ASGSB 1998 Annual Meeting Abstracts

[19]
FUNCTIONAL AND MOLECULAR EXPRESSION OF CALCIUM CHANNELS IN OSTEOBLASTS IN RESPONSE TO MECHANICAL LOADING.   K.D. Ryder¹, J. Bergh², M.C. Farach-Carson² and R.L. Duncan¹. ¹Dept. of Orthopaedic Surgery, Physiology and Biophysics, Indiana University Medical Center, Indianapolis, IN and ²Dept. of Biological Sciences, University of Delaware, Newark, DE.

We previously characterized a mechanosensitive, cation-selective channel (MSCC) in osteoblasts that we have tentatively identified as an alternatively spliced isoform of the _1C subunit of the L-type voltage-sensitive Ca²⁺ channel (VSCC). We postulate that this channel plays an important role in the response of osteoblasts to mechanical stimulation. Patch clamp studies indicate that non-loaded MC3T3-E1 cells exhibit little activation of currents when challenged with a hypotonic solution. However, when MC3T3-E1 cells are subjected to fluid forces induced by a four point bending device, a 7-fold increase in whole cell inward currents is observed. These data correlate with intracellular Ca²⁺ ([Ca²⁺]_i) imaging studies that show that approx. 50% (54/106) of non-loaded MC3T3-E1 cells respond to hypotonic challenge with a peak [Ca²⁺]_i transient of 180nM. Mechanically loaded MC3T3-E1 cells exhibited a more vigorous response (peak [Ca²⁺]_I=240nM) with 88% (67/76) of the cells responding to challenge. To ascertain if the MSCC was involved in this response, 10 µM GdCl₃ (a known MSCC blocker) was added prior to hypotonic challenge. Gd³⁺ totally abolished the [Ca²⁺]_i response to challenge. rt-PCR studies using primers designed to amplify all _1C subunit isoforms of the L-type VSCC demonstrated that non-loaded MC3T3-E1 cells express low levels of this subunit. Yet when cells are mechanically loaded as described above, a 3-5 fold increase in _1C subunit expression is observed within 4 hr after the initiation of loading. These data indicate that loading increases functional and molecular expression of this VSCC isoform which may be integral in the [Ca²⁺]_i response to mechanical stimulation. (Supported by NASA: NAG5-4917, NASA/TMC.)

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