Abstract
In previous studies we have characterised various properties of capacitative Ca 2+ entry (CCE) in different epithelia. After Ca 2+ store depletion with PLC/InsP 3-coupled agonists or by inhibition of store Ca 2+ uptake, with for example thapsigargin, Ca 2+ influx is activated. This leads to a sustained cellular response (e.g. NaCl secretion). In the present study, we have investigated CCE in polarised MDCK-C7 cells grown on permeable supports in a chamber allowing for separate luminal and basolateral perfusion. The transepithelial resistance (R te) and voltage (V te) were measured simultaneously to verify the tightness of the epithelia] monolayers. MDCK-C7 cells grew to very tight monolayers (R te > 3000 Ω.cm 2). Apical ATP (100 μmol/I) led to a biphasic [Ca 2+] i increase. Removal of apical Ca 2+ in the continuous presence of ATP did not reduce the stimulated plateau. However, removal of Ca 2+ from the basolateral side rapidly and completely interrupted the [Ca 2+] i plateau to below basal values ([Ca 2+] i decrease during plateau phase after removal of basolateral Ca 2+ = 213 ± 15 nmol/I, n = 9). Furthermore, MDCK-C7 responded to basolateral ATP (100 μmol/I) with a biphasic [Ca 2+] i transient. Again the plateau phase of the ATP-induced [Ca 2+] i effect was fully dependent on the presence of basolateral but not apical Ca 2+ ([Ca 2+] i decrease during plateau phase after removal of basolateral Ca 2+ = 196 ± 5 nmol/I, n = 10). Receptor-independent depletion of cytosolic Ca 2+ stores with thapsigargin from both sides led to a rise in [Ca 2+] i, which was also exclusively dependent on the presence of basolateral Ca 2+ ( n = 8). These data indicate that MDCK-C7 cells express luminal and basolateral P2-receptors coupled to PLC/InsP 3/Ca 2+. ATP applied from both sides induced a sustained [Ca 2+] i plateau which was due to transmembrane Ca 2+ influx. The ATP- and thapsigargin-induced Ca 2+ influx pathway was exclusively located in the basolateral membrane.
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