Coupling of²²Na and³⁶C1 uptake in cultured pigmented ciliary epithelial cells: A proposed role for the isoenzymes of carbonic anhydrase

Abstract

Uptake studies with 22Na and 36Cl were performed in cultured bovine pigmented ciliary epithelial cells (PE) to investigate interdependence of Na+ and Cl- transport. (1) 22Na uptake into NaCl depleted cells was stimulated by Cl-. This stimulation was abolished by the simultaneous application of amiloride (1 mM) and bumetanide (0.1 mM), indicating two independent mechanism for Cl- stimulated Na+ uptake: loop diuretic sensitive Na+/Cl- symport and an indirect stimulation of Na+/H+ exchange by Cl-. The latter component of Cl- stimulated Na+ uptake was HCO3- dependent. (2) 36Cl uptake was increased by extracellular Na+. Na+-stimulated Cl- uptake also consisted of two components. One was bumetanide sensitive and the other was blockable by amiloride and partly inhibited by the carbonic anhydrase (CA) inhibitor methazolamide (0.1 mM). (3) Homogenized PE cells were tested for biochemical CA activity using an electrometric method. The cytoplasmic as well as the membrane fraction contained specific CA activity. (4) A model is presented for Na+ and Cl- transport into PE: in addition to Na+/Cl- symport, Na+/H+ and Cl-/HCO3- double exchange may operate in the ciliary epithelium. The latter mechanism provides NaCl uptake into the cell in exchange for H+ and HCO3-, which recycle as CO2 across the membrane. This recycling of CO2 and HCO3-/H+ (and hence indirectly NaCl uptake) is facilitated by the cooperation between membrane bound and cytoplasmic CA.

Cytoplasmic pH Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit Nonpigmented Ciliary Epithelium

“…tion. In different tissues it has been suggested that CA might facilitate ion transport mediated by the Na-H exchanger (Knickelbein, Aronson & Schron, 1985;Dagher, Egnor & Charney, 1993;Helbig et al, 1989). In studies here with cultured rabbit NPE, partial inhibition of the Na-H exchanger when CA-IV is inhibited could explain why the magnitude of the pH i response to amiloride was reduced in the presence of either DBI or ACTZ.…”

“…Inhibition of CA-IV with DBI or ACTZ could lower pH in the unstirred layer and since H + and Na + compete for the same extracellular binding site (Aronson, 1985) this could inhibit the activity of the Na-H exchanger. In the PE cell layer, Na-H exchange might have a specialized role, importing sodium from the plasma which subsequently passes to the NPE where Na,K-ATPase shifts it across the basolateral membrane (Helbig et al, 1989). However, the NPE is not thought to absorb sodium from the aqueous humor.…”

Fluid transport phenomena in ocular epithelia

“…Following this, several investigators were able to isolate the ciliary body epithelia of toad, shark, rabbit, and bovine with which active transport mechanisms for Cl -, Na + and HCO 3 -were described . Complementing this work, other investigators have used the isolated tissue to look for transporters with intracellular electrodes (Chu and Green, 1990;Wiederholt and Zadunaisky, 1986), fluorescent probes (Butler et al, 1994;Wolosin et al, 1991), electron probe (Bowler et al, 1996;Macknight et al, 2000), and patch clamp techniques (Chen and Sears, 1997;Chen et al, 1998;Edelman et al,1995;Wang et al, 2000), or have used cultured CE cells to look for channels using microelectrodes (Helbig et al, 1989c) or patch-clamping CoCa-Prados et al,1995,1996Yantorno et al, 1992), and for transporters using radioisotopes (Helbig et al 1988(Helbig et al ,1989a(Helbig et al ,1989b, or fluorescent techniques (Tao et al, 1998;Hou et al, 1998Hou et al, ,2000. As a result of this wealth of information, several models have been proposed to explain aqueous humor production by the ciliary epithelium (Civan, 1997;Civan and Macknight, 2004;Jacob and Civan, 1996).…”

The loci of carbonic anhydrase activitiy in the ciliary epithelium of the rabbit eye: Electrophysiological study with isolated ciliary epithelial bilayer.

“…Biochemical studies of carbonic anhydrase for the separate cell layers of the ciliary epithelium and its membranes have not been reported, although, for cultured bovine PE, using an electrometric method, Helbig et al found specific carbonic anhydrase activity in both cytoplasmic and membrane fractions (9). Histochemical studies of the eye for localization of the enzyme done with the cobalt method of Hansson (19,20), presumably specific for carbonic anhydrases, suffer from a low level of resolution.…”