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

Abstract

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.

Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity

“…So far, no sufficient evidence has been documented suggesting that V-ATPase is directly related to metastasis. Our laboratory's previous studies showed that human melanoma cells with different metastatic potential have distinct pH cyt regulatory mechanisms (27). Furthermore, there is a positive correlation between metastatic potential and the level of V-ATPase activity at the plasma membrane (26), suggesting that V-ATPases provide the acidic extracellular environment necessary for invasion.…”

“…The ability to upregulate proton extrusion may be essential for tumor cell survival. The influence of pH cyt on many cellular functions has been studied with respect to cell growth (16), cell motility (27), tumorigenesis (36), metastasis (45), apoptosis (14), and drug resistance in cancer cells (28,50).…”

“…V-ATPase is functionally expressed in plasma membranes of human tumor cells and may have specialized functions in cell growth, differentiation, angiogenesis, and metastasis (25)(26)(27). Furthermore, pH cyt is critical for the cytotoxicity of anticancer agents, and V-ATPase has been implicated in the acquisition of the multidrug resistance phenotype (20,28,40).…”

“…The J H ϩ in these cases were significantly inhibited by the V-ATPase inhibitors concanamycin and bafilomycin. These earlier experiments were performed at pH ex 8.0 because our laboratory's previous studies in other tumor cell types showed a maximal proton pumping activity at an alkaline pH ex (26,27,29). To further evaluate the physiological significance of pmV-ATPase, we performed experiments at "cell culture pH ex ϭ 7.4" in both highly and lowly metastatic cells.…”

Angiostatin Generation by Cathepsin D Secreted by Human Prostate Carcinoma Cells

“…However, recent evidence has indicated that the proton pump of the vacuolar H ϩ -ATPase at the plasma membrane of cancer cells contributes to the formation and maintenance of acidic pH in the extracellular space of tumors (37). This enzyme normally resides in acidic organelles such as endosomes and lysosomes and contributes to the maintenance of acidic environments.…”