Steffen Hamann

Aquaporins in complex tissues: distribution of aquaporins 1–5 in human and rat eye

Multiple physiological fluid movements are involved in vision. Here we define the cellular and subcellular sites of aquaporin (AQP) water transport proteins in human and rat eyes by immunoblotting, high-resolution immunocytochemistry, and immunoelectron microscopy. AQP3 is abundant in bulbar conjunctival epithelium and glands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering the trabecular meshwork and Schlemms canal. Although AQP1 is plentiful in ciliary nonpigmented epithelium, it is not present in ciliary pigmented epithelium. Posterior and anterior epithelium of the iris and anterior lens epithelium also contain significant amounts of AQP1, but AQP0 (major intrinsic protein of the lens) is expressed in lens fiber cells. Retinal Müller cells and astrocytes exhibit notable concentrations of AQP4, whereas neurons and retinal pigment epithelium do not display aquaporin immunolabeling. These studies demonstrate selective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocular epithelia, predicting specific roles for each in the complex network through which water movements occur in the eye.Multiple physiological fluid movements are involved in vision. Here we define the cellular and subcellular sites of aquaporin (AQP) water transport proteins in human and rat eyes by immunoblotting, high-resolution immunocytochemistry, and immunoelectron microscopy. AQP3 is abundant in bulbar conjunctival epithelium and glands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering the trabecular meshwork and Schlemms canal. Although AQP1 is plentiful in ciliary nonpigmented epithelium, it is not present in ciliary pigmented epithelium. Posterior and anterior epithelium of the iris and anterior lens epithelium also contain significant amounts of AQP1, but AQP0 (major intrinsic protein of the lens) is expressed in lens fiber cells. Retinal Müller cells and astrocytes exhibit notable concentrations of AQP4, whereas neurons and retinal pigment epithelium do not display aquaporin immunolabeling. These studies demonstrate selective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocular epithelia, predicting specific roles for each in the complex network through which water movements occur in the eye.

Measurement of Cell Volume Changes by Fluorescence Self-Quenching

At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (μM) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.

Water transport in the brain: Role of cotransporters

It is generally accepted that cotransporters transport water in addition to their normal substrates, although the precise mechanism is debated; both active and passive modes of transport have been suggested. The magnitude of the water flux mediated by cotransporters may well be significant: both the number of cotransporters per cell and the unit water permeability are high. For example, the Na(+)-glutamate cotransporter (EAAT1) has a unit water permeability one tenth of that of aquaporin (AQP) 1. Cotransporters are widely distributed in the brain and participate in several vital functions: inorganic ions are transported by K(+)-Cl(-) and Na(+)-K(+)-Cl(-) cotransporters, neurotransmitters are reabsorbed from the synaptic cleft by Na(+)-dependent cotransporters located on glial cells and neurons, and metabolites such as lactate are removed from the extracellular space by means of H(+)-lactate cotransporters. We have previously determined water transport capacities for these cotransporters in model systems (Xenopus oocytes, cell cultures, and in vitro preparations), and will discuss their role in water homeostasis of the astroglial cell under both normo- and pathophysiologal situations. Astroglia is a polarized cell with EAAT localized at the end facing the neuropil while the end abutting the circulation is rich in AQP4. The water transport properties of EAAT suggest a new model for volume homeostasis of the extracellular space during neural activity.

Molecular mechanisms of water transport in the eye.

The four major sites for ocular water transport, the corneal epithelium and endothelium, the ciliary epithelium, and the retinal pigment epithelium, are reviewed. The cornea has an inherent tendency to swell, which is counteracted by its two surface cell layers, the corneal epithelium and endothelium. The bilayered ciliary epithelium secretes the aqueous humor into the posterior chamber, and the retinal pigment epithelium transports water from the retinal to the choroidal site. For each epithelium, ion transport mechanisms are associated with fluid transport, but the exact molecular coupling sites between ion and water transport remain undefined. In the retinal pigment epithelium, a H+-lactate cotransporter transports water. This protein could be the site of coupling between salt and water in this epithelium. The distribution of aquaporins does not suggest a role for these proteins in a general model for water transport in ocular epithelia. Some water-transporting membranes contain aquaporins, others do not. The ultrastructure is also variable among the cell layers and cannot be fitted into a general model. On the other hand, the direction of cotransport in symporters complies with the direction of fluid transport in both the corneal epi- and endothelium, as well as the ciliary epithelium and retinal pigment epithelium.

Cotransport of H+, lactate and H2O by membrane proteins in retinal pigment epithelium of bullfrog.

1. The interaction between H+, lactate and H2O fluxes in the retinal membrane of the pigment epithelium from bullfrog Rana catesbiana was studied by means of ion‐selective micro‐electrodes. 2. Changes in intracellular pH and cell volume were recorded in response to abrupt changes in retinal solution concentration and/or osmolarity. 3. Two parallel pathways for water transport were identified across the retinal membrane, an osmotic one with a hydraulic water permeability of 3.2 x 10(‐4) cm s‐1 (osmol l‐1)‐1 and one which depended on the presence of lactate. 4. Addition of sodium lactate to the retinal solution caused cell shrinkages that were small compared with those produced by mannitol. The reflection coefficient for sodium lactate was 0.25. 5. Isosmotic replacement of Cl‐ with lactate caused an influx of water. Simultaneous acidification of the retinal solution from pH 7.4 to 6.4 enhanced the effect. The influx of water could proceed against osmotic gradients elicited by mannitol. 6. The interdependence of the fluxes of H+, lactate and H2O can be described as cotransport: the fluxes had a fixed ratio of about 109 mmol of lactic acid per litre of water, the flux of one species was able to energize the flux of the other two, and the fluxes exhibited saturation for increasing driving forces. 7. The Gibbs equation gives an accurate quantitative description of these coupled fluxes.

Cotransport of water by the Na+ −K+ −2Cl− cotransporter NKCC1 in mammalian epithelial cells

Water transport by the Na+–K+–2Cl− cotransporter (NKCC1) was studied in confluent cultures of pigmented epithelial (PE) cells from the ciliary body of the fetal human eye. Interdependence among water, Na+ and Cl− fluxes mediated by NKCC1 was inferred from changes in cell water volume, monitored by intracellular self‐quenching of the fluorescent dye calcein. Isosmotic removal of external Cl− or Na+ caused a rapid efflux of water from the cells, which was inhibited by bumetanide (10 μm). When returned to the control solution there was a rapid water influx that required the simultaneous presence of external Na+ and Cl−. The water influx could proceed uphill, against a transmembrane osmotic gradient, suggesting that energy contained in the ion fluxes can be transferred to the water flux. The influx of water induced by changes in external [Cl−] saturated in a sigmoidal fashion with a Km of 60 mm, while that induced by changes in external [Na+] followed first order kinetics with a Km of about 40 mm. These parameters are consistent with ion transport mediated by NKCC1. Our findings support a previous investigation, in which we showed water transport by NKCC1 to be a result of a balance between ionic and osmotic gradients. The coupling between salt and water transport in NKCC1 represents a novel aspect of cellular water homeostasis where cells can change their volume independently of the direction of an osmotic gradient across the membrane. This has relevance for both epithelial and symmetrical cells.

Water permeability of Na+–K+–2Cl− cotransporters in mammalian epithelial cells

Water transport properties of the Na+–K+–2Cl− cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity. The anatomy of the cultured cell layer was investigated by light and electron microscopy. The transport rate of the cotransporter was determined from the bumetanide‐sensitive component of 86Rb+ uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (Lp) of the membrane was halved by the specific inhibitor bumetanide. The bumetanide‐sensitive component of the water transport exhibited apparent saturation at osmotic gradients higher than 200 mosmol l−1. Cell shrinkages produced by NaCl or KCl were smaller than those elicited by equi‐osmolar applications of mannitol, indicating reflection coefficients for these salts close to zero. The activation energy of the bumetanide‐sensitive component of the Lp was 21 kcal mol−1, which is four times higher than that of an aqueous pore. The data suggest that osmotic transport via the cotransporter involves conformational changes of the cotransporter and interaction with Na+, K+ and Cl−. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide‐sensitive 86Rb+, the NKCCs of this tissue did not contribute any bumetanide‐sensitive Lp. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.

Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells.

The retinal pigment epithelium (RPE) of the eye transports water and lactate ions in the direction from retina to choroid. The water transport is important in maintenance of retinal adhesion and the transport of lactate ions serves to regulate the lactate levels and pH of the subretinal space. This study investigates by means of a non-invasive technique the mechanism of coupling between transport of H(+), lactate ion, and water in the monocarboxylate transporter (MCT1) located in the apical (retinal) membrane of a mammalian RPE. Primary cultures of porcine RPE cells were grown to confluence and placed in a perfusion chamber in which the solution facing the retinal membrane could be changed rapidly. Two types of experiments were performed: Changes in cell water volume were measured by self-quenching of the fluorescent dye Calcein, and changes in intracellular pH were measured ratiometrically using the fluorescent dye BCECF. In lactate-free solutions, mannitol addition to the retinal bath caused intracellular acidification and cell shrinkage, given by a single osmotic water permeability of 1.2+/-0.1 x 10(-4)cmsec(-1) (osmoll(-1))(-1). In solutions containing 50 mmoll(-1) lactate, however, the mannitol-induced cell shrinkage was faster and the cells alkalinized. These effects were not linear functions of the magnitude of the imposed osmotic gradients: Both volume effects and changes in intracellular pH showed apparent saturation with increasing gradients. Abrupt isosmotic replacement of Cl(-) with lactate in the concentration range from 3 to 50 mmoll(-1) caused an immediate cell swelling as well as an immediate intracellular acidification; both effects showed apparent saturation with increasing lactate concentration. The K(m) values were: 11+/-2 mmoll(-1) for the water fluxes and 13+/-4 mmoll(-1) for the H(+) and lactate fluxes. The data suggest that H(2)O is cotransported along with H(+) and lactate ions in MCT1 localized to the retinal membrane. The study emphasizes the importance of this cotransporter in the maintenance of water homeostasis and pH in the subretinal space of a mammalian tissue and supports our previous study performed by an invasive technique in an amphibian tissue.

The course of headache in idiopathic intracranial hypertension: a 12-month prospective follow-up study.

Our aim was to prospectively describe the course of headache during the first year of idiopathic intracranial hypertension (IIH).

Aquaporins 6–12 in the human eye

Purpose:  Aquaporins (AQPs) are widely expressed and have diverse distribution patterns in the eye. AQPs 0–5 have been localized at the cellular level in human eyes. We investigated the presence of the more recently discovered AQPs 6–12 in the human eye.