Expression of rat, renal NHE2 and NHE3 during postnatal development

In apical membrane vesicles from beef tracheal epithelia expressing up to 30% of the proteins as functional cystic fibrosis transmembrane conductance regulator (CFTR)-- i.e. a voltage-independent and PKA-sensitive 36Cl- flux--an ATPase activity, different from P, F0F1 and V types, was reproducibly detected. Its specific activity averaged 20 micromol Pi h(-1) mg(-1) with an apparent affinity for ATP of 530 +/- 30 microM. Its possible involvement in CFTR functions was supported by (1) the linear relationship between the ATPase activity and the magnitude of 36Cl- fluxes (turnover rate: 3 ATP hydrolyzed per CFTR per second), (2) the same rank of potency of ATP, ITP, GTP, UTP and CTP to be hydrolyzed and to open CFTR chloride channels, (3) the similar and parallel inhibition of the ATPase and CFTR Cl- fluxes by NS004 (IC50: 60 microM) and (4) the potency of anti-R domain antibodies to increase by 18% the ATPase activity.     

Polarized cylindrical body in the epididymis of the flying squirrel

Spermatozoa in the head of the epididymis of the flying squirrel have large cup-shaped acrosomal heads with two ventral ridges. The cytoplasmic droplet contains an ovoid body and a group of large granules. These structures may be related to the chromatoid body of spermatids. The spermatozoa form polarized cylindrical bodies with centrally placed tails and peripheral heads. The tips of acrosomes protrude into concavities of acrosomal cups of neighboring spermatozoa. Peripheral portions of acrosomes are in contact with microvilli (stereocilia) of epididymal cells. Polarized cylindrical bodies are present in five species of Sciuridae.     

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.     

The presence and longitudinal distribution of the glutathione S-transferase in rat epididymis and vas deferens

The presence of the glutathione S-transferases, enzymes that catalyse the conjugation of glutathione with a variety of compounds, is reported here, for the first time, in the mammalian epididymis–vas deferens. These glutathione S-transferases, approx. 50% of those from rat liver on a per-mg-of-protein basis, are resolved by isoelectric focusing into six peaks, each with a characteristic isoelectric point and substrate specificity. By these same criteria, the first three peaks (pI 8.9, 8.2 and 7.8) can be identified as transferases B, A and C respectively. The fifth peak (pI7.2) may correspond to transferase M; the fourth (pI7.5) and sixth (pI7.0) peaks do not correspond to previously described transferases. The distribution of transferase activity towards any one substrate studied differs in sequential sections of the epididymis and vas deferens; in addition, the longitudinal-distribution pattern differs for each of the three substrates studied. Isoelectric focusing of the cytosol fractions of the different sections further substantiates these observations. The potential significance of these enzymes and of their distribution in terms of epididymal function, maturation of spermatozoa, is discussed.     

Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Intestinal NaCl transport in NHE2 and NHE3 knockout mice

Sodium/proton exchangers [Na(+)/H(+) (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na(+) and Cl(-) flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na(+) (approximately 6 microeq/cm(2) x h) and Cl(-) (approximately 3 microeq/cm(2) x h) absorption. In contrast, NHE3(-) jejuna had reduced net Na(+) absorption (approximately 2 microeq/cm(2) x h) but absorbed Cl(-) at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na(+) and Cl(-) absorption in all genotypes. Studies of the Na(+) absorptive flux (J) indicated that J in NHE(+) jejunum was not sensitive to 1 microM EIPA, whereas J in NHE3(-) jejunum was equally sensitive to 1 and 100 microM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP(i)) abolished net NaCl absorption and stimulated electrogenic Cl(-) secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na(+) absorption; 2) an amiloride-sensitive Na(+) transporter partially compensates for Na(+) absorption in NHE3(-) jejunum; 3) cAMP(i) stimulation abolishes net Na(+) absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl(-) absorption is not directly dependent on either NHE2 or NHE3.     

IFN-gamma downregulates expression of Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells

Diarrhea associated with inflammatory bowel diseases has traditionally been attributed to stimulated secretion. The purpose of this study was to determine whether chronic stimulation of intestinal mucosa by interferon-gamma (IFN-gamma) affects expression and function of the apical membrane Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells expressing NHE2 and NHE3 were treated with IFN-gamma for 2, 24, and 48 h. Adult rats were injected with IFN-gamma intraperitoneally for 12 and 48 h. NHE2 and NHE3 activities were measured by unidirectional (22)Na influx across C2 cells and in rat brush-border membrane vesicles. NHE protein and mRNA were assessed by Western and Northern blotting. IFN-gamma treatment of C2 monolayers caused a >50% reduction in NHE2 and NHE3 activities and protein expression. In rats, region-specific, time- and dose-dependent reductions of NHE2 and NHE3 activities, protein expression, and mRNA were observed after exposure to IFN-gamma. Chronic exposure of intestinal epithelial cells to IFN-gamma results in selective downregulation of NHE2 and NHE3 expression and activity, a potential cause of inflammation-associated diarrhea.     

Polarized distribution of endogenous Rac1 and RhoA at the cell surface.

Rac1 and RhoA regulate membrane ruffling and stress fiber formation. Both molecules appear to exert their control from the plasma membrane. In fibroblasts stimulated with platelet-derived growth factor or lysophosphatidic acid, the reorganization of the cytoskeleton begins at specific sites on the cell surface. We now report that endogenous Rac1 and RhoA also have a polarized distribution at the cell surface. Cell fractionation and immunogold labeling show that in quiescent fibroblasts both of these molecules are concentrated in caveolae, which are plasma membrane domains that are associated with actin-rich regions of the cell. Treatment of these cells with platelet-derived growth factor stimulated the recruitment of additional Rac1 and RhoA to caveolae fractions, while lysophosphatidic acid only caused the recruitment of RhoA. We could reconstitute the recruitment of RhoA using either whole cell lysates or purified caveolae. Surprisingly, pretreatment of the lysates with exoenzyme C3 shifted both resident and recruited RhoA from caveolae to noncaveolae membranes. The shift in location was not caused by inactivation of the RhoA effector domain. Moreover, chimeric proteins containing the C-terminal consensus site for Rac1 and RhoA prenylation were constitutively targeted to caveolae fractions. These results suggest that the polarized distribution of Rho family proteins at the cell surface involves an initial targeting of the protein to caveolae and a mechanism for retaining it at this site.     

Androgens and androgen-binding protein in the rat epididymis

The levels of testosterone, dihydrotestosterone (DHT), 5alpha-androstan-3alpha,17beta-diol and androgen-binding protein (ABP) were measured in various segments of the epididymis from adult rats which had been unilaterally orchidectomized for 4 weeks. On the 'intact' side, ABP concentrations were highest in the caput region. The segmental distribution of DHT closely followed that of ABP with the highest concentration in the caput (40 ng/g tissue) and lowest in the cauda (10 ng/g tissue) epididymidis. There was a high degree of correlation (r = 0.98) between the concentration of DHT in the epididymis and ABP levels. 'Castration' completely abolished the DHT gradient. The levels of testosterone and androstanediol were lower than those of DHT; most was present in the corpus epididymidis. The relative differences were reduced after 'castration'. It is concluded that ABP in the rat epididymis is the primary factor for determining the concentration of DHT in the epididymal fluid.     

Immunolocalization of anion exchanger AE2, Na(+)/H(+) exchangers NHE1 and NHE4, and vacuolar type H(+)-ATPase in rat pancreas.

We have studied the expression and localization of several H(+) and HCO(3)(-) transporters, whose presence in the rat pancreas is still unclear. The Cl(-)/HCO(3)(-) exchanger AE2, the Na(+)/H(+) exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H(+)-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at approximately 160 kD for AE2, at approximately 90 kD and approximately 103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl(-) uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO(3)(-) secretion. The molecular identity of the ductal Cl(-)/HCO(3)(-) exchanger remains unclear.     

Immunolocalization of AE2 anion exchanger in rat and mouse epididymis.

A low-bicarbonate concentration and an acidic pH in the luminal fluid of the epididymis and vas deferens are important for sperm maturation. These factors help maintain mature sperm in an immotile but viable state during storage in the cauda epididymidis and vas deferens. Two proton extrusion mechanisms, an Na(+)/H(+) exchanger and an H(+)ATPase, have been proposed to be involved in this luminal acidification process. The Na(+)/H(+) exchanger has not yet been localized in situ, but we have reported that H(+)ATPase is expressed on the apical membrane of apical (or narrow) and clear cells of the epididymis. These cells are enriched in carbonic anhydrase II, indicating the involvement of bicarbonate in the acidification process and suggesting that the epididymis is a site of bicarbonate reabsorption. Previous unsuccessful attempts to localize the Cl/HCO(3) anion exchanger AE1 in rat epididymis did not investigate other anion exchanger (AE) isoforms. In this report, we used a recently described SDS antigen unmasking treatment to localize the Cl/HCO(3) exchanger AE2 in rat and mouse epididymis. AE2 is highly expressed in the initial segment, intermediate zone, and caput epididymidis, where it is located on the basolateral membrane of epithelial cells. The cauda epididymidis and vas deferens also contain basolateral AE2, but in lower amounts. The identity of the AE2 protein was further confirmed by the observation that basolateral AE2 expression was unaltered in the epididymis of AE1-knockout mice. Basolateral AE2 may participate in bicarbonate reabsorption and luminal acidification, and/or may be involved in intracellular pH homeostasis of epithelial cells of the male reproductive tract.     

Cyclooxygenase-2 regulates apoptosis in rat epididymis through prostaglandin D2.

In previous studies, cyclooxygenase (COX)-1 and COX-2 isozymes have been detected in the rat epididymis. COX-1 mediates electrolyte and fluid secretion induced by a number of peptide hormones, including bradykinin, angiotensin, and endothelin, via local formation of prostaglandin (PG) E2; however, the physiological role of COX-2 remains largely unknown. Marked apoptotic cell death in the rat epididymis following androgen depletion has been reported. Because expression of both COX isozymes is dependent on androgen, we investigated whether these isozymes control apoptosis in the epididymis. Apoptosis was detected in rat epididymal epithelial cells by in situ staining using the TUNEL method and by the presence of internucleosomal DNA fragmentation using capillary electrophoresis with laser-induced fluorescence detection. Specific COX inhibitors were used to delineate the roles of the 2 isozymes. There was no significant apoptotic cell death in normal and specific COX-1 inhibitor (SC-560)-treated epididymal cells. However, application of a specific COX-2 inhibitor (NS-398) induced apoptosis in a dose- and time-dependent manner. A similar apoptotic effect of COX-2 inhibitor was seen in the in vivo study. The drastic DNA fragmentation induced by COX-2 inhibitor could be reversed completely by PGD2 and partially by PGE2. In addition, the protective effect of PGD2 against COX-2 inhibition was significantly blocked by a PGDP-receptor antagonist, BWA868C. These results indicate that the COX-2 products PGD2 and, to a lesser extent, PGE2 control apoptosis in cultured rat epididymal cells in vitro.     

Resorption versus secretion in the rat epididymis

Micropuncture samples were taken from the rete testis, caput epididymidis and cauda epididymidis of anaesthetized adult rats and assayed for total protein, sodium and potassium concentrations. Intraluminal sperm concentrations were determined and used to calculate the amount of fluid resorbed from the efferent duct and epididymal lumen. It was demonstrated that large amounts of protein (30.2 mg/ml cauda volume) and sodium (241.8 mequiv./l) and smaller amounts of potassium (19.4 mequiv./l) are resorbed from the rat epididymal lumen between the caput and corpus epididymidis. This occurs despite increases in intraluminal concentrations of protein (from 22 to 28 mg/ml) and potassium (from 16 to 50 mequiv./l). Resorption is an important aspect of epididymal control of the intraluminal environment.     

Androgen-dependence of ornithine decarboxylase in the rat epididymis

Ornithine decarboxylase (ODC) activity was measured in epididymides of 45-day-old rats. Higher ODC activity was detected in the corpus and cauda than in the caput epididymidis. Bilateral castration for 7 days caused epididymal ODC to fall to undetectable values, whereas testosterone restored activity to normal values. The effect of the androgen was significantly inhibited by cyproterone acetate. The caput was more sensitive to the action of testosterone than were the corpus and caudal segments. Unilateral castration for 4 or 8 days did not affect ODC on the control or castrated side, but the activity fell in epididymides of both sides after removal of the remaining testis. These results show that epididymal ODC activity is androgen-dependent.