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 ␣12␥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 ...
Previous studies on pulmonary arterial smooth muscle cells have shown that nicotinic acid adenine dinucleotide phosphate (NAADP) evokes highly localized intracellular Ca 2؉ signals by mobilizing thapsigargininsensitive stores. Such localized Ca 2؉ signals may initiate global Ca 2؉ waves and contraction of the myocytes through the recruitment of ryanodine receptors on the sarcoplasmic reticulum via Ca 2؉ -induced Ca 2؉ release. Here we show that NAADP evokes localized Ca 2؉ signals by mobilizing a bafilomycin A1-sensitive, lysosome-related Ca 2؉ store. These lysosomal stores facilitate this process by co-localizing with a portion of the sarcoplasmic reticulum expressing ryanodine receptors to comprise a highly specialized trigger zone for NAADPdependent Ca 2؉ signaling by the vasoconstrictor hormone, endothelin-1. These findings further advance our understanding of how the spatial organization of discrete, organellar Ca 2؉ stores may underpin the generation of differential Ca 2؉ signaling patterns by different Ca 2؉ -mobilizing messengers.
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.
In pulmonary arterial smooth muscle, Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) may induce constriction and dilation in a manner that is not mutually exclusive. We show here that the targeting of different sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and RyR subtypes to discrete SR regions explains this paradox. Western blots identified protein bands for SERCA2a and SERCA2b, whereas immunofluorescence labeling of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA2a and SERCA2b and RyR1, RyR2, and RyR3, respectively. Almost all SERCA2a and RyR3 labeling was restricted to a region within 1.5 μm of the nucleus. In marked contrast, SERCA2b labeling was primarily found within 1.5 μm of the plasma membrane, where labeling for RyR1 was maximal. The majority of labeling for RyR2 lay in between these two regions of the cell. Application of the vasoconstrictor endothelin-1 induced global Ca2+ waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca2+ stores by preincubation of cells with the SERCA inhibitor thapsigargin but remained unaffected after preincubation of cells with a second SERCA antagonist, cyclopiazonic acid. We conclude that functionally segregated SR Ca2+ stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca2+ via SERCA2b, and likely releases Ca2+ via RyR1 to mediate vasodilation. The other is located centrally, receives Ca2+ via SERCA2a, and likely releases Ca2+ via RyR3 and RyR2 to initiate vasoconstriction.
In artery smooth muscle, adenylyl cyclase-coupled receptors such as -adrenoceptors evoke Ca 2؉ signals, which open Ca 2؉ -activated potassium (BK Ca ) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca 2؉ signal is evoked via ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to the plasma membrane. We show here that cyclic adenosine diphosphate-ribose (cADPR), by activating RyRs, mediates, in part, hyperpolarization and vasodilation by -adrenoceptors. Thus, intracellular dialysis of cADPR increased the cytoplasmic Ca 2؉ concentration proximal to the plasma membrane in isolated arterial smooth muscle cells and induced a concomitant membrane hyperpolarization. Smooth muscle hyperpolarization mediated by cADPR, by -adrenoceptors, and by cAMP, respectively, was abolished by chelating intracellular Ca 2؉ and by blocking RyRs, cADPR, and BK Ca channels with ryanodine, 8-amino-cADPR, and iberiotoxin, respectively. The cAMP-dependent protein kinase A antagonist N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP, respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a downstream element in this signaling cascade. Importantly, antagonists of cADPR and BK Ca channels, respectively, inhibited -adrenoreceptorinduced artery dilation. We conclude, therefore, that relaxation of arterial smooth muscle by adenylyl cyclase-coupled receptors results, in part, from a cAMP-dependent and protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca 2؉ release via RyRs, which leads to activation of BK Ca channels and membrane hyperpolarization.
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