Electrogenic Cl− secretion does not occur in the ileum of the Australian common brushtail possum, Trichosurus vulpecula, due to low levels of expression of the NaK2Cl cotransporter, NKCC1

Abstract

The colon of the brushtail possum does not have an electrogenic secretory response. Given the functional significance of electrogenic Cl(-) secretion in the intestine of eutherian mammals, we have investigated the secretory response in the small intestine of this marsupial. In the Ussing chamber cAMP-dependent secretagogues stimulated a sustained increase in ileal short-circuit current (Isc), whereas Ca(2+)-dependent secretagogues induced a transient increase. Both the responses were inhibited by mucosal addition of the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 mciromol l(-1)), consistent with an anion secretory response. However, the responses were not inhibited by serosal bumetanide (10 mciromol l(-1)) and were independent of bath Cl(-), indicating that the stimulated ileal Isc does not involve electrogenic Cl(-) secretion driven by the NaK2Cl cotransporter, NKCC1. Consistent with this, there were low levels of NKCC1 expression in the ileal epithelium. In particular, NKCC1 expression in the ileal crypt cells was comparable to that of the villous cells. This differs from eutherian mammals where high levels of NKCC1 expression in the ileal crypt cells are associated with their role in Cl(-) secretion. The cAMP- and Ca(2+)-dependent secretory responses were inhibited by the removal of HCO(3) (-) suggesting that these responses were due to electrogenic HCO(3) (-) secretion. We conclude that the ileum of the possum does not secrete Cl(-) due to low levels of NKCC1 expression. It does however appear to secrete HCO(3) (-). These results are further significant examples of differences in the transport function of the possum intestinal epithelium compared with eutherian mammals.

The distribution and expression of CFTR restricts electrogenic anion secretion to the ileum of the brushtail possum, Trichosurus vulpecula

“…They had a live mass >2kg and were collected and maintained as previously described (Butt et al, 2002b). Animals were killed by an intracardiac injection of barbiturate (Euthal TM ; Delta Veterinary Laboratories, Hornsby, NSW, Australia) administered under halothane-induced anaesthesia (Fluothane; ICI New Zealand Ltd, Lower Hutt, New Zealand), and collection of tissues for RNA and protein isolation, in situ hybridisation studies and Ussing chamber experiments were carried out as previously described (Bartolo et al, 2009a;Bartolo et al, 2009b).…”

“…Crude membrane extracts were obtained as described previously (Bartolo et al, 2009a) and the samples were separated by SDSpolyacrylamide gel electrophoresis in 7.5% polyacrylamide gels and electroblotted onto a PVDF membrane (GE Healthcare Biosciences, Auckland, New Zealand). Membranes were blocked with 5% (w/v) skimmed milk powder in Tris-buffered saline containing 0.1% Nonidet-P40 (TBS-NP40) for 1h, and then incubated with specific antibodies; CFTR mouse anti-human (Ab-4) [1:100 primary, 1:3000 rabbit anti-mouse horseradish peroxidase (HRP) conjugate secondary] purchased from Lab Vision (Fremont, CA, USA); NKCC1 (N-16) goat polyclonal IgG (1:800 primary, 1:5000 donkey anti-goat IgG-HRP conjugated secondary) purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); NaHCO 3 cotransporter (NBC) rabbit anti-rat NBC serum (1:2000 primary, 1:6000 antirabbit HRP conjugate), for 2h.…”

“…RNA isolation and cDNA synthesis have been described previously (Bartolo et al, 2009a;Bartolo et al, 2009b). To quantify the level of pCFTR mRNA expression between the different regions of the intestine, the expression of 18S rRNA was used as an internal control.…”

“…In the Australian common brushtail possum, Trichosurus vulpecula, a metatherian mammal, the mechanisms of electrolyte transport in the intestinal tract differ from those seen in the eutherian intestine (Butt et al, 2002a;Butt et al, 2002b). This is particularly evident in the ileum where secretagogues stimulate electrogenic anion secretion, but this involves electrogenic HCO 3 -secretion, not electrogenic Cl -secretion as seen in eutherian mammals (Bartolo et al, 2009a;Bartolo et al, 2009b). The absence of Cl -secretion in the possum ileum is primarily due to low levels of expression of NKCC1, the major Cl -uptake pathway of secretory epithelia in eutherian mammals (Bartolo et al, 2009a).…”

“…This is particularly evident in the ileum where secretagogues stimulate electrogenic anion secretion, but this involves electrogenic HCO 3 -secretion, not electrogenic Cl -secretion as seen in eutherian mammals (Bartolo et al, 2009a;Bartolo et al, 2009b). The absence of Cl -secretion in the possum ileum is primarily due to low levels of expression of NKCC1, the major Cl -uptake pathway of secretory epithelia in eutherian mammals (Bartolo et al, 2009a). In contrast, there are high levels of pNBCe1 expression in the basolateral membrane of the possum ileum and HCO 3 -secretion is driven by this transporter (Bartolo et al, 2009b).…”