Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells

Abstract

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca2+ oscillations from an inositol 1,4,5-triphosphate-sensitive cytoplasmic Ca2+ store. In this study, Ca2+ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca2+ video imaging and the Fura-2/Mn2+ quenching technique. Oscillations were dependent on extracellular Ca2+ concentration and were inhibited by extracellularly applied La3+, Co2+ and Ni2+. Depolarization of the cell membrane with high K+ concentrations and the L-type Ca2+ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca2+ channels. Mn2+ quenching experiments disclosed significant Ca2+ influx into MDCK-F cells. The rate of this influx was constant between Ca2+ spikes, but markedly increased during the spontaneous Ca2+ spikes. Similar transient increases in Ca2+ entry could be mimicked by agents triggering intracellular Ca2+ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca2+ permeability permitting Ca2+ entry into the cytoplasm. The rate of Ca2+ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca2+ concentration.

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system

“…[Ca2+]j could serve as this signal, analogous to its role in th e regulation of solute transport in various epithelia [18,19]. For instance, in intestine and MDCK-F cells, [Ca2+]j has been implicated in the regulation of Ca2+ entry [2,20]. In the present study, reduction of th e basolateral efflux capacity evoked by reversal of the operation of the Na+/Ca2+ exchanger was paralleled by a m arked increase in [Ca2+].. At first sight, this observation is in agreem ent w ith the idea th at an overall increase in [Ca2'^ is responsible for the inhibition of the apical Ca2+ influx.…”

Oscillating activity of a Ca(2+)-sensitive K+ channel. A prerequisite for migration of transformed Madin-Darby canine kidney focus cells.

“…In the present study we looked as selectively as possible at the effect of K+ channels on migration. Therefore, a key experiment was to superfuse the cells with 50 mmol/liter KCI Ringer's solution (24). Ca2+ oscillations are not affected, but migration of MDCK-F cells is inhibited nonetheless.…”

“…Ca2+ oscillations of MDCK-F cells depend on Ca2+ influx from the extracellular space (24). Ca2+ influx is ofgreat importance for migration too (38).…”

“…As shown in Fig. 1 In this way we also tested the effect of the following K+ channel blockers on migration: Ba2+ ( nels are still triggered by oscillations of intracellular Ca2+ (24) there is no K+ net movement. Migration is reduced to 0.15±0.1 um/min (22±13% of control; n = 11 ).…”

Capacitative calcium entry

“…Measurements of entry during spiking, using the Mn2+ quench technique, have given conflicting results. In endothelial cells there was no evidence for oscillations in entry [94] but this was found to occur in synchrony with Ca2+ spikes in AR42J cells [110] and in Mardin-Darby canine kidney-focus cells [111]. The second mechanism is more direct in that the entry mechanism oscillates autonomously to set up periodic fluctuations in cytosolic calcium.…”