Abstract
In the intestine of eutherian mammals, such as human, rabbit, rat and mouse, fluid secretion is primarily driven by Cl- transport. However, in the ileum of possum (Trichosurus vulpecula), a metatherian mammal, the fluid secretion is independent of Cl-, but is dependent on serosal HCO3- and inhibited by serosal 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). This suggests that the secretion is driven by the pancreatic type Na+-HCO3- cotransporter (pNBCe1). Initially, the aim of the project was to confirm that a NBC was functionally expressed in the ileal epithelium using measurements of intracellular pH (pHi). Intact villi were acutely isolated by calcium chelation and loaded with a pHi-sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Then, H+ and HCO3- transporter activity was determined from the initial rate of pHi (ΔpHi) recovery following acid loading with the ammonium prepulse technique. Alternatively, the steadystate activity of the transporters was investigated from the effect of specific inhibitors on pHi in the absence of acid loading, and it was determined whether the activity could be regulated by cAMP-dependent secretagogues.
In HCO3--free HEPES buffered Ringer’s, recovery from acid loading was dependent on Na+ and inhibited by ethyl-isopropyl amiloride (EIPA, 10 μM), consistent with the activity of a Na+/H+ exchanger (NHE). In contrast, in CO2/HCO3- buffered Ringers, although the recovery from acid loading was Na+-dependent, the rate of recovery was only partially inhibited by EIPA (ΔpHi control = 0.0072 ± 0.0003, n=5; ΔpHi EIPA = 0.0034 ± 0.0002, n=5, P<0.001). However, addition of EIPA and DIDS, an inhibitor of NBC (0.5 mM), largely abolished the recovery (ΔpHi EIPA+DIDS = 0.0008 ± 0.0002, n=4, P<0.001). In the steady state, EIPA (ΔpHi = 0.00003 ± 0.0002, n=6) and DIDS (ΔpHi = -0.0006 ± 0.0002, n=6) only induced slow changes in pHi. However, following stimulation with secretagogues, while EIPA continued to have little effect (ΔpHi = -0.0005 ± 0.0001, n=6), DIDS induced a significantly faster rate of acidification (ΔpHi = -0.0017 ± 0.0002, n=6, P<0.001).
Moreover, two variants of possum pNBCe1 (long-pNBCe1 and short-pNBCe1) were revealed through re-cloning from the ileal epithelium. While long-pNBCe1 is the same length as the human pNBCe1, short-pNBCe1 has a nine-amino acid deletion. Both variants have the identical post-translational modification sites, except long-pNBCe1 has one additional casein kinase II phosphorylation site. Then, the activity of the two variants was investigated heterologously in HEK 293 cells. The long-pNBCe1 transfected cells had a significantly (P<0.001) faster ΔpHi from acid loading, but the short-pNBCe1 transfected cells had similar ΔpHi when compared to the control. This suggests that the transfected long-pNBCe1 was functionally active, but not short-pNBCe1, despite both forms being detected by Western blot and immunostaining. In addition, the transfected long-pNBCe1 exhibited an IC50 of 0.94 mM for DIDS.
In summary, these findings demonstrate the functional expression of a NBC in possum ileal villous cells, and the transporter can be stimulated by secretagogues. This is consistent with the involvement of NBC in the secretion of HCO3- by the ileal epithelium. This study may provide an insight into the gastrointestinal secretory physiology of the possum.