Effects of Calcium on Gap Junctions Between Osteoblast-Like Cells in Culture

Abstract.

The present study investigated the effects of elevated cytoplasmic free calcium concentrations ([Ca²⁺]_i) on the permeability of gap junctions between cultured osteoblast-like (OB) cells derived from calvarial and periosteal fragments of newborn rats. This was studied using the double whole cell patch clamp technique and intracellular dye injections. To increase [Ca²⁺]_i, patch pipette solutions contained 100 μmol/liter Ca²⁺. About 1–2 minutes after whole cell modes had been attained, the total number of gap junction channels was reduced from an average of 400 in normal Ca²⁺ to 20 in high Ca²⁺. Thereafter, remaining gap junction channels were active for up to 8 minutes. In normal rat kidney (NRK) cells, gap junction channels were closed by high Ca²⁺ within 1 minute, pointing to a similar sensitivity of Connexin43 gap junction channels in OB and NRK cells. To study the effects of elevated [Ca²⁺]_i on the dye permeability of gap junctions between extended OB cells, the spread of Lucifer Yellow to neighboring cells was evaluated. [Ca²⁺]_i was gradually increased from 1.5- to 14-fold the normal value by application of either ouabain, Na⁺-free/ouabain, or A23187. Reduced dye spread correlated with the increase of [Ca²⁺]_i measured by analyzing the fluorescence of fura-2. These data show that gap junctions in OB cells are sensitive to Ca²⁺.