Korean J Physiol Pharmacol.  2013 Feb;17(1):57-64. 10.4196/kjpp.2013.17.1.57.

Acute Hypoxia Activates an ENaC-like Channel in Rat Pheochromocytoma (PC12) Cells

• Bae YJ
• Yoo JC
• Park N
• Kang D
• Han J
• Hwang E
• Park JY
• Hong SG

• KMID: 1432764
• DOI: http://doi.org/10.4196/kjpp.2013.17.1.57

Abstract

Cells can resist and even recover from stress induced by acute hypoxia, whereas chronic hypoxia often leads to irreversible damage and eventually death. Although little is known about the response(s) to acute hypoxia in neuronal cells, alterations in ion channel activity could be preferential. This study aimed to elucidate which channel type is involved in the response to acute hypoxia in rat pheochromocytomal (PC12) cells as a neuronal cell model. Using perfusing solution saturated with 95% N2 and 5% CO2, induction of cell hypoxia was confirmed based on increased intracellular Ca2+ with diminished oxygen content in the perfusate. During acute hypoxia, one channel type with a conductance of about 30 pS (2.5 pA at -80 mV) was activated within the first 2~3 min following onset of hypoxia and was long-lived for more than 300 ms with high open probability (Po, up to 0.8). This channel was permeable to Na+ ions, but not to K+, Ca+, and Cl- ions, and was sensitively blocked by amiloride (200 nM). These characteristics and behaviors were quite similar to those of epithelial sodium channel (ENaC). RT-PCR and Western blot analyses confirmed that ENaC channel was endogenously expressed in PC12 cells. Taken together, a 30-pS ENaC-like channel was activated in response to acute hypoxia in PC12 cells. This is the first evidence of an acute hypoxia-activated Na+ channel that can contribute to depolarization of the cell.

Keyword

Acute hypoxia; Amiloride; Epithelial Na+ channel; PC12 Cells; Rats

MeSH Terms

Amiloride
Animals
Anoxia
Blotting, Western
Cell Hypoxia
Epithelial Sodium Channels
Ion Channels
Ions
Neurons
Oxygen
PC12 Cells
Pheochromocytoma
Rats
Amiloride
Epithelial Sodium Channels
Ion Channels
Ions
Oxygen

Figure

• 

  Fig. 1 Single channel currents activated by acute hypoxia. (A) Single channel currents recorded at +80 mV (upper) and -80 mV (lower) under normoxic conditions. Channel currents in expanded scale were quoted from each representative recording. (B) All-point histograms analyzed from activities shown in (A). Histograms at +80 mV and -80 mV were separately presented. (C) Channel currents recorded under hypoxic conditions. These activities were recorded in the same cell after switching to hypoxic solution (HS). (D) Histograms analyzed with recording data shown in (C). Arrowheads indicate the peaks activated by infusion of HS. Current recordings were made in cell-attached (CA) configuration with voltage waveforms as presented at the top of (A). Dashed lines (in A, C) denote closed level. Calibration bars are equal to 100 ms and 2 pA.

• 

  Fig. 2 Hypoxia-activated channel currents impermeable to anions. (A) Preservation of channel activity induced by hypoxia in excised cell-free membrane patch. This channel was silent upon return to normoxia (upper tracings) after switching to inside-out (IO) configuration. This channel was reactivated following perfusion of HS (lower tracings). (B) All-point histograms analyzed from activities recorded in IO patch before (left) and after perfusion of HS (right). These experiments were performed on membrane patches, which were found to contain hypoxia-activated channels in CA mode. Arrowhead shows the peak activated by hypoxia. (C) Effects of potassium and choride ions on hypoxia-activated channel. Right panel shows inhibition of 30-pS channels by application of potassium glutamate (K-glu). (D) Ineffectiveness of Cl- on hypoxia-elicited activation of 30-pS channel. Right panel shows no effect without Cl- in the pipette. For experiments in (C) and (D), membrane patches were clamped at +80 mV in order to record currents elicited either by internal (bath) K+ or external (pipette) Cl-. Dashed lines (in A, C) denote closed level. Calibration bars are equal to 100 ms and 2 pA

• 

  Fig. 3 Hypoxia-activated channel selective to Na+. (A) Representative tracings of 30-pS channels blocked by removal of Na+. Upper two traces were recorded in the presence of Na+ in normal (upper) and hypoxic bathing solution (middle), respectively, whereas the lower one was recorded in the presence of impermeable NMDG instead of Na+ as indicated by the drawings. (B) Na+-dependence of peaks corresponding to activation of channels under hypoxic conditions. Arrowhead designates typical peak for channels with an amplitude of 2.5 pA (at 80 mV) under hypoxic conditions. Note that removal of Na+ abolished typical peaks in the histogram labeled as Hypoxia+NMDG-Cl. (C) Summarized bar chart representing the channel activity reduced by removal of Na+ under hypoxic conditions. All data were obtained from single channel recordings in IO patches clamped at +80 mV. Asterisk (*) stands for differences from control (p<0.001).

• 

  Fig. 4 Hypoxia-activated channels sensitive to amiloride. (A) Representative tracings of 30-pS channels blocked by amiloride, a neuronal Na+ channel blocker. Tracings represent that channel activity due to hypoxia (second tracing) was sensitively inhibited by 200 nM amiloride (third tracing). (B) Summarized bar chart representing the channel activity reduced by amiloride under hypoxic conditions. All data were obtained from single channel recordings at -80 mV. (C) All-point histogram showing the abolishment of peaks typically activated under hypoxic conditions. Note that the middle histogram shows hypoxia-elicited peaks in regular 2.5-pA intervals. Asterisk (*) stands for differences from control (p<0.01).

• 

  Fig. 5 Expression of ENaC in PC12 cells. (A) mRNA expression levels of ENaC-α subunit (747 bp), β subunit (178 bp), and γ subunit (405 bp) in PC12 cells. (B) Detection of ENaC-α subunit protein by Western blotting in rat lung (left lane) and PC12 cells (right lane). ENaC-α subunit detected by human polyclonal antibody.

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