Korean J Anesthesiol.  2003 Apr;44(4):544-554. 10.4097/kjae.2003.44.4.544.

Effects of Reactive Oxygen Species on ATP-induced intracellular Ca2+ Activity in Osteoblasts

• Kim JH
• Kwon TD
• Nam SH
• Suh CK
• Hong YW

• KMID: 1860817
• DOI: http://doi.org/10.4097/kjae.2003.44.4.544

Abstract

BACKGORUND: The physiological activity of osteoblsts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+ ]i) in osteoblasts. The cellular regulation of ([Ca2+ ]i) in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or as toxins in cells.
METHODS
Osteoblasts were isolated from the femurs and tibias of neonatal Sprague-Dawley rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+ -sensitive fluorescent dye, Fura-2 AM ester, and fluorescence images were monitored using a cooled CCD camera. Ca-spike changes upon ATP application were checked for (1) osteoblasts in Ca2+ -free and 2.5 mM CaCl2 normal Tyrode solution, (2) osteoblasts in which the Ca2+ of the endoplastic reticulumin had been depleted with ryanodine, thapsigargin ord caffein, and (3) osteoblasts pretreated with H2O2, in which the expression of iP3 receptor was checked by Western blotting.
RESULTS
ATP increased intracellular free Ca2+ regardless of extracellular Ca2+ concentration. When the intracellular Ca2+ store was depleted, the level of increased Ca2+ activity by ATP was suppressed. H2O2 sustained the Ca2+ increase induced by ATP. The expression of iP3 receptor was enhanced by H2O2.
CONCLUSiONS
H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.

Keyword

ATP; Ca2 activity; H2O2; iP3 receptor; osteoblast

MeSH Terms

Adenosine Triphosphate
Blotting, Western
Calcium-Transporting ATPases
Femur
Fluorescence
Fura-2
Inositol 1,4,5-Trisphosphate Receptors
Osteoblasts*
Rats, Sprague-Dawley
Reactive Oxygen Species*
Ryanodine
Thapsigargin
Tibia
Adenosine Triphosphate
Calcium-Transporting ATPases
Fura-2
Inositol 1,4,5-Trisphosphate Receptors
Reactive Oxygen Species
Ryanodine
Thapsigargin