Desensitization of rat osteoblast-like cells (ROS 17/2.8) to parathyroid hormone uncouples the adenosine 3',5'-monophosphate and cytosolic ionized calcium response limbs.
We have investigated the effects of PTH-induced 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-(1-34) [rPTH-(1-34); 10(-8) M for 48 h, then 10(-7) M for 24 h] dramatically reduced (by 85%) the cAMP response to fresh challenge [2 min; 10(-9)-10(-7) M rPTH-(1-34)], 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 (greater than 80% reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (less than 35% reduction). Treated cells also had a blunted [Ca2+]i response to PTH in the presence of low extracellular calcium (greater than 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 (greater than 50%). Low extracellular Ca2+ had no influence on the peak [Ca2+]i responses of treated cells to bradykinin or prostaglandin F2 alpha. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-maximum [Ca2+]i concentration was significantly increased (greater than 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 F2 alpha. Thus, 1) the cAMP and [Ca2+]i responses of ROS 17/2.8 cells to rPTH-(1-34) 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-(1-34) 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.
Bidwell
J P
JP
Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905.
Fryer
M J
MJ
Firek
A F
AF
Donahue
H J
HJ
Heath
H
H
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D007477:Ions; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology; D051381:Rats; D015290:Second Messenger Systems; D019379:Teriparatide
D007477:Ions; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D019379:Teriparatide; D003994:Bucladesine; D000242:Cyclic AMP; D002118:Calcium