No abstract

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K ϩ and caffeine. K ϩ -evoked exocytosis was entirely dependent on Ca 2ϩ influx through voltage-gated Ca 2ϩ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K ϩ -evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca 2ϩ . Application of caffeine in Ca 2ϩ -free solutions evoked large, transient rises of [Ca 2ϩ ] i , but did not trigger exocytosis. When Ca 2ϩ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca 2ϩ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca 2ϩ channels, but less than that caused by Ca 2ϩ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca 2ϩ influx through pathways other than voltage-gated Ca 2ϩ channels.

1Ï3, determined by the integration of amperometric events, indicating an increase in quantal size: this reflects either an increase in vesicular dimensions or vesicular catecholamine concentration. 4. Exocytotic frequency evoked by bath application of tetraethylammonium (1-10 mÒ) was significantly enhanced following chronic hypoxia. 5. In both control and chronically hypoxic PC12 cells, exocytosis in response to acute hypoxia was completely abolished in Ca¥-free solutions. Cd¥ (200 ìÒ) completely inhibited exocytosis from control cells, but left a significant residual release in chronically hypoxic PC12 cells. 6. The Cd¥-resistant release evoked by acute hypoxia in chronically hypoxic PC12 cells was inhibited by inorganic ions (0·01-10 mÒ) in a potency order of LaŤ > GdŤ > Zn¥. Ni¥ (10 mÒ) was without effect. 7. Our results suggest that chronic hypoxia enhances the secretory response of PC12 cells in part by increasing the depolarization mediated by an oxygen-sensitive K¤ channel. In addition, acute hypoxia activates a Cd¥-resistant Ca¥ influx pathway in chronically hypoxic PC12 cells.8518

The carotid body is the major arterial chemoreceptor and responds to physiological stimuli (hypoxia, hypercapnia, acidosis) by increasing the discharge frequency of afferent chemosensory neurons, thereby initiating corrective changes in breathing pattern (Fidone & Gonzalez, 1986;Gonzalez et al. 1994). Type I (glomus) cells within the carotid body are central to this chemoreceptive process, releasing transmitters -particularly catecholamines -in response to physiological stimuli in a manner which correlates well with increased chemosensory nerve activity (reviewed by Gonzalez et al. 1994). Patch-clamp recordings have shown that type I cells possess Oµ-sensitive K¤ channels whose activity is reduced under hypoxic conditions (Lopez-Barneo et al. 1988;Delpiano & Hescheler, 1989;Peers, 1990a;Stea & Nurse, 1991). Such an effect causes membrane depolarization, opening of voltage-gated Ca¥ channels (Buckler & Vaughan-Jones, 1994a;Wyatt & Peers, 1995) and thus transmitter release (Urena et al. 1994). In the rat model, evidence suggests that acidicÏhypercapnic stimuli evoke transmitter release via a similar mechanism (Peers, 1990b;Peers & Green, 1991; Buckler & Vaughan-Jones, 1994b) although in the rabbit model, a completely different mechanism has been put forward to account for transduction of acidic stimuli (Rocher et al. 1991). Hypoxic and acidic stimuli are well-known to be multiplicative in their ability to increase afferent chemosensory nerve discharge (Fitzgerald & Parks, 1971;Lahiri & Delaney, 1975), an effect which may account for the well-known postnatal maturation of this sensory organ (Pepper et al. 1995). However, no explanation has been forwarded to account for this interactive effect at the cellular level, and it remains unknown whether this interaction occurs within the type I cell or involves other cellular elements of the carotid body. Recently, evidence has emerged that the rat phaeochromocytoma (PC12) cell line responds to hypoxia in a manner which is remarkably similar to that of the type I carotid body cell. Thus, hypoxia inhibits K¤ channels in these cells, causing membrane depolarization and a subsequent rise in [Ca¥]é (Zhu et al. 1996;Conforti & Millhorn, 1997). In addition, we have shown that hypoxia evokes quantal secretion of catecholamines from PC12 cells, which is entirely dependent on Ca¥ influx through voltage-

Amperometry and microfluorimetry were employed to investigate the Ca 2ϩ -dependence of catecholamine release induced from PC12 cells by cholinergic agonists. Nicotine-evoked exocytosis was entirely dependent on extracellular Ca 2ϩ but was only partly blocked by Cd 2ϩ , a nonselective blocker of voltagegated Ca 2ϩ channels. Secretion and rises of [Ca 2ϩ ] i observed in response to nicotine could be almost completely blocked by methyllycaconitine and ␣-bungarotoxin, indicating that such release was mediated by receptors composed of ␣7 nicotinic acetylcholine receptor subunits. Secretion and [Ca 2ϩ ] i rises could also be fully blocked by co-application of Cd 2ϩ and Zn 2ϩ . Release evoked by muscarine was also fully dependent on extracellular Ca 2ϩ . Muscarinic receptor activation stimulated release of Ca 2ϩ from a caffeine-sensitive intracellular store, and release from this store induced capacitative Ca 2ϩ entry that could be blocked by La 3ϩ and Zn 2ϩ . This Ca 2ϩ entry pathway mediated all secretion evoked by muscarine. Thus, activation of acetylcholine receptors stimulated rises of [Ca 2ϩ ] i and exocytosis via Ca 2ϩ influx through voltage-gated Ca 2ϩ channels, ␣7 subunit-containing nicotinic acetylcholine receptors, and channels underlying capacitative Ca 2ϩ entry.