The effects of hypoxia on K+ current (IK), resting membrane potential, and cytosolic free Ca2+ in rat pheochromocytoma (PC-12) cells were studied. Whole cell voltage- and current-clamp experiments were performed to measure IK and membrane potential, respectively. Cytosolic free Ca2+ level was measured using the Ca(2+)-sensitive fluorescent dye fura 2. Depolarizing voltage steps to +50 mV from a holding potential of -90 mV elicited a slowly inactivating, tetraethylammonium chloride-sensitive, and Ca(2+)-insensitive IK that was reversibly inhibited by reduced O2 tension. Graded reduction in PO2 (from 150 to 0 mmHg) induced a graded inhibition of O2-sensitive IK [IK(O2)] up to 46% at 0 mmHg. Moreover, hypoxia induced a 19-mV membrane depolarization and a twofold increase in cytosolic free Ca2+. In Ca(2+)-free condition, inhibition of IK(O2) induced an 8-mV depolarization, suggesting that inhibition of IK(O2) was responsible for initiating depolarization. The effect of reduced PO2 on the current-voltage relationship showed a reduction of outward current and a 14-mV shift in the reversal potential comparable with the amount of depolarization measured in current clamp experiments. Neither Ca(2+)-activated IK nor inwardly rectifying IK are responsible for the hypoxia-induced depolarization. In conclusion, PC-12 cells express an IK(O2), inhibition of which leads to membrane depolarization and increased intracellular Ca2+, making the PC-12 clonal cell line a useful model for studying the molecular and biophysical mechanisms that mediate O2 chemosensitivity.

Carotid body type I cells and the O2 sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2 tension causes inhibition of an outward rectifying the O2-sensitive potassium (K) channel in the O2-sensitive pheochromocytoma (PC12) cell line, which leads to membrane depolarization and increased intracellular free Ca2+. We found that removal of Ca2+ from the extracellular milieu, inhibition of voltage-dependent Ca2+ channels, and chelation of intracellular Ca2+ prevents full activation of the TH gene expression during hypoxia. These findings suggest that membrane depolarization and regulation of intracellular free Ca2+ are critical signal transduction events that regulate expression of the TH gene in PC12 cells during hypoxia. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O2 tension in both type I cells and PC12 cells. The increase in TH gene expression in PC12 cells during hypoxia is due to increases in both the rate of transcription and mRNA stability. Analysis of reporter-gene constructs revealed that increased transcription of the TH gene during hypoxia is regulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of the gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays revealed that hypoxia-induced binding occurs at both the AP1 and HIF-1 sites. Results from super-shift and shift Western assays showed that a heterodimer consisting of c-Fos and JunB binds to the AP1 site during hypoxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also found that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O2 tension.

PC-12 cells depolarize during hypoxia and release dopamine. The hypoxia-induced depolarization is due to inhibition of an O2-sensitive K+ current. The role of dopamine released during hypoxia is uncertain, but it could act as an autocrine to modulate membrane conductance during hypoxia. The current study was undertaken to investigate this possibility. Reverse transcription-polymerase chain reaction and sequence analysis revealed that the D2 isoform of the dopamine receptor is expressed in rat PC-12 cells. Exogenously applied dopamine and the D2agonist quinpirole elicited inhibition of a voltage-dependent K+ current ( I K) that was prevented by sulpiride, a D2 receptor antagonist. Dopamine and quinpirole applied during hypoxia potentiated the inhibitory effect of hypoxia on I K. We also found that quinpirole caused reversible inhibition of a voltage-dependent Ca2+ current ( I Ca) and attenuation of the increase in intracellular free Ca2+ during hypoxia. Our results indicate that dopamine released from PC-12 cells during hypoxia acts via a D2 receptor to “autoregulate” I K and I Ca.

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We have previously shown that pheochromocytoma (PC12) cells rapidly depolarize and undergo Ca2+ influx through voltage-dependent Ca2+ channels in response to moderate hypoxia and that intracellular free Ca2+ is modulated by activation of dopamine D2 receptors in this cell type. The present study shows that D2 (quinpirole-mediated) inhibition of a voltage-dependent Ca2+ current (ICa) in PC12 cells is dramatically attenuated after chronic exposure to moderate hypoxia (24 h at 10% O2). Pretreatment of cells with pertussis toxin abolished D2-mediated inhibition of ICa. The D2-induced inhibition of ICa did not depend on protein kinase A (PKA), as it persisted both in the presence of a specific PKA inhibitor (PKI) and in PKA-deficient PC12 cells. Prolonged exposure to hypoxia (24 h) significantly reduced the level of Gi/o alpha immunoreactivity, but did not alter G beta levels. Furthermore, dialysis of recombinant G(o) alpha protein through the patch pipette restored the inhibitory effect of quinpirole in cells chronically exposed to hypoxia. We conclude that the attenuation of the D2-mediated inhibition of ICa by chronic hypoxia is caused by impaired receptor-G protein coupling, due to reduced levels of G(o) alpha protein. This attenuated feedback modulation of ICa by dopamine may allow for a more sustained Ca2+ influx and enhanced cellular excitation during prolonged hypoxia.