Nicholas P. Kinnear

Does AMP-activated Protein Kinase Couple Inhibition of Mitochondrial Oxidative Phosphorylation by Hypoxia to Calcium Signaling in O2-sensing Cells?

Specialized O2-sensing cells exhibit a particularly low threshold to regulation by O2 supply and function to maintain arterial pO2 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 O2-sensing cells, thereby mediating, in part, cell activation. However, the mechanism by which this process couples to Ca2+ 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 Ca2+ signals in O2-sensing cells. Co-immunoprecipitation identified three possible AMP-activated protein kinase subunit isoform combinations in pulmonary arterial myocytes, with α1β2γ1 predominant. Furthermore, their tissue-specific distribution suggested that the AMP-activated protein kinase-α1 catalytic isoform may contribute, via amplification of the metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMP-activated protein kinase-α1 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 AMP-activated protein kinase by phenformin or 5-aminoimidazole-4-carboxamide-riboside elicited discrete Ca2+ signaling mechanisms in each cell type, namely cyclic ADP-ribose-dependent Ca2+ mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial myocytes and transmembrane Ca2+ influx into carotid body glomus cells. Thus, metabolic sensing by AMP-activated protein kinase may mediate chemotransduction by hypoxia.

Identification of Functionally Segregated Sarcoplasmic Reticulum Calcium Stores in Pulmonary Arterial Smooth Muscle

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.

The SMN Protein is a Key Regulator of Nuclear Architecture in Differentiating Neuroblastoma Cells

The cell nucleus contains two closely related structures, Cajal bodies (CBs) and gems. CBs are the first site of accumulation of newly assembled splicing snRNPs (small nuclear ribonucleoproteins) following their import into the nucleus, before they form their steady‐state localization in nuclear splicing speckles. Gems are the nuclear site of accumulation of survival motor neurons (SMNs), an insufficiency of which leads to the inherited neurodegenerative condition, spinal muscular atrophy (SMA). SMN is required in the cytoplasm for the addition of core, Sm, proteins to new snRNPs and is believed to accompany snRNPs to the CB. In most cell lines, gems are indistinguishable from CBs, although the structures are often separate in vivo. The relationship between CBs and gems is not fully understood, but there is evidence that symmetrical dimethylation of arginine residues in the CB protein coilin brings them together in HeLa cells. During neuronal differentiation of the human neuroblastoma cell line SH‐SY5Y, CBs and gems increase their colocalization, mimicking changes seen during foetal development. This does not result from alterations in the methylation of coilin, but from increased levels of SMN. Expression of exogenous SMN results in an increased efficiency of snRNP transport to nuclear speckles. This suggests different mechanisms are present in different cell types and in vivo that may be significant for the tissue‐specific pathology of SMA.

Does AMP-activated Protein Kinase Couple Inhibition of Mitochondrial Oxidative Phosphorylation by Hypoxia to Pulmonary Artery Constriction?

Pulmonary arteries constricts in response to hypoxia and thereby aid ventilation-perfusion matching in the lung. Although O2-sensitive mechanisms independent of mitochondria may also play a role, it is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation and that this underpins, at least in part, cell activation. Despite this consensus, the mechanism by which inhibition of mitochondrial oxidative phosphorylation couples to Ca-dependent vasoconstriction has remained elusive. To date, the field has focussed on the role of the cellular energy status (ATP), reduced redox couples and reactive oxygen species, respectively, but investigation of these hypotheses has delivered conflicting data and failed to unite the field. Recently, the AMPK cascade has come to prominence as a sensor of metabolic stress that appears to be ubiquitous throughout eukaryotes. AMPK complexes are heterotrimers comprising a catalytic subunit and regulatory and subunits, which monitor the cellular AMP/ATP ratio as an index of metabolic stress. Binding of AMP to two sites in the subunits triggers activation of the kinase via phosphorylation of the subunit at Thr-172, an effect antagonized by high concentrations of ATP. This phosphorylation is catalyzed by upstream kinases (AMPK kinases) the major form of which is a complex between the tumour suppressor kinase, LKB1, and two accessory subunits, STRAD and MO25. Given that inhibition of mitochondrial oxidative phosphorylation by hypoxia would be expected to promote a rise in the AMP/ATP ratio we considered the proposal that AMPK activation may mediate, in part, pulmonary artery constriction by hypoxia.