Shafeena C. Taylor

Hypoxic Enhancement of Quantal Catecholamine Secretion EVIDENCE FOR THE INVOLVEMENT OF AMYLOID β-PEPTIDES

Prolonged exposure to hypoxia (10% O2) enhanced quantal catecholamine release evoked from O2-sensing pheochromocytoma (PC12) cells, as monitored using single-cell amperometric recordings. The enhancement of exocytosis was apparent after 12 h of hypoxia and was maximal at 24 h. Elevated levels of secretion were due to the emergence of a Ca2+ influx pathway that persisted during complete blockade of known voltage-gated Ca2+ channels. Secretion triggered by this Ca2+ influx was severely reduced by known inhibitors of Alzheimers amyloid β-peptides (AβPs), including an N terminus-directed monoclonal antibody. The enhancing effect on secretion of chronic hypoxia was mimicked closely by direct application of AβP to cells under normoxic conditions, although the effects of AβP were more rapid at onset, being maximal after only 6 h. The present results suggest that prolonged hypoxia can induce formation of Ca2+-permeable AβP channels and that such induction can lead directly to excessive neurosecretion. This is a potential contributory factor to AβP pathophysiology following cerebral ischemia.

Store-operated Ca2+ influx and voltage-gated Ca2+channels coupled to exocytosis in pheochromocytoma (PC12) cells

Abstract : Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+‐evoked exocytosis was entirely dependent on Ca2+ influx through voltage‐gated Ca2+ 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+‐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 Ca2+. Application of caffeine in Ca2+‐free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store‐operated Ca2+ 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 Ca2+ channels, but less than that caused by Ca2+ influx through N‐type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage‐gated Ca2+ channels.

Chronic hypoxia enhances the secretory response of rat phaeochromocytoma cells to acute hypoxia

1 Amperometric recordings were made from individual phaeochromocytoma (PC12) cells using carbon fibre microelectrodes to investigate the effects of chronic hypoxia (10% O2) on the secretory responses evoked by acute hypoxia. 2 Exposure to chronic hypoxia for 21–26 h increased the frequency of exocytotic events evoked in response to acute hypoxia (P  O 2ca 10–60 mmHg). 3 Chronic hypoxia increased the value of Q1/3, 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 mm) 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 Ca2+‐free solutions. Cd2+ (200 μm) completely inhibited exocytosis from control cells, but left a significant residual release in chronically hypoxic PC12 cells. 6 The Cd2+‐resistant release evoked by acute hypoxia in chronically hypoxic PC12 cells was inhibited by inorganic ions (0.01–10 mm) in a potency order of La3+ > Gd3+ > Zn2+. Ni2+ (10 mm) 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 Cd2+‐resistant Ca2+ influx pathway in chronically hypoxic PC12 cells.

Three Distinct Ca2+ Influx Pathways Couple Acetylcholine Receptor Activation to Catecholamine Secretion from PC12 Cells

Abstract: Amperometry and microfluorimetry were employed to investigate the Ca2+‐dependence of catecholamine release induced from PC12 cells by cholinergic agonists. Nicotine‐evoked exocytosis was entirely dependent on extracellular Ca2+ but was only partly blocked by Cd2+, a nonselective blocker of voltagegated Ca2+ channels. Secretion and rises of [Ca2+]i observed in response to nicotine could be almost completely blocked by methyllycaconitine and α‐bungarotoxin, indicating that such release was mediated by receptors composed of α7 nicotinic acetylcholine receptor subunits. Secretion and [Ca2+]i rises could also be fully blocked by co‐application of Cd2+ and Zn2+. Release evoked by muscarine was also fully dependent on extracellular Ca2+. Muscarinic receptor activation stimulated release of Ca2+ from a caffeine‐sensitive intracellular store, and release from this store induced capacitative Ca2+ entry that could be blocked by La3+ and Zn2+. This Ca2+ entry pathway mediated all secretion evoked by muscarine. Thus, activation of acetylcholine receptors stimulated rises of [Ca2+]i and exocytosis via Ca2+ influx through voltage‐gated Ca2+ channels, α7 subunit‐containing nicotinic acetylcholine receptors, and channels underlying capacitative Ca2+ entry.

Acid-evoked quantal catecholamine secretion from rat phaeochromocytoma cells and its interaction with hypoxia-evoked secretion

1 Amperometric recordings using polarized carbon fibre microelectrodes were used to detect exocytosis of catecholamines from rat phaeochromocytoma (PC12) cells in response to a reduction in pHo. 2 Exocytosis was detected at pHo levels of between 7.2 and 6.8. This was probably due to intracellular acidification, since acid‐evoked secretion was enhanced by the Na+‐H+ exchange blocker ethylisopropylamiloride (30 μm), and was mimicked by sodium propionate (10 mM), which causes selective intracellular acidosis. 3 Acid‐evoked exocytosis was abolished by removal of Ca2+o or application of 200 μm Cd2+. It was unaffected by nifedipine, but significantly reduced by either ω‐conotoxin GVIA (1 μm) or ω‐agatoxin GIVA (200 nM). The two toxins applied together almost completely abolished (> 97 %) acid‐evoked secretion. 4 Hypoxia‐evoked catecholamine release was potentiated under acidic conditions and suppressed under alkaline conditions in a manner which indicated a greater than additive interaction of these two stimuli. 5 Our results indicate that, like carotid body arterial chemoreceptors, PC12 cells represent model chemoreceptor cells for both hypoxia and acidity and that the release of catecholamines in response to these physiological stimuli is dependent on Ca2+ influx through voltage‐gated N‐ and P/Q‐type Ca2+ channels.

A central role for ROS in the functional remodelling of L-type Ca2+ channels by hypoxia

Periods of prolonged hypoxia are associated clinically with an increased incidence of dementia, the most common form of which is Alzheimers disease. Here, we review recent studies aimed at providing a cellular basis for this association. Hypoxia promoted an enhanced secretory response of excitable cells via formation of a novel Ca2+ influx pathway associated with the formation of amyloid peptides of Alzheimers disease. More strikingly, hypoxia potentiated Ca2+ influx specifically through L-type Ca2+ channels in three distinct cellular systems. This effect was post-transcriptional, and evidence suggests it occurred via increased formation of amyloid peptides which alter Ca2+ channel trafficking via a mechanism involving increased production of reactive oxygen species by mitochondria. This action of hypoxia is likely to contribute to dysregulation of Ca2+ homeostasis, which has been proposed as a mechanism of cell death in Alzheimers disease. We suggest, therefore, that our data provide a cellular basis to account for the known increased incidence of Alzheimers disease in patients who have suffered prolonged hypoxic episodes.