Abstract
Electrolyte transport in the intestine of the common brushtail possum (Trichosurus vulpecula) differs from that observed in eutherian mammals. This study has used molecular physiology to identify and characterise the expression and distribution of membrane transporters potentially responsible for these differences in electrolyte transport in the possum intestine.
In the possum ileum, secretagogues stimulate an electrogenic Cl--independent HCO3- secretory response but secretagogue-stimulated Cl- secretion does not occur in this tissue.
Based on the ion dependence and pharmacology of the stimulated secretory response, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR), pancreatic Na+ HCO3- cotransporter (pNBC) and Na+ K+ 2Cl- cotransporter (NKCC1) were investigated in the ileum. Reverse transcription PCR experiments showed that CFTR, pNBC and NKCC1 mRNA transcripts were expressed in the ileal epithelium. It was then demonstrated by in situ hybridisation that both CFTR and pNBC were localised predominantly in the crypts and the levels of expression decreased along the crypt-villous axis towards the lumen. Significantly, the in situ hybridisation results showed that there were low levels of NKCC1 transcript in the ileal epithelium. Western blot studies confirmed that mature CFTR and pNBC proteins were expressed in the ileum, while NKCC1 protein was not detected. The findings of the present study suggest that the absence of Cl- secretion in the ileum is because NKCC1 expression is not elevated in the epithelium. The expression of mature CFTR and pNBC protein suggest that these membrane transporters are involved in the stimulated electrogenic HCO3- secretory response. The evidence also suggests that CFTR may mediate HCO3- efflux in the ileum.
In contrast, secretagogues do not stimulate an electrogenic secretory response in the proximal and distal colon. This study has shown that CFTR, NKCC1 and pNBC proteins are expressed in the proximal and distal colon. Both NKCC1 and pNBC transcripts were localised to the crypt base in the proximal colon. However, it was shown that CFTR has a punctuate distribution and the transcript was predominantly observed in the upper crypt and surface cell region. This study indicated that NKCC1 and pNBC were distributed in a different region of the epithelium compared to CFTR. It was concluded that the distribution of these membrane transporters in different regions of the epithelium accounts for the absence of a stimulated electrogenic secretory response in the possum colon. Given that no stimulated electrogenic secretory response is observed in the colon, it is suggested that HCO3- secretion by the ileum may have an important physiological role in maintaining an appropriate fluid and pH composition for fermentation in the colonic lumen.