Identification and Characterization of the 02sensitive Ion Channels in AMC

Ligand-gated nicotinic ACh receptors mediate CA secretion from AMC in mature animals via splanchnic nerve stimulation. AMC also express several types ofvoltage-gated ion channels that are intimately involved in the regulation ofCA secretion, and that are candidates for the hypoxia-sensitive ion channels. In addition to K+ channels (see below), AMC express a TTX-sensitive Na+ channel and three types of voltage-dependent Ca2+ channels: the dihydropyridine-sensitive L-type, the conotoxin-sensitive N-type, and agatoxin-sensitive P-type Ca2+channels (4). Interestingly ,theL-typechannel appears most efficiently coupled to depolarization-induced CA secretion in adult AMC (4), and the hypoxia-induced CA secretion from neonatal AMC could be blocked by the L-type channel blocker nifedipine (1, 20, 33).

Several voltage-dependent K+ channels are found in AMC, including delayed rectifier (Kv) and large conductance Ca2+-dependent (BK) K+ channels. Several of these classes of ion channels are known to be regulated by hypoxia in various cell types (18). For example, BK channels are thought to be a major component of the Oj-sensitive K+ (Ko2) current in carotid body glomus cells (23) and the delayed rectifier K+ channels, Kv1.5 and Kv2.1 are thought to mediate the hypoxia-sensitive outward currents in pulmonary myocytes (3). In order to identify the types of K+ currents that mediate the hypoxia-sensitive outward currents in AMC, we tested the ability of inhibitors of different classes of K+ channels to block the hypoxia-induced suppression of outward currents in neonatal AMC (31). The major component (-65%) of the K02 current (IK02) was blocked by removal of extracellular Ca2+ or by 50-100 nM iberiotoxin (IbTx), suggesting that BK channels are a major contributor. Thus the remaining -35 % of IK02 was attributable mainly to delayed rectifier Kv channels which are sensitiveto20mMtetraethylammonium(TEA)(31). Additionally, AMCexpress a pinacidil-activated and glibenclamide-sensitive, ATP-sensitive K+ current (Katp) that is augmented by hypoxia (31). This current is reminiscent ofthe one carried by 02-sensitive channels in neurons of the substantia nigra, which are thoughtto limit membrane depolarization during hypoxia (13).

What is the molecular identity of the 02-sensitive Kv channels in AMC? Recent work from our laboratory raises the possibility that these channels are Kv 1.2/KV1.5 heteromultimers, though this requires validation. Both Kv1.2 and Kv1.5 subunits appear to be expressed in neonatal AMC based on immunocytochemistry, and the heteromultimer likely comprises one of the 02-sensitive K+ channels expressed by the immortalized chromaffin cell line, i.e., v-myc, adrenal-derived HNK1+(MAH) cells, which are derived from embryonic AMC (8). These Kv channel subunits are also potential molecular components of the 02-sensitive outward current in pulmonary myocytes (3, 29) and PC 12 cells (Kv1.2 only) (6). The molecular characterization ofthe 02-sensitive K+

channels in neonatal AMC need to be confirmed using expression systems and mutagenesis of the candidate proteins.

It should be noted that none of the 02-sensitive channels described above were responsible for generation of the receptor potential in neonatal AMC, although Katp channels could modulate its magnitude. The hypoxia-induced membrane depolarization persisted in the presence IbTx, Cd2+, TEA, and 4-aminopyridine (31). Though the resting membrane potential of AMC hyperpolarized in a Na+- and Ca2+-free solution, the receptor potential remained constant. This argues against a role for cation selective channels in generating the receptor potential in rat AMC, as has been suggested for adult guinea pig cells (12). Interestingly, glibenclamide augmented the magnitude of the receptor potential in our studies, suggesting that KATP channels may play a similar protective role to limit the extent of membrane depolarization during hypoxia in AMC as in central neurons. Consistent with this notion, cromakalim, an activator of Katp channels, reversed the hypoxia-induced stimulation of CA secretion in adult AMC maintained in long-term culture (20). There is recent evidence that the receptor potential may be mediated by small-conductance Ca2+-dependent K+ channels (SK) because apamin, an inhibitor of SK channels, can depolarize AMC and block the receptor potential (14, 16, 22).

Figure 3. The mitochondrial electron transport chain complex I inhibitor, rotenorie, mimics and attenuates the effects of hypoxia on neonatal adrenomedullary chromaffin cells. A: Nystatin perforated patch clamp recordings (step to +30 mV from -60 mV) from a neonatal AMC exposed to control (C), hypoxia (H), 10 |iM rotenone (Rot), hypoxia+rotenone (Rot+H), and washout (W). Both: hypoxia and rotenone inhibited outward currents and the combined effects of rotenone and hypoxia did not exceed the suppression seen by rotenone alone. B: Current-voltage plots for the cell currents shown in A. C: Current-clamp recording from a single AMC in a small cluster of -10 cells. Both spontaneous action potential generation and a receptor potential of ~ 12 mV are visible. All current-clamp recordings from the same cell are 500 ms in duration and illustrate that 10 nM rotenone depolarizes the cell and blocks the hypoxia-induced receptor potential.

Figure 3. The mitochondrial electron transport chain complex I inhibitor, rotenorie, mimics and attenuates the effects of hypoxia on neonatal adrenomedullary chromaffin cells. A: Nystatin perforated patch clamp recordings (step to +30 mV from -60 mV) from a neonatal AMC exposed to control (C), hypoxia (H), 10 |iM rotenone (Rot), hypoxia+rotenone (Rot+H), and washout (W). Both: hypoxia and rotenone inhibited outward currents and the combined effects of rotenone and hypoxia did not exceed the suppression seen by rotenone alone. B: Current-voltage plots for the cell currents shown in A. C: Current-clamp recording from a single AMC in a small cluster of -10 cells. Both spontaneous action potential generation and a receptor potential of ~ 12 mV are visible. All current-clamp recordings from the same cell are 500 ms in duration and illustrate that 10 nM rotenone depolarizes the cell and blocks the hypoxia-induced receptor potential.

The hypoxic inhibition of voltage-dependent K+ channels suggests that hypoxia may induce CA secretion via modulation of the action potential waveform. We recorded membrane potential in neonatal rat and mouse AMC from singly-isolated and small clusters of cells. Interestingly, single isolated cells were often quiescent and fired action potentials superimposed on the receptor potential (Fig. 2C) (33), whereas those in small clusters (>8 cells) frequently fired spontaneous rhythmic action potentials at ~ 1 Hz (Fig. 3C) (32). Exposure to hypoxia did not significantly modulate the frequency of action potentials in clustered AMC, but caused a reversible broadening ofthe spike duration that was primarily associated with a prolongation of the decay phase, as would be expected if K+ channels were inhibited (Fig. 3C) (32). However, we also observed a slight increase in the rise time of the action potential that could arise from inhibition of a conductance that is active at the resting membrane potential. Candidates for these channels include non-selective cation conductances (12) and SK channels (14, 16, 22).

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