Capacitative Ca2+ entry (CCE) induced by luminal and basolateral ATP in polarised MDCK-C7 cells is restricted to the basolateral membrane.
In previous studies we have characterised various properties of capacitative Ca2+ entry (CCE) in different epithelia. After Ca2+ store depletion with PLC/InsP3-coupled agonists or by inhibition of store Ca2+ uptake, with for example thapsigargin, Ca2+ 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 (Rte) and voltage (Vte) were measured simultaneously to verify the tightness of the epithelial monolayers. MDCK-C7 cells grew to very tight monolayers (Rto > 3000 omega.cm2). Apical ATP (100 mumol/l) led to a biphasic [Ca2+]i increase. Removal of apical Ca2+ in the continuous presence of ATP did not reduce the stimulated plateau. However, removal of Ca2+ from the basolateral side rapidly and completely interrupted the [Ca2+]i plateau to below basal values ([Ca2+]i decrease during plateau phase after removal of basolateral Ca2+ = 213 +/- 15 nmol/l, n = 9). Furthermore, MDCK-C7 responded to basolateral ATP (100 mumol/l) with a biphasic [Ca2+]i transient. Again the plateau phase of the ATP-induced [Ca2+]i effect was fully dependent on the presence of basolateral but not apical Ca2+ ([Ca2+]i decrease during plateau phase after removal of basolateral Ca2+ = 196 +/- 5 nmol/l, n = 10). Receptor-independent depletion of cytosolic Ca2+ stores with thapsigargin from both sides led to a rise in [Ca2+]i, which was also exclusively dependent on the presence of basolateral Ca2+ (n = 8). These data indicate that MDCK-C7 cells express luminal and basolateral P2-receptors coupled to PLC/InsP3/Ca2+. ATP applied from both sides induced a sustained [Ca2+]i plateau which was due to transmembrane Ca2+ influx. The ATP- and thapsigargin-induced Ca2+ influx pathway was exclusively located in the basolateral membrane.
Gordjani
N
N
Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Germany.
Nitschke
R
R
Greger
R
R
Leipziger
J
J
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology*; D015194:Diffusion Chambers, Culture; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials; D018048:Receptors, Purinergic P2 / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology
D015220:Calcium Channels; D005456:Fluorescent Dyes; D018048:Receptors, Purinergic P2; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2