4:24 Human annulus cell response to vibratory loading and modulation of the effect by interleukin-1B

Purpose of study: Chronic exposure to whole body vibration correlates with increased risk of lumbar degenerative disorders. Our laboratory has demonstrated rabbit annulus cells respond to vibratory stimulation with an influx of extracellular calcium through stretch-activated Ca2+ channels. The objective of this study is to determine if human annular cells react similarly to vibratory loading, and what affect the presence of an inflammatory mediator interleukin (IL)-1B, has on the response. Methods used: Annulus tissue was obtained from subjects having surgery for herniated nucleus pulposus. Cells were isolated by means of collagenase digestion and cultured. Cultures were loaded with a Ca2+ sensitive fluorescent dye and fixed to a vibration jig. A computer-operated microscope was used to determine intracellular calcium concentrations ([Ca2+]ic). Vibration trials were for 15, 30 45 or 60 seconds. Ten mM ATP was used as a positive control. Separate cultures were vibrated in Ca2+ free EBSS to determine the need for extracellular calcium. Additional cultures were pretreated with 10 mM ATP, a purinoceptor agonist, or either 10 mM verapamil or 10 mM gadolinium, Ca2+ channel antagonists. All of the vibration trials were then repeated after incubation in 1 nM IL-1B. Data were analyzed with one-way analysis of variance and paired Student t test. of findings: Baseline [Ca2+]ic was 185 nM. Increases in [Ca2+]ic were seen after the 30- and 45-second vibration trials (p<.01). No change in [Ca2+]ic was seen after 15- or 60-second trials. Trials in Ca2+ free EBSS demonstrated no change in [Ca2+]ic with vibration but maintained response to ATP. Pretreatment with 10 uM ATP inhibited the response to vibration. Incubation of the culture in 10 mM verapamil, a slow Ca2+ channel antagonist, did not inhibit the response to vibration or ATP. Incubation in 10 mm gadolinium, a stretch-activated Ca2+ channel antagonist, inhibited the response to vibration but not the response to ATP. Vibration trials after IL-1B incubation demonstrated increased [Ca2+]ic at all vibration intervals (p<.01). In addition, the response in each trial was significantly greater than the corresponding trials not pretreated with IL-1B (p<.05). Again, gadolinium, but not verapamil, blocked the response to vibration in the IL-1B groups. Relationship between findings and existing knowledge: This study demonstrates human annulus fibrosus cells respond to vibratory stimuli with increases in [Ca2+]ic by means of an influx of extracellular calcium through stretch-activated Ca2+ channels. Exposure to ATP results in an increase in [Ca2+]ic by means of the release of intracellular Ca2+ stores. This suggests that vibration exerts a direct cellular effect on annulus cells with the activation of membrane Ca2+ channels. Activation of annulus cell surface purinoceptors by ATP inhibits the mechanisms triggered by vibration. However, the trials performed after pretreatment with the inflammatory mediator IL-1B clearly demonstrate that inflammation exaggerates the response of annulus cells to vibratory load. Overall significance of findings: These results suggest mechanisms involving intracellular calcium are involved in the response of intervertebral disc tissues to vibration, and extracellular purines and inflammatory mediators modulate the signaling cascade. As the mechanism of increasing intracellular calcium is different for ATP and vibration, this does not simply represent a depletion of calcium stores. We are currently working to delineate the downstream effects of this signaling mechanism and if purinoceptor and inflammatory modulation is protective or deleterious. Disclosures: No disclosures. Conflict of interest: Anthony Russo, grant research support. CREF 99-016NIH-AR38121.