ABSTRACT
BACKGROUND: ASIC1a, the predominant acid-sensing ion channels (ASICs), is implicated in neurological disorders including stroke, traumatic spinal cord injury, and ALS. Potent ASIC1a inhibitors should have promising therapeutic potential for ASIC1a-related diseases. AIMS: We examined the inhibitory effects of a number of amiloride analogs on ASIC1a currents, aimed at understanding the structure-activity relationship and identifying potent ASIC1a inhibitors for stroke intervention. METHODS: Whole-cell patch-clamp techniques and a mouse model of middle cerebral artery occlusion (MCAO)-induced focal ischemia were used. Surflex-Dock was used to dock the analogs into the pocket with default parameters. RESULTS: Amiloride and its analogs inhibit ASIC1a currents expressed in Chinese hamster ovary cells with a potency rank order of benzamil > phenamil > 5-(N,N-dimethyl)amiloride (DMA) > amiloride > 5-(N,N-hexamethylene)amiloride (HMA) ≥ 5-(N-methyl-N-isopropyl)amiloride (MIA) > 5-(N-ethyl-N-isopropyl)amiloride (EIPA). In addition, amiloride and its analogs inhibit ASIC currents in cortical neurons with the same potency rank order. In mice, benzamil and EIPA decreased MCAO-induced infarct volume. Similar to its effect on the ASIC current, benzamil showed a much higher potency than EIPA. CONCLUSION: Addition of a benzyl group to the terminal guanidinyl group resulted in enhanced inhibitory activity on ASIC1a. On the other hand, the bulky groups added to the 5-amino residues slightly decreased the activity. Among the tested amiloride analogs, benzamil is the most potent ASIC1a inhibitor.
Subject(s)
Acid Sensing Ion Channels/physiology , Amiloride/analogs & derivatives , Amiloride/pharmacology , Amiloride/chemistry , Amiloride/therapeutic use , Animals , Biophysics , CHO Cells , Cells, Cultured , Cerebral Cortex/cytology , Cricetinae , Cricetulus , Disease Models, Animal , Dose-Response Relationship, Drug , Electric Stimulation , Embryo, Mammalian , Infarction, Middle Cerebral Artery/drug therapy , Inhibitory Concentration 50 , Male , Membrane Potentials/drug effects , Mice , Mice, Inbred C57BL , Models, Molecular , Neurons/drug effects , Patch-Clamp Techniques , TransfectionABSTRACT
BACKGROUND: Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a dismal prognosis. Despite intensive study on tumor biology, the underlying mechanisms of the unlimited proliferation and progressive local invasion are still poorly understood, and no effective treatment has been developed for GBM patients. AIMS: We determine the role of TRPM7 channels in the growth, migration, and infiltration of malignant glioma cells. METHODS: Using a combination of RT-PCR, Western blot, and patch-clamp techniques, we demonstrated the expression of functional TRPM7 channels of A172 cells, a human glioma cell line, as well as in human glioma tissues. Furthermore, we evaluated the role of TRPM7 in growth, migration, and infiltration of A172 cells with MTT and transwell migration and invasion assays. RESULTS: We showed the expression of functional TRPM7 channels in both A172 cells and human glioma tissues. Suppression of TRPM7 expression with TRPM7-siRNA dramatically reduced the proliferation, migration, and invasion of A172 cells. Pharmacological inhibition of TRPM7 channel with 2-aminoethoxydiphenyl borate (2-APB) showed a similar effect as TRPM7-siRNA. CONCLUSION: We demonstrate that human glioma cells express functional TRPM7 channel and that activation of this channel plays an important role in the proliferation, migration, and invasion of malignant glioma cells. TRPM7 channel may represent a novel and promising target for therapeutic intervention of malignant glioma.
Subject(s)
Brain Neoplasms/physiopathology , Cell Movement/drug effects , Cell Proliferation/drug effects , Glioblastoma/physiopathology , Neoplasm Invasiveness/physiopathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , TRPM Cation Channels/antagonists & inhibitors , Blotting, Western , Boron Compounds/pharmacology , Brain Neoplasms/drug therapy , Cell Line, Tumor , Cell Movement/physiology , Cell Proliferation/physiology , Cells, Cultured , Glioblastoma/drug therapy , Humans , Patch-Clamp Techniques , Polymerase Chain Reaction , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering , TRPM Cation Channels/genetics , TRPM Cation Channels/metabolismABSTRACT
Brain ischemia is a leading cause of death and long-term disabilities worldwide. Unfortunately, current treatment is limited to thrombolysis, which has limited success and a potential side effect of intracerebral hemorrhage. Searching for new cell injury mechanisms and therapeutic interventions has become a major challenge in the field. It has been recognized for many years that intracellular Ca(2+) overload in neurons is essential for neuronal injury associated with brain ischemia. However, the exact pathway(s) underlying the toxic Ca(2+) loading remained elusive. This review discusses the role of two Ca(2+)-permeable cation channels, TRPM7 and acid-sensing channels, in glutamate-independent Ca(2+) toxicity associated with brain ischemia.
