We showed recently that the initial peak cyto-solic ionized calcium ([Ca2+]i) response to PTH (2-min exposure) is preserved relative to the cAMP response in osteoblast-like rat osteosarcoma cells (ROS 17/2.8) desensitized by 72-h exposure to PTH. We attempted in the present studies to determine the mechanisms for preservation of the [Ca2+]iresponse and to explore the effects of longer PTH rechallenges. The [Ca2+]iresponse to a 20-min perifusion with rat PTH [rPTH-(l-34)] was monitored by aequorin luminescence in both naive and PTH-desensitized ROS 17/2.8 cells. The responses of both naive and desensitized cells consisted of two phases: An initial peak, followed by an intermediate plateau that was sustained in the presence of PTH. We observed in the naive cell populations synchronous oscillations in [Ca2+]iconcentration during this second phase (amplitude, 10-60 nM; frequency, 1-3/100 sec). These oscillations were maintained through extracellular calcium (EC Ca2+) entry; the initial peak was the result of Ca2+release from intracellular stores. In desensitized cells, these two phases could not be clearly separated with respect to Ca2+source, but, as we showed before, exhibited an enhanced dependence on EC Ca2+entry for the response to PTH. Nevertheless, in the desensitized cells, the sustained [Ca2+]iresponse was diminished in magnitude and showed little oscillatory behavior. Brief exposure to neomycin sulfate, an inhibitor of phosphoinositide turnover, attenuated the PTH-induced [Ca2+]irise in both naive and desensitized cells. Protein kinase-C activity did not appear to be required for either phase of the PTH-induced [Ca2+]i response. Exposure to cholera toxin attenuated the [Ca2+]iresponse to hormone in both naive and desensitized cells, more markedly in the latter. Cholera toxin treatment dramatically increased basal cAMP levels in both cell preparations; PTH-stimulated cAMP production was unchanged in naive cells, but increased nearly 4-fold in desensitized cells. We propose that the preserved PTH-induced peak [Ca2+]irise in desensitized cells results primarily from the diminished regulation of EC Ca2+entry by the cAMP response limb. The attenuated sustained oscillatory behavior observed in desensitized cells upon rechallenge with hormone may be the result of reduced phosphoinositide turnover and reduced Ca2+-stimulated Ca2+release. Thus, the [Ca2+]iresponse to PTH in osteoblast-like cells is complex and modulable and seems to provide a number of ways to regulate intracellular metabolism under various conditions. We speculate that this plasticity of the [Ca2+]iresponse to PTH is related to the pleiotropic actions of the hormone on cells of the osteoblast lineage.
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