Ca2+ mobilization from intracellular stores represents an important cell signaling process 1 which is regulated, in mammalian cells, by inositol 1,4,5-trisphosphate (InsP3), cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP3 and cADPR release Ca2+ from sarco / endoplasmic reticulum (S/ER) stores through activation of InsP3 and ryanodine receptors (InsP3Rs and RyRs). By contrast, the nature of the intracellular stores targeted by NAADP and molecular identity of the NAADP receptors remain controversial 1,2, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments 3,4. Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with TPC1 and TPC3 being expressed on endosomal and TPC2 on lysosomal membranes. Membranes enriched with TPC2 exhibit high affinity NAADP binding and TPC2 underpins NAADP-induced Ca2+ release from lysosome-related stores that is subsequently amplified by Ca2+-induced Ca2+ release via InsP3Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but only attenuated by depleting ER Ca2+ stores or blocking InsP3Rs. Thus, TPCs form NAADP receptors that release Ca2+ from acidic organelles, which can trigger additional Ca2+ signals via S/ER. TPCs therefore provide new insights into the regulation and organization of Ca2+ signals in animal cells and will advance our understanding of the physiological role of NAADP.

Specialized O 2 -sensing cells exhibit a particularly low threshold to regulation by O 2 supply and function to maintain arterial pO 2 within physiological limits. For example, hypoxic pulmonary vasoconstriction optimizes ventilation-perfusion matching in the lung, whereas carotid body excitation elicits corrective cardio-respiratory reflexes. It is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation in O 2 -sensing cells, thereby mediating, in part, cell activation. However, the mechanism by which this process couples to Ca 2؉ signaling mechanisms remains elusive, and investigation of previous hypotheses has generated contrary data and failed to unite the field. We propose that a rise in the cellular AMP/ATP ratio activates AMP-activated protein kinase and thereby evokes Ca 2؉ signals in O 2 -sensing cells. Co-immunoprecipitation identified three possible AMP-activated protein kinase subunit isoform combinations in pulmonary arterial myocytes, with ␣1␤2␥1 predominant. Furthermore, their tissue-specific distribution suggested that the AMP-activated protein kinase-␣1 catalytic isoform may contribute, via amplification of the metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMPactivated protein kinase-␣1 to be located throughout the cytoplasm of pulmonary arterial myocytes. In contrast, it was targeted to the plasma membrane in carotid body glomus cells. Consistent with these observations and the effects of hypoxia, stimulation of AMPactivated protein kinase by phenformin or 5-aminoimidazole-4-carboxamide-riboside elicited discrete Ca 2؉ signaling mechanisms in each cell type, namely cyclic ADP-ribose-dependent Ca 2؉ mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial myocytes and transmembrane Ca 2؉ influx into carotid body glomus cells. Thus, metabolic sensing by AMP-activated protein kinase may mediate chemotransduction by hypoxia.Specialized O 2 -sensing cells within the body have evolved as vital homeostatic mechanisms that monitor O 2 supply and alter respiratory and circulatory function, as well as the capacity of the blood to transport O 2 . By these means, arterial pO 2 is maintained within physiological limits. Two key systems involved are the pulmonary arteries and the carotid body. Constriction of pulmonary arteries by hypoxia optimizes ventilation-perfusion matching in the lung (1), whereas carotid body excitation by hypoxia initiates corrective changes in breathing patterns via increased sensory afferent discharge to the brain stem (2). Although O 2 -sensitive mechanisms independent of mitochondria may also play a role (3-5), it is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation and that this underpins, at least in part, cell activation (2, 6 -10). Despite this consensus, the mechanism by which inhibition of mitochondrial oxidative phosphorylation couples to discrete cell-specific Ca 2ϩ signaling ...

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SummaryIn arterial myocytes the Ca 2+ mobilizing messenger NAADP evokes spatially restricted Ca 2+ bursts from a lysosome-related store that are subsequently amplified into global Ca 2+ waves by Ca 2+ -induced Ca 2+ -release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) αlgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5 μm of the nucleus) was ~4-fold greater than in the sub-plasmalemmal (within 1.5 μm of the plasma membrane) and ~2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was ~4-and ~14-fold greater than that in the extraperinuclear and sub-plasmalemmal regions, and ~2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with αlgp120 labelling within the perinuclear region was ~2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between αlgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30μM) abolished global Ca 2+ waves but not Ca 2+ bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca 2+ signalling by NAADP.

Early detection of an O 2 deficit in the bloodstream is essential to initiate corrective changes in the breathing pattern of mammals. Carotid bodies serve an essential role in this respect; their type I cells depolarize when O 2 levels fall, causing voltage-gated Ca 2؉ entry. Subsequent neurosecretion elicits increased afferent chemosensory fiber discharge to induce appropriate changes in respiratory function (1). Although depolarization of type I cells by hypoxia is known to arise from K ؉ channel inhibition, the identity of the signaling pathway has been contested, and the coupling mechanism is unknown (2). We tested the hypothesis that AMP-activated protein kinase (AMPK) is the effector of hypoxic chemotransduction. AMPK is co-localized at the plasma membrane of type I cells with O 2 -sensitive K ؉ channels. In isolated type I cells, activation of AMPK using 5-aminoimidazole-4-carboxamide riboside (AICAR) inhibited O 2 -sensitive K ؉ currents (carried by large conductance Ca 2؉ -activated (BK Ca ) channels and TASK (tandem pore, acidsensing potassium channel)-like channels, leading to plasma membrane depolarization, Ca 2؉ influx, and increased chemosensory fiber discharge. Conversely, the AMPK antagonist compound C reversed the effects of hypoxia and AICAR on type I cell and carotid body activation. These results suggest that AMPK activation is both sufficient and necessary for the effects of hypoxia. Furthermore, AMPK activation inhibited currents carried by recombinant BK Ca channels, whereas purified AMPK phosphorylated the ␣ subunit of the channel in immunoprecipitates, an effect that was stimulated by AMP and inhibited by compound C. Our findings demonstrate a central role for AMPK in stimulus-response coupling by hypoxia and identify for the first time a link between metabolic stress and ion channel regulation in an O 2 -sensing system.Chronic and intermittent deficits in O 2 supply to the body precipitate a variety of pathologies including dementia (3) and pulmonary hypertension (4). To develop effective therapies, it is necessary to understand the homeostatic mechanisms that monitor O 2 supply to the body and elicit corrective changes in respiratory and circulatory function to maintain O 2 levels. O 2 -sensitive ion channels, which were first identified in the carotid body type I cell, play a pivotal role in this respect and have now been reported in a diverse range of highly specialized O 2 -sensing tissues (5). Within the carotid body, clusters of type I cells lie in presynaptic contact with afferent sensory fibers, whose discharge increases in proportion to the degree of systemic arterial O 2 deficit, providing information concerning blood O 2 levels to the central respiratory centers (1, 2). This occurs subsequent to hypoxic inhibition of type I cell K ϩ channels, membrane depolarization, voltage-gated Ca 2ϩ influx (6), and consequent neurotransmitter release. For many years, there has existed compelling evidence that mitochondria serve an important role in O 2 sensing by type I cells (2, 7). Inde...

Voltage-dependent calcium channels and currents in native neurons and other cells have been divided into high voltage activated (HVA) and low voltage activated (LVA) (Carbone & Lux, 1984;Nowycky et al. 1985). LVA currents can be distinguished by their activation at smaller depolarizations, near to the resting potential, and by their rapid inactivation (Huguenard, 1996). At the single channel level, channels with a small unitary conductance activate over the same voltage range (Carbone & Lux, 1984). Native T-type channels are heterogeneous (Kobrinsky et al. 1994;Huguenard, 1996), suggesting that they comprise more than one subtype of channel. A new subfamily of voltage-dependent calcium channel á1 subunit genes (comprising á1G, á1H and á1I) has recently been cloned, whose structure is superficially similar to the previously cloned HVA á1 subunits A, B, C, D, E and S (Perez-Reyes et al. 1998;Lee et al. 1999), having four domains, each with a voltage sensor and a pore-forming P loop. However, there are a number of regions where the homology is very low, particularly in the intracellular linkers and the N and C termini. These novel channels, when expressed, form rapidly inactivating LVA currents that also have a small single channel conductance and slowly deactivating tail currents like native T-type currents (Carbone & Lux, 1984;Armstrong & Matteson, 1985). Recently, using an antisense approach, evidence has been obtained that T-type currents in primary sensory neurons are generated by the á1G, H and I family (Lambert et al. 1998). The HVA channels are all thought to form heteromeric channels with the accessory subunits á2-ä, â and possibly ã. The accessory subunits, particularly â subunits, have marked effects on the assembly of functional channels at the plasma membrane. In expression systems, the â subunits increase the number of plasma membrane channels (Chien et al. 1995;Shistik et al. 1995;Brice et al. 1997) and also affect the voltage dependence and kinetics of activation and inactivation (Jones et al. 1998). Inactivation kinetics are

Key pointsr Hypoglycaemia is counteracted by release of hormones and an increase in ventilation and CO 2 sensitivity to restore blood glucose levels and prevent a fall in blood pH.r The full counter-regulatory response and an appropriate increase in ventilation is dependent on carotid body stimulation.r We show that the hypoglycaemia-induced increase in ventilation and CO 2 sensitivity is abolished by preventing adrenaline release or blocking its receptors.r Physiological levels of adrenaline mimicked the effect of hypoglycaemia on ventilation and CO 2 sensitivity.r These results suggest that adrenaline, rather than low glucose, is an adequate stimulus for the carotid body-mediated changes in ventilation and CO 2 sensitivity during hypoglycaemia to prevent a serious acidosis in poorly controlled diabetes.Abstract Hypoglycaemia in vivo induces a counter-regulatory response that involves the release of hormones to restore blood glucose levels. Concomitantly, hypoglycaemia evokes a carotid body-mediated hyperpnoea that maintains arterial CO 2 levels and prevents respiratory acidosis in the face of increased metabolism. It is unclear whether the carotid body is directly stimulated by low glucose or by a counter-regulatory hormone such as adrenaline. Minute ventilation was recorded during infusion of insulin-induced hypoglycaemia (8-17 mIU kg −1 min −1 ) in Alfaxan-anaesthetised male Wistar rats. Hypoglycaemia significantly augmented minute ventilation (123 ± 4 to 143 ± 7 ml min −1 ) and CO 2 sensitivity (3.3 ± 0.3 to 4.4 ± 0.4 ml min −1 mmHg −1 ). These effects were abolished by either β-adrenoreceptor blockade with propranolol or adrenalectomy. In this hypermetabolic, hypoglycaemic state, propranolol stimulated a rise in P aCO 2 , suggestive of a ventilation-metabolism mismatch. Infusion of adrenaline (1 μg kg −1 min −1 ) increased minute ventilation (145 ± 4 to 173 ± 5 ml min −1 ) without altering P aCO 2 or pH and enhanced ventilatory CO 2 sensitivity (3.4 ± 0.4 to 5.1 ± 0.8 ml min −1 mmHg −1 ). These effects were attenuated by either resection of the carotid sinus nerve or propranolol. Physiological concentrations of adrenaline increased the CO 2 sensitivity of freshly dissociated carotid body type I cells in vitro. These findings suggest that adrenaline release can account for the ventilatory hyperpnoea observed during hypoglycaemia by an augmented carotid body and whole body ventilatory CO 2 sensitivity.

Carotid body-mediated ventilatory increases in response to acute hypoxia are attenuated in animals reared in an hypoxic environment. Normally, 02-sensitive K+ channels in neurosecretory type I carotid body cells are intimately involved in excitation of the intact organ by hypoxia. We have therefore studied K+ channels and their sensitivity to acute hypoxia The ventilatory responses to acute hypoxia of animals and humans change dramatically from fetal to adult life: in fetal animals, exposure to hypoxia is inhibitory to breathing movements (1), whereas in the adult, hypoxia causes a sustained increase in ventilation (2). Neonatal animals produce an intermediate biphasic response, with ventilation increasing and then falling again during sustained hypoxia (3). This transient increase, along with the sustained increase seen in adults, is a result of stimulation of peripheral chemoreceptors, primarily the carotid body (4, 5). Ventilatory responses to acute hypoxia of neonatal animals born and raised in hypoxic environments are blunted or absent (6), and a similar lack of ventilatory response to hypoxia has also been noted in adult animals exposed to hypoxia chronically (7) and in high-altitude residents (8). It is conceivable that common mechanisms underlie the lack of ventilatory response to hypoxia of chronically hypoxic neonatal animals and high-altitude residents.The carotid bodies of chronically hypoxic animals or humans show dramatic morphological changes following prolonged hypoxia: most notably, type I carotid body cellsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. undergo hyperplasia and hypertrophy (9, 10). Type I cells are widely accepted as the chemosensory element of the carotid body, and various stimuli including hypoxia stimulate Ca2+-dependent release of neurotransmitters from these cells in a manner that correlates with increased discharge of afferent chemosensory fibers (5, 11). In recent years, several groups have used patchclamp techniques to investigate ion channels in type I cells, and there are several reports describing 02-sensitive K+ channels in these cells (12)(13)(14)(15)(16). These findings have given rise to a proposed mechanism for hypoxic chemotransduction in which inhibition of K+ channels by hypoxia leads to depolarization and increased excitability of type I cells sufficient to activate voltage-gated Ca2+ channels. This leads to Ca2+ influx and triggering of neurosecretion, an essential step in the chemotransductive pathway (5). Here we have compared ionic channels and their modulation by acute hypoxia in type I cells isolated from neonatal rats born and raised in normoxia and hypoxia in order to investigate whether the lack of chemoreceptor-mediated increases in ventilation seen in animals reared under chronically hypoxic conditions can be attributed to altered electrophysiological properties of type I cells. ...