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 ...

The hypoxic constriction of isolated pulmonary vessels is composed of an initial transient phase (phase 1) followed by a slowly developing increase in tone (phase 2). We investigated the roles of the endothelium and of intracellular Ca2+ stores in both preconstricted and unpreconstricted intrapulmonary rabbit arteries when challenged with hypoxia (PO2 16-21 Torr). Removing the endothelium did not affect phase 1, but phase 2 appeared as a steady plateau. Removing extracellular Ca2+ had essentially the same effect as removing the endothelium. Depletion of sarcoplasmic reticulum Ca2+ stores with caffeine and ryanodine abolished the hypoxic response. Omitting preconstriction reduced the amplitude of the hypoxic response but did not qualitatively affect any of the above responses. We conclude that hypoxia releases intracellular Ca2+ from ryanodine-sensitive stores by a mechanism intrinsic to pulmonary vascular smooth muscle without the need for Ca2+ influx across the plasmalemma or an endothelial factor. Our results also suggest that extracellular Ca2+ is required for the release of an endothelium-derived vasoconstrictor.

We have examined the eects of Y-27632, a speci®c inhibitor of Rho-activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y-27632 (100 nM ± 3 mM) was found to cause a concentration-dependent inhibition of acute sustained HPV in rat IPA. Application of Y-27632 (10 ± 600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration-dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y-27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho-associated kinase may be a pivotal step in the generation of sustained HPV.

Abstract-Hypoxic pulmonary vasoconstriction (HPV) is unique to pulmonary arteries, and it aids ventilation/perfusion matching. However, in diseases such as emphysema, HPV can promote hypoxic pulmonary hypertension. We recently showed that hypoxia constricts pulmonary arteries in part by increasing cyclic ADP-ribose (cADPR) accumulation in the smooth muscle and, thereby, Ca 2ϩ release by ryanodine receptors. We now report on the role of cADPR in HPV in isolated rat pulmonary arteries and in the rat lung in situ. In isolated pulmonary arteries, the membrane-permeant cADPR antagonist, 8-bromo-cADPR, blocked sustained HPV by blocking Ca 2ϩ release from smooth muscle ryanodine-sensitive stores in the sarcoplasmic reticulum. Most importantly, we showed that 8-bromo-cADPR blocks HPV induced by alveolar hypoxia in the ventilated rat lung in situ. Inhibition of HPV was achieved without affecting (1) constriction by membrane depolarization and voltage-gated Ca 2ϩ influx, (2) the release (by hypoxia) of an endothelium-derived vasoconstrictor, or (3) endothelium-dependent vasoconstriction. Our findings suggest that HPV is both triggered and maintained by cADPR in the rat lung in situ. Key Words: cADP-ribose Ⅲ pulmonary artery Ⅲ hypoxia S ince it was first described, hypoxic pulmonary vasoconstriction (HPV) has been recognized as the critical and distinguishing characteristic of pulmonary arteries 1 ; systemic arteries dilate in response to hypoxia. Physiologically, HPV contributes to ventilation-perfusion matching in the lung. However, when alveolar hypoxia is global, as it is in disease states such as emphysema and cystic fibrosis, it results in pulmonary hypertension and, eventually, right heart failure. 2 In isolated pulmonary arteries, HPV is biphasic. A transient constriction (phase 1) is followed by slow tonic constriction (phase 2). It was thought that phase 1 was initiated by a reduction in membrane K ϩ conductance in the smooth muscle 3,4 and voltage-gated Ca 2ϩ influx. 5,6 In addition, the primary mediator of phase 2 of HPV was thought to be an endothelium-derived vasoconstrictor. 7,8 Contrary to this, we and others discovered that hypoxia promotes phases 1 and 2 by releasing Ca 2ϩ from ryanodine-sensitive stores in the sarcoplasmic reticulum (SR) by a mechanism intrinsic to the smooth muscle. 9 -11 Recently, we showed that the ␤-NAD ϩ metabolite cyclic ADP-ribose (cADPR), 12,13 which increases Ca 2ϩ release by ryanodine receptors, 14 plays a role in this process. Thus, hypoxia increases cADPR accumulation and SR Ca 2ϩ release in pulmonary artery smooth muscle, leading to constriction in isolated rabbit pulmonary arteries. 15 In the present investigation, we demonstrate that HPV is triggered and maintained by cADPR in isolated rat pulmonary arteries and in the rat lung in situ. Materials and Methods DissectionMale Wistar rats (250 to 350 g) were anesthetized with 4% enflurane and exsanguinated. The heart and lungs were removed and placed in chilled physiological saline solution A containing (in mmol/L): 118 NaCl...