Activation of cAMP-dependent Cl− currents in guinea-pig Paneth cells without relevant evidence for CFTR expression
Corresponding author Y. Okada: Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444-8585, Japan. Email: firstname.lastname@example.org
- 1To determine whether Paneth cells exhibit functional expression of cAMP-activated Cl− currents and molecular expression of the cystic fibrosis transmembrane conductance regulator (CFTR), we applied whole-cell patch clamp and single-cell mRNA analysis by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification to single Paneth cells in crypts isolated from the guinea-pig small intestine.
- 2Prominent activation of Cl− currents was consistently observed after stimulation with dibutyryl cAMP and forskolin or with vasoactive intestinal polypeptide (VIP). The cAMP-activated Cl− current was inhibited by removal of intracellular ATP or administration of an inhibitor of protein kinase A.
- 3Many of the biophysical and pharmacological properties of the currents were phenotypically similar to those of the CFTR Cl− channel, such as the ohmic current-voltage relationship, the anion selectivity with a Type III sequence (Br− > Cl− > I−≫ F−≥ gluconate−), I−-induced blockage, insensitivity to a stilbene-derivative Cl− channel blocker, and sensitivity to a carboxylate analogue Cl− channel blocker. The sensitivity of the current to glibenclamide was, however, much weaker than that reported for the CFTR Cl− channel current. In contrast to the time independence of CFTR currents, the inward component of the Paneth cell Cl− currents exhibited inactivation kinetics.
- 4Expression of CFTR mRNA could not be detected by RT-PCR analysis in almost all single Paneth cells, although its expression was consistently detected at the whole-crypt level. The presence of a small number of CFTR-expressing epithelial cells, which were scattered both in villi and crypts but not at the crypt base where Paneth cells were located, was demonstrated by immunocytochemistry.
- 5Taken together, it appears that guinea-pig Paneth cells functionally express cAMP-activated Cl− conductance without relevant evidence for molecular expression of CFTR. Functional expression of VIP receptors in the Paneth cells was also demonstrated.