Xin Li1, George Gorodeski. 1. Departments of Reproductive Biology, Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA.
Abstract
OBJECTIVE: To understand myosin regulation of epithelial permeability. METHODS: This was an experimental study, using human cervical epithelial cells CaSki. End points were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B-actin interaction (determined in vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in vitro using purified NMM-II-B filaments in terms of filaments disassembly/assembly ratios. RESULTS: Treatment of cells with the Rho-associated kinase (ROCK) inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the resistance of the lateral intercellular space (R(LIS)), and increased phosphorylation of NMM-II-B on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, a casein kinase-II (CK2) inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole (DRB) did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the R(LIS) was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in vivo with Y-27632, okadaic acid, or DRB. CONCLUSIONS: The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the R(LIS) and increase the paracellular permeability.
OBJECTIVE: To understand myosin regulation of epithelial permeability. METHODS: This was an experimental study, using human cervical epithelial cells CaSki. End points were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B-actin interaction (determined in vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in vitro using purified NMM-II-B filaments in terms of filaments disassembly/assembly ratios. RESULTS: Treatment of cells with the Rho-associated kinase (ROCK) inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the resistance of the lateral intercellular space (R(LIS)), and increased phosphorylation of NMM-II-B on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, a casein kinase-II (CK2) inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole (DRB) did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the R(LIS) was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in vivo with Y-27632, okadaic acid, or DRB. CONCLUSIONS: The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the R(LIS) and increase the paracellular permeability.
Authors: G I Gorodeski; D Merlin; B J De Santis; K A Frieden; U Hopfer; R L Eckert; W H Utian; M F Romero Journal: J Soc Gynecol Investig Date: 1994 Jul-Sep
Authors: M Uehata; T Ishizaki; H Satoh; T Ono; T Kawahara; T Morishita; H Tamakawa; K Yamagami; J Inui; M Maekawa; S Narumiya Journal: Nature Date: 1997-10-30 Impact factor: 49.962
Authors: G Hecht; L Pestic; G Nikcevic; A Koutsouris; J Tripuraneni; D D Lorimer; G Nowak; V Guerriero; E L Elson; P D Lanerolle Journal: Am J Physiol Date: 1996-11