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 K m of 60 mm, while that induced by changes in external [Na + ] followed first order kinetics with a K m 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 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 86 Rb + uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (L p ) 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 L p 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 86 Rb + , the NKCCs of this tissue did not contribute any bumetanide-sensitive L p . This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.

Estradiol increases tryptophan hydroxylase-2 and serotonin transporter expression and decreases the expression of serotonin 1A receptor and monoamine oxidase A and B through the interaction with its intracellular receptors. The understanding of molecular mechanisms of estradiol regulation on the protein expression that modulates serotonin neurotransmission will be helpful for development of new and more effective treatment for women with depression.

Regulation and maintenance of cell water volume and intracellular pH (pHi) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pHi. Disruption of either function underlies various pathologies. To study the interaction and kinetics of these two mechanisms, we developed and validated a quantitative fluorescence imaging microscopy method to measure simultaneous changes in pHi and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pHi is determined ratiometrically. The method has a time resolution of <1 s and sensitivity to osmotic changes of approximately 1%. It can be applied in real time to virtually any cell type attached to a coverslip, independently of cellular shape and geometry. Calibration procedures and algorithms developed to transform fluorescence signals into changes in cell water volume (CWV) and examples of applications are presented.

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