| Abstract |
Mechanical stress is an important regulatory factor in bone homeostasis. Mechanical stimulation of osteoblasts has been shown to elicit an increase in the concentration of intracellular free Ca2+ ([Ca2+]i). The pattern of functional expression of mechanosensitive ion channels remains unclear, however. Therefore, the purpose of this study was to investigate the pharmacological characteristics of [Ca2+]i in response to direct mechanical stimulation in osteoblasts. The morphological expression of mechanosensitive ion channels was also examined. Mouse osteoblast-like cells (MC3T3-E1 cells) were loaded with fura-2-acetoxymethyl ester, after which [Ca2+]i was measured. Increased levels of [Ca2+]i were observed in MC3T3-E1 cells in response to direct mechanical stimulation by means of a glass micropipette, but no desensitization. Application of a hypotonic solution also induced an increase in [Ca2+]i but was accompanied by a desensitizing effect. Extracellular Gd3+, GsMTx4, or RN-1734 reversibly inhibited this mechanical stimulation-induced increase in [Ca2+]i, whereas no inhibitory effect was observed with HC030031 or clemizole. When osteoblasts were stimulated with Yoda1, an increase was observed in [Ca2+]i together with a significant desensitizing effect. Immunoreactivity against Piezo1 and TRPV4 channel antibodies was detected in MC3T3-E1 cells. These results suggest that osteoblasts express Piezo1 and TRPV4 channels, which are involved in mechanosensitive processes during mechanical stress.
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