Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells

We previously reported a 120-kDa phosphoprotein that translocated from cytosol to the apical membrane of gastric parietal cells in association with stimulation of HCl secretion. To determine the molecular identity of the protein, we performed molecular cloning and expression of the protein. Immunoblot analysis showed that this protein was highly enriched in tissues that secrete water, such as parietal cell, choroid plexus, salivary duct, lacrimal gland, kidney, airway epithelia, and chorioretinal epithelia. We named this protein "parchorin" based on its highest enrichment in parietal cells and choroid plexus. We obtained cDNA for parchorin from rabbit choroid plexus coding a protein consisting of 637 amino acids with a predicted molecular mass of 65 kDa. The discrepancy in size on 6% SDS-polyacrylamide gel electrophoresis is considered to be due to its highly acidic nature (pI = 4.18), because COS-7 cells transfected with parchorin cDNA produced a protein with apparent molecular mass of 120 kDa on 6% SDS-polyacrylamide gel electrophoresis. Parchorin is a novel protein that has significant homology to the family of chloride intracellular channels (CLIC), especially the chloride channel from bovine kidney, p64, in the C-terminal 235 amino acids. When expressed as a fusion protein with green fluorescent protein (GFP) in the LLC-PK1 kidney cell line, GFP-parchorin, unlike other CLIC family members, existed mainly in the cytosol. Furthermore, when Cl(-) efflux from the cell was elicited, GFP-parchorin translocated to the plasma membrane. These results suggest that parchorin generally plays a critical role in water-secreting cells, possibly through the regulation of chloride ion transport.     

17-AAG,a Hsp90 inhibitor,attenuates the hypoxia-induced expression of SDF-1α and ILK in mouse RPE cells

The aim of this study was to investigate the changes of SDF-1α and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1α and ILK. RPE cells were cultured with 200 μmol/L cobalt chloride (CoCl₂) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1α and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1α and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1α and ILK expression in vitro. Our results indicated that SDF-1α and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1α and ILK expression.     

Identification of a synergistic interaction between endothelial cells and retinal pigment epithelium

The retinal pigment epithelium located between the neurosensory retina and the choroidal vasculature is critical for the function and maintenance of both the photoreceptors and underlying capillary endothelium. While the trophic role of retinal pigment epithelium on choroidal endothelial cells is well recognized, the existence of a reciprocal regulatory function of endothelial cells on retinal pigment epithelium cells remained to be fully characterized. Using a physiological long‐term co‐culture system, we determined the effect of retinal pigment epithelium‐endothelial cell heterotypic interactions on cell survival, behaviour and matrix deposition. Human retinal pigment epithelium and endothelial cells were cultured on opposite sides of polyester transwells for up to 4 weeks in low serum conditions. Cell viability was quantified using a trypan blue assay. Cellular morphology was evaluated by H&E staining, S.E.M. and immunohistochemistry. Retinal pigment epithelium phagocytic function was examined using a fluorescent bead assay. Gene expression analysis was performed on both retinal pigment epithelium and endothelial cells by quantitative PCR. Quantification of extracellular matrix deposition was performed on decellularized transwells stained for collagen IV, fibronectin and fibrillin. Our results showed that presence of endothelial cells significantly improves retinal pigment epithelium maturation and function as indicated by the induction of visual cycle‐associated genes, accumulation of a Bruch's membrane‐like matrix and increase in retinal pigment epithelium phagocytic activity. Co‐culture conditions led to increased expression of anti‐angiogenic growth factors and receptors in both retinal pigment epithelium and endothelial cells compared to monoculture. Tube‐formation assays confirmed that co‐culture with retinal pigment epithelium significantly decreased the angiogenic phenotype of endothelial cells. These findings provide evidence of critical interdependent interactions between retinal pigment epithelium and endothelial cell involved in the maintenance of retinal homeostasis.     

The congenital chloride diarrhea gene is expressed in seminal vesicle, sweat gland, inflammatory colon epithelium, and in some dysplastic colon cells

Congenital chloride diarrhea (CLD) is an autosomal recessive disorder of intestinal electrolyte transportation caused by mutations in the anion transporter protein encoded by the down-regulated in adenoma (DRA), or CLD, gene. In this study, in situ hybridization and immunohistochemistry were performed to investigate the expression of CLD in extraintestinal normal epithelia and in intestinal inflammatory and neoplastic epithelia. The expression of the closely related anion transporter diastrophic dysplasia sulfate transporter, DTDST, was also examined and compared with that of CLD in colon. The only extraintestinal tissues showing CLD expression were eccrine sweat glands and seminal vesicles. In inflammatory bowel disease and ischemic colitis, expression of CLD mRNA in colon epithelium was similar to histologically normal colon epithelium, but the protein was found deeper in crypts, including proliferative epithelial cells. In intestinal tumors, the expression pattern of CLD was dependent on the differentiation status of the tissue studied: epithelial polyps with no or minor dysplasia showed abundant expression, whereas adenocarcinomas were negative. The DTDST gene was abundantly expressed in the upper crypt epithelium of colonic mucosa.     

Potential, Current, and Ionic Fluxes across the Isolated Retinal Pigment Epithelium and Choroid

A flux chamber was utilized for in vitro studies of a membrane formed by the retinal pigment epithelium and choroid of the eye of the toad (Bufo arenarum and Bufo marinus). A transmembrane potential of 20 to 30 mv was found, the pigment epithelium surface positive with respect to the choroidal surface. Unidirectional fluxes of chloride, sodium, potassium, and calcium were determined in the absence of an electrochemical potential difference. A net transfer of chloride from pigment epithelium to choroid accounted for a major fraction of the mean short-circuit current. A small net flux of sodium from choroid to pigment epithelium was detected in Bufo marinus. In both species of toads, however, about one-third of the mean short-circuit current remained unaccounted for. Manometric determinations of bicarbonate suggested an uptake of this ion at the epithelial surface of the membrane but did not provide evidence of a relationship between this process and the short-circuit current.     

Anatomy and ultrastructure of dermal glands in an adult water mite,Teutonia cometes (Koch, 1837) (Acariformes: Hydrachnidia: Teutoniidae)

Organization of dermal glands in adult water mites Teutonia cometes (Koch, 1837) was studied using light-optical, SEM and TEM methods for the first time. These glands are large and occur in a total number of ten pairs at the dorsal, ventral and lateral sides of the body. The slit-like external openings of the glands (glandularia) are provided with a cone-shaped sclerite, and are combined with a single small trichoid seta (hair sensillum), which is always situated slightly apart from the anterior aspect of the gland opening. Each gland is formed by an epithelium encompassing a very large lumen (central cavity) normally filled with secretion that stains in varying intensity on toluidine blue stained sections. The epithelium is composed of irregularly shaped secretory cells with an electron-dense cytoplasm and infolded basal portions. The cells possess a large irregularly shaped nucleus and are filled with tightly packed slightly dilated cisterns and vesicles of rough endoplasmic reticulum (RER) with electron lucent contents. Dense vesicles are also present in the apical cell zone. Some cells undergo dissolution, occupy an upper position within the epithelium and have a lighter cytoplasm with disorganized RER. Muscle fibers are regularly present in the deep folds of the basal cell portions and may serve to squeeze the gland and eject the secretion into the external milieu. The structure of these dermal glands is compared with the previously described idiosomal glands of the same species and a tentative correlation with the glandularia system of water mites is given. Possible functions of the dermal glands of T. cometes are discussed.     

Inhibition of high glucose-induced VEGF and ICAM-1 expression in human retinal pigment epithelium cells by targeting ILK with small interference RNA

The aim of this study was to investigate the change of Integrin-linked kinase (ILK) expression of human retinal pigment epithelium (RPE) cells in response to high glucose, and the effect of targeting ILK with small interference RNA (siRNA) on the high glucose-induced expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1). The ILK mRNA and protein expression in human RPE cells were analyzed with RT-PCR and western blot after exposure to 5.5, 30, 40, 50 mM glucose, or 5.5 mM glucose + 45.5 mM mannitol for 48 h. The expression of VEGF and ICAM-1 was also determined. Cells were treated with ILK siRNA, to determine the effect of ILK on VEGF and ICAM-1 expression following treatment with high glucose. High concentrations of glucose significantly up-regulated ILK mRNA and protein expression, and the ILK expression increased along with the glucose concentration. The changes of VEGF and ICAM-1 expression were similar to that of ILK expression. Knocking down ILK gene expression with siRNA inhibited the elevation of VEGF and ICAM-1 induced by high glucose treatment. These results suggested that ILK was involved in the response of RPE cells to high glucose and may therefore play a role in the pathogenesis of diabetic ophthalmology.     

Histochemical and immunohistochemical adaptations on the gastroesophageal tube of the blue-fronted parrot (Amazona aestiva Linnaeus, 1758)

Amazona aestiva Linnaeus, 1758 is known as the blue-fronted parrot. These animals are considered seed dispersion, contributing to ecological balance. Despite its ecological importance, A. aestiva is listed as an imminent danger of becoming extinct, as it is one of the most illegally traded native species in Brazil, besides losing its habitat due to deforestation and forest fires. The cranial oesophagus mucosa is lined with a non-keratinized stratified squamous epithelium, and the basal layer presents melanocyte accumulation. No mucous glands were observed in the lamina propria/submucosa. The ingluvium two different muscular layer patterns were observed, one internal and circular and one very thick, external and longitudinal. The proventriculum submucosa consists of polygonal proventricular glands and contents into a broad central duct lined with simple columnar epithelium Periodic acid-reactive Schiff positive (PAS+) and Alcian blue positive (AB+). The 5-HT cells are dispersed in the proventricular glands located at base and apex. The ventricle mucous layer contains numerous branched folds lined by a simple columnar epithelium. The epithelium was negative reaction for PAS and positive for AB only on the fold surfaces. The 5-HT cells were scarce and observed only at the base of the ventricular gland, as well as in the adjacent connective tissue.     

Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium

Retinal pigment epithelium (RPE)possesses regulated chloride channels that are crucial fortransepithelial fluid and ion transport. At present, little is knownabout the molecular nature of chloride channels in human adult RPE(haRPE) or the effects of oxidative stress on membrane conductanceproperties. In the present study, we assessed ClC channel and cysticfibrosis transmembrane conductance regulator (CFTR) expression andmembrane chloride conductance properties in haRPE cells. ClC-5, ClC-3,ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, andprotein expression was detected with Western blot analysis andimmunofluorescence microscopy. Whole cell recordings of primarycultures of haRPE showed an outwardly rectifying chloride current thatwas inhibited by the oxidant H₂O₂. Theinhibitory effects of H₂O₂ were reduced incultured human RPE cells that were incubated with precursors ofglutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate apossible role for ClC channels in haRPE cells and suggest possibleredox modulation of human RPE chloride conductances.

     

Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation

Background  

Mammary glands harbor a profound burden of apoptotic cells (ACs) during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs) regulating macrophage-mediated efferocytosis in monocytes. MerTK expression, apoptosis and the accumulation of apoptotic debris were examined in histological sections of MerTK-deficient, Axl/Tyro3-deficient, and wild-type mammary glands harvested at specific time points during lactation and synchronized involution. The ability of primary mammary epithelial cells (MECs) to engulf ACs was assessed in culture. Transplant of MerTK-deficient mammary epithelium into cleared WT mammary fat pads was used to assess the contribution of WT mammary macrophages to post-lactational efferocytosis.     

Challenging accepted ion channel biology: p64 and the CLIC family of putative intracellular anion channel proteins (Review)

Parchorin, p64 and the related chloride intracellular channel (CLIC) proteins are widely expressed in multicellular organisms and have emerged as candidates for novel, auto-inserting, self-assembling intracellular anion channels involved in a wide variety of fundamental cellular events including regulated secretion, cell division and apoptosis. Although the mammalian phosphoproteins p64 and parchorin (49 and 65K, respectively) have only been indirectly implicated in anion channel activity, two CLIC proteins (CLIC1 and CLIC4, 27 and 29K, respectively) appear to be essential molecular components of anion channels, and CLIC1 can form anion channels in planar lipid bilayers in the absence of other cellular proteins. However, these putative ion channel proteins are controversial because they exist in both soluble and membrane forms, with at least one transmembrane domain. Even more surprisingly, soluble CLICs share the same glutaredoxin fold as soluble omega class glutathione-S-transferases. Working out how these ubiquitous, soluble proteins unfold, insert into membranes and then refold to form integral membrane proteins, and how cells control this potentially dangerous process and make use of the associated ion channels, are challenging prospects. Critical to this future work is the need for better characterization of membrane topology, careful functional analysis of reconstituted and native channels, including their conductances and selectivities, and detailed structure/function studies including targeted mutagenesis to investigate the structure of the putative pore, the role of protein phosphorylation and the role of conserved cysteine residues.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418     

Headspace measurements of irradiated in vitro cultured cells using PTR-MS

A pilot study was performed to evaluate a new concept for a radiation biodosimetry method. Proton transfer reaction-mass spectrometry (PTR-MS) was used to find out whether radiation induces changes in the composition of volatile organic compounds (VOCs) in the headspace of in vitro cultured cells. Two different cell lines, retinal pigment epithelium cells hTERT-RPE1 and lung epithelium cells A-549, were irradiated with gamma radiation at doses of 4 Gy and 8 Gy. For measuring the cell-specific effects, the VOC concentrations in the headspace of flasks containing cells plus medium, as well as of flasks containing pure medium were analyzed for changes before and after irradiation. No significant radiation-induced alterations in VOC concentrations in the headspace could be observed after irradiation.     

Ciliary epithelia of the mammalian eye in cultured explants

The ultrastructural characteristics of ciliary epithelium from bovine, pigmented rabbit, and fetal albino rabbit were studied in cultured explants. The tips of ciliary processes were cultured in plastic dishes with Dulbecco Modified Eagle Medium (DMEM) containing 5% fetal bovine serum. More than half of the explants adhered to the plastic culture dish, and epithelial cells spread as monolayers within a few days. Initially the explant contains two layers, the outer (nonpigmented cells) and the inner (pigmented cells). Later the explant exhibits three layers: 1) outermost lightly pigmented flattened cells, 2) an outer layer of non-pigmented cells, and 3) an inner layer of densely pigmented cuboidal cells. The cells of the outermost layer are continuous with the cells of the inner layer. A narrow space lies between the outermost layer and the outer layer. The columnar cells in the outer layer contain well developed organelles but no pigment granules; they possess a basement membrane, lateral interdigitations, and junctional complexes near their apices. Numerous focal junctions and some ciliary channel-like structures were detected between the columnar cells of the outer layer and the cuboidal cells of the inner layer. The cuboidal cells of the inner layer are filled with pigment granules. These observations suggest that the columnar cells of the outer layer are nonpigmented epithelium, the cuboidal cells of the inner layer are pigmented epithelium, and the flattened cells in the outermost layer are derived from pigmented epithelium.     

Identification,purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19?±?0.26 mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333 IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

     

Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

Morphological characterisation of the digestive tract of the catfish Lophiosilurus alexandri Steindachner, 1876 (Siluriformes,Pseudopimelodidae)

The anatomical arrangement of the digestive tract and the length (cm) of the oesophagus and intestine of the catfish Lophiosilurus alexandri were described, and the intestinal coefficient was determined. L. alexandri oesophagus is short, in median position, and presents longitudinally folded mucosa, whilst its epithelium is stratified and non-keratinised, with mucous, claviform and epithelial cells. Stomach has “C” shape, with folded mucosa along cardiac region, disordered in the fundic region, and directed to the sphincter in the pyloric region. Its epithelium is simple prismatic, and cardiac and fundic portions have gastric glands. Cranial intestine is formed by pyloric flexure and descending loop attached to the right side of stomach. Middle intestine is winding and positioned to the right of caudal portion of stomach. Caudal intestine is linear and with a median position up to the anus. Intestinal coefficient was 1.39 ± 0.30 cm. Epithelium is simple prismatic with brush border and contains epithelial and goblet cells. Caudal region has highest concentration of goblet cells. Were detected neutral glycoproteins, carboxylated and sulphated acid glycoconjugates for mucous cells and goblet cells, and neutral glycoproteins for the apical region of gastric epithelial cells. Morphological features could be related to piscivorous species feeding habit.     

Characterization of bovine fetal Leydig cells by KIT expression

The origin of fetal Leydig cells (FLC) and whether they share a common lineage with adult Leydig cells (ALC) is still under debate, and a marker to reliably track and isolate fetal Leydig precursor cells remains to be identified. We analyzed KIT positive (KIT+) cells in gonads from bovine fetuses with crown-rump-length (CRL) 2.5–85 cm by immunohistochemistry, and found that KIT expression was gender-specific. In female gonads, expression was mainly associated with epithelial cell cords, which extended from the surface epithelium towards the KIT-negative inner stroma. In male gonads of fetuses, after CRL 2.9 cm, KIT expression was strikingly strong in interstitial cells (IC). Only a few KIT+ cells were detected in the epithelial cell cords and in the stromal layer under the surface epithelium after CRL 3.5 cm. In the male fetuses, KIT expression in IC was a continuous and characteristic feature until full term. At all developmental stages KIT+ areas alternated with anti-Müllerian hormone-positive areas. Platelet-derived growth factor receptor α production was initiated after the expression of KIT at CRL 4.5 cm. Detection of cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein in KIT+ IC identified them as FLC. KIT+ cells, isolated from testes by magnetic-activated cell sorting, retained their steroidogenic capacity in vitro. Together, these findings show that KIT+ IC of fetal testis correspond to FLC, which can be successfully cultivated for advanced studies.     

Immunolocalization of Na+/K+‐ATPase,Na+/K+/2Cl− co‐transporter (NKCC) and mRNA expression of Na+/K+‐ATPase α‐subunit during short‐term salinity transfer in the gills of Persian sturgeon (Acipenser persicus,Borodin, 1897) juveniles

The study tests the physiological responses of Persian sturgeon, Acipenser persicus, during the abrupt release of juveniles from freshwater (FW) into brackish waters (BW = 11‰) of the Caspian Sea. Fish weight at release was 2‐3 g (2.55 ± 0.41 g; 8.8 ± 0.58 cm TL). Totals of 160 individuals were randomly distributed into four fiber‐glass aerated tanks (volume 60‐L). Two tanks served as controls (FW groups), and two as exposure tanks for BW (Caspian Sea water = CSW). Fish were sampled at 0, 3, 6, 12, 24, 48 and 96 hr after abrupt transfer to CSW. Plasma osmolality, immunolocalization of Na⁺, K⁺ ‐ATPase (NKA) and Na⁺/K⁺/2Cl^– (NKCC) Co‐transporter, NKA activity and the NKA α‐subunit mRNA expression were analyzed. Blood osmolality of fish transferred from FW to CSW increased significantly within hours post‐transfer (p < .05) and remained at a high level for up to 96 hr. Immunolocalization of NKCC indicated co‐localization with NKA in the chloride cells in the gill epithelium. A partial sequence of the NKA α‐subunit (632 bp) is described. Its expression levels were up‐regulated at 12 and 48 hr following salinity transfer (p < .05). However, NKA activity sharply increased in CSW specimens by almost 2.8‐fold (p < .05) between 48 and 96 hr after transfer. Gill NKCC co‐transporter abundance increased, coinciding with increased gill NKA activity. The increased activity of NKCC during salt excretion in CSW may lead to an influx of Na⁺ into the chloride cells. Consequently, NKA activity increases to maintain intracellular Na⁺ homeostasis.