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Parathyroid hormone (PTH)-Induced Intracellular Ca²⁺ Signalling in Naive and PTH-Desensitized Osteoblast-Like Cells (ROS 17/2.8): Pharmacological Characterization and Evidence for Synchronous Oscillation of Intracellular Ca^2+*

JOSEPH P. BIDWELL, W. BRADFORD CARTER, MICHAEL J. FRYER and HUNTER HEATH, III

Endocrine Research Unit, Division of Endocrinology, Metabolism, and Internal Medicine, Mayo Clinic Rochester, Minnesota 55905

Address requests for reprints to: Dr. Hunter Heath III, University of Utah Medical Center, 4C216 SOM, 50 N. Medical Drive, Salt Lake City, Utah 84132.

Abstract

We showed recently that the initial peak cytosolic 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+]_i response and to explore the effects of longer PTH rechallenges. The [Ca²+]_i; response 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+]_i concentration during this second phase (amplitude, 10β60 nM; frequency, 1β3/100 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+]i response was diminished in magnitude and showed little oscillatory behavior. Brief exposure to neomycin sulfate, an inhibitor of phosphoinositide turnover, attenuated the PTH-induced [Ca2+]i rise 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+]i response 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; PTHstimulated cAMP production was unchanged in naive cells, but increased nearly 4-fold in desensitized cells. We propose that the preserved PTH-induced peak [Ca2+]j rise 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+]_ii response 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+]_i response to PTH is related to the pleiotropic actions of the hormone on cells of the osteoblast lineage. (Endocrinology 129: 2993β3000,1991)

Footnotes

^* This work was supported in part by grants from the USPHS, NIH (DK-38855 and DK-03752).

Current address: Department of Cell Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655.

Current address: Department of Surgery, Thomas Jefferson University, 1025 Walnut Street, Philadelphia, Pennsylvania 19107.

Received June 12, 1991.

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