Distinct regulation of pHin and [Ca2+]in in human melanoma cells with different metastatic potential.
We investigated whether alterations in the mechanisms involved in intracellular pH (pHin) and intracellular calcium ([Ca2+]in) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pHin and [Ca2+]in simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steady-state pHin and [Ca2+]in between these cell types were not significantly different. Treatment of cells with NH4Cl resulted in larger pHin increases in highly than in poorly metastatic cells, suggesting that C8161 cells have a lower H+ buffering capacity than A375P. NH4Cl treatment also increased [Ca2+]in only in C8161 cells. To determine if the changes in [Ca2+]in triggered by NH4Cl treatment were due to alterations in either H+- or Ca2+-buffering capacity, cells were treated with the Ca2+-ionophore 4Br-A23187, to alter [Ca2+]in. The magnitude of the ionophore-induced [Ca2+]in increase was slightly greater in C8161 cells than in A375P. Moreover, A375P cells recover from the ionophore-induced [Ca2+]in load, whereas C8161 cells did not, suggesting that A375P may exhibit distinct [Ca2+]in regulatory mechanisms than C8161 cells, to recover from Ca2+ loads. Removal of extracellular Ca2+ ([Ca2+]ex) decreased [Ca2+]in in both cell types at the same extent. Ionophore treatment in the absence of [Ca2+]ex transiently increased [Ca2+]in in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca2+-ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady-state [Ca2+]in only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca2+ stores for [Ca2+]in homeostasis is greater in highly than in poorly metastatic cells. Bafilomycin treatment, to inhibit V-type H+-ATPases, corroborated our previous results that V-H+-ATPases are functionally expressed at the plasma membranes of highly metastatic, but not in poorly metastatic cells (Martínez-Zaguilán et al., 1993). Collectively, these data suggest that distinct pHin and [Ca2+]in regulatory mechanisms are present in poorly and highly metastatic human melanoma cells.
Martínez-Zaguilán
R
R
Department of Physiology, Texas Tech University Health Sciences Center, Lubbock 79430, USA. phyrmz@ttuhsc.edu
Martinez
G M
GM
Gomez
A
A
Hendrix
M J
MJ
Gillies
R J
RJ
D000643:Ammonium Chloride / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001578:Benzopyrans; D000001:Calcimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D018942:Macrolides; D008545:Melanoma / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism; D009362:Neoplasm Metastasis / Q000503:physiopathology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D012235:Rhodamines / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000900:Anti-Bacterial Agents; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D007476:Ionophores; D018942:Macrolides; D009284:Naphthols; D012235:Rhodamines; C091666:seminaphthorhodaminefluoride; D000643:Ammonium Chloride; C057620:bafilomycin A; D000001:Calcimycin; D019284:Thapsigargin; C033170:4-bromo-A-23187; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2; C000543:cyclopiazonic acid