Cellular redox change regulates pulmonary vascular tone by affecting function of membrane and cytoplasmic proteins, enzymes, and second messengers. This study was designed to test the hypothesis that functional modulation of ion channels by thiol oxidation contributes to regulation of excitation-contraction coupling in isolated pulmonary artery (PA) rings. Acute treatment with the thiol oxidant diamide produced a dose-dependent relaxation in PA rings; the IC50 was 335 and 58 microM for 40 mM K+ - and 2 microM phenylephrine-induced PA contraction, respectively. The diamide-mediated pulmonary vasodilation was affected by neither functional removal of endothelium nor 8-bromoguanosine-3'-5'-cyclic monophosphate (50 microM) and HA-1004 (30 microM). A rise in extracellular K+ concentration (from 20 to 80 mM) attenuated the thiol oxidant-induced PA relaxation. Passive store depletion by cyclopiazonic acid (50 microM) and active store depletion by phenylephrine (in the absence of external Ca2+ both induced PA contraction due to capacitative Ca2+ entry. Thiol oxidation by diamide significantly attenuated capacitative Ca2+ entry-induced PA contraction due to active and passive store depletion. The PA rings isolated from left and right PA branches appeared to respond differently to store depletion. Although the active tension induced by passive store depletion was comparable, the active tension induced by active store depletion was 3.5-fold greater in right branches than in left branches. These data indicate that thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels, which subsequently attenuate Ca2+ influx and decrease cytosolic free Ca2+ concentration in pulmonary artery smooth muscle cells. The mechanisms involved in thiol oxidation-mediated pulmonary vasodilation or activation of K+ channels and inhibition of store-operated Ca2+ channels appear to be independent of functional endothelium and of the cGMP-dependent protein kinase pathway.

In recent years, transgenic mouse models have been developed to examine the underlying cellular and molecular mechanisms of lung disease and pulmonary vascular disease, such as asthma, pulmonary thromboembolic disease, and pulmonary hypertension. However, there has not been systematic characterization of the basic physiological pulmonary vascular reactivity in normal and transgenic mice. This represents an intellectual "gap", since it is important to characterize basic murine pulmonary vascular reactivity in response to various contractile and relaxant factors to which the pulmonary vasculature is exposed under physiological conditions. The present study evaluates excitation-and pharmacomechanical-contraction coupling in pulmonary arteries (PA) isolated from wild-type BALB/c mice. We demonstrate that both pharmacoand electromechanical coupling mechanisms exist in mice PA. These arteries are also reactive to stimulation by ␣ 1-adrenergic agonists, serotonin, endothelin-1, vasopressin, and U-46619 (a thromboxane A 2 analog). We conclude that the basic vascular responsiveness of mouse PA is similar to those observed in PA of other species, including rat, pig, and human, albeit on a different scale and to varying amplitudes. pharmacology; store depletion; excitation-contraction coupling; G protein-coupled receptors MICE, ESPECIALLY TRANSGENIC or knockout variants, are increasingly being used to study disease mechanisms. With respect to pulmonary hypertension, transgenic or knockout mouse models have been developed to evaluate the modulation of pulmonary vasoconstriction and remodeling due to 1) endothelial nitric oxide synthase disruption (18,47,57); 2) vasoactive and mitogenic agonists, such as hypoxia-inducible factors-1␣ (63) and -2␣ (2), calcitonin gene-related peptide (5), serotonin (5-HT) (9,32,34,45), matrix metalloproteinases (67), transforming growth factor-␤ (35), and vasoactive intestinal peptide (51); 3) bone morphogenetic protein receptor type II gene mutations and altered bone morphogenetic protein signaling (19,28,40,62); 4) altered function of ion channels (12) and transporters (49); and 5) altered superoxide production (36,38). Despite these studies, there is still relatively little information regarding the basic characterization of the pulmonary vascular reactivity in normal mice. Nor have the excitationcontraction coupling mechanisms, pharmacological properties of vasoactive response to agonists, been evaluated completely in wild-type mice. This represents an intellectual gap, since it is important to characterize basic murine pulmonary vascular reactivity in response to various contractile and relaxant factors to which the pulmonary vasculature is exposed under physiological conditions. The aim of this study was to characterize pulmonary arterial (PA) excitation-contraction coupling and pharmacological properties of PA using isolated PA rings from mice. METHODS AND MATERIALSIsolation of PA rings. Male 5-to 8-wk-old BALB/c mice were used in this study. Use of mice for the experiments presented in...

Sustained pulmonary vasoconstriction contributes to the elevated pulmonary vascular resistance observed in pulmonary arterial hypertension. A rise in cytosolic Ca 2+ in pulmonary artery smooth muscle cells (PASMCs) is major trigger for pulmonary vasoconstriction. One family of drugs currently being pursued as a potential treatment for pulmonary hypertension are the statins, which act by depleting cholesterol and reducing the number of caveolae. This study aimed at investigating the role of caveolae, membrane receptors, and ion channels (that are potentially located in the caveolae) in agonist-mediated pulmonary vasoconstriction in order to gain a greater understanding of the signaling mechanisms involved in the regulation of pulmonary vascular tone. Chronic treatment of PASMCs with the cholesterol-depleting agent, methyl-β-cyclodextrin (MβCD), significantly reduced the number of cholesterol rich caveolae regions in the membrane. This disruption of cholesterol in caveolae significantly inhibited pharmacomechanical (induced by phenylephrine), but not electromechanical (induced by elevated extracellular potassium concentration), rat pulmonary artery contraction. These results indicate that receptors may Address correspondence to Jason X.J. Yuan, MD, PhD, Division of Pulmonary and Critical Care Medicine, Department of Medicine, MC 0725, University of California, San Diego, 9100 Gilman Drive, LaJolla, CA 92093-0725, USA. xiyuan@ucsd.edu. The current address of Christian Schach is Clinic for Internal Medicine II, University of Regensburg, Regensburg, Germany. In pulmonary vascular smooth muscle and endothelial cells, caveo-lae and the signaling cascades localized in the caveolae may have differential effects on pulmonary vascular tone. In this study, we investigated the role of caveolae in electromechanical and pharmacomechanical coupling mechanisms involved in regulating pulmonary vasoconstriction and vasodilation. HHS Public Access Methods and Materials Tension Measurement in Isolated Pulmonary Artery RingsPulmonary arteries, 2nd to 3rd divisions with an internal diameter of ∼400 μm, were isolated from male Sprague-Dawley rats (100 to 250 g) in accordance with the animal use protocol approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. Adipose and connective tissues were carefully removed, and the remaining arteries were cut into 1 to 2 mm long rings and mounted on stainless steel wire (100 μm in diameter) to a force transducer (Harvard Apparatus, Holliston, MA) in an organ bath (the volume is approximately 0.75 mL). Isometric tension was continuously monitored and recorded using DATAQ data acquisition software (DATAQInstruments). Isolated PA rings were superfused (2 to 2.5 mL/min) with modified Krebs solution (MKS; at 37°C) consisting of (in mM): 138 NaCl, 1.8 CaCl 2 , 4.7 KCl, 1.2 MgSO 4 , 1.2 NaH 2 PO 4 , 5 HEPES, and 10 glucose (pH 7.4, with 2 M NaOH). For Ca 2+ -free (0 Ca) MKS, CaCl 2 was replaced by equimolar MgCl 2 and 1 mM EGTA was added to chelate ...

JXJ. p75 neurotrophin receptor regulates agonist-induced pulmonary vasoconstriction. Am J Physiol Heart Circ Physiol 295: H1529-H1538, 2008. First published August 8, 2008 doi:10.1152/ajpheart.00115.2008.-A member of the TNF receptor family, the p75 neurotrophin receptor (p75 NTR ) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of p75 NTR in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of p75 NTR in mouse pulmonary arteries and the putative role of p75 NTR in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and p75 NTR knockout (p75 Ϫ/Ϫ ) mice. Our data indicated that p75 NTR is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery rings from p75 Ϫ/Ϫ mice exhibited significantly elevated active tension due to endothelin-1-mediated Ca 2ϩ influx. Furthermore, the contraction due to capacitative Ca 2ϩ entry (CCE) in response to phenylephrine-mediated active depletion of intracellular Ca 2ϩ stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and p75 Ϫ/Ϫ rings. Active tension induced by serotonin, U-46619 (a thromboxane A 2 analog), thrombin, 4-aminopyridine (a K ϩ channel blocker), and high extracellular K ϩ in p75 Ϫ/Ϫ rings was similar to that in WT rings. Deletion of p75 NTR did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact p75 NTR signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion. mouse; pulmonary artery; pharmacology; vasoconstriction; store depletion NEUROTROPHINS are unique growth factors that interact with two separate types of transmembrane receptors: tropomyosin receptor kinases (Trk) and the p75 neurotrophin receptor (p75 NTR ), with the latter being a member of the TNF superfamily of membrane receptors (38). A wealth of in vitro experiments has shown that neurotrophins and pro-neurotrophins can induce apoptosis via p75 NTR (15). In vivo, p75 NTR expression and induction are associated with increased apoptosis in vascular smooth muscle cells (SMCs) from atherosclerotic lesions (15, 41) as well as in promoting apoptosis in oligodendrocytes and neurons in injury models in the nervous system (1, 2, 26). Conversely, Chu et al. (9)