Activation of cAMP-dependent C1- currents in guinea-pig paneth cells without relevant evidence for CFTR expression.
1. To 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. 2. Prominent 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. 3. Many 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. 4. Expression 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. 5. Taken 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.
Department of Internal Medicine, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan.
T
T
Tsumura
A
A
Hazama
T
T
Miyoshi
S
S
Ueda
Y
Y
Okada
D000818:Animals; D001711:Biotransformation / Q000502:physiology; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000502:physiology; D004558:Electric Stimulation; D004594:Electrophysiology; D006168:Guinea Pigs; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000166:cytology* / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D010641:Phenotype; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D018118:Chloride Channels; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP