]. There is evidence that in the epididymis, which epitomizes the gut and the kidney tubule in electrolyte and water transport, Na⁺/H⁺ exchangers are involved in the reabsorption of sodium [5], secretion of hydrogen [6] and HCO₃^- ions [7], and regulation of intracellular pH [8]. In this study we used immunohistochemical method to detect the apical versus basolateral membrane distribution of two of the isoforms, NHE1 and NHE2, in the intact rat epididymis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antibody Production

Both polyclonal NHE1 and NHE2 antibodies were raised in rabbits. NHE1 antibody was raised against a purified fusion protein of maltose-binding protein and the putative cytoplasmic C-terminus of human NHE1 (amino acids 503–815), whereas NHE2 antibody was raised against a purified fusion protein of glutathione-S-transferase and the last 87 amino acids (amino acids 723–809) of rabbit NHE2 as previously described [1, 9]. It has been shown that NHE2 antibody does not cross-react with NHE1 or NHE3 [9]. Also, NHE1 antibody did not cross-react with NHE2 or NHE3. NHE1 labeled the basolateral surface but not the apical surface where NHE2 and NHE3 reside.

Immunohistochemistry

The animals used in this study were adult Sprague-Dawley (Harlan Industries, Indianapolis, IN) rats weighing between 350 and 400 g. They were killed by asphyxiation by CO₂. The epididymides (n = 6; 3 for NHE1 and 3 for NHE2) were carefully dissected and snap frozen with isopentane in liquid nitrogen. The weight of each epididymis was about 350 mg. Frozen sections of 8 µm and paraffin sections of 3 µm were prepared from these specimens.

Cryostat sections were air dried and fixed with acetone at -20°C for 10 min. They were then rinsed twice with pure water obtained from the NANOpure Bioresearch Deionization System (Barnstead/Thermolyne, Dubuque, IA) and incubated in methanol containing 3% H₂O₂ for 15 min, after which time they were rinsed again with pure water and PBS. Sections were incubated in normal blocking serum (Vectastain Elite ABC kit, Vector PK-6101; Vector Labs., Burlingame, CA) and then with NHE1 or NHE2 antibodies, diluted 1:100 with diluting buffer (PBS with 0.01% Triton X-100, 0.01% Tween 20, and 0.1% BSA) at 37°C for 1 h. Sections were washed with PBS and incubated with biotinylated secondary antibody (ABC kit) for 30 min, washed with PBS, incubated with Vectastain Elite ABC reagent (ABC kit) for 30 min, and finally washed with PBS again. Visualization was achieved by immersing sections in a peroxidase substrate solution (Vector VIP substrate kit) until desired stain intensity developed. Slides were rinsed with pure water for 5 min, counterstained with Lillie-Mayer's hematoxylin (Merck, Darmstadt, Germany), dehydrated, and mounted for observation. Negative controls were obtained by omission of primary antibodies.

Paraffin sections of Bouin's-fixed epididymides were dewaxed and hydrated. Antigen was retrieved by treatment in 0.01 M citrate buffer for 5 min in a microwave oven. The endogenous peroxidase was blocked by 3% H₂O₂ in methanol. Immunostaining proceeded as for the cryostat sections.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Immunoreactive NHE1 (irNHE1) was detected on the basolateral surface of the pseudostratified epithelial cells of the rat epididymis. Staining for NHE1 was observed from the initial segment to the tail of the epididymis. The initial segment and the caput regions were lightly stained (Fig. 1, a and b). In the corpus region, irNHE1 started to show a more prominent appearance (Fig. 1c) that extended to the cauda and the vas deferens. In the cauda epididymidis and the vas deferens, irNHE1 was clearly seen to be localized on the basal side (Fig. 1, d and e) of the epithelial cells. No irNHE1 was seen on the apical surface. This pattern of immunoreactivity was observed in all the epididymides used.

View larger version (149K): [in this window] [in a new window]   FIG. 1. Frozen sections of immunolocalization of NHE1 at various regions of rat epididymis. IrNHE1 was localized on the basal side of the tubular cells, which were more prominently stained in the corpus and the cauda, as well as in the vas deferens. L, Tubular lumen

NHE2 immunoreactivity (irNHE2) was also detected in the epididymis (Fig. 2, b–d). However, the regional distribution, as well as the cellular localization, was different from that of irNHE1. The initial segment of the epididymis was consistently negatively stained (Fig. 2a). Immunoreactive NHE2 was localized in the caput, corpus, and cauda epididymidis (Fig. 2, b–d). In contrast to irNHE1, irNHE2 was notably present on the apical (luminal) surface of the principal cells. As shown in Figure 2, b and d, the apical surfaces of the principal cells were highly stained with NHE2 antibody, while those of the clear cells and basal cells were not stained at all. The apical surface of the rat vas deferens exhibited similar results (not shown). Negative controls are shown in Figure 3, a and b. These are results from sections from which the NHE1 or NHE2 antibodies were omitted.

View larger version (114K): [in this window] [in a new window]   FIG. 2. Paraffin sections of immunolocalization of NHE2 in rat epididymis. a) The initial segment was negatively stained. b) Portion of the caput epididymidis with antibody concentration of 1:2000. The apical surface of the principal cells was intensely stained. The clear cells (arrow) and basal cells (arrowhead) were not stained. c) Apical irNHE2 expression (arrows) of the corpus region. d) The cauda region. Arrows indicate two clear cells with negative irNHE2. The smooth muscle layers were also unstained. L, Tubular lumen; Sm, smooth muscle layer

View larger version (107K): [in this window] [in a new window]   FIG. 3. Representatives of control sections

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Previous work in our laboratory has shown that the epididymis reabsorbs NaCl and water under basal conditions [5] but secretes NaCl and NaHCO₃ under stimulated conditions [7, 10, 11]. Reabsorption of Na⁺ is linked to secretion of hydrogen ions [6] and is inhibited by the apical addition of the diuretic drug, amiloride [5, 12]. It was from these studies that an apical Na⁺/H⁺ exchanger was implicated. Since NHE2 can be expressed in either the apical or the basolateral membranes of epithelial cells (see Introduction), we determined whether, in the epididymis, NHE2 is localized in the apical membrane where it could participate in Na⁺ reabsorption (as in the intestine) or in the basolateral membrane where it could regulate intracellular pH and cell volume (as in renal IMCD3 cells).

The detection of irNHE2 protein on the apical surface of the epididymal epithelium may suggest that this isoform is the likely candidate for Na⁺ and water reabsorption (Fig. 2). NHE2 is known to be amiloride sensitive [13, 14], as is Na⁺ reabsorption in the rat epididymis [5, 12, 15]. There is evidence also that in the intact rat epididymis, the luminal fluid becomes more acidic as it flows from the caput to the cauda epididymidis [16]. Luminal acidification may involve an apical NHE, as the process depends on the presence of intraluminal sodium and is amiloride sensitive [6]. These pieces of evidence, albeit circumstantial, lend support to the contention that NHE2 isoform is responsible for Na⁺ and fluid reabsorption and hydrogen ion secretion by the epididymal epithelium. However, it is not known why irNHE2 is not detected in the initial segment, which also reabsorbs fluid [17]. One possible explanation is that the initial segment may express NHE3, which is the main renal isoform of the Na⁺/H⁺ exchanger. Further study involving the anti-NHE3 antibody is needed to substantiate this point.

In contrast to irNHE2, irNHE1 was concentrated in the basal side of the epithelium (Fig. 1). This finding is therefore in line with previous reports that NHE1 is distributed in the basolateral membranes of many mammalian epithelial cells [18–20]. Short-circuit current measurement in cultured rat epididymal epithelia has shown that the cells secrete HCO₃^- electrogenically when stimulated by cAMP-elevating agents, forskolin, or cAMP [7, 21]. According to the secretion model, carbonic anhydrase in the cells catalyses the hydration of CO₂ to carbonic acid, which is dissociated into H⁺ and HCO₃^-. H⁺ is exported out of the cell across the basolateral membrane via a Na⁺/H⁺ exchanger, while HCO₃^- is secreted into the lumen through an apical cystic fibrosis transmembrane conductance regulator [22]. Secretion of HCO₃^- is blocked by basolateral addition of amiloride [7], which also inhibits the cloned NHE1 stably expressed in PS120 cells [13]. Basolateral NHE1 activity has been shown to be stimulated by protein kinase C [14], which also stimulates HCO₃^- secretion in epididymal epithelia [7]. It is plausible that the NHE1-immunoreactive protein detected in the basolateral membrane of the cells is the Na⁺/H⁺ exchanger responsible for HCO₃^- secretion. The present study therefore correlated the cellular distribution of irNHE1 and irNHE2 with previous functional studies performed in the same laboratory and provided evidence that in the epididymis, an apical NHE2 may be involved in NaCl reabsorption whereas a basolateral NHE1 may be involved in HCO₃^- secretion.

	FOOTNOTES

 
First decision: 19 February 1999.

¹ Correspondence. FAX: 852 2603 5022; patrickwong{at}cuhk.edu.hk

Accepted: October 22, 1999.

Received: December 21, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Hoogerwerf W, Tsao SC, Devuyst O, Levine SA, Yun CHC, Yip JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, Donowitz M. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am J Physiol 1996; 270:G29-G42.
2. Wormmeester L, Sanchez de Medina F, Kokke F, Tse CM, Khurana S, Bowser J, Cohan ME, Donowitz M. Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na⁺/H⁺ exchange. Am J Physiol 1998; 274:C1261-C1272.
3. Sun AM, Liu Y, Dworkin LD, Tse CM, Donowitz M, Yip KP. Na⁺/H⁺ exchanger isoform 2 (NHE2) is expressed in the apical membrane of the medullary thick ascending limb. J Membr Biol 1997; 160:85–90.[CrossRef][Medline]
4. Sun AM, Liu Y, Centracchio J, Dworkin LD. Expression of Na⁺/H⁺ exchanger isoforms in inner segment of inner medullary collecting duct. J Membr Biol 1998; 164:293–300.[CrossRef][Medline]
5. Wong PYD, Yeung CH. Absorptive and secretory functions of the perfused rat cauda epididymidis. J Physiol 1978; 275:13–26.[Abstract/Free Full Text]
6. Au CL, Wong PYD. Luminal acidification by the perfused rat cauda epididymidis. J Physiol 1980; 309:419–428.[Abstract/Free Full Text]
7. Wong PYD. Mechanism of adrenergic stimulation of anion secretion in cultured rat epididymal epithelium. Am J Physiol 1988; 254:F121-F133.
8. Wong PYD, Huang SJ. Intracellular pH measurement in primary monolayer cultures of rat epididymal cells. Pfluegers Arch Eur J Physiol 1989; 413:414–421.[CrossRef][Medline]
9. Tse CM, Levine SA, Yun CH, Khurana S, Donowitz M. Na⁺/H⁺ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein. Biochemistry 1994; 33:12954–12961.[CrossRef][Medline]
10. Wong PYD. Control of anion and fluid secretion by P₂-purinoceptor in the rat epididymis. Br J Pharmacol 1988; 95:1315–1321.[Medline]
11. Wong PYD, Fu WO, Huang SJ. Endothelin stimulates anion secretion in a cultured epithelium. Br J Pharmacol 1989; 98:191–196.
12. Wong PYD, Yeung CH. Inhibition by amiloride of the sodium dependent fluid reabsorption in isolated rat cauda epididymidis. Br J Pharmacol 1976; 58:529–532.[Medline]
13. Yun CHC, Little PJ, Nath SK, Levine SA, Pouysségur J, Tse CM. Donowitz M. Leu 143 in the putative fourth membrane-spanning domain is critical for amiloride inhibition of an epithelial Na⁺/H⁺ exchanger isoform (NHE2). Biochem Biophys Res Commun 1993; 193:532–539.[CrossRef][Medline]
14. Tse CM, Levine SA, Yun CHC, Brant SR, Pouysségur J, Montrose MH, Donowitz M. Functional characteristics of a cloned epithelial Na⁺/H⁺ exchanger (NHE3): resistance to amiloride and inhibition by protein kinase C. Proc Natl Acad Sci USA 1993; 90:9110–9114.[Abstract/Free Full Text]
15. Wong PYD, Au CL, Ngai HK. Some characteristics of salt and water transport in the rat epididymis. In: Fawcett DW, Bedford JM (eds.), Spermatozoon. Baltimore-Munich: Urban-Schwarzenberg; 1978: 57–63.
16. Caflisch CR, DuBose TD Jr. Direct evaluation of acidification by rat testis and epididymis: role of carbonic anhydrase. Am J Physiol 1990; 258:E143-E150.
17. Hohlbrugger G. Post-vasectomy impairment of transepithelial water reabsorption in the initial segment of the epididymis. Arch Androl 1983; 11:265–270.[Medline]
18. Watson AJM, Levine S, Donowitz M, Montrose MH. Kinetics and regulation of polarized Na⁺/H⁺ exchanger from Caco-2 cells, a human intestinal cell line. Am J Physiol 1991; 261:G229-G238.
19. Reilly RF, Hildebrandt F, Biemesderfer D, Sardet C, Pouysségur J, Aronson PS, Slayman CN, Igarashi P. cDNA cloning and immunolocalization of a Na⁺/H⁺ exchange in LLC-Pk_i renal epithelial cells. Am J Physiol 1991; 261:F1088-F1094.
20. Biemesderfer D, Reilly R, Exner M, Igarashi P, Aronson P. Immunocytochemical characterization of Na⁺/H⁺ exchanger isoform NHE1 in rabbit kidney. Am J Physiol 1992; 263:F833-F840.
21. Chan HC, Ko WH, Zhao W, Fu WO, Wong PYD. Evidence for independent Cl^- and HCO₃^- secretion and involvement of an apical Na⁺-HCO₃^- cotransporter in cultured rat epididymal epithelia. Exp Physiol 1996; 81:515–524.[Abstract]
22. Wong PYD. CFTR gene and male fertility. Mol Hum Reprod 1998; 4:107–110.[Abstract/Free Full Text]

This article has been cited by other articles:

				W. W. C. Shum, N. Da Silva, D. Brown, and S. Breton Regulation of luminal acidification in the male reproductive tract via cell-cell crosstalk J. Exp. Biol., June 1, 2009; 212(11): 1753 - 1761. [Abstract] [Full Text] [PDF]

				S. Seenundun and B. Robaire Time-Dependent Rescue of Gene Expression by Androgens in the Mouse Proximal Caput Epididymidis-1 Cell Line after Androgen Withdrawal Endocrinology, January 1, 2007; 148(1): 173 - 188. [Abstract] [Full Text] [PDF]

				N. Pastor-Soler, C. Pietrement, and S. Breton Role of Acid/Base Transporters in the Male Reproductive Tract and Potential Consequences of Their Malfunction Physiology, December 1, 2005; 20(6): 417 - 428. [Abstract] [Full Text] [PDF]

				C. L. Brett, M. Donowitz, and R. Rao Evolutionary origins of eukaryotic sodium/proton exchangers Am J Physiol Cell Physiol, February 1, 2005; 288(2): C223 - C239. [Abstract] [Full Text] [PDF]

				X.F. Wang, M.K. Yu, S.Y. Lam, K.M. Leung, J.L. Jiang, P.S. Leung, W.H. Ko, P.Y. Leung, S.B.C. Chew, C.Q. Liu, et al. Expression, Immunolocalization, and Functional Activity of Na+/H+ Exchanger Isoforms in Mouse Endometrial Epithelium Biol Reprod, January 1, 2003; 68(1): 302 - 308. [Abstract] [Full Text] [PDF]

				K.-H. Lee, C. Finnigan-Bunick, J. Bahr, and D. Bunick Estrogen Regulation of Ion Transporter Messenger RNA Levels in Mouse Efferent Ductules Are Mediated Differentially Through Estrogen Receptor (ER) {alpha} and ER{beta} Biol Reprod, November 1, 2001; 65(5): 1534 - 1541. [Abstract] [Full Text] [PDF]

				Q. Zhou, L. Clarke, R. Nie, K. Carnes, L.-W. Lai, Y.-H. H. Lien, A. Verkman, D. Lubahn, J. S. Fisher, B. S. Katzenellenbogen, et al. Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function PNAS, October 31, 2001; (2001) 241245898. [Abstract] [Full Text] [PDF]

				C. M. Herak-Kramberger, S. Breton, D. Brown, O. Kraus, and I. Sabolic Distribution of the Vacuolar H+ATPase Along the Rat and Human Male Reproductive Tract Biol Reprod, June 1, 2001; 64(6): 1699 - 1707. [Abstract] [Full Text]

				C. Bagnis, M. Marsolais, D. Biemesderfer, R. Laprade, and S. Breton Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis Am J Physiol Renal Physiol, March 1, 2001; 280(3): F426 - F436. [Abstract] [Full Text] [PDF]

				G.P.H. Leung, K.H. Cheung, C.M. Tse, and P.Y.D. Wong Na+ Reabsorption in Cultured Rat Epididymal Epithelium via the Na+/Nucleoside Cotransporter Biol Reprod, March 1, 2001; 64(3): 764 - 769. [Abstract] [Full Text]

				G.P.H. Leung, C.M. Tse, S.B. Cheng Chew, and P.Y.D. Wong Expression of Multiple Na+/H+ Exchanger Isoforms in Cultured Epithelial Cells from Rat Efferent Duct and Cauda Epididymidis Biol Reprod, February 1, 2001; 64(2): 482 - 490. [Abstract] [Full Text]

				Q. Zhou, L. Clarke, R. Nie, K. Carnes, L.-W. Lai, Y.-H. H. Lien, A. Verkman, D. Lubahn, J. S. Fisher, B. S. Katzenellenbogen, et al. From the Cover: Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function PNAS, November 20, 2001; 98(24): 14132 - 14137. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chew, S.B. C. Articles by Wong, P.Y.D. Search for Related Content PubMed PubMed Citation Articles by Chew, S.B. C. Articles by Wong, P.Y.D. Agricola Articles by Chew, S.B. C. Articles by Wong, P.Y.D.