Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2-dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.

No abstract

Alzheimer's disease is recognized post mortem by the presence of extracellular senile plaques, made primarily of aggregation of amyloid β peptide (Aβ). This peptide has consequently been regarded as the principal toxic factor in the neurodegeneration of Alzheimer's disease. As such, intense research effort has been directed at determining its source, activity and fate, primarily with a view to preventing its formation or its biological activity, or promoting its degradation. Clearly, much progress has been made concerning its formation by proteolytic processing of the amyloid precursor protein, and its degradation by enzymes such as neprilysin and insulin degrading enzyme. The activities of Aβ, however, are numerous and yet to be fully elucidated. What is currently emerging from such studies is a diffuse but steadily growing body of data that suggests Aβ has important physiological functions and, further, that it should only be regarded as toxic when its production and degradation are imbalanced. Here, we review these data and suggest that physiological levels of Aβ have important physiological roles, and may even be crucial for neuronal cell survival. Thus, the view of Aβ being a purely toxic peptide requires re-evaluation.

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 ...

Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/ threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca 2؉ currents and the recombinant ␣ 1C subunit of the human cardiac L-type Ca 2؉ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca 2؉ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.CO is an established and important signaling molecule in both the heart and vasculature as well as other tissues (1, 2). Cardiac atrial and ventricular myocytes express heme oxygenases HO-1 4 and HO-2, which generate CO along with biliverdin and free Fe 2ϩ by heme catabolism, and HO-1 levels can be increased by various stress factors (3), including myocardial infarction (4). CO limits the cellular damage of ischemia/reperfusion injury in the heart (5). Indeed, greater cardiac damage is seen following ischemia/reperfusion injury in HO-1 knock-out mice (6). Conversely, HO-1 overexpression in the heart reduces infarct size and other markers of damage following ischemia/ reperfusion injury (7). CO also improves cardiac blood supply through coronary vessel dilation (8, 9) and reduces cardiac contractility (9). However, the mechanisms underlying this cardioprotective effect of CO are not understood.In the vasculature, CO also exerts numerous beneficial effects. Its ability to dilate blood vessels is long established (9 -11) and endothelium-independent (12) and not due to development of hypoxia through displacement of O 2 (see Ref. 13). CO has clear, protective effects in various vascular diseases, such as systemic and pulmonary hypertension, development of atherosclerosis, and neointimal hyperplasia due to proliferation of vascular smooth muscle cells following vascular injury (all reviewed in Refs. 2, 13, and 14). Import...

Substance P (SP) is a prominent neuromodulator, which is produced and released by peripheral damage-sensing (nociceptive) neurons; these neurons also express SP receptors. However, the mechanisms of peripheral SP signaling are poorly understood. We report a signaling pathway of SP in nociceptive neurons: Acting predominantly through NK1 receptors and G i/o proteins, SP stimulates increased release of reactive oxygen species from the mitochondrial electron transport chain. Reactive oxygen species, functioning as second messengers, induce oxidative modification and augment M-type potassium channels, thereby suppressing excitability. This signaling cascade requires activation of phospholipase C but is largely uncoupled from the inositol 1,4,5-trisphosphate sensitive Ca 2+ stores. In rats SP causes sensitization of TRPV1 and produces thermal hyperalgesia. However, the lack of coupling between SP signaling and inositol 1,4,5-trisphosphate sensitive Ca 2+ stores, together with the augmenting effect on M channels, renders the SP pathway ineffective to excite nociceptors acutely and produce spontaneous pain. Our study describes a mechanism for neurokinin signaling in sensory neurons and provides evidence that spontaneous pain and hyperalgesia can have distinct underlying mechanisms within a single nociceptive neuron.G protein coupled receptors | inflammatory pain | intracellular signaling | KCNQ | M current E xcitation of peripheral terminals of sensory neurons is a primary event in somatosensation, including pain. A large proportion of sensory neurons (mostly TRPV1 + damage-sensing or "nociceptive" neurons) produce neuropeptides such as substance P (SP), which are released in response to nociceptive stimulation both at spinal cord synapses and in the periphery (1). In the spinal cord SP acts as an excitatory neurotransmitter or cotransmitter (2, 3), whereas peripheral SP release is thought to underlie the inflammatory response known as "neurogenic inflammation" (4). Many peripheral nociceptors also express receptors for SP [neurokinin receptors (NKR) 1-3 (NK1-3)]; this expression may suggest paracrine or autocrine actions of this peptide. However, the nature of such actions is controversial, and the molecular events triggered by NKR in peripheral nociceptors are not well established. The NKR traditionally are classed as G q/11 -coupled G protein-coupled receptors (GPCR) that activate phospholipase C (PLC), with subsequent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and release of inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG) (5, 6). Common downstream steps of G q/11 signaling include IP 3 -mediated release of Ca 2+ from intracellular stores and DAG-mediated activation of PKC. However, it was hitherto unknown whether NKR in peripheral sensory neurons couple fully to this signaling cascade, because a lack of coupling between NKR and Ca 2+ release in dorsal root ganglia (DRG) neurons has been noted (ref. 7 and 8, but cf. ref. 9). Nevertheless, it is accepted that peripherally act...

No abstract