Inhibition of hypoxia-induced calcium responses in pulmonary arterial smooth muscle by acetazolamide is independent of carbonic anhydrase inhibition

2007 ◽  
Vol 292 (4) ◽  
pp. L1002-L1012 ◽  
Author(s):  
Larissa A. Shimoda ◽  
Trevor Luke ◽  
J. T. Sylvester ◽  
Hui-Wen Shih ◽  
Ahamindra Jain ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Carbonic Anhydrase ◽  
Vascular Smooth Muscle ◽  
High Altitude ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Fluorescent Microscopy ◽  
Arterial Smooth Muscle ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle

Hypoxic pulmonary vasoconstriction (HPV) occurs with ascent to high altitude and can contribute to development of high altitude pulmonary edema (HAPE). Vascular smooth muscle contains carbonic anhydrase (CA), and acetazolamide (AZ), a CA inhibitor, blunts HPV and might be useful in the prevention of HAPE. The mechanism by which AZ impairs HPV is uncertain. Originally developed as a diuretic, AZ also has direct effects on systemic vascular smooth muscle, including modulation of pH and membrane potential; however, the effect of AZ on pulmonary arterial smooth muscle cells (PASMCs) is unknown. Since HPV requires Ca2+ influx into PASMCs and can be modulated by pH, we hypothesized that AZ alters hypoxia-induced changes in PASMC intracellular pH (pHi) or Ca2+ concentration ([Ca2+]i). Using fluorescent microscopy, we tested the effect of AZ as well as two other potent CA inhibitors, benzolamide and ethoxzolamide, which exhibit low and high membrane permeability, respectively, on hypoxia-induced responses in PASMCs. Hypoxia caused a significant increase in [Ca2+]i but no change in pHi. All three CA inhibitors slightly decreased basal pHi, but only AZ caused a concentration-dependent decrease in the [Ca2+]i response to hypoxia. AZ had no effect on the KCl-induced increase in [Ca2+]i or membrane potential. N-methyl-AZ, a synthesized compound lacking the unsubstituted sulfonamide group required for CA inhibition, had no effect on pHi but inhibited hypoxia-induced Ca2+ responses. These results suggest that AZ attenuates HPV by selectively inhibiting hypoxia-induced Ca2+ responses via a mechanism independent of CA inhibition, changes in pHi, or membrane potential.

Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry

2005 ◽  
Vol 288 (6) ◽  
pp. L1059-L1069 ◽  
Author(s):  
Jian Wang ◽  
Larissa A. Shimoda ◽  
Letitia Weigand ◽  
Wenqian Wang ◽  
Dejun Sun ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Acute Hypoxia ◽  
Primary Cultures ◽  
Fluorescent Microscopy ◽  
Pulmonary Arteries ◽  
Arterial Smooth Muscle ◽  
Fura 2 ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle

Hypoxic pulmonary vasoconstriction (HPV) requires influx of extracellular Ca2+ in pulmonary arterial smooth muscle cells (PASMCs). To determine whether capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCCs) contributes to this influx, we used fluorescent microscopy and the Ca2+-sensitive dye fura-2 to measure effects of 4% O2 on intracellular [Ca2+] ([Ca2+]i) and CCE in primary cultures of PASMCs from rat distal pulmonary arteries. In PASMCs perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels (VOCCs), hypoxia markedly enhanced both the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] and the rate at which extracellular Mn2+ quenched fura-2 fluorescence. These effects, as well as the increased [Ca2+]i caused by hypoxia in PASMCs perfused with normal salt solutions, were blocked by the SOCC antagonists SKF-96365, NiCl2, and LaCl3 at concentrations that inhibited CCE >80% but did not alter [Ca2+]i responses to 60 mM KCl. In contrast, the VOCC antagonist nifedipine inhibited [Ca2+]i responses to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca2+]i caused by hypoxia was completely reversed by perfusion with Ca2+-free KRBS. LaCl3 increased basal [Ca2+]i during normoxia, indicating effects other than inhibition of SOCCs. Our results suggest that acute hypoxia enhances CCE through SOCCs in distal PASMCs, leading to depolarization, secondary activation of VOCCs, and increased [Ca2+]i. SOCCs and CCE may play important roles in HPV.

scholarly journals Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia

2008 ◽  
Vol 295 (1) ◽  
pp. L104-L113 ◽  
Author(s):  
Wenju Lu ◽  
Jian Wang ◽  
Larissa A. Shimoda ◽  
J. T. Sylvester
Keyword(s):  
Smooth Muscle ◽  
Transient Receptor Potential ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Quantitative Polymerase Chain Reaction ◽  
Primary Cultures ◽  
Fluorescent Microscopy ◽  
Arterial Smooth Muscle ◽  
Fura 2 ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle

Hypoxic pulmonary vasoconstriction (HPV) requires Ca2+ influx through store-operated Ca2+ channels (SOCC) in pulmonary arterial smooth muscle cells (PASMC) and is greater in distal than proximal pulmonary arteries (PA). SOCC may be composed of canonical transient receptor potential (TRPC) proteins and activated by stromal interacting molecule 1 (STIM1). To assess the possibility that HPV is greater in distal PA because store-operated Ca2+ entry (SOCE) is greater in distal PASMC, we measured intracellular Ca2+ concentration ([Ca2+]i) and SOCE in primary cultures of PASMC using fluorescent microscopy and the Ca2+-sensitive dye fura 2. Both hypoxia (4% O2) and KCl (60 mM) increased [Ca2+]i. Responses to hypoxia, but not KCl, were greater in distal cells. We measured SOCE in PASMC perfused with Ca2+-free solutions containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels. Under these conditions, the increase in [Ca2+]i caused by restoration of extracellular Ca2+ and the decrease in fura 2 fluorescence caused by Mn2+ were greater in distal PASMC, indicating greater SOCE. Moreover, the increase in SOCE caused by hypoxia was also greater in distal cells. Real-time quantitative polymerase chain reaction analysis of PASMC and freshly isolated deendothelialized PA tissue demonstrated expression of STIM1 and five of seven known TRPC isoforms (TRPC1 > TRPC6 > TRPC4 ≫ TRPC3 ≈ TRPC5). For both protein, as measured by Western blotting, and mRNA, expression of STIM1, TRPC1, TRPC6, and TRPC4 was greater in distal than proximal PASMC and PA. These results provide further support for the importance of SOCE in HPV and suggest that HPV is greater in distal than proximal PA because greater numbers and activation of SOCC in distal PASMC generate bigger increases in [Ca2+]i.

Graded response of K+ current, membrane potential, and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle

2002 ◽  
Vol 283 (5) ◽  
pp. L1143-L1150 ◽  
Author(s):  
Andrea Olschewski ◽  
Zhigang Hong ◽  
Daniel P. Nelson ◽  
E. Kenneth Weir
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Cortical Neurons ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Resting Membrane Potential ◽  
Type I ◽  
Body Type ◽  
Arterial Smooth Muscle ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle

Many studies indicate that hypoxic inhibition of some K+ channels in the membrane of the pulmonary arterial smooth muscle cells (PASMCs) plays a part in initiating hypoxic pulmonary vasoconstriction. The sensitivity of the K+ current ( I k), resting membrane potential ( E m), and intracellular Ca2+ concentration ([Ca2+]i) of PASMCs to different levels of hypoxia in these cells has not been explored fully. Reducing Po 2 levels gradually inhibited steady-state I k of rat resistance PASMCs and depolarized the cell membrane. The block of I k by hypoxia was voltage dependent in that low O2 tensions (3 and 0% O2) inhibited I k more at 0 and −20 mV than at 50 mV. As expected, the hypoxia-sensitive I k was also 4-aminopyridine sensitive. Fura 2-loaded PASMCs showed a graded increase in [Ca2+]i as Po 2 levels declined. This increase was reduced markedly by nifedipine and removal of extracellular Ca2+. We conclude that, as in the carotid body type I cells, PC-12 pheochromocytoma cells, and cortical neurons, increasing severity of hypoxia causes a proportional decrease in I k and E m and an increase of [Ca2+]i.

scholarly journals Hypoxia-induced migration in pulmonary arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1

2012 ◽  
Vol 303 (4) ◽  
pp. L343-L353 ◽  
Author(s):  
Kyle Leggett ◽  
Julie Maylor ◽  
Clark Undem ◽  
Ning Lai ◽  
Wenju Lu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Arterial Smooth Muscle ◽  
Aquaporin 1 ◽  
Protein Levels ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle ◽  
And Migration

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca2+]i) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca2+ dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca2+]i, and migration in PASMCs. Blockade of Ca2+ entry through voltage-dependent Ca2+ or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca2+]i, AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca2+]i that results in increased AQP1 protein levels and cell migration.

Association of pulmonary artery photorelaxation with H2O2 metabolism by catalase

1991 ◽  
Vol 261 (4) ◽  
pp. H1141-H1147 ◽  
Author(s):  
M. S. Wolin ◽  
H. A. Omar ◽  
M. P. Mortelliti ◽  
P. D. Cherry
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Soluble Guanylate Cyclase ◽  
Uv Light ◽  
Organ Bath ◽  
Arterial Smooth Muscle ◽  
Photochemical Reduction ◽  
Oxidation Of Methanol ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle

We have examined the mechanism governing guanosine 3',5'-cyclic monophosphate (cGMP)-associated photoinduced relaxation elicited by long-wavelength ultraviolet (UV) light of endothelium-removed, isolated bovine pulmonary arteries. Hypoxia, produced by gassing of the organ bath solution with 95% N2-5% CO2, inhibited photorelaxation. Photorelaxation was also inhibited by cyanide (1 mM NaCN) but was potentiated by lactate (5 mM). Irradiation of bovine pulmonary arterial smooth muscle with UV light (or exposure to exogenous H2O2) stimulated cyanide-inhibitable oxidation of methanol to formaldehyde, suggesting that UV light increased H2O2 metabolism via catalase. The UV light-induced oxidation of methanol by pulmonary arterial smooth muscle was also inhibited by hypoxia. Consumption of O2 was detected when pulmonary arterial tissue was exposed to UV light, but cyanide failed to interfere with this effect, consistent with the photochemical reduction of O2 within vascular smooth muscle in a manner independent of mitochondrial respiration. We propose that photorelaxation is associated with the intracellular photochemical reduction of O2 to form H2O2, which elicits increases of vascular smooth muscle cGMP levels via the catalase-dependent activation of soluble guanylate cyclase. In addition, we hypothesize that the photooxidation of NAD(P)H could contribute to the generation of H2O2, since the enhancement of photorelaxation by lactate may originate from increased levels of NADH.

scholarly journals Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle

2009 ◽  
Vol 297 (1) ◽  
pp. L17-L25 ◽  
Author(s):  
Wenju Lu ◽  
Jian Wang ◽  
Gongyong Peng ◽  
Larissa A. Shimoda ◽  
J. T. Sylvester
Keyword(s):  
Smooth Muscle ◽  
Acute Hypoxia ◽  
Fluorescent Microscopy ◽  
Pcr Analysis ◽  
Arterial Smooth Muscle ◽  
Real Time Quantitative Pcr ◽  
Stromal Interaction Molecule 1 ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle ◽  
Membrane Spanning

Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal Ca2+ in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Ca2+-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca2+ depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in Ca2+ signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca2+-sensitive dye, fura 2, to measure basal intracellular Ca2+ concentration ([Ca2+]i), store-operated Ca2+ entry (SOCE), and increases in [Ca2+]i caused by acute hypoxia (4% O2) or depolarization (60 mmol/l KCl) in cells treated with small interfering RNA targeted to STIM1 (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative PCR analysis (qPCR), STIM1 mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM1 and siSTIM2 caused specific and significant knockdown of both mRNA measured by qPCR and protein measured by Western blotting. siSTIM1 markedly inhibited SOCE and abolished the sustained [Ca2+]i response to hypoxia but did not alter the initial transient [Ca2+]i response to hypoxia, the [Ca2+]i response to depolarization, or basal [Ca2+]i. The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC and that the increase in [Ca2+]i induced by acute hypoxia in these cells required SR Ca2+ release and STIM1-dependent activation of SOC.

Inhibition of glycolysis by 2-DG increases [Ca2+]i in pulmonary arterial smooth muscle cells

1995 ◽  
Vol 269 (2) ◽  
pp. L203-L208 ◽  
Author(s):  
R. T. Bright ◽  
C. G. Salvaterra ◽  
L. J. Rubin ◽  
X. J. Yuan
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Cyclopiazonic Acid ◽  
Muscle Cells ◽  
Arterial Smooth Muscle ◽  
Inhibition Of Glycolysis ◽  
Pulmonary Arterial ◽  
Arterial Smooth Muscle Cells ◽  
Pulmonary Arterial Smooth Muscle

Inhibition of glycolysis depolarizes single pulmonary arterial smooth muscle cells (PASMC) and potentiates hypoxic pulmonary vasoconstriction (HPV) in isolated perfused rat lungs. Whether glycolytic inhibition causes an increase in the intracellular Ca2+ concentration ([Ca2+]i) in PASMC was determined in this study. [Ca2+]i was measured in primary cultured rat PASMC using the Ca2+ -sensitive fluorescent indicator, fura 2, and quantitative fluorescence microscopy. Extracellular application of the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), significantly and reversibly increased [Ca2+]i in PASMC. Removal of extracellular Ca2+ and application of the Ca2+ channel blocker, verapamil (10 microM), attenuated, but did not eliminate, the 2-DG-induced rise in [Ca2+]i. In the absence of extracellular Ca2+, however, depletion of inositol triphosphate-sensitive intracellular Ca2+ stores by 10 microM cyclopiazonic acid (CPA) completely abolished the 2-DG-induced increase in [Ca2+]i. The data suggest that 2-DG-induced increases in [Ca2+]i result from both Ca2+ influx through the verapamil-sensitive voltage-gated Ca2+ channels and Ca2+ release from the CPA-sensitive intracellular Ca2+ stores.

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