Cellular distribution of parchorin, a chloride intracellular channel-related protein, in various tissues

Yumiko Mizukawa1, Tomohiro Nishizawa1, Taku Nagao1, Ken Kitamura2, and Tetsuro Urushidani1

1 Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033; and 2 Department of Otolaryngology, School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cellular distribution of parchorin, a new chloride intracellular channel family member, was investigated in rabbit tissues by immunohistochemistry using an antibody recognizing the sequence containing a parchorin-specific repeat. Parchorin preferentially resides in the epithelium of the ducts of the lacrymal, parotid, submandibular, and mammary glands and the pancreas, prostate, and testis. In the trachea and lung, parchorin was found in the airway epithelium and type II alveolar cells. In the kidney, parchorin was distributed mainly from the thick ascending limb to the distal convoluted tubule. In the eye, both pigment and nonpigment epithelia of the ciliary body were positive, whereas only the pigment epithelium was positive in the retina. Parchorin was also present in the cochlea and semicircular canal. The amount of parchorin in the gastric mucosa, but not in the submandibular glands, increased after weaning. In the mammary gland, parchorin expression was greater in a lactating rabbit (1 wk after delivery) compared with a pregnant (3 wk) rabbit. The cellular distribution and changes in expression indicate that parchorin plays an important role, possibly in chloride transport, in the cells that create an ion gradient for water movement.

chloride channel; duct; rabbit; immunohistochemistry


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN THE VARIOUS ORGANS OF THE body, a huge amount of water moves as cerebrospinal fluid, urine, or digestive fluid. It has been considered that the water is transepithelially or laterally driven by the osmotic pressure created by ions (including Na+, K+, and Cl-) and that their movement is the consequence of activation of various ion pumps, transporters, and channels (1, 26, 30). The regulation of fluid movement in the body is very important for homeostasis. This has been proven by natural or artificial diseases in which aquaporins (35, 36), cystic fibrosis transmembrane conductance regulator (CFTR; 9), Na+-K+-2Cl- cotransporter 1 (NKCC1; 5, 7), or ClC-K1 (19) is mutated or deleted. Subsequent abnormalities occur in urine reabsorption and secretions from airway epithelium, sweat glands, the pancreas, and the inner ear. Among these, Cl- conductance afforded by CFTR is considered essential for transepithelial water transport, since the defect in CFTR function causes CF due to an impairment in water movement in various tissues. In addition to CFTR, ClC-2 and other volume-sensitive Ca2+-dependent voltage-gated Cl- channels have been suggested (1) to play important roles in water movement in the apical side of epithelial cells.

Recently, we (21) cloned a new protein, parchorin, which has homology to the Cl- intracellular channel (CLIC) family. In contrast to other CLIC family members, most of which are considered to be Cl- channels in intracellular vesicles (32), parchorin is a soluble cytosolic phosphoprotein and translocates to the plasma membrane under stimulation. When acid-secreting parietal cells are stimulated, tubulovesicles containing H+-K+-ATPase fuse with the apical membrane, and both K+ and Cl- permeability in the apical membrane are increased. Consequently, HCl secretion is elicited (35). During this activation process, it was observed (21, 33) that parchorin translocated from the cytosol to the apical membrane. It was also observed (21) that parchorin, transfected to LLC-PK1 cells, translocated to the plasma membrane and accelerated the Cl- efflux rate when Cl- efflux was caused by elimination of extracellular Cl- concentration. Parchorin is preferentially expressed in tissues related to water movement, i.e., the gastric mucosa, choroid plexus, salivary gland, and kidney (21, 33). Parchorin was named according to its characteristic distribution, i.e., the highest expression being in the parietal cell and choroid plexus. These observations suggest strongly that parchorin plays an important role in the regulated movement of body fluid via Cl- transport.

In the present study, we examined the cellular localization of parchorin in various tissues using immunohistochemistry under light microscopy. We also observed development-related changes of parchorin expression in some exocrine glands.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Production of parchorin fragments as green fluorescent protein fusion protein. Full-length rabbit parchorin (22) or its fragments cut out with NaeI-EcoRI (73-637), AccI-EcoRI (159-637), BstXI-EcoRI (262-637), XhoI-EcoRI (404-637), BamHI-SalI (1-404) were subcloned into pEGFP vector (Clontech). Of these, XhoI and SalI sites were artificially introduced by PCR. COS-7 cells were transfected with these cDNA by the DEAE-dextran method. The cells were then homogenized with 120 mM NaCl and 20 mM HEPES-Tris, pH 7.4, and the 100,000 g supernatant was concentrated with Centriprep 30 (Amicon). The fusion protein was lastly purified with HPLC using a gel-filtration column (Diol-150).

Western blotting. To estimate the contents of parchorin by immunoblotting, various rabbit tissues were homogenized with 120 mM NaCl and 20 mM HEPES-Tris, pH 7.4, and centrifuged at 800 g for 15 min, and the supernatant was harvested. After the protein assay, the sample was separated by SDS-PAGE according to the method of Laemmli (15) and blotted to a polyvinylidene difluoride membrane (Bio-Rad) using a semidry apparatus at 1 mA/cm2 for 50 min. The membrane was blocked with 5% skim milk in 0.5% Tween-PBS at room temperature for 1 h. We incubated the membrane with anti-parchorin mouse monoclonal antibody (33) or anti-green fluorescent protein (GFP) rabbit polyclonal antibody (Clontech) (1:5,000 dilution each) at 4°C overnight. The membrane was then further incubated with a second antibody, horseradish peroxidase-conjugated anti-mouse IgG (Sigma, 1:2,000 dilution) or anti-rabbit IgG (Santa Cruz, 1:2,000 dilution), at 37°C for 1 h and visualized using the Renaissance Western blot chemiluminescence reagent kit (NEN).

Immunohistochemistry. Each organ dissected from a Japanese White rabbit (Shiraishi Tokyo) was immediately fixed with 10% formalin in PBS at 4°C for 2 h to overnight, depending on the thickness of the tissue. The tissue was then immersed for 24 h in 15% (wt/vol) sucrose in 0.1 M phosphate buffer for cryoprotection and frozen in Tissue-Tek OCT compound (Sakura Finetechnical). Cryostat sections (10 µm) were obtained and mounted on slides coated by 3-aminopropyltriethoxysilane. For the inner ear sample, the tissue was decalcified in 10% EDTA-Tris for 7 days before freezing. Endogenous peroxidase activity was inhibited by 30 min preincubation in 0.3% H2O2-methanol followed by a 10-min wash in PBS, and blocking was performed with 8% skim milk in PBS for 1 h. The section was probed with anti-parchorin mouse monoclonal antibody (33) (1:1,000 dilution) in PBS-Tween at 4°C overnight. Except for mammary glands in which endogenous biotin appeared to be high, the section was incubated with biotin-conjugated anti-mouse IgG (Sigma; 1:3,000 dilution) and subsequently with horseradish peroxidase-conjugated biotin-avidin complex (Vectastain ABC kit, Vector Laboratories) at 37°C, both for 50 min. For mammary glands, horseradish peroxidase-conjugated anti-mouse IgG (Sigma; 1:50 dilution) was used as the second antibody. The section was developed for an appropriate time in 0.025% diaminobenzidine, 1 µM H2O2, and 10 mM Tris · HCl, pH 7.5, and counterstained with 1% methyl green and 0.1 M barbital-acetate, pH 4.0, at room temperature for 2 h. The images were taken by microscopy (Olympus BX-50) connected to a digital charge-coupled device camera (Fujix HC-2500 3CCD, Fujifilm).

For double staining, the section was permeabilized with 1% Triton X-100 in PBS at room temperature for 30 min and then blocked with 5% skim milk in PBS-Tween for 1 h. To detect parchorin and Tamm-Horsfall glycoprotein, the section was incubated with anti-parchorin mouse monoclonal antibody (1:200 dilution) and anti-Tamm-Horsfall glycoprotein (COSMO-BIO; 1:100 dilution) at 4°C overnight, followed by Cy3-anti-mouse IgG (Amersham Pharmacia Biotech; 1:50 dilution) and FITC-anti-sheep IgG (Sigma; 1:20 dilution) at 4°C overnight. For parchorin and cytokeratin, only mouse antibodies were available. Therefore, the section was sequentially incubated with anti-cytokeratin (AE1/AE3, Dako, ready to use), FITC-anti-mouse IgG (Sigma; 1:50 dilution), anti-parchorin, and Cy3-anti-mouse IgG. We took special care to ensure that the section with anti-cytokeratin, FITC-anti-mouse IgG, and Cy3-anti-mouse IgG (but without anti-parchorin) did not show any signal for Cy3. This confirmed that the second antibody for parchorin did not bind to uncovered mouse IgG probed for cytokeratin. The sections were examined with a microscope (Nikon Eclipse) with a confocal laser scanning system (µRadiance, Bio-Rad). FITC was excited at 488 nm (argon-ion laser) and detected with HQ 515/30 filter, and Cy3 was excited at 543 nm (green helium-neon laser) and detected with E570LP. A sequential acquisition mode was employed to avoid "bleed through" of the staining.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of epitope of anti-parchorin antibody. To ensure the specificity of the antibody for recognizing parchorin among the CLIC family members, we first identified the epitope of the presently used monoclonal anti-parchorin antibody (33). As shown in Fig. 1A, we created full-length and deletion mutants of parchorin as GFP fusion protein, transiently expressed them in COS-7 cells, and partially purified them. All the fragments were successfully expressed, and it was confirmed that they were recognized by anti-GFP antibody (Fig. 1B, right). As shown in Fig. 1B, left, the monoclonal anti-parchorin antibody recognized GFP-parchorin1-637 (full length), GFP-parchorin73-637, GFP-parchorin159-637, and GFP-parchorin1-404, as well as native parchorin partially purified from rabbit gastric mucosa, but it did not recognize GFP-parchorin262-637 or GFP-parchorin404-637 (CLIC homology domain). It is obvious that the antibody recognized any fragments having the amino acid sequence 159-262, but it did not recognize fragments without that sequence. This indicates that the epitope for the antibody exists in the sequence that contains the characteristic GGSVDA repeat and is specific for parchorin (21). We conclude that the antibody could specifically distinguish parchorin from other CLIC family members reported to be widely distributed in the organs.


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Fig. 1.   Monoclonal antibody to parchorin (Ref. 33) recognizes the sequence containing the characteristic GGSVDA repeat. A: generated green fluorescent protein (GFP)-parchorin mutant. The shaded area at left indicates the position of 15 repeats of GGSVDA or similar sequence. The shaded area at right indicates the Cl- intracellular channel (CLIC) homology domain. B: full-length or truncated mutant of GFP-parchorin was transiently transfected in COS-7 cells. Supernatant (100,000 g) of these cells was purified by gel filtration, separated on SDS-12% PAGE (30 µg/lane), and blotted to a polyvinylidene difluoride (PVDF) membrane. The membrane was probed with anti-parchorin (1:5,000 dilution) or anti-GFP (1:5,000 dilution). Lane 1, native parchorin partially purified from rabbit gastric mucosa. Lanes 2 and 8, full-length GFP-parchorin (GFP-parchorin1-637). Lanes 3 and 9, GFP-parchorin73-637. Lanes 4 and 10, GFP-parchorin159-637. Lanes 5 and 11, GFP-parchorin262-637. Lanes 6 and 12, GFP-parchorin404-637. Lanes 7 and 13, GFP-parchorin1-404. Molecular mass (MW) standards are shown at right.

In a previous report (21), we described the high expression of parchorin in tissues involved in fluid movement, e.g., the brain, gastric mucosa, salivary glands, kidney, chorioretinal epithelium, and airway epithelium, using immunoblot. To elucidate the expression of parchorin in a comprehensive way, we examined various tissues and organs by immunohistochemistry.

Cardiovascular system. Parchorin immunoreactivity was completely absent in the aorta and heart (data not shown). Cardiac tissue is reported to be enriched in other CLIC family members (2, 6, 17, 24, 32), and thus parchorin is exceptional in that regard. The arteriole, capillary, and vascular endothelium were all negative for parchorin, which is obvious from staining of the various tissues mentioned below.

Digestive system. As previously reported (21), within the fundic mucosa, only parietal cells showed highly positive staining for parchorin antibody (Fig. 2A). In the pyloric mucosa where parietal cells are absent, no reactivity was noticed (data not shown). In the intestine, from duodenum to rectum, no positive staining was obtained, as shown in the case of the rectum (Fig. 2B).


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Fig. 2.   Immunolocalization of parchorin in digestive organs. Cryosections (10 µm) of fixed organs were probed with anti-parchorin mouse monoclonal antibody (1:1,000 dilution), biotin-conjugated anti-mouse IgG (1:3,000 dilution), and horseradish peroxidase-conjugated biotin-avidin complex (Vectastain ABC kit). The immunocomplex was developed by diaminobenzidine (brown), and the sections were counterstained with methyl green (blue green). A: gastric mucosa. Parietal cells were exclusively stained within the gastric wall. A, inset: magnified image of a gastric gland. B: rectum. Parchorin-positive cells were absent. C: submandibular gland. Intercalated ductal cells were strongly positive and thicker ducts (arrow) were weakly positive. D: parotid gland. Parchorin exists in intercalated ductal cells. Acinar cells were negative for parchorin. E-H: pancreas. The epithelial cells of the largest part of the duct were weakly positive (E). The epithelial cells of the midsized duct show more intense staining (F). Although parchorin is present in the midsized duct (arrowhead), it is absent in the intercalated ductal cells (arrow; G). Pancreatic acinar cells were absolutely negative for parchorin (E-G) compared with the control staining without first antibody (H). I: gallbladder. The apical side of the epithelial cells was parchorin positive. A, inset, and C-I: bar = 20 µm; A and B: bar = 100 µm.

Intense parchorin immunoreactivity was found in the intercalated duct of the submandibular gland, and weak staining appeared in the thicker duct, whereas it was almost absent in the acinar cells (Fig. 2C). Parchorin also was found in the intercalated duct of the parotid gland (Fig. 2D). This observation supports our opinion that parchorin participates in electrolyte movement since the intercalated ducts of the salivary glands actively operate reabsorption of luminal NaCl. (9).

In our previous report (21), parchorin was not detectable in the pancreas by Western blotting. However, by the immunohistochemical technique, positive staining was observed in the epithelium of the largest part of the duct (Fig. 2E). Staining in the midsized duct was more intensely positive (Fig. 2, F and G), whereas staining in the smallest part of the duct (Fig. 2, F and G) and acinar cells (Fig. 2, E-G) was absolutely negative compared with control staining (Fig. 2H).

In the gallbladder, epithelium that elicits water reabsorption from bile (24) was strongly positive for parchorin, whereas the smooth muscle layer was negative. Within the epithelial cells, parchorin appeared to be present in the apical membrane (Fig. 2I).

Respiratory system. In the trachea, parchorin was exclusively expressed in the mucous epithelial cells that secrete airway fluid (Fig. 3, A and B). In previous reports (21, 33), parchorin was not detectable in the lung by Western blotting, but in the present study it was found by immunohistochemistry, in the surface epithelium of bronchiole and type II alveolar surface cells (Fig. 3, C and D). It was reported (10) that these cells drive the absorption of body fluid within the alveolae using osmotic pressure created by ion transport in which Na+-K+-ATPase in the basolateral membrane and the amiloride-sensitive epithelial Na+ channel in the apical membrane are involved.


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Fig. 3.   Immunolocalization of parchorin in respiratory organs. Cryosections (10 µm) of fixed organs were probed with anti-parchorin, visualized with diaminobenzidine (brown), and counterstained with methyl green (blue green). A: trachea. Surface epithelial cells were parchorin positive (arrow). B: magnified image of tracheal epithelial cells. C: lung. Parchorin was found in bronchiolar (arrowhead) and tracheal (arrow) epithelial cells and alveoli. D: magnified image of alveoli showing type II alveolar epithelial cells that were parchorin positive (arrows). A and C: bar = 100 µm; B and D: bar = 10 µm.

Urinary system. In the kidney, staining for parchorin was strongly positive in the outer part of the medulla, and dense positive dots were also visible in the cortex. Some weak staining was present near the calyx in the inner medulla (Fig. 4A). Under higher magnification, it was found that some of the tubular cells, including the macula densa, was parchorin positive (Fig. 4, B and C). Within the medulla, some of the papillary ductal cells near the calyx and the epithelium of the calyx were parchorin positive (Fig. 4D).


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Fig. 4.   Immunolocalization of parchorin in the kidney. Cryosection (10 µm) of fixed kidney was probed with anti-parchorin mouse monoclonal antibody, visualized with diaminobenzidine (brown), and counterstained with methyl green (blue green). A: low magnification. Outer medulla is strongly positive, and many positive dots are also visible in the cortex. Some weak staining is present near the calyx in the inner medulla. B: cortex. Some of the tubular cells, including the macula densa (arrow), are parchorin positive. C: outer medulla. Some of the tubular cells are parchorin positive. D: inner medulla. Some of the papillary ductal cells near the calyx (arrows) and the epithelium of calyx (arrowhead) are parchorin positive. A: bar = 1 mm; B-D: bar = 100 µm.

To identify the parchorin-positive portions in the tubular cells, we performed double staining with parchorin and region-specific markers. As a marker for the thick ascending limb of Henle's loop, we chose anti-Tamm-Horsfall glycoprotein (14). It is obvious from Fig. 5, A-C, that parchorin colocalized with Tamm-Horsfall glycoprotein, which suggests that parchorin is present in the thick ascending limb of Henle's loop.


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Fig. 5.   Colocalization of parchorin with markers for the kidney. Cryosections (10 µm) of the kidney were double stained with indirect immunofluorescence and examined with confocal microscopy. The section was stained by anti-parchorin (1:200 dilution) in addition to anti-Tamm-Horsfall glycoprotein (1:100 dilution) or anti-cytokeratin (AE1/AE3). Anti-parchorin, anti-Tamm-Horsfall glycoprotein, and anti-cytokeratin were visualized with Cy3-conjugated anti-mouse IgG (1:50 dilution), FITC-conjugated anti-sheep IgG (1:20 dilution), and FITC-conjugated anti-mouse IgG (1:50 dilution), respectively. A: staining for parchorin. B: staining for Tamm-Horsfall glycoprotein, marker of the thick ascending limb of Henle's loop. C: combination of A and B. Most of the cells expressing parchorin overlap with those expressing Tamm-Horsfall glycoprotein. D: staining for parchorin. E: staining for cytokeratin, which is relatively high in Henle's loop and collecting ducts. F: combination of D and E. Some of the positive cells overlap (yellow). However, parchorin-positive, cytokeratin-negative cells (red) and parchorin-negative, cytokeratin-positive cells (green) are also seen. G and H: negative control for parchorin/cytokeratin double staining. I: combination of G and H. The section was sequentially incubated with anti-cytokeratin, FITC-anti-mouse IgG, and Cy3-anti-mouse IgG, omitting anti-parchorin. The FITC signal for detecting cytokeratin is obvious (H), whereas the specific Cy3 signal is absent when anti-parchorin is omitted (G), confirming that Cy3-anti-mouse IgG for detection of parchorin did not bind to anti-cytokeratin in D-F. The bright red spots in G and I are nonspecific staining due to the aggregation of Cy3-anti-mouse IgG. The field containing relatively large numbers of these spots was selected to show that neither excitation for FITC at 488 nm nor that for Cy3 at 543 nm visualized the other dye under the present conditions. Bar = 100 µm.

It was reported (8) that cytokeratin-positive cells were relatively enriched in Henle's loop and collecting ducts of the rabbit kidney. As shown in Fig. 5, D-F, cytokeratin-positive cells were overlapped with parchorin-positive cells, suggesting that some parts of the collecting duct and Henle's loop were parchorin positive. The section had to be sequentially incubated with anti-cytokeratin, FITC-anti-mouse IgG, anti-parchorin, and Cy3-anti-mouse IgG, since the antibodies available for this double staining were both raised in the mouse. We took special care that the fourth antibody, Cy3-anti-mouse IgG, did not show any signal when the third antibody, anti-parchorin, was absent. Using the same protocol, we confirmed that Cy3-anti-mouse IgG for the detection of parchorin did not bind to uncovered mouse IgG probed for cytokeratin (Fig. 5, G-I).

These results indicate that parchorin is distributed mainly from the thick ascending limb to the distal convoluted tubule and somewhat in the collecting duct. The thick ascending limb of Henle's loop is characteristic in its absence of water permeability and thus has an important role in the mechanism of urine concentration by reabsorption of NaCl and the subsequent increase in osmolarity (11).

In the bladder, neither the epithelium nor the smooth muscle layer contained parchorin-positive cells (data not shown).

Genital organs. In the prostate, glandular epithelial cells of the urethra were positively stained with anti-parchorin antibody (Fig. 6, A and B). Also, in the testis, the epithelial cells of rete testis were strongly positive (Fig. 6, C and D).


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Fig. 6.   Immunolocalization of parchorin in genital organs. Cryosections (10 µm) of fixed organs were probed with anti-parchorin mouse monoclonal antibody. A: prostate. Glandular epithelial cells of urethra are parchorin positive (arrows). B: magnified image of A. C: testis. Epithelial cells of rete testis are parchorin positive (arrows). D: magnified image of C. E: mammary gland of a pregnant rabbit. Parchorin exists in ductal cells (arrowheads) and glandular epithelial cells (arrows). F: magnified image of E showing part of the ductal cells. A, C, and E: bar =100 µm; B, D, and F: bar = 20 µm.

Tissue sections from the uterus (including the placenta and amnion) of a pregnant (3 wk) rabbit were stained with anti-parchorin but no positive cells were identified (data not shown). The mammary gland of a pregnant rabbit was isolated and examined (Fig. 6, E and F). Parchorin staining was evident in the apical side of the glandular epithelial cells that secrete milk as well as in the ductal cells. A similar staining pattern was observed in mammary gland obtained from a lactating rabbit 1 wk after delivery (data not shown).

Sensory organs. In the eyeball, both pigment and nonpigment cell layers of the ciliary body were positive for parchorin (Fig. 7, A and B). In the retina, pigment epithelium, which is known to deliver water to the side of the choreal membrane, was exclusively positive (Fig. 7, A and C), whereas no positive staining was noted in the visual nerves or photoreceptor cell layer.


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Fig. 7.   Immunolocalization of parchorin in the eye. Cryosection (10 µm) of fixed eyeball or lacrymal gland was probed with anti-parchorin. A: low magnification of the iris, ciliary body, and retina. The ciliary body epithelium (small arrows) and scleral side of retina (large arrow) are parchorin positive, whereas the epithelium of the iris (arrowhead) is negative. B: higher magnification of the ciliary body epithelium. Parchorin exists in both the pigment and nonpigment epithelium. C: higher magnification of the retina. Parchorin exists in the retinal pigment epithelium (arrow). D: lacrymal gland. Parchorin exists in the apical side of the ductal cells (arrow). A: bar = 100 µm; B-D: bar = 20 µm.

In the lacrymal gland, the apical side of the ductal cells was exclusively stained (Fig. 7D), as in the other exocrine glands.

In the inner ear, several cell types were found to be positive for parchorin antibody. In the cochlea (Fig. 8, A-C), parchorin was found to be abundant in supporting cells in the organ of Corti, including Hensen's and Claudius' cells. Positive staining was also evident in the outer and inner hair cells and tectorial membrane. The spiral ligament was also parchorin positive, whereas the stria vascularis, which secretes endolymph, and the afferent nerves were parchorin negative. In the cochlea, putative K+-recycling pathways have been postulated (27-29). When hair cells are excited, K+ is taken up by the hair cells and transported via different routes by, for instance, Hensen's cells, Claudius' cells, outer sulcus cells, interdental cells, tectorial membrane, and fibrocytes and finally back to endolymph via the stria vascularis (27-29). In the present study, parchorin was shown to be present in most of the cells involved in those routes, with the exception of the stria vascularis.


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Fig. 8.   Immunolocalization of parchorin in the inner ear. Cryosection (10 µm) of fixed and decalcified inner ear was probed with anti-parchorin mouse monoclonal antibody. A: cochlea. Parchorin exists in the organ of Corti (OC), spiral ligament (SL), and Reissner's membrane (RM). B: magnified image of A. Spiral ligament is parchorin positive, whereas stria vascularis (SV; arrow) is negative. C: magnified image of the organ of Corti. Parchorin exists in Claudius' cells (arrowhead), Hensen's cells (arrow), outer and inner hair cells, and tectorial membrane. Afferent nerves (open arrowhead) are negative for parchorin. D: ampulla of semicircular canal. Parchorin exists in hair cells (arrow). E: higher magnification of ampulla hair cells. Bar = 100 µm.

In the semicircular canal, it was found that ampulla hair cells were strongly positive for parchorin antibody (Fig. 8, D and E).

In the brain, including the cerebrum, cerebellum, and oblongata, no positive staining was found, except for the choroid plexus, in which parchorin is highly enriched as previously reported (21).

Correlation between levels of parchorin and secretory capacity. In general, the acid-secreting capacity of the mammalian stomach is scant after birth until weaning, and thereafter it drastically increases. It has been shown (4) that H+-K+-ATPase activity parallels this pattern of increased acid secretion in the rabbit stomach. To examine whether developmental changes in acid secretory capacity are parallel to parchorin expression, we quantified parchorin in the 800 g supernatant of mucosal homogenate obtained from rabbit fetus (3 wk of gestation), suckling rabbits (1 wk after birth), and adult rabbits by Western blotting. As shown in Fig. 9A, the content of parchorin increased with development. In contrast, no difference was observed between suckling and adult in the submandibular glands, reflecting the fact that salivary secretion does not increase after weaning (17).


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Fig. 9.   Correlation between secretory level and expression of parchorin. A: gastric mucosa from fetus (3 wk of pregnancy), suckling (1 wk after birth), or adult rabbits or submandibular gland from suckling and adult rabbits was homogenized and centrifuged at 800 g. Supernatants were separated on SDS-7.5% PAGE (30 µg protein/lane) and blotted to PVDF membrane. Parchorin was probed with anti-parchorin antibody. B: mammary glands were obtained from pregnant (3 wk) or lactating (1 wk) rabbits, and parchorin content was analyzed as in A.

We could not detect parchorin in the mammary gland in the normal female rabbit by immunoblotting (data not shown). The mammary gland markedly develops during gestation but actual milk secretion starts after delivery. To examine the parchorin content relative to the milk secretory function, we quantified parchorin in the 800 g supernatant of mammary gland homogenate obtained from a pregnant (3 wk) rabbit and a lactating (1 wk after birth) rabbit by Western blotting. As shown in Fig. 9B, parchorin expression was greater in the mammary gland of the lactating rabbit compared with the pregnant rabbit.

These results indicate that the levels of parchorin in the exocrine glands are proportional to their secretory capacity.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The CLIC family proteins have a highly conserved region in the COOH terminus (ca. 230 amino acids), which is considered to be involved in Cl- conductance (32). At present, more than seven members of the CLIC family have been identified. Among them, CLIC-1 and CLIC-3 have short NH2-terminal regions with this consensus sequence. CLIC-1 (NCC27) was first identified (31) as a nuclear Cl- channel but then was found in extranuclear vesicular structures in the cells, with relatively widespread distribution. CLIC-3 was identified (23) as a mitogen-activated protein kinase-associated intracellular Cl- channel, and it was found in the heart and lung with highest levels in the placenta. Of the CLIC family proteins with longer NH2-terminal regions, CLIC-4 (p64H1) was found to be relatively widespread in distribution and to exist in the intracellular vesicular structure (6). CLIC-5 was originally purified from placental microvilli as a protein that bound to actin and ezrin, and its expression was reported (2) to be high in striated muscle. However, recent work (Shanks RA, Berryman M, Edwards JC, Navarre J, Urushidani T, and Goldenning JR, unpublished observations) revealed that the highest level of this molecule (now termed as CLIC-5A) was in the lung. The first discovered member, p64, is widely distributed to various tissues and localized mainly in intracellular vesicles (16). There has been no success in discerning any common physiological role for the CLIC family of proteins based on their tissue distribution. Recently, a putative human homolog of bovine p64 was identified (Shanks et al., unpublished observations) as a splicing variant of CLIC-5 and designated as CLIC-5B. This protein is exclusively present in the Golgi apparatus of intestinal mucosal cells, suggesting it has a specialized role in intestinal function. The intestinal mucosa, which is known to transport a huge amount of water, lacks parchorin, indicating that CLIC-5B may play the role instead of parchorin.

Among the members of the CLIC family, parchorin has a characteristic feature. This protein has an exceptionally long, hydrophilic, and acidic NH2-terminal sequence and a peculiar quality in that it translocates from the cytosol to the plasma membrane in association with stimulation. Recent work (32) demonstrated that CLIC-1, which only has a little of this NH2-terminal sequence, worked as a Cl- channel alone when it was expressed in Escherichia coli and reconstituted in an artificial membrane. This means that the consensus sequence itself has the ability to form a Cl- channel. This argues against the hypothesis that CLIC family proteins work together to activate a Cl- channel. However, parchorin is definitely a soluble protein because of its highly hydrophilic nature. Further investigation is necessary to elucidate the molecular mechanism by which parchorin itself forms an ion channel within the membrane, if indeed this is really possible. It might be more likely that the protein binds either directly or via other proteins to the channel proteins. In any event, the cellular distribution of parchorin revealed in the present study strongly suggests its specialized physiological role in ion transport as related to water movement.

In the present study, we elucidated that the epitope of anti-parchorin monoclonal antibody exists in the sequence containing the parchorin-specific GGSVDA repeat in the middle of the NH2-terminal region. This ensures specific recognition of parchorin to distinguish it from the other CLIC members present in the tissues. In previous reports (21, 33), parchorin was not detectable in the lung and pancreas by Western blotting. In the present study, however, the expression of parchorin was high but quite restricted to the epithelial cells of the bronchiole and type II alveolar cells in the lung and to the epithelial cells of the pancreatic duct. Consequently, the content of parchorin in their total homogenate is lower than the detection limit by Western blotting. Care should be taken in discussing the physiological role of proteins based on their tissue distribution according to Western or Northern blot analysis. To our knowledge, the present work is the first report for the cell-specific expression of the CLIC members by histochemistry of normal tissues. It would be worthwhile to perform the histochemical analysis of other CLIC family members to clarify their physiological roles.

Parchorin was found in the apical membrane of epithelial cells in various organs, including the gallbladder, pancreas, airway, and prostate, and it also exists mainly in the cytosol of cells such as type II alveolar cells and the retinal pigment epithelium in the eye. The observation (21, 33) that parchorin translocates from the cytosol to the membrane in association with stimulation in parietal or transfected LLC-PK1 cells might reflect the degree of activation of water movement in each tissue. In the present study, the rabbits were simply killed under anesthesia without any pretreatments, so the physiological state of the tissues might have varied. We postulate that parchorin appears in the apical membrane in tissue where water transport is constitutively or accidentally activated. To obtain conclusive results, detailed examination by electron microscopy in which the stimulatory level of the tissues in vivo would be completely controlled is necessary.

Currently, CFTR is considered to be one of the most important Cl- channels in the apical membrane of water-transporting epithelium (1). Although the tissue distribution of CFTR has something in common with that of parchorin, that is, in the airway epithelium, salivary gland, pancreas, and gallbladder (9), there were obvious differences. The heart, one of the main organs for CFTR, completely lacked parchorin. Comparing cellular distribution, CFTR was found in both acinar and ductal cells in the submandibular glands and pancreas (9), whereas the expression of parchorin was restricted to ductal cells in both tissues. CFTR is known to be present at high levels in the base of the submandibular gland but only slightly present in the surface epithelium of the airway (22), whereas parchorin was exclusively present in the mucous epithelial cells. Therefore, it is suggested that parchorin plays a different role than CFTR in Cl- transport, whether its function is actually as channel or activator. Parchorin was found to be present in ductal cells in all the exocrine glands tested (submandibular glands, parotid glands, lacrymal glands, pancreas, prostate, testis, and mammary glands), suggesting that it plays a role in the transport system specific for ducts. It is evident that parchorin is a good marker for ducts.

In the kidney, parchorin was found mainly in the thick ascending limb of Henle's loop and the distal convoluted tubule. Various ion transporters and channels working in the kidney comprise the complex system for urine concentration. NKCC was reported (11) as a Cl- transporter in the apical side of the thick ascending limb of Henle's loop, whereas the Na+-Cl- cotransporter acts in the distal convoluted tubule. It was also reported (24) that the distal convoluted tubule contained CFTR. Further study is required to find out how parchorin cooperates with these proteins. It could be strongly suggested at this stage that parchorin involves physiological Cl- absorption, since the corresponding regions have a very high level of Cl- absorption in the kidney.

The inner ear has a highly controlled water-movement system to ensure hearing and body balance. NKCC1 is well known as a Cl- transporter expressed in the stria vascularis (5, 25). It has also been reported (12, 25) that ClC-1, ClC-2, ClC-3, and ClC-K1 were present in outer hair cells. Parchorin was highly expressed in epithelial cells near the organ of Corti in the cochlea. This strongly suggests that parchorin is involved in the transport of endolymph fluid. On the basis of the study (5) that NKCC1-deficient mice exhibited diseases due to impediments in the inner ear, it can be inferred that ion transport in the inner ear must be quite important in maintaining ear function. The inner ear should be an interesting area of study in future investigations of parchorin.

Recently (3), the ClC2-deficient mouse was reported. Although a diverse phenotype had been expected from the wide distribution of this channel, tissue-specific diseases emerged: male infertility and blindness, because of the loss of cells maintaining the blood-testis barrier and the blood-retina barrier, respectively. These results suggest the pathophysiological significance of Cl- transport in Sertoli's cells and retinal pigment epithelium. Considering the finding that parchorin exists in both the epithelium of the testis and the retinal pigment epithelium, the male genital organs and the eye should also be interesting areas for future study.

To elucidate the physiological significance of parchorin in secretion, we studied its level of expression in organs in which secretory capacity drastically changes. We chose the gastric mucosa and mammary gland for this purpose. In the gastric mucosa, the content of parchorin increased after weaning. This was in contrast to an unchanged amount of parchorin in the submandibular gland before and after weaning, reflecting the fact that salivary secretion does not change with weaning. In the mammary gland, parchorin content was increased in the lactating period compared with the pregnant period. These results were consistent with our idea that the amount of parchorin correlated with the secretory capacity of the exocrine glands. It would be important for future study to know whether expression of parchorin is essential for an increase of secretory capacity.

In summary, we showed that parchorin is specifically expressed in cells that are thought to be involved in ion transport for various organs participating in water movement. Along with the observation that its expression was proportional to physiological function, we suggest that parchorin plays an indispensable role in ion transport, possibly in Cl- transport, essential for water movement. The molecular mechanism by which parchorin participates in ion transport, possibly in Cl- channel activity, requires further study to be understood. Future study should also probe the physiological status of the organs expressing parchorin and include the development of a gene-targeting animal.


    ACKNOWLEDGEMENTS

We thank J. H. Jennings for manuscript editing.


    FOOTNOTES

This study was supported in part by the Japanese Ministry of Education, Science, Sports, and Culture Grants 13470511 and 13557220.

Address for reprint requests and other correspondence: T. Urushidani, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The Univ. of Tokyo, Tokyo 113-0033, Japan (E-mail: urushi{at}mol.f.u-tokyo.ac.jp).

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. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00239.2001

Received 29 May 2001; accepted in final form 19 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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