Growth Hormone Induced[Ca²⁺]_i- Signaling in Growth Plate Chondrocytes 124

Background: Growth hormone (GH) stimulates proliferation and differtiation of growth cartilage cells both, in vivo and in vitro. However, postreceptor events like change of free ionized intracellular calcium concentration, [Ca²⁺]_i, in chondrocytes after GH stimulation have not been investigated.

Subjects: Cultured single epiphyseal growth plate chondrocytes(60- 80g SD- rats).

Interventions: [Ca²⁺]_i measurements using microfluorometric imageing (dual wavelength excitation technique, Ca²⁺-sensitive fluorescence dye Fura-2 AM); Cell proliferation assessment by [³H]-thymidine incorporation assays.

Results: GH stimulation of chondroctes increased[Ca²⁺]_i from baseline levels of 88 ± 26 nmol/l (mean± SD) during 21 ± 4 minutes to peak levels between 0.5 and 1.2μmol/l, reaching baseline levels again after 16.5 ± 4 minutes(Δ = 0.3 ± 0.15 nmol/l/s for GH 40 ng/ml; variation of baseline±18%; n=20). Occasionally [Ca²⁺]_i oscillations were observed in response to GH stimuli. The [Ca²⁺]_i-increase was inhibited by the Ca²⁺ channel blocker I-Verapamil (10⁶ M) and EGTA buffered Ca²⁺-free superfusion. Addional cell culture experiments showed dependence of GH stimulated chondrocyte proliferation on medium Ca²⁺- concentration with maximal [³H]-thymidine incorporation at 1.2 nM Ca²⁺. Coincubation with Verapamil (10⁶ M) decreased GH stimulated DNA synthesis significantly.

Conclusion: A transient rise in [Ca²⁺]_i caused by transmembranous influx of Ca²⁺ via L-type Ca²⁺ channels represents a GH induced signal transduction event in growth plate chondrocytes with a possible physiological role in cell proliferation and growth.