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

Joseph Bidwell, Michael J. Fryer, Anthony F. Firek, Henry J. Donahue, Hunter Heath

Research output: Contribution to journalArticle

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Abstract

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 ([Ca-2+]i) concentrations were measued 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 (>80% reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (2+]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 F. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-medium [Ca2+]i concentration was significantly increased (>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 F. 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.

Original languageEnglish (US)
Pages (from-to)1021-1028
Number of pages8
JournalEndocrinology
Volume128
Issue number2
StatePublished - Feb 1991
Externally publishedYes

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Parathyroid Hormone
Osteoblasts
Adenosine
Extremities
Calcium
Dinoprost
Bradykinin
Luminescent Proteins
Aequorin
Second Messenger Systems
Osteosarcoma
Adenylyl Cyclases
Hormones
Ions
Cell Line

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

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. / Bidwell, Joseph; Fryer, Michael J.; Firek, Anthony F.; Donahue, Henry J.; Heath, Hunter.

In: Endocrinology, Vol. 128, No. 2, 02.1991, p. 1021-1028.

Research output: Contribution to journalArticle

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title = "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",
abstract = "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 ([Ca-2+]i) concentrations were measued 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 (>80{\%} reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (2+]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 F2α. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-medium [Ca2+]i concentration was significantly increased (>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 F2α. 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.",
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N2 - 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 ([Ca-2+]i) concentrations were measued 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 (>80% reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (2+]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 F2α. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-medium [Ca2+]i concentration was significantly increased (>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 F2α. 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.

AB - 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 ([Ca-2+]i) concentrations were measued 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 (>80% reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (2+]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 F2α. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-medium [Ca2+]i concentration was significantly increased (>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 F2α. 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.

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