Dihydropyridine-sensitive Ca²⁺ channels in human glomerular mesangial cells

In mesangial cells (MC), the response of intracellular Ca²⁺ concentration ([C²⁺](i)) to a contractile agonist is biphasic with a large, transient increase in [Ca²⁺](i) followed by a smaller but sustained elevation as Ca²⁺ flows into the cell from the extracellular fluid. It has been postulated that membrane depolarization precedes opening of Ca²⁺ channels in the plasmalemmal membrane. However, a role for voltage-gated Ca²⁺ channels (VGCC) in human MC has been controversial, and their existence has not been verified with single-channel analysis. We used fura 2 fluorescence and patch-clamp techniques to determine the properties of the Ca²⁺ entry pathway responsible for the sustained response of [Ca²⁺](i) in human MC. We found that ANG II at 10 nM, 100 nM, and 1 μM increased [Ca²⁺](i) to sustained levels of 22%, 35%, and 49%, respectively, above baseline. The sustained response to 1 μM ANG II was attenuated by diltiazem and was reduced to a value less than baseline in the absence of external Ca²⁺. None of the peak responses (due to release of intracellular stores of Ca²⁺) were affected by removal of external Ca²⁺ or addition of diltiazem. Upon elevating the extracellular [K⁺] from 5 mM to 75 mM, [Ca²⁺](i) reached a sustained level of 48% greater than baseline. This effect of high K⁺ was attenuated by either Ca²⁺ removal or addition of diltiazem. In the presence of 75 or 140 mM K⁺, the dihydropyridine agonist BAY K 8644 (1 μM and 10 μM) initiated sustained [Ca²⁺](i) responses averaging 18% and 25%, respectively, greater than baseline. With <10 nM Ca²⁺ in the external solution, BAY K 8644 did not significantly affect [Ca²⁺](i). In separate patch-clamp experiments, barium-selective channels were found in cell- attached patches with 90 mM BaCl₂ and 10 μM BAY K 8644 in the pipette solution. The single-channel conductance was 11.2 pS, and the open probability increased steeply at membrane potentials between -30 mV and 0 mV. It is concluded that human glomerular MC contain dihydropyridine-sensitive Ca²⁺ channels responsible for the voltage-regulated entry of Ca²⁺ into the cell during an agonist-induced contraction.