Our previous studies using mouse renal inner medullary collecting duct cells (mIMCD-K2 cell-line) have shown the presence of sodium-potassium-2 chloride (NKCC1) cotransport at the basolateral membranes (Glanville et al. 2001). However forskolin-stimulated secretory short-circuit current in a bicarbonate-containing medium was largely insensitive to 0.2 mM furosemide suggesting that alternative mechanisms must exist at this membrane to accumulate cytosolic Cl^– above its electrochemical equilibrium value (Glanville et al. 2001). Accordingly we have sought evidence for expression and activity of alternative transporters such as the Na⁺-dependent bicarbonate cotransporter (NBC) and anion exchanger (AE) isoforms.

Isoform-specific polymerase chain reaction (PCR) primers were designed for mouse NBC1-3 and AE1-3 isoforms. Reverse-transcription/PCR and agrarose gel electrophoresis of products showed products of the expected size for only NBC3 and AE2/3. The molecular identity of each product was confirmed by T/A cloning (Invitrogen) and sequencing of each product.

Using BCECF-loaded mIMCD-K2 cell monolayers perfused separately across apical and basal surfaces (Glanville et al. 2001), a change in the apical superfusion from a Na⁺-rich bicarbonate-free Krebs solution to one containing 10 mM bicarbonate resulted in cell acidification by 0.35 ± 0.06 pH units (mean ± S.E.M., n = 4), in contrast at the basolateral surface cell alkalinisation (0.13 ± 0.02 pH units, n = 4) was observed. In Na⁺-free conditions, apical superfusion with 10 mM bicarbonate Krebs still resulted in cell acidification, but at the basolateral surface superfusion with 10 mM bicarbonate Krebs now resulted in cell acidification (0.26 ± 0.01 pH units). These data are consistent with basolateral expression of NBC-3. Upon superfusion of the basolateral surface with a low Cl^– (20 mM, Cl^– replaced by gluconate) bicarbonate-containing (10 mM) Krebs solution a cell alkalinisation of 0.14 ± 0.02 pH units (n = 5) was observed whereas at the apical surface a small acidification was observed (0.06 ± 0.01 pH units (n = 5)). These data are consistent with AE expression only at the basolateral surface.

In conclusion we propose that the co-expression of NBC-3 activity together with AE activity at the basolateral surface of mIMCDK2 cells is likely to provide a mechanism for intracellular Cl^– accumulation to support transepithelial Cl^– secretion independent of NKCC1 activity in mIMCDK2 epithelial layers.

This work was supported by the BHF and the NKRF.