We have investigated the effects of PTH-in- duced desensitization on second messenger interactions in the rat osteosarcoma cell line ROS 17/2.8. Adenylate cyclase activation was assessed by accumulation of immunoassayable cAMP, and cytosolic calcium ion ([Ca2+]i) concentrations were measured in adherent perifused cells loaded with the Ca2+- sensitive bioluminescent protein aequorin. Preexposure to rat PTH-U-34) [rPTH-(l-34); 10-8M for 48 h, then 10-7M for 24 h] dramatically reduced (by 85%) the cAMP response to fresh challenge [2 min; 10-9-10-7M rPTH-(l-34)], but the peak PTH- induced rise of [Ca2+]i was not diminished significantly (0-20%). Nevertheless, we did observe other changes in the PTH-induced [Ca2+]i response. Exposure of treated cells to (Bu)2cAMP nearly abolished the [Ca2+]i response to PTH (>80% reduction), but had muchless effect on the PTH-stimulated [Ca2+]i increment of the naive cells (<35% reduction). Treated cells also had a blunted[Ca2+]i response to PTH in the presence of low extracellular calcium (>60% reduction), but in the naive cells, low extracellular Ca2+did not significantly diminish the peak PTH- induced [Ca2+]i rise, although low extracellular Ca2+dramatically reduced the area under this [Ca2+]i transient (>50%). Low extracellular Ca2+had no influence on the peak [Ca2+]i responses of treated cells to bradykinin or prostaglandin F2ɑ. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+medium was not significantly attenuated, the time to halfmaximum [Ca2+]i concentration was significantlyincreased (>100%), and the area under the [Ca2+]i transient was diminished. These alterations in the [Ca2+]i response of treated cells were not observed upon challenge with bradykinin or prostaglandin F2a. Thus, 1) the cAMP and [Ca2+]i responses of ROS 17/2.8 cells to rPTH-(l-34) are not obligatorily coupled; 2) the response of naive cells to PTH includes both the release of Ca2+from intracellular stores and the entry of extracellular Ca2+; and 3) pretreatment of these cells with rPTH-(l-34) augments the dependence on Ca2+entry during hormone rechallenge. We propose that the preserved PTH-stimulated [Ca2+]i rise in treated cells results partly from loss of cAMP-mediated inhibition of extracellular Ca2+entry.
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