Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique.
Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca2+ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10(-5) mol/l)-induced intracellular Ca2+ transient, (2) a reduced fura-2 Mn(2+)-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca2+ as a function of membrane voltage, we measured simultaneously the intracellular Ca2+ activity ([Ca2+]i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT29 cells. Ca2+ influx was either stimulated by ATP (10(-5) mol/l) or thapsigargin (TG, 10(-8) mol/l). After [Ca2+]i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca2+]i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca2+]i (ATP: 27.5 +/- 10 nmol/l per 10 mV, n = 6; TG: 19 +/- 7.9 nmol/l per 10 mV, n = 12). The summarised data show a linear relationship between the delta fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca2+]i (n = 9). Under extracellular Ca(2+)-free conditions [Ca2+]i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca2+ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)
Leipziger
J
J
Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Germany.
Fischer
K G
KG
Greger
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000032:analysis*; D015220:Calcium Channels / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D003106:Colon / Q000737:chemistry / Q000166:cytology* / Q000502:physiology; D004558:Electric Stimulation; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000502:physiology; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D009761:Nystatin / Q000494:pharmacology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D015220:Calcium Channels; D013729:Terpenes; D009761:Nystatin; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium