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1. We characterized chloride currents in freshly isolated rabbit osteoclasts using whole‐cell and single channel patch‐clamp recording configurations. Depolarization activated an outwardly rectifying current in 40‐50% of cells, distinct from the inwardly rectifying K+ current we have previously reported in osteoclasts. 2. The outwardly rectifying current persisted under conditions where all K+ currents were blocked. Furthermore, the outward current was reversibly inhibited by Cl‐ transport blockers 4‐acetamido‐4'‐isothiocyanostilbene‐2,2'‐disulphonic acid (SITS); 4,4'‐diisothiocyanostilbene‐2,2'‐disulphonic acid (DIDS); 4,4'‐dinitrostilbene‐2,2'‐disulphonic acid (DNDS); and niflumic acid. The blocked current had a reversal potential close to the predicted chloride equilibrium potential and was dependent on the chloride concentration gradient. 3. In those osteoclasts in which outwardly rectifying current was not initially apparent, exposure to hyposmotic extracellular solution resulted in its reversible activation. The induced current was due to Cl‐, based on its reversal close to the chloride equilibrium potential and sensitivity to blockade by Cl‐ channel inhibitors. The hyposmotically induced current could be activated in Ca(2+)‐free solutions containing 0.2 mM EGTA. 4. When studied in the current‐clamp configuration, hyposmotic stimulation caused depolarization from ‐76 +/‐ 5 to ‐5 +/‐ 6 mV (mean +/‐ S.D., n = 7). 5. Unitary Cl‐ currents were recorded in the cell‐attached patch configuration at positive potentials. Single channels had a slope conductance of 19 +/‐ 3 pS (n = 5). Reduction of the external [Cl‐] shifted the current‐voltage relationship in the positive direction, supporting the conclusion that these were Cl‐ currents. Like the whole‐cell currents, single channel Cl‐ currents were activated by exposure of cells to hyposmotic bathing solution. 6. We conclude that rabbit osteoclasts express an outwardly rectifying Cl‐ current that can be activated by osmotic stress. Cl‐ channels may play a role in cell volume regulation and may also provide conductive pathways for dissipating the potential difference that arises from electrogenic proton transport during bone resorption.

Extracellular nucleotides, through interaction with specific cell-surface receptors, mediate a variety of biological responses, including elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in a number of cell types. The effects of nucleotides on [Ca2+]i in the rat osteoblastic cell line UMR-106 were studied by fluorescence spectrophotometry of indo-1-loaded cells. In response to ATP (100 microM), [Ca2+]i rose to peaks 228 +/- 16 nM (n = 59) above baseline (85 +/- 3 nM) before returning to near basal levels. Half-maximal elevation of [Ca2+]i was observed at an ATP concentration of 3 +/- 1 microM, consistent with a high-affinity interaction. The response arose primarily by release of Ca2+ from internal stores. UTP, ADP, and 2-methylthioadenosine 5'-triphosphate also induced Ca2+ transients, whereas adenosine, AMP, CTP, and TTP did not, demonstrating specificity. Responsiveness to adenosine 5'-O-(3-thiotriphosphate) and inhibition by Mg2+ of the response to ATP indicated that signaling was not dependent on nucleotide hydrolysis. Ca2+ responses to ADP, ATP, and UTP, added sequentially or simultaneously, were consistent with the presence of two distinct P2-purinoceptor subtypes, both linked to Ca2+ mobilization. ADP appeared to interact selectively with one receptor, whereas ATP and UTP interacted selectively with the other. After maximal stimulation with ATP, subsequent responses to ATP were abolished. However, removal of ATP from the extracellular medium rapidly restored responsiveness, suggesting that, with continued receptor occupation, there is time-dependent inactivation of the Ca2+ signaling pathway. Our findings indicate that extracellular nucleotides elevate [Ca2+]i in osteoblastic cells through interaction with two receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Electromagnetic fields have been reported to cause a variety of biological effects. It has been hypothesized that many of these phenomena are mediated by a primary effect on the concentration of cytosolic free calcium ([Ca2+]i). We investigated the effects of exposure to electromagnetic fields on [Ca2+]i in HL-60 cells using the Ca2(+)-sensitive fluorescent indicator indo-1. Indo-1-loaded cell samples were exposed to a radiofrequency electromagnetic field, a static magnetic field, and a time-varying magnetic field, which were generated by a magnetic resonance imaging (MRI) unit. We found that a 23-min exposure to all three fields, in combination, induced a significant increase in [Ca2+]i of 31 +/- 8 (SE) nM (P less than 0.01, n = 13) from a basal level of 121 +/- 8 nM. Also, cells exposed to only the time-varying magnetic field had a mean [Ca2+]i that was 34 +/- 10 nM (P less than 0.01, n = 11) higher than parallel control samples. Separate exposure to the radio-frequency (6.25 MHz) or static field (0.15 T) had no detectable effects. These results demonstrate that time-varying magnetic fields alter [Ca2+]i and suggest that at least some of the reported biological effects of time-varying magnetic fields may arise from elevation of [Ca2+]i.