1.MLCK and PKC Involvements via Gi and Rho A Protein in Contraction by the Electrical Field Stimulation in Feline Esophageal Smooth Muscle.
Sun Young PARK ; Jae Ho SHIM ; Mina KIM ; Yih Hsiu SUN ; Hyun Soo KWAK ; Xiangmei YAN ; Byung Chul CHOI ; Chaeuk IM ; Sang Soo SIM ; Ji Hoon JEONG ; In Kyeom KIM ; Young Sil MIN ; Uy Dong SOHN
The Korean Journal of Physiology and Pharmacology 2010;14(1):29-35
We have shown that myosin light chain kinase (MLCK) was required for the off-contraction in response to the electrical field stimulation (EFS) of feline esophageal smooth muscle. In this study, we investigated whether protein kinase C (PKC) may require the on-contraction in response to EFS using feline esophageal smooth muscle. The contractions were recorded using an isometric force transducer. On-contraction occurred in the presence of NG-nitro-L-arginine methyl ester (L-NAME), suggesting that nitric oxide acts as an inhibitory mediator in smooth muscle. The excitatory composition of both contractions was cholinergic dependent which was blocked by tetrodotoxin or atropine. The on-contraction was abolished in Ca2+-free buffer but reappeared in normal Ca2+-containing buffer indicating that the contraction was Ca2+ dependent. 4-aminopyridine (4-AP), voltage-dependent K+ channel blocker, significantly enhanced on-contraction. Aluminum fluoride (a G-protein activator) increased on-contraction. Pertussis toxin (a Gi inactivator) and C3 exoenzyme (a rhoA inactivator) significantly decreased on-contraction suggesting that Gi or rhoA protein may be related with Ca2+ and K+ channel. ML-9, a MLCK inhibitor, significantly inhibited on-contraction, and chelerythrine (PKC inhibitor) affected on the contraction. These results suggest that endogenous cholinergic contractions activated directly by low-frequency EFS may be mediated by Ca2+, and G proteins, such as Gi and rhoA, which resulted in the activation of MLCK, and PKC to produce the contraction in feline distal esophageal smooth muscle.
4-Aminopyridine
;
Aluminum
;
Aluminum Compounds
;
Atropine
;
Azepines
;
Benzophenanthridines
;
Contracts
;
Esophagus
;
Fluorides
;
GTP-Binding Proteins
;
Muscle, Smooth
;
Myosin-Light-Chain Kinase
;
NG-Nitroarginine Methyl Ester
;
Nitric Oxide
;
Pertussis Toxin
;
Protein Kinase C
;
rhoA GTP-Binding Protein
;
Tetrodotoxin
;
Transducers
2.Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity
Chih-Hsin LIN ; Yu-Shao HSIEH ; Ying-Chieh SUN ; Wun-Han HUANG ; Shu-Ling CHEN ; Zheng-Kui WENG ; Te-Hsien LIN ; Yih-Ru WU ; Kuo-Hsuan CHANG ; Hei-Jen HUANG ; Guan-Chiun LEE ; Hsiu Mei HSIEH-LI ; Guey-Jen LEE-CHEN
Biomolecules & Therapeutics 2023;31(1):127-138
Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer’s disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine’s screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.