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Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca(2+)-mediated secretagogue-induced volume
M A Valverde1, J A O'Brien, F V Sepúlveda
1AFRC Institute of Animal Physiology and Genetics Research, Cambridge Research Station, UK.
Abstract:
The cellular volume of crypts isolated from 2- to 3-week-old mouse small intestine has been measured to assess the capacity of the epithelial cells to respond to secretagogues. Vasoactive intestinal polypeptide (VIP) or carbachol, respectively cAMP- and calcium-mediated secretagogues, produced a reduction crypt volume attributed to KCl loss through channels activated by the agonists. Consistent with the participation of separate chloride channels, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) blocked the carbachol- but not the VIP-induced volume decrease, whilst glibenclamide abolished the VIP effect without affecting the carbachol-induced volume decrease. Animals homozygous for a disrupted cftr gene, introduced by gene targeting, were also used as the source for crypt isolation. In these CFTR (-/-) crypts. VIP failed to elicit any reduction in cellular volume, while the response to carbachol was indistinguishable from that seen in crypts from age-matched control animals. These results are consistent with murine CFTR being a cAMP-activated chloride channel inhibited by glibenclamide and resistant to DIDS. A separate chloride conductance activated by calcium mobilization in small-intestinal crypts appears to be independent of CFTR.
Insights
Mouse intestinal crypts reveal distinct chloride channel functions. Cystic fibrosis transmembrane conductance regulator (CFTR) mediates cAMP-dependent secretion, while a separate calcium-activated channel is CFTR-independent, offering insights into intestinal fluid balance.
Area of Science:
- Gastroenterology
- Cell Biology
- Molecular Physiology
Background:
- Epithelial cells in the small intestine regulate fluid and electrolyte transport.
- Secretagogues like vasoactive intestinal polypeptide (VIP) and carbachol stimulate cellular responses.
- Understanding the specific ion channels involved is crucial for comprehending intestinal function.
Purpose of the Study:
- To investigate the distinct roles of chloride channels in mouse small intestinal crypts.
- To characterize the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in secretagogue-induced volume changes.
- To differentiate between cAMP- and calcium-mediated chloride conductances.
Main Methods:
- Measurement of cellular volume in isolated mouse small intestinal crypts.
- Application of secretagogues vasoactive intestinal polypeptide (VIP) and carbachol.
- Pharmacological inhibition using 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and glibenclamide.
- Utilizing gene-targeted CFTR (-/-) knockout mice.
Main Results:
- VIP and carbachol induced crypt volume reduction via KCl loss.
- DIDS inhibited carbachol-induced but not VIP-induced volume decrease.
- Glibenclamide abolished VIP-induced volume decrease but not carbachol-induced.
- CFTR (-/-) crypts lacked VIP-induced volume reduction, while carbachol response remained intact.
Conclusions:
- Murine CFTR functions as a cAMP-activated chloride channel, inhibited by glibenclamide and resistant to DIDS.
- A distinct calcium-activated chloride conductance in small intestinal crypts is independent of CFTR.
- These findings elucidate the differential regulation of chloride transport in intestinal epithelial cells.