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Anion exchanger isoform 2 operates in parallel with Na(+)/H(+) exchanger isoform 1 during regulatory volume decrease
Meng Ru Shen1, Robert J Wilkins, Cheng Yang Chou
1University Laboratory of Physiology, Oxford, UK.
Abstract:
Intracellular pH (pH(i)) homeostasis was investigated in human cervical cancer SiHa cells undergoing regulatory volume decrease (RVD) to determine which transport systems were involved. Using isoform-specific primers, mRNA transcripts of Na(+)/H(+) exchanger isoform 1 (NHE1) and isoform 3 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) and the results confirmed by Western immunoblotting. From anion exchanger isoforms 1-3 (AE1-3), only the mRNA transcript of AE2 was identified by RT-PCR and the identity was confirmed by digestion with a specific restriction endonuclease. SiHa cells loaded with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and resuspended in isotonic media showed a stable pH(i). In contrast, a gradual internal acidification took place following resuspension in hypotonic media. The NHE inhibitors, HOE694 (10 microM) and amiloride (1 mM), showed a similar potency in enhancing the rate and extent of the hypotonicity-induced internal acidification. The absence of extracellular Na(+) also substantially enhanced the acidification during RVD. These results suggest that internal acidification during RVD is mainly compensated by the operation of NHE1. Extracellular Cl(-) was critically necessary for the pH(i) acidification during RVD. The hypotonicity-induced acidification was significantly attenuated by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, a concentration inhibiting more than 90% AE activity. This indicates that AE2 mediates a net Cl(-) influx with compensating HCO(3)(-) efflux during RVD. We conclude that AE2 operates in parallel with NHE1 to regulate pH(i) during RVD of human cervical cancer cells.
Insights
Intracellular pH regulation in cervical cancer cells during volume changes involves Na+/H+ exchanger 1 (NHE1) and anion exchanger 2 (AE2). These transporters work together to maintain pH balance in SiHa cells experiencing regulatory volume decrease (RVD).
Area of Science:
- Cell Biology
- Physiology
- Biochemistry
Background:
- Intracellular pH (pH(i)) homeostasis is crucial for cell function.
- Regulatory Volume Decrease (RVD) is a cellular response to osmotic stress.
- Understanding ion transport during RVD in cancer cells is important for disease research.
Purpose of the Study:
- To investigate the role of specific ion transport systems in regulating intracellular pH during RVD in human cervical cancer SiHa cells.
- To identify the key transporters involved in maintaining pH(i) homeostasis under hypotonic conditions.
Main Methods:
- Reverse transcriptase polymerase chain reaction (RT-PCR) to detect mRNA transcripts of Na+/H+ exchanger (NHE) and anion exchanger (AE) isoforms.
- Western immunoblotting to confirm protein expression of NHE1 and AE2.
- Fluorescent dye measurements of intracellular pH changes in SiHa cells subjected to hypotonic stress.
- Pharmacological inhibition of NHE and AE transporters.
Main Results:
- SiHa cells express mRNA for NHE1, NHE3, and AE2.
- Hypotonic stress induced intracellular acidification, which was enhanced by NHE inhibitors and absence of extracellular Na+, indicating NHE1's role in compensation.
- Extracellular chloride was essential for pH(i) acidification during RVD.
- AE2 inhibition significantly attenuated hypotonicity-induced acidification, suggesting its role in mediating Cl- influx and HCO3- efflux.
Conclusions:
- The Na+/H+ exchanger isoform 1 (NHE1) plays a primary role in compensating for intracellular acidification during RVD in SiHa cells.
- The anion exchanger isoform 2 (AE2) functions in parallel with NHE1, mediating Cl- influx to regulate pH(i) homeostasis.
- Both NHE1 and AE2 are critical for maintaining pH(i) during regulatory volume decrease in human cervical cancer cells.