Distinct regulation of pHin and [Ca2+]in in human melanoma cells with different metastatic potential

J Cell Physiol. 1998 Jul;176(1):196-205. doi: 10.1002/(SICI)1097-4652(199807)176:1<196::AID-JCP21>3.0.CO;2-4.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Ammonium Chloride / pharmacology
  • Anti-Bacterial Agents / pharmacology
  • Benzopyrans
  • Calcimycin / analogs & derivatives
  • Calcimycin / pharmacology
  • Calcium / metabolism*
  • Calcium-Transporting ATPases / antagonists & inhibitors
  • Enzyme Inhibitors / pharmacology
  • Fluorescent Dyes / metabolism
  • Fura-2 / metabolism
  • Homeostasis / physiology
  • Humans
  • Hydrogen-Ion Concentration
  • Indoles / pharmacology
  • Ionophores / pharmacology
  • Macrolides*
  • Melanoma / metabolism*
  • Naphthols / metabolism
  • Neoplasm Metastasis / physiopathology*
  • Proton-Translocating ATPases / antagonists & inhibitors
  • Rhodamines / metabolism
  • Thapsigargin / pharmacology
  • Tumor Cells, Cultured


  • Anti-Bacterial Agents
  • Benzopyrans
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Indoles
  • Ionophores
  • Macrolides
  • Naphthols
  • Rhodamines
  • seminaphthorhodaminefluoride
  • Ammonium Chloride
  • bafilomycin A
  • Calcimycin
  • Thapsigargin
  • 4-bromo-A-23187
  • Proton-Translocating ATPases
  • Calcium-Transporting ATPases
  • Calcium
  • Fura-2
  • cyclopiazonic acid