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The high mortality rate in patients with chronic obstructive pulmonary disease (COPD) may be due to pulmonary hypertension (PH). These diseases are highly associated with cigarette smoke and its key component nicotine. Here, we created a novel animal model of PH using coexposure to nicotine (or cigarette smoke) and hypoxia. This heretofore unreported model showed significant early-onset pulmonary vasoremodeling and PH. Using newly generated mice with complementary smooth muscle–specific Rieske iron-sulfur protein (RISP) gene knockout and overexpression, we demonstrate that RISP is critically involved in promoting pulmonary vasoremodeling and PH, which are implemented by oxidative ataxia telangiectasia-mutated–mediated DNA damage and NF-κB–dependent inflammation in a reciprocal positive mechanism. Together, our findings establish for the first time an animal model of hypoxia-induced early-onset PH in which mitochondrial RISP-dependent DNA damage and NF-κB inflammation play critical roles in vasoremodeling. Specific therapeutic targets for RISP and related oxidative stress–associated signaling pathways may create unique and effective treatments for PH, chronic obstructive pulmonary disease, and their complications. [ABSTRACT FROM AUTHOR]

Mitochondria are essential organelles for energy production, calcium homeostasis, redox signaling, and other cellular responses involved in pulmonary vascular biology and disease processes. Mitochondrial homeostasis depends on a balance in mitochondrial fusion and fission (dynamics). Mitochondrial dynamics are regulated by a viable circadian clock. Hypoxia and nicotine exposure can cause dysfunctions in mitochondrial dynamics, increases in mitochondrial reactive oxygen species generation and calcium concentration, and decreases in ATP production. These mitochondrial changes contribute significantly to pulmonary vascular oxidative stress, inflammatory responses, contractile dysfunction, pathologic remodeling, and eventually pulmonary hypertension. In this review article, therefore, we primarily summarize recent advances in basic, translational, and clinical studies of circadian roles in mitochondrial metabolism in the pulmonary vasculature. This knowledge may not only be crucial to fully understanding the development of pulmonary hypertension, but also greatly help to create new therapeutic strategies for treating this devastating disease and other related pulmonary disorders. [ABSTRACT FROM AUTHOR]

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitterand hypoxia-induced [Ca.sup.2+] release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors ([IP.sub.3]Rs) with noradrenaline induced eqnivalent increases in [[Ca.sup.2+]]i and [Ca.sup.2+-] activated [C1.sup.-] currents in freshly isolated rat PASMCs. Following maximal [IP.sub.3]-induced Ca9+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked [IP.sub.3]-induced [Ca.sup.2+] release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [[Ca.sup.2+]]i in the presence and absence of extracellular [Cd.sup.2+]. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced [Ca.sup.2+] release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced [Ca.sup.2-] release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluoreseence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout ([RyR3.sup.-/-]) mice, hypoxia-induced, but not submaximal noradrenaline-induced, [Ca.sup.2+] release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [[[Ca.sup.2+]].sub.i] in [RyR3.sup. -/-] mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced [Ca.sup.2+] release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced [Ca.sup.2+] and contractile responses in PASMCs. KEY WORDS: ryanodine receptor * inositol triphosphate receptor * hypoxia * neurotransmitter * pulmonary artery smooth muscle cell

Intracellular [Ca.sup.2+] release through ryanodine receptors (RyRs) plays important roles in smooth muscle excitation-contraction coupling, but the underlying regulatory mechanisms are poorly understood. Here we show that FK506 binding protein of 12.6 kDa (FKBP12.6) associates with and regulates type 2 RyRs (RyR2) in tracheal smooth muscle. FKBP12.6 binds to RyR2 but not other RyR or inositol 1,4,5-trisphosphate receptors, and FKBP12, known to bind to and modulate skeletal RyRs, does not associate with RyR2. When dialyzed into tracheal myocytes, cyclic ADP-ribose (cADPR) alters spontaneous [Ca.sup.2+] release at lower concentrations and produces macroscopic [Ca.sup.2+] release at higher concentrations; neurotransmitter-evoked [Ca.sup.2+] release is also augmented by cADPR. These actions are mediated through FKBP12.6 because they are inhibited by molar excess of recombinant FKBP12.6 and are not observed in myocytes from FKBP12.6-knockout mice. We also report that force development in FKBP12.6-null mice, observed as a decrease in the concentration/tension relationship of isolated trachealis segments, is impaired. Taken together, these findings point to an important role of the FKBP12.6/RyR2 complex in stochastic (spontaneous) and receptor-mediated [Ca.sup.2+] release in smooth muscle. FK506 binding protein 12.6; ryanodine receptor type 2; calcium sparks; calcium-activated chloride currents

We examined the effects of metabolic inhibition on intracellular [Ca.sup.2+] release in single pulmonary arterial smooth muscle cells (PASMCs). Severe metabolic inhibition with cyanide (CN, 10 mM) increased intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) and activated [Ca.sup.2+]-activated [Cl.sup.-] currents [[I.sub.Cl(Ca)]] in PASMCs, responses that were greatly inhibited by BAPTA-AM or caffeine. Mild metabolic inhibition with CN (1 mM) increased spontaneous transient inward currents and [Ca.sup.2+] sparks in PASMCs. In Xenopus oocytes, CN also induced [Ca.sup.2+] release and activated [I.sub.Cl(Ca)], and these responses were inhibited by thapsigargin and cyclopiazonic acid to deplete sarcoplasmic reticulum (SR) [Ca.sup.2+], whereas neither heparin nor anti-inositol 1,4,5-trisphosphate receptor (I[P.sub.3]R) antibodies affected CN responses. In both PASMCs and oocytes, CN-evoked [Ca.sup.2+] release was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin or CCCP and thapsigargin. Whereas hypoxic stimuli resulted in [Ca.sup.2+] release in pulmonary but not mesenteric artery myocytes, CN induced release in both cell types. We conclude that metabolic inhibition with CN increases [[[Ca.sup.2+]].sub.i] in both pulmonary and systemic artery myocytes by stimulating [Ca.sup.2+] release from the SR and mitochondria. sarcoplasmic reticulum; mitochondria