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Carbachol activates IkappaB kinase in isolated canine gastric parietal cells
A Todisco1, S Ramamoorthy, N Pausawasdi
1Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan, 48109-0682, USA.
Insights
Carbachol activates IkappaB kinase (IKK) in gastric parietal cells through calcium and PKC signaling. This novel pathway regulates nuclear factor-kappaB (NF-kappaB) activation via G-protein-coupled receptors.
Area of Science:
- Cellular signaling
- Molecular biology
- Gastroenterology
Background:
- IkappaB kinase (IKK) complex, comprising IKKalpha and IKKbeta, is vital for nuclear factor-kappaB (NF-kappaB) activation.
- Gastric parietal cells play a key role in digestion and are regulated by various signaling pathways.
Purpose of the Study:
- To investigate the regulation of IKK by carbachol in isolated gastric parietal cells.
- To elucidate the signaling mechanisms involved in carbachol-induced IKK activation.
Main Methods:
- Immune complex kinase assays were used to measure IKKalpha and IKKbeta activity.
- Inhibition studies utilized BAPTA-AM (Ca(++) chelator), GF109203X (PKC inhibitor), and PDTC (NF-kappaB inhibitor).
- NF-kappaB-luciferase reporter gene assays in COS-7 cells assessed reporter gene activity.
Main Results:
- Carbachol induced IKKalpha and IKKbeta activity in a time-dependent manner, peaking at 5 minutes.
- Carbachol-induced IKK activation and IkappaBalpha degradation were inhibited by BAPTA-AM, GF109203X, and PDTC.
- Carbachol stimulated NF-kappaB-luciferase reporter gene activity in cells expressing the M3 muscarinic receptor.
Conclusions:
- Carbachol activates IKK in gastric parietal cells through intracellular Ca(++)- and PKC-dependent pathways.
- This study reveals a novel mechanism for NF-kappaB regulation mediated by seven transmembrane G-protein-coupled receptors.
- The findings provide new insights into the molecular mechanisms governing gastric function and inflammation.
Abstract:
IkappaB kinase (IKK) is a recently discovered kinase complex composed of the kinases IKKalpha and beta, which plays a crucial role in the activation of NF-kappaB. In this study we examined the regulation of IKK by carbachol in isolated gastric parietal cells. IKKalpha and beta activities were measured by immune complex kinase assay. Carbachol induced both IKK alpha and beta in a time-dependent fashion, with a maximal stimulatory effect detected after 5 min of incubation. The action of carbachol was inhibited by the intracellular Ca(++) chelator BAPTA-AM, the PKC inhibitor GF109203X, and the NF-kappaB inhibitor PDTC. Carbachol also induced degradation of IkappaBalpha, which was reversed by addition of both GF109203X and PDTC and stimulated the activity of a NF-kappaB-luciferase reporter gene plasmid in COS-7 cells stably expressing the human M3 muscarinic receptor. In conclusion, carbachol induces IKK in the parietal cells via intracellular Ca(++)- and PKC-dependent signaling pathways. This observation represents a novel mechanism for the regulation of NF-kappaB through the activation of seven transmembrane G-protein-coupled receptors.