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

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.

H2O2 Enhances Ca2+ Release from Osteoblast Internal Stores

The physiological activity of osteoblasts 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 toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.

Effects of Na+ and Ca2+ concentration in cardioplegic and reperfusion solutions on the intracellular Ca2+ of cardiac muscle cells

The removal of Ca2+ from the cardioplegic solutions could cause the danger of inducing a "calcium paradox" during reperfusion. Since intracellular Ca2+ activities are coupled to Na+ activities via Na(+)-Ca2+ exchange, an increase in intracellular Na+ activities during the cardioplegia could cause an abrupt Ca2+ influx when reperfused. To study the effects of Na+ and Ca2+ concentrations in cardioplegic solutions on intracellular Ca2+ activities during the cardioplegia and subsequent recovery period, the membrane potential and intracellular Na+ and Ca2+ activities of guinea pig ventricular papillary were measured. 1) A cardioplegia with low Ca2+ cardioplegic solution significantly decreased the overshoot and duration of the first action potential after cardioplegia, but the changes in action potential configuration were minimized after a cardioplegia with Ca2+ concentration adjusted according to the Na(+)-Ca2+ exchange mechanism. 2) Intracellular Na+ activity was continuously decreased during the cardioplegia, and the intracellular Na+ activity 20 minutes after cardioplegia was the highest with low Ca2+ cardioplegic solution. 3) Intracellular Na+ and Ca2+ activities were continuously decreased during the cardioplegia with Ca2+ concentration adjusted according to the Na(+)-Ca2+ exchange mechanism. 4) During a reperfusion of Tyrode solution after cardioplegia intracellular Na+ and Ca2+ activities were increased. Intracellular Ca2+ activity was increased more rapidly than intracellular Na+ activity. 5) The rate of increase in intracellular Ca2+ activity with reperfusion of Tyrode solution was dependent upon intracellular Na+ activity during cardioplegia, in such a way that the higher the intracellular Na+ activity was, the faster the intracellular Ca2+ activity increased. These data suggest that Na(+)-Ca2+ exchange mechanism may play an important role in the regulation of intracellular Ca2+ activity during recovery after cardioplegia.

Effects of Vanadate on Cellular Ca2+ Movements in Guinea Pig Papillary Muscles

The effects of vanadate on cellular Ca2+ movements across the sarcolemma of cardiac muscle cells were investigated by measuring the intracellular and extracellular Ca2+ activities of guinea pig papillary muscle with Ca2+-selective electrodes. During the rest period following a steady-state of 2 contractions per second the extracellular Ca2+ concentration was increased over the basal level within a minute. During the rest period Ca2+ was transported across the sarcolemma into the extracellular space. Vanadate decreased the change in extracellular Ca2+ concentration during the rest period implying that the Ca2+ efflux across the sarcolemma was decreased by vanadate. Vanadate increased intracellular Ca2+ activities significantly (from 1.9 X 10(-7) M to 10(-6)M) resulting in an increase in resting tension. These results suggest that vanadate decreases Ca2+ efflux from the cells into the extracellular space by blocking Ca2+ transport across the sarcolemma, possibly blocking the Na+-Ca2+ exchange transport.