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Ionomycin and PDBU increase MDCK monolayer permeability independently of myosin light chain phosphorylation
D M Shasby1, J M Kamath, A B Moy
1Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, USA.
Abstract:
It has been hypothesized that modulation of epithelial paracellular permeability may be mediated by initiation of contraction of a band of actin and myosin located at the tight junction. Phosphorylation of myosin light chain (MLC) is an important determinant of actomyosin contraction. We asked if ionomycin (iono) and phorbol 12,13-dibutyrate (PDBU), which increase paracellular permeability of Madin-Darby canine kidney (MDCK) cell monolayers, increased MLC phosphorylation in MDCK cells. MDCK cell MLC was constitutively phosphorylated by myosin light chain kinase (MLCK), and after PDBU and iono > 99% of MLC continued to be phosphorylated by MLCK. Neither iono or PDBU, nor the combination of iono and PDBU, increased MLC phosphorylation. In contrast, the phosphatase inhibitor okadaic acid did increase MLC phosphorylation. Adenosine 3',5'-cyclic monophosphate (cAMP) and forskolin decreased MLC phosphorylation in control MDCK cells and in cells exposed to iono and PDBU. In contrast, cAMP and forskolin did not blunt the decrease in transepithelial resistance caused by iono and PDBU. Iono and PDBU increase MDCK monolayer permeability independently of an increase in MLC phosphorylation.
Insights
Epithelial barrier function is crucial. This study found that increased permeability in kidney cell monolayers was not mediated by myosin light chain (MLC) phosphorylation, suggesting alternative mechanisms regulate epithelial tight junctions.
Area of Science:
- Cell Biology
- Physiology
- Epithelial Biology
Background:
- Epithelial paracellular permeability is regulated by actomyosin contraction at tight junctions.
- Myosin light chain (MLC) phosphorylation is a key factor in actomyosin contraction.
Purpose of the Study:
- To investigate if ionomycin (iono) and phorbol 12,13-dibutyrate (PDBU) increase MLC phosphorylation in Madin-Darby canine kidney (MDCK) cells.
- To determine if MLC phosphorylation mediates the increase in paracellular permeability induced by iono and PDBU.
Main Methods:
- MDCK cell monolayers were treated with iono and PDBU.
- MLC phosphorylation levels were assessed.
- Transepithelial electrical resistance was measured.
- The effects of okadaic acid, forskolin, and cyclic adenosine monophosphate (cAMP) on MLC phosphorylation and permeability were evaluated.
Main Results:
- MLC phosphorylation in MDCK cells was constitutive and primarily mediated by myosin light chain kinase (MLCK).
- Iono and PDBU did not increase MLC phosphorylation, even in the presence of the phosphatase inhibitor okadaic acid.
- Adenosine 3',5'-cyclic monophosphate (cAMP) and forskolin decreased MLC phosphorylation but did not prevent the iono- and PDBU-induced decrease in transepithelial resistance.
Conclusions:
- Ionomycin and PDBU increase MDCK cell monolayer permeability independently of changes in MLC phosphorylation.
- The mechanisms regulating epithelial paracellular permeability in response to these stimuli likely involve pathways other than MLCK-mediated actomyosin contraction.