The cell-wide web coordinates cellular processes by directing site-specific Ca2+ flux across cytoplasmic nanocourses.

Ca2+ coordinates diverse cellular processes, yet how function-specific signals arise is enigmatic. We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca2+ diffusion and by nanocourse-specific Ca2+-pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca2+ flux through RyR2/3 clusters selects for rapid propagation of Ca2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.

Identification of functionally segregated sarcoplasmic reticulum calcium stores in pulmonary arterial smooth muscle.

In pulmonary arterial smooth muscle, Ca(2+) 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 Ca(2+)-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 microm of the nucleus. In marked contrast, SERCA2b labeling was primarily found within 1.5 microm 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 Ca(2+) waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca(2+) 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 Ca(2+) stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca(2+) via SERCA2b, and likely releases Ca(2+) via RyR1 to mediate vasodilation. The other is located centrally, receives Ca(2+) via SERCA2a, and likely releases Ca(2+) via RyR3 and RyR2 to initiate vasoconstriction.

AMP-activated protein kinase and hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction is a vital homeostatic mechanism that aids ventilation-perfusion matching in the lung, for which the underlying mechanism(s) remains controversial. However, our most recent investigations strongly suggest that hypoxic pulmonary vasoconstriction is precipitated, at least in part, by the inhibition of mitochondrial oxidative phosphorylation by hypoxia, an increase in the AMP/ATP ratio and consequent activation of AMP-activated protein kinase (AMPK). Unfortunately, these studies lacked the definitive proof that can only be provided by selectively blocking AMPK-dependent signalling cascades. The aim of the present study was, therefore, to determine the effects of the AMPK inhibitor compound C upon: (1) phosphorylation in response to hypoxia of a classical AMPK substrate, acetyl CoA carboxylase, in rat pulmonary arterial smooth muscle and (2) hypoxic pulmonary vasoconstriction in rat isolated intrapulmonary arteries. Acetyl CoA carboxylase phosphorylation was increased approximately 3 fold in the presence of hypoxia (pO(2) = 16-21 mm Hg, 1 h) and 5-aminoimidazole-4-carboxamide riboside (AICAR; 1 mM; 4 h) and in a manner that was significantly attenuated by the AMPK antagonist compound C (40 microM). Most importantly, pre-incubation of intrapulmonary arteries with compound C (40 microM) inhibited phase II, but not phase I, of hypoxic pulmonary vasoconstriction. Likewise, compound C (40 microM) inhibited constriction by AICAR (1 mM). The results of the present study are consistent with the activation of AMPK being a key event in the initiation of the contractile response of pulmonary arteries to acute hypoxia.

Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle.

In 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) alphalgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5mum of the nucleus) was approximately 4-fold greater than in the sub-plasmalemmal (within 1.5mum of the plasma membrane) and approximately 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was approximately 4- and approximately 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and approximately 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 alphalgp120 labelling within the perinuclear region was approximately 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alphalgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30muM) 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.

Pyridine nucleotides and calcium signalling in arterial smooth muscle: from cell physiology to pharmacology.

It is generally accepted that the mobilisation of intracellular Ca2+ stores plays a pivotal role in the regulation of arterial smooth muscle function, paradoxically during both contraction and relaxation. However, the spatiotemporal pattern of different Ca2+ signals that elicit such responses may also contribute to the regulation of, for example, differential gene expression. These findings, among others, demonstrate the importance of discrete spatiotemporal Ca2+ signalling patterns and the mechanisms that underpin them. Of fundamental importance in this respect is the realisation that different Ca2+ storing organelles may be selected by the discrete or coordinated actions of multiple Ca2+ mobilising messengers. When considering such messengers, it is generally accepted that sarcoplasmic reticulum (SR) stores may be mobilised by the ubiquitous messenger inositol 1,4,5 trisphosphate. However, relatively little attention has been paid to the role of Ca2+ mobilising pyridine nucleotides in arterial smooth muscle, namely, cyclic adenosine diphosphate-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review will therefore focus on these novel mechanisms of calcium signalling and their likely therapeutic potential.