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Articles in PresS, published online ahead of print February 13, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00544.2001
Submitted on November 14, 2001
Accepted on February 6, 2002
1 Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA
2 Department of Ophthalmology, University of Arizona, Tucson, AZ, USA
3 Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA; Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA
* To whom correspondence should be addressed. E-mail: civan{at}mail.med.upenn.edu.
Volume of trabecular meshwork (TM) cells may modify outflow resistance of aqueous humor, thereby altering intraocular pressure. This study examines the contribution that Na+/H+, Cl-/HCO3- exchange and K-Cl efflux mechanisms have on the volume of TM cells. Volume, Cl- currents and intracellular Ca2+ activity of cultured human TM cells were studied with calcein fluorescence, whole-cell patch clamping and fura-2 fluorescence, respectively. At physiologic bicarbonate concentration, the selective Na+/H+ antiport inhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicity triggered a regulatory volume decrease (RVD), which could be inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), K+ channel blockers Ba2+ and tetramethylammonium (TEA), and by the K+-Cl- symport blocker [(dihydro-indenyl)oxy]alkanoic acid (DIOA). The fluid uptake mechanism in isotonic conditions was dependent upon bicarbonate; at physiologic levels, the Na+/H+ exchange inhibitor dimethylamiloride (DMA) reduced cell volume while at low levels the Na+-K+-2Cl- symport inhibitor bumetanide had the predominant effect. Patch-clamp measurements showed that hypotonicity activated an outwardly-rectifying, NPPB-sensitive Cl- channel displaying the permeability ranking Cl- > methylsulfonate > aspartate. 2,3-Butanedione 2-monoxime (BDM) antagonized actomyosin activity, and both increased baseline [Ca2+] and abolished swelling-activated increase in [Ca2+], but did not affect RVD. Results indicate that human TM-cells display a Ca2+-independent RVD and that volume is regulated by swelling-activated K+ and Cl- channels, Na+/H+ antiports and possibly K+-Cl- symports in addition to Na+-K+-2Cl- symports.
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