Oxygen Sensitivity of PC12 Cells

An initial critical step in the process of 02-sensing common to chemosensitive cells is membrane depolarization upon exposure to hypoxia (21, 22). This event is essential for activating voltage-sensitive Ca2+ channels and producing the increase in [Ca2+], necessary to trigger many physiological responses such as constriction (in pulmonary artery) and neurotransmitter release (in CB). Like other oxygen-chemosensitive cells, PC12 cells respond to acute exposure to hypoxia with membrane depolarization. Whole-cell current-clamp studies of membrane potential show that hypoxia causes membrane depolarization in PC 12 cells (Fig. 1A) and that the degree of membrane depolarization is proportional to the severity of the hypoxic stimulus and is independent of external space Ca2+ (31). The membrane depolarization is necessary to activate voltage-dependent Ca2+ channels, thereby increasing cytosolic Ca2+. Indeed, Fura-2 experiments indicated that hypoxia induces a 2-3 fold increase in [Ca2+]s in PC12 cells (Fig. IB). A similar effect ofhypoxia on Ca2+ homeostasis has been reported for CB type I cells and pulmonary artery smooth muscle cells (21, 22, 25). Ultimately, these responses lead to transmitter release. Indeed, amperometric measurements indicated that hypoxia evokes dopamine and norepinephrine release in PC 12 cells (19). The hypoxia-induced exocytosis occurs via depolarization, leading to Ca2+ influx primarily via N-type Ca2+ channels (30). Overall, a primary response to acute hypoxia in PC12 cells is facilitation of release of neurotransmitters such as dopamine (DA), norepinephrine (NE) and adenosine (18, 19, 30, 31). Although the role of these transmitters in transduction of the hypoxic stimulus is still controversial, recent findings show that DA and adenosine exert a feedback control on cellular excitability and function in PC 12 cells during hypoxia via stimulation of D2 and A2 receptors, respectively (18, 32).

Figure /. 02-sensing mechanisms triggered by acute hypoxia in PC 12 cells. A: Acute hypoxia induces cell depolarization. Membrane potential (£m) was recorded in current-clamp mode. B: Acute hypoxia increases [CaJ+]j. Arrows indicate point of introduction of hypoxia (Hyp; Po2<10 mmHg) and return to normoxic conditions (Ree; Po2= 150 mmHg) in pianels A and B. C: Acute hypoxia inhibits a Ko2 current. Superimposed current traces were recorded in normoxia (Nor), after steady-state inhibition by hypoxia (Hyp) and after returning to normoxia (Ree). D: The effect of hypoxia on the outward K+ current, elicited by a 800-ms test pulse of+50 mV, was determined under control conditions with a holding potential (HP) of -90 mV (open bar, standard bath solution with 2 raM CaCl2 and Ca2+-free pipette solution, n=l 1), in the presence of 5 mM TEA (n=4) or 20 nM CTX (n=7), in Cai+-free external medium (n=5) or using a HP of-30 mV (n=5).

Figure /. 02-sensing mechanisms triggered by acute hypoxia in PC 12 cells. A: Acute hypoxia induces cell depolarization. Membrane potential (£m) was recorded in current-clamp mode. B: Acute hypoxia increases [CaJ+]j. Arrows indicate point of introduction of hypoxia (Hyp; Po2<10 mmHg) and return to normoxic conditions (Ree; Po2= 150 mmHg) in pianels A and B. C: Acute hypoxia inhibits a Ko2 current. Superimposed current traces were recorded in normoxia (Nor), after steady-state inhibition by hypoxia (Hyp) and after returning to normoxia (Ree). D: The effect of hypoxia on the outward K+ current, elicited by a 800-ms test pulse of+50 mV, was determined under control conditions with a holding potential (HP) of -90 mV (open bar, standard bath solution with 2 raM CaCl2 and Ca2+-free pipette solution, n=l 1), in the presence of 5 mM TEA (n=4) or 20 nM CTX (n=7), in Cai+-free external medium (n=5) or using a HP of-30 mV (n=5).

All the above series of events necessary to produce the functional response to hypoxia are triggered by the initial inhibition of a K+ conductance (20). PC12 cells, like other chemosensitive cells, express an Ko2 channel that is inhibited by hypoxia (21). Exposure to hypoxia (Po2<10 mmHg) induces inhibition of an outward slow-inactivating voltage-dependent K+ current, and this effect is reversible upon returning to normoxia (PO2=150 mmHg) (Fig. 1C). The magnitude of hypoxia-induced inhibition of the K current depends on the severity of hypoxia. Perfusion with progressively lower Po2 reduces the K

current in a step-wise fashion (31). The Ko2 current in PC 12 cells is present at the voltage range of their resting potential (ca. -35 to -45 mV), thus its inhibition results in membrane depolarization (31). Detailed whole-cell voltage-clamp studies have indicated that the Ko2 current in PC 12 cells is a slowly-inactivating voltage-dependent K+ current which is blocked by tetraethylammonium (TEA), a blocker of voltage-dependent K (Kv) channels (31). Taylor and Peers (30) have confirmed that inhibition of a TEA-sensitive K+ conductance is indeed responsible for the hypoxia-evoked depolarization and consequent exocytosis in PC 12 cells. Figure ID illustrates the characteristics ofthe Ko2 current in PC12 cells by showing the relative inhibition of the K+ current induced by hypoxia under various experimental conditions. In control conditions, hypoxia inhibits the K+ current by approximately 20%. Exposure of cells to 5 mM external TEA results in loss of the hypoxia-induced inhibition of the K current. Furthermore, the Ko2 current in PC 12 cells is inhibited by charybdotoxin, a potent blocker of Kv channels (in particular Kv 1.2 and KV1.3) and Ca2+-activated K (KCa) channels. Although KCa channels are expressed in PC 12 cells, they do not appear to be 02-sensitive. Indeed, the Ko2 current is not sensitive to Ca2+. Furthermore, the Ko2 current is not sensitive to the holding voltage. In fact, the same percentage inhibition ofthe K+ current by hypoxia was observed in experiments performed in medium and in experiments where the holding potential was kept at

-30 mV. Therefore the Ko2 current in PC12 cells does not appear to be either a or a transient current.

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