Carbachol-activated calcium entry into HT-29 cells is regulated by both membrane potential and cell volume.
Intracellular Ca2+ ([Ca2+]i) was measured in single Cl(-)-secretory HT-29/B6 colonic carcinoma cells with the Ca2+ probe fura-2 and digital imaging microscopy. Resting [Ca2+]i was 63 +/- 3 nM (n = 62). During treatment with the muscarinic agonist carbachol, [Ca2+]i rapidly increased to 901 +/- 119 nM and subsequently reached a stable level of 309 +/- 23 nM, which depended on Ca2+ entry into the cells from the extracellular solution. The goal of this study was to characterize the Ca2+ entry pathway across the cell membrane with respect to its dependence on membrane potential and cell volume. Under resting conditions [Ca2+]i showed no apparent dependence on either potential or cell volume. After stimulating Ca2+ entry with carbachol (100 microM), [Ca2+]i increased with hyperpolarization (low-K+ or valinomycin treatment) and decreased with depolarization (high-K+ or gramicidin treatment) of the cell, as expected from changes in driving force for Ca2+ entry. In stimulated cells, hypotonic solutions caused [Ca2+]i to increase, whereas hypertonic solutions blocked Ca2+ entry. The shrinkage-induced decreases in [Ca2+]i were only slightly affected when the membrane potential was increased with valinomycin, suggesting that shrinkage directly affects the carbachol-activated Ca2+ conductance. In contrast, the swelling-induced increase in [Ca2+]i was significantly reduced in valinomycin-treated cells, suggesting an indirect dependence on a swelling-activated K+ conductance. Thus, carbachol-stimulated Ca2+ entry is under the dual control of membrane potential and cell volume. This mechanism may serve as a regulatory influence that determines the extent of Ca2+ influx during cholinergic stimulation.
Fischer
H
H
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Illek
B
B
Negulescu
P A
PA
Clauss
W
W
Machen
T E
TE
D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D018118:Chloride Channels; D004553:Electric Conductivity; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000502:physiology; D011188:Potassium / Q000502:physiology; D015221:Potassium Channels / Q000502:physiology; D014407:Tumor Cells, Cultured; D014634:Valinomycin / Q000494:pharmacology; D014882:Water-Electrolyte Balance
D015220:Calcium Channels; D018118:Chloride Channels; D008565:Membrane Proteins; D015221:Potassium Channels; D014634:Valinomycin; D002217:Carbachol; D011188:Potassium; D002118:Calcium