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The role of intracellular pH in cell growth arrest induced by ATP
Sandrine Humez1, Michaël Monet, Fabien van Coppenolle
1Laboratoire de Physiologie Cellulaire, INSERM EMI 0228, Université des Sciences et Technologies de Lille, Bât. SN3, 59655 Villeneuve d'Ascq Cedex, France. sandrine.humez@univ-lille1.fr
Abstract:
In this study, we investigated ionic mechanisms involved in growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells. Extracellular ATP reversibly induced a rapid and sustained intracellular pH (pH(i)) decrease from 7.41 to 7.11. Inhibition of Ca(2+) influx, lowering extracellular Ca(2+), and buffering cytoplasmic Ca(2+) inhibited ATP-induced acidification, thereby demonstrating that acidification is a consequence of Ca(2+) entry. We show that ATP induced reuptake of Ca(2+) by the mitochondria and a transient depolarization of the inner mitochondrial membrane. ATP-induced acidification was reduced after the dissipation of the mitochondrial proton gradient by rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, after inhibition of Ca(2+) uptake into the mitochondria by ruthenium red, and after inhibition of the F(0)F(1)-ATPase with oligomycin. ATP-induced acidification was not induced by either stimulation of the Cl(-)/HCO(3)(-) exchanger or inhibition of the Na(+)/H(+) exchanger. In addition, intracellular acidification, induced by an ammonium prepulse method, reduced the amount of releasable Ca(2+) from the endoplasmic reticulum, assessed by measuring change in cytosolic Ca(2+) induced by thapsigargin or ATP in a Ca(2+)-free medium. This latter finding reveals cross talk between pH(i) and Ca(2+) homeostasis in which the Ca(2+)-induced intracellular acidification can in turn regulate the amount of Ca(2+) that can be released from the endoplasmic reticulum. Furthermore, pH(i) decrease was capable of reducing cell growth. Taken together, our results suggest that ATP-induced acidification in DU-145 cells results from specific effect of mitochondrial function and is one of the major mechanisms leading to growth arrest induced by ATP.
Insights
Extracellular ATP causes prostate cancer cell growth arrest by decreasing intracellular pH (pH(i)). This acidification results from calcium influx and mitochondrial activity, impacting cell growth.
Area of Science:
- Cell Biology
- Cancer Research
- Biochemistry
Background:
- Androgen-independent prostate cancer (PCa) poses a significant therapeutic challenge.
- Extracellular ATP is implicated in various cellular processes, including cancer cell behavior.
- Understanding the ionic mechanisms underlying PCa growth arrest is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the ionic mechanisms driving growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells.
- To elucidate the role of intracellular pH (pH(i)) and calcium (Ca(2+)) homeostasis in ATP-mediated growth inhibition.
- To determine the contribution of mitochondrial function to ATP-induced cellular responses.
Main Methods:
- Measurement of intracellular pH (pH(i)) using standard techniques.
- Manipulation of extracellular and intracellular Ca(2+) levels.
- Assessment of mitochondrial membrane potential and Ca(2+) uptake.
- Pharmacological inhibition of ion transporters and mitochondrial enzymes (rotenone, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, ruthenium red, oligomycin).
- Induction of intracellular acidification using an ammonium prepulse method.
- Measurement of Ca(2+) release from the endoplasmic reticulum.
- Cell growth assays.
Main Results:
- Extracellular ATP induced a rapid and sustained decrease in pH(i) from 7.41 to 7.11.
- ATP-induced acidification was dependent on Ca(2+) influx and subsequent mitochondrial Ca(2+) reuptake.
- Mitochondrial proton gradient dissipation and F(0)F(1)-ATPase inhibition reduced ATP-induced acidification.
- Intracellular acidification reduced releasable Ca(2+) from the endoplasmic reticulum, indicating cross-talk between pH(i) and Ca(2+) homeostasis.
- A decrease in pH(i) was directly correlated with reduced cell growth.
Conclusions:
- Extracellular ATP-induced growth arrest in androgen-independent prostate cancer cells is mediated by intracellular acidification.
- This acidification is primarily driven by Ca(2+) influx and subsequent mitochondrial dysfunction.
- The interplay between pH(i) and Ca(2+) homeostasis is critical for regulating cancer cell proliferation.
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