Cellular distribution and differential gene expression of the three alpha subunit isoforms of the Na,K-ATPase in the ocular ciliary epithelium.

We have investigated the localization and pattern of expression of the three alpha subunit isoforms of Na,K-ATPase in the transporting ciliary epithelium of the bovine eye. Using specific cDNA probes and antisera to the alpha 1, alpha 2, and alpha 3 isoforms of Na,K-ATPase, we demonstrated that mRNAs and polypeptides for the three distinct forms of the Na,K-ATPase alpha subunit (alpha 1, alpha 2, and alpha 3) were expressed in the ciliary epithelium in vivo. Immunochemical localization of the three alpha isoforms of Na,K-ATPase in two ultrastructurally different regions of the ciliary epithelium (namely, the pars plicata and pars plana) revealed that the three alpha isoforms of Na,K-ATPase were distributed in a distinct fashion in the basolateral plasma membrane domains of nonpigmented (NPE) and pigmented (PE) cells. The NPE cells in the pars plicata showed an immunoreactive signal to all the three alpha isoforms; in the pars plana, they showed immunoreactive signals only for the alpha 1 and alpha 2 isoforms but not for alpha 3. The PE cells, in both the pars plana and pars plicata regions, showed an immunoreactive signal only for the alpha 1 isoform; immunoreactive signals were not detected for alpha 2 and alpha 3. To verify the differential immunostaining patterns of NPE and PE cells, specific antibodies for each of the three alpha subunit isoforms of Na,K-ATPase were applied to immunoblots containing microsomal fractions from flow cytometric-sorted cells (NPE and PE). Our results indicate that alpha 1, alpha 2, and alpha 3 polypeptides were present in microsomal fractions of NPE cells of the pars plicata and pars plana and that the alpha 1 polypeptide was the only polypeptide present in the PE cells from both regions of the ciliary epithelium. These results also revealed that the alpha 3 isoform epitope recognized by the monoclonal antibody McB-X3.1 in the pars plicata is not readily accessible in the pars plana. A cell line was established from the ciliary epithelium of a bovine eye by viral transformation with simian virus 40. In culture, this cell line expressed all three alpha isoforms at the mRNA and polypeptide levels, suggesting that the line may have derived from the NPE layer.     

Development of a procedure for autoradiographic localization of carbonic anhydrase at the electron microscope level

A method for preparing tissue suitable for electron microscope autoradiographic localization of carbonic anhydrase is described. Radioactivity is in the form of 3H-acetazolamide, a specific inhibitor of carbonic anhydrase. Tissues fixed in glutaraldehyde, dehydrated in ethylene glycol followed by cellosolve as a transition fluid, and embedded in epoxy resin, were found to retain most (74%) of the label. Electron micrographs of avian gastric mucosa prepared in this manner are shown. Other methods of preparation were explored and resulted in considerable losses of label or in inadequately preserved tissue. Light microscope autoradiographic localization of gastric mucosa, shell gland, chorioallantoic membrane, and skeletal muscle compare well with previous localizations.     

Inhibition of CA V decreases glucose synthesis from pyruvate

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.     

High glucose-6-phosphatase activity in non-pigmented epithelial cells of rabbit ciliary body.

For study of the origin of glucose in the aqueous humor, glucose-6-phosphatase (G6Pase) and hexokinase activities, and glycogen, were cytochemically examined in the ciliary body (CB) of rabbit. G6Pase activity was also assayed biochemically. The staining reaction for G6Pase activity was strong in the non-pigmented epithelium (NPE) in the pars plana and tips of ciliary processes in the region containing large ciliary pockets within the pars plicata. NPE cells contained abundant reaction product for G6Pase activity in the endoplasmic reticulum (ER) and nuclear envelope. However, NPE in other regions of the CB and pigmented epithelium (PE) of CB, and other areas surrounding the anterior and (PE) of CB, and other areas surrounding the anterior and posterior chambers, showed weak or no G6Pase staining reaction. Biochemical G6Pase activity in the whole ciliary body was relatively high. Both NPE and PE in the pars plana and the tips showed strong staining reaction for hexokinase activity but no staining for glycogen. Furthermore, NPE cells in the tips bore large aggregates of smooth ER and many Golgi apparati. These suggest that the high G6Pase activity in NPE cells in the pars plana and the tips is related to glucose release into the aqueous humor.     

Regional differences in ciliary epithelial cell transport properties

Experiments were performed to determine whether the transport properties of the ciliary epithelium vary over different regions. Rabbit iris-ciliary bodies were incubated under experimental or control conditions for 30 min before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. Cryosections were cut from three regions along the major axis of the iris-ciliary body, i.e., the anterior, middle and posterior (pars plicata) regions. In bicarbonate/CO₂ solution, the epithelial cells of the anterior and middle regions contained more Cl and K than did those of the posterior region. These higher levels of Cl and K were reduced by the carbonic anhydrase inhibitor acetazolamide. Application of bumetanide, an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, resulted in significant increases in Cl and K in the anterior and middle regions but not in the posterior region. In bicarbonate-free solution, the ratio for K/Na contents was higher in the posterior than in the two more anterior regions; Na, K and Cl contents of epithelial cells in the three regions were otherwise similar. Cell composition did not differ significantly between the crests and valleys of the posterior region. The divergent responses to perturbation of epithelial transport in the different regions provide the first demonstration of functional heterogeneity along the major axis of the iris-ciliary body. The response to inhibition of carbonic anhydrase raises the possibility that the anterior aspect of the ciliary epithelium may be the major site of aqueous humor secretion. Received: 4 December 2000/Revised: 24 April 2001     

Presence and distribution of Na+/K+-ATPase in the ciliary epithelium of the rabbit

Regional differences in the localization of Na+/K+-ATPase in the ciliary epithelium of albino rabbits were studied histochemically using the method of Chayen et al. and ultra-histochemically using a cerium-based method. In addition, the incubation time necessary to achieve first signs of staining was investigated as an indication of Na+/K+-ATPase activity. In the entire pars plicata: prelenticular, postlenticular, as well as tips and valleys, staining was seen in the lateral infoldings of the non pigmented epithelium (NPE) after short incubation periods. Somewhat later, the apical cell membranes also stained. The ultrastructure of these cells, together with the staining pattern, point towards a functional significance of the NPE in active fluid secretion. The pigmented epithelium (PE) did not stain. In the iridial processes and in the area of the ciliary ridges staining first appeared in the apical cell membranes of the NPE, which form the typical ciliary channels. The basolateral infoldings of the NPE also stained, whilst the PE remained unstained. The difference in morphology and staining between pars plicata and iridial processes could indicate a difference in function, e.g. reabsorption of freshly secreted aqueous humour. In the pars plana, only the basolateral infoldings of the PE stained. A functional significance of this area in connection with the blood retina barrier is discussed.     

Carbonic anhydrase in human platelets.

The carbonic anhydrase activity of human platelets was investigated by measuring the kinetics of CO2 hydration in supernatants of platelet lysates by using a pH stopped-flow apparatus. An average carbonic anhydrase concentration of 2.1 microM was determined for pellets of human platelets. Analysis of the kinetic properties of this carbonic anhydrase yielded a Km value of 1.0 mM, a catalytic-centre activity kcat. of 130000 s-1 and an inhibition constant Ki towards ethoxzolamide of 0.3 nM. From these values, CO2 hydration inside platelets is estimated to be accelerated by a factor of 2500. When platelet lysates were subjected to affinity chromatography, only the high-activity carbonic anhydrase II could be eluted from the affinity column, whereas the carbonic anhydrase isoenzyme I, which is known to occur in high concentrations in human erythrocytes, appeared to be absent.     

Presence and distribution of Na+/K+-ATPase in the ciliary epithelium of the rabbit

Summary Regional differences in the localization of Na⁺/K⁺-ATPase in the ciliary epithelium of albino rabbits were studied histochemically using the method of Chayen et al. and ultra-histochemically using a cerium-based method. In addition, the incubation time necessary to achieve first signs of staining was investigated as an indication of Na⁺/K⁺-ATPase activity. In the entire pars plicata: prelenticular, postlenticular, as well as tips and valleys, staining was seen in the lateral infoldings of the non pigmented epithelium (NPE) after short incubation periods. Somewhat later, the apical cell membranes also stained. The ultrastructure of these cells, together with the staining pattern, point towards a functional significance of the NPE in active fluid secretion. The pigmented epithelium (PE) did not stain. In the iridial processes and in the area of the ciliary ridges staining first appeared in the apical cell membranes of the NPE, which form the typical ciliary channels. The basolateral infoldings of the NPE also stained, whilst the PE remained unstained. The difference in morphology and staining between pars plicata and iridial processes could indicate a difference in function, e.g. reabsorption of freshly secreted aqueous humour. In the pars plana, only the basolateral infoldings of the PE stained. A functional significance of this area in connection with the blood retina barrier is discussed.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Carbonic anhydrase C in white-skeletal-muscle tissue.

We investigated the activity of carbonic anhydrase in blood-free perfused white skeletal muscles of the rabbit. Carbonic anhydrase activities were measured in supernatants and in Triton extracts of the particulate fractions of white-skeletal-muscle homogenate by using a rapid-reaction stopped-flow apparatus equipped with a pH electrode. An average carbonic anhydrase concentration of about 0.5 microM was determined for white skeletal muscle. This concentration is about 1% of that inside the erythrocyte. Some 85% of the muscle enzyme was found in the homogenate supernatant, and only 15% appeared to be associated with membranes and organelles. White-skeletal-muscle carbonic anhydrase was characterized in terms of its Michaelis constant and catalytic-centre activity (turnover number) for CO2 and its inhibition constant towards ethoxzolamide. These properties were identical with those of the rabbit erythrocyte carbonic anhydrase C, suggesting that a type-C enzyme is present in white skeletal muscle. Affinity chromatography of muscle supernatant and of lysed erythrocytes showed that, whereas rabbit erythrocytes contain about equal amounts of carbonic anhydrase isoenzymes B and C, the B isoenzyme is practically absent from white skeletal muscle. Similarly, ethoxzolamide-inhibition curves suggested that white skeletal muscle contains no carbonic anhydrase A. It is concluded that white skeletal muscle contains essentially one carbonic anhydrase isoenzyme, the C form, most of which is probably of cytosolic origin.     

Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined

The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.     

The effect of carbonic anhydrase inhibition on the velocity of thrombin-stimulated platelet aggregation under physiological conditions

We have studied the effect of ethoxzolamide, a specific carbonic anhydrase inhibitor, on the velocity of thrombin-stimulated platelet aggregation. After preincubation of platelet rich plasma with 10^?6 M ethoxzolamide the velocity of platelet aggregation was reduced by about 40%. Between 10^?11 M and 10^?10M ethoxzolamide was necessary to achieve a half-maximal diminution of the aggregation velocity. An identical maximal reduction of the velocity of aggregation as with ethoxzolamide could be achieved by a nearly complete removal of CO₂ from the platelet rich plasma. These results suggest that the intracellular CO₂ hydration-dehydration reaction is involved in the activation of human platelets by thrombin. It is possible that the cytosolic carbonic anhydrase of platelets provides a rapid source of the protons that are transferred across the plasma membrane during the activation process.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

Role of anions and carbonic anhydrase in epithelia

The existence of carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1) in blood was suspected and sought because the rates of spontaneous hydration and dehydration of CO2 and carbonic acid were slow compared with the rates of exchange of CO2 with blood. The existence of the enzyme in absorbing and secreting epithelial tissues has, in contrast, often been sought because its presence was required for the operations of theoretical models for the movements of H+ ions or HCO-3 into or out of epithelial cells. In addition to the HCl-secreting gastric mucosal epithelium, the enzyme was subsequently found in the rumen, in the kidney, especially those of species that produce acid urine, and salivary gland, the liver and biliary duct system, the mucosa of the small intestine, caecum and colon, the choroid plexuses and ciliary body of mammals, in toad urinary bladder and in the Cl-secreting cells of fish gill. The presence of carbonic anhydrase in exocrine pancreas does not seem to be well established. The enzyme, of molecular mass about 30kDa and containing one zinc atom, exists in three related forms: one of high specific activity and two of low specific activity, one of which is found in red skeletal muscle. Although most, but not all, types of erythrocyte contain both varieties, epithelia usually contain only the high-activity enzyme; however, ox rumen contains large quantities of the low-activity variety as do guinea-pig caecal and colonic mucosae. Salt transport in the intestinal tract is associated with movements of HCO-3 and H+ ions, yet although carbon dioxide stimulates solute and fluid transport in the gall bladder in jejunum, and inhibitors of carbonic anhydrase reduce fluid and ion transport across many epithelia the role of the enzyme in epithelial transport is not clearly understood. Knowledge of the rates of hydration and dehydration of CO2/HCO-3 in the fraction of the tissue water responsible for the H+-HCO-3 movements in many secretory epithelia is currently lacking.     

Pulmonary vasodilation by acetazolamide during hypoxia is unrelated to carbonic anhydrase inhibition

Acute hypoxic pulmonary vasoconstriction can be inhibited by high doses of the carbonic anhydrase inhibitor acetazolamide. This study aimed to determine whether acetazolamide is effective at dosing relevant to human use at high altitude and to investigate whether its efficacy against hypoxic pulmonary vasoconstriction is dependent on carbonic anhydrase inhibition by testing other potent heterocyclic sulfonamide carbonic anhydrase inhibitors. Six conscious dogs were studied in five protocols: 1) controls, 2) low-dose intravenous acetazolamide (2 mg.kg(-1).h(-1)), 3) oral acetazolamide (5 mg/kg), 4) benzolamide, a membrane-impermeant inhibitor, and 5) ethoxzolamide, a membrane-permeant inhibitor. In all protocols, unanesthetized dogs breathed spontaneously during the first hour (normoxia) and then breathed 9-10% O(2) for the next 2 h. Arterial oxygen tension ranged between 35 and 39 mmHg during hypoxia in all protocols. In controls, mean pulmonary artery pressure increased by 8 mmHg and pulmonary vascular resistance by 200 dyn.s.cm(-5) (P <0.05). With intravenous acetazolamide, mean pulmonary artery pressure and pulmonary vascular resistance remained unchanged during hypoxia. With oral acetazolamide, mean pulmonary artery pressure increased by 5 mmHg (P < 0.05), but pulmonary vascular resistance did not change during hypoxia. With benzolamide and ethoxzolamide, mean pulmonary artery pressure increased by 6-7 mmHg and pulmonary vascular resistance by 150-200 dyn.s.cm(-5) during hypoxia (P < 0.05). Low-dose acetazolamide is effective against acute hypoxic pulmonary vasoconstriction in vivo. The lack of effect with two other potent carbonic anhydrase inhibitors suggests that carbonic anhydrase is not involved in the mediation of hypoxic pulmonary vasoconstriction and that acetazolamide acts on a different receptor or channel.     

Histochemical detection of carbonic anhydrase with diemthylaminonaphthalene-5-sulfonamide.

A new specific method for the detection of carbonic anhydrase, EC 4.2.1.1, in tissues is described. The reaction of carbonic anhydrase with dimethylaminonaphthalene-5-sulfonamide (DNSA) forms a highly fluorescent complex. The specificity of the method is proved by the quenching of this fluorescence with ethoxzolamide (6-ethoxybenzothiazole-5-sulfonamide). The difference in the wavelength makes it possible to absorb the fluorescence of the unbound dimethylaminonaphthalene-5-sulfonamide by filters. Kidney, proventriculus, and bone from chicken have been examined. Carbonic anhydrase has been detected in the cytoplasm of the columnar lining cells, proximal tubule cells, and osteoclasts.     

Immunohistochemical demonstration of carbonic anhydrase isoenzyme C in the epithelium of the human ciliary processes

Extravascular location of two main carbonic anhydrase isoenzymes was immunohistochemically investigated in human ciliary processes. The high-activity carbonic anhydrase isoenzyme C was clearly demonstrated in the ciliary epithelium, but was absent from the ciliary stroma. The low-activity isoenzyme B was evident neither in the epithelium nor in the stroma.     

Comparison of 18O exchange and pH stop-flow assays for carbonic anhydrase

The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25 degrees C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (Km) and the inhibition constants for chloride (Ki,Cl) and ethoxzolamide (Ki,ez) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same. Km, maximal reaction velocity (Vmax), Ki,ez, and Ki,Cl obtained from normal human hemolysate at 37 degrees C and pH 7.2 by methods 2 and 3 were the same. Km and Vmax of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of Km and Vmax obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.     

Carbonic anhydrase in rat liver and rabbit skeletal muscle: further evidence for the specificity of the histochemical cobalt-phosphate method of Hansson

Rat liver and rabbit skeletal muscle were studied by Hansson's method for histochemical demonstration of carbonic anhydrase activity. In histochemical model experiments purified male rat liver carbonic anhydrase was much more resistant to acetazolamide than rat erythrocyte carbonic anhydrase. Male rat liver slices showed cytoplasmic staining, which was about 1000 times more resistant to acetazolamide and ethoxzolamide than that of female rat liver or erthyrocytes of either sex. Rabbit skeletal muscle slices showed staining at the sarcolemma of all fibers, whereas the staining of the sarcoplasm varied. The walls of capillaries situated within the muscle bundles were intensely stained. The sarcoplasmic staining of a certain number of fibers was at least 1000 times less sensitive to acetazolamide than the other staining. These findings, which are in good agreement with biochemical data, show that the sulfonamides inhibit histochemical staining in a specific way. This is strong evidence for the specificity of the method.     

Localization and ontogeny of aquaporin-1 and -4 expression in iris and ciliary epithelial cells in rats

The precise localization of aquaporin (AQP)1 and AQP4 was studied in iris and ciliary epithelial cells, in both mature and developing rats, to elucidate the molecular mechanisms underlying aqueous humor balance. Anterior segments of eyes dissected from embryonic day (E)13, E15, E18, and E20, postnatal day (P)0, P7, and P14, and postnatal week 8 rats were subjected to immunofluorescence analysis with AQP isoform-specific antibodies. In adult rat eye, AQP1 was localized to the apical and basolateral plasma membranes of iris epithelial cell layers and of anterior ciliary non-pigmented epithelial (NPE) cells. Conversely, AQP4 was localized to the basolateral plasma membrane of NPE cells in ciliary epithelium and the posterior iris. Developmentally, AQP1 was detected as early as E15 in immature iris and ciliary epithelial cells, and expression persisted throughout development up to adulthood. In contrast, AQP4 was first observed at P7 in the developing pars plicata, and the AQP4-positive area gradually spread to cover the entire pars plicata as development proceeded. These findings indicate that both AQP1 and AQP4 contribute to aqueous humor secretion in the rat eye, thereby maintaining proper intraocular pressure. Moreover, AQP appears to play a major role in aqueous humor secretion in early eye development. This study thus provides a basis for understanding the molecular mechanisms of aqueous humor secretion in pathological and physiological conditions.