Membrane depolarization in NRK fibroblasts by bradykinin is mediated by a calcium-dependent chloride conductance

Abstract

The effects of the phosphoinositide-mobilizing agonist bradykinin (BK) on membrane potential and intracellular calcium in monolayers of normal rat kidney (NRK) fibroblasts were investigated. BK induced a rapid transient depolarization in these cells, which was mimicked by other phosphoinositide-mobilizing factors such as prostaglandin F2alpha (PGF2alpha), lysophosphatidic acid (LPA), platelet-derived growth factor (PDGF-BB), and serum. Depolarization by BK was independent of extracellular Ca2+ or Na+. It was shown using extracellular Cl- substitutions that the depolarization was caused by an increased Cl- conductance. Depolarization was inhibited by 5-nitro-2-3-phenylpropyl(amino)benzoic acid (NPPB), niflumic acid, and flufenamic acid, inhibitors of calcium-dependent chloride channels. The depolarization provoked by BK could be mimicked by raising intracellular calcium with ionomycin or thapsigargin and could be blocked with geneticin, a blocker of phospholipase C. When intracellular calcium was buffered by loading the cells with 1,2-bis(2-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA), depolarization was prevented. We conclude that in NRK fibroblasts extracellular stimuli that increase intracellular calcium, depolarize the cells via the activation of a calcium-dependent chloride conductance. In addition to an increase in intracellular calcium, depolarization may be an important effector pathway in response to extracellular stimuli in fibroblasts. It is hypothesized that, in electrically coupled cells such as NRK fibroblasts, intercellular transmission of these depolarizations may represent a mechanism to coordinate uniform multicellular responses to Ca2+-mobilizing agonists.

Synchronized calcium spiking resulting from spontaneous calcium action potentials in monolayers of NRK fibroblasts

“…This indicates the involvement of a Ca2h-activated chloride conductance. Recently, we showed that BK depolarises NRK cells to -15 mV by a Ca2+-activated chloride conductance [25], which is in agreement with the results obtained here. Thus, the influx of Ca2+, concomitant with every Ca2+ action potential, activates a Ca2+-dependent chloride conductance, which prolongs the action potential.…”

“…This result indicates that gap junction-mediated intercellular communication plays an important role in spontaneous Ca2+ spiking of the densityarrested cells, although an aspecific effect of octanol on other membrane proteins such as ion channels cannot be excluded. Figure 5B shows that 10 nM bradykinin, which causes a long-lasting depolarisation (up to 15 min; [25]), also blocked the repetitive Ca2+ spikes. In addition, spikes were blocked by depolarisation of the cells with 60 mM K+ (results not shown).…”

Prostaglandin F2α induces unsynchronized intracellular calcium oscillations in monolayers of gap junctionally coupled NRK fibroblasts

“…Previous studies have shown that the arachidonic acid metabolite prostaglandin F2a (PGF2a), which activates the Fprostanoid (FP)-subtype of prostanoid receptor [35], induces phosphoinositide turnover in NRK fibroblasts thereby increasing the intracellular calcium concentration in these cells [26]. In confluent NRK cell monolayers, this is accompanied by cell membrane depolarization, due to opening of calcium-activated chloride channels [10], thus reflecting the increase in the intracellular calcium concentration following stimulation with PGF2a.…”

Autocrine production of prostaglandin F2α enhances phenotypic transformation of normal rat kidney fibroblasts

“…In contrast to quiescent monolayers, which have a stable, low membrane potential, monolayers of density-arrested NRK fibroblasts repetitively fire action potentials (8,9). Such action potentials are accompanied by transient increases in intracellular Ca 2ϩ levels.…”