AJP - Lung Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 290: L517-L525, 2006. First published October 21, 2005; doi:10.1152/ajplung.00308.2004
1040-0605/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/3/L517    most recent
00308.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jernigan, N. L.
Right arrow Articles by Resta, T. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jernigan, N. L.
Right arrow Articles by Resta, T. C.

Impaired NO-dependent inhibition of store- and receptor-operated calcium entry in pulmonary vascular smooth muscle after chronic hypoxia

Nikki L. Jernigan, Brad R. S. Broughton, Benjimen R. Walker, and Thomas C. Resta

Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico

Submitted 16 August 2004 ; accepted in final form 17 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have recently demonstrated that chronic hypoxia (CH) attenuates nitric oxide (NO)-mediated decreases in pulmonary vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca2+]i) and promotes NO-dependent VSM Ca2+ desensitization. The objective of the current study was to identify potential mechanisms by which CH interferes with regulation of [Ca2+]i by NO. We hypothesized that CH impairs NO-mediated inhibition of store-operated (capacitative) Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) in pulmonary VSM. To test this hypothesis, we examined effects of the NO donor, spermine NONOate, on SOCE resulting from depletion of intracellular Ca2+ stores with cyclopiazonic acid, and on UTP-induced ROCE in isolated, endothelium-denuded, pressurized pulmonary arteries (213 ± 8 µm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. We found that the change in [Ca2+]i associated with SOCE and ROCE was significantly reduced in vessels from CH animals. Furthermore, spermine NONOate diminished SOCE and ROCE in vessels from control, but not CH animals. We conclude that NO-mediated inhibition of SOCE and ROCE is impaired after CH-induced pulmonary hypertension.

pulmonary hypertension; capacitative calcium entry; uridine 5'-triphosphate; protein kinase G; NiCl2; SKF-96365; nitric oxide

ENDOTHELIUM-DERIVED NITRIC OXIDE (EDNO) may play an important protective role in diminishing the severity of chronic hypoxia (CH)-induced pulmonary hypertension (9, 35). CH is associated with enhanced EDNO-dependent vasodilation, a function of elevated endothelial nitric oxide synthase expression (9, 20, 28, 29, 32, 37) and increased pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) (16). Moreover, this enhanced VSM reactivity to NO following CH correlates with increased expression and activity of the downstream effector, protein kinase G (PKG) (15). Interestingly, the augmented NO-dependent vasodilation after CH is coupled to a smaller decrease in VSM intracellular free calcium ([Ca2+]i) compared with vessels from control animals (16), suggesting that CH promotes a change in NO signaling resulting in less dependence on Ca2+ handling pathways such as Ca2+ sequestration, influx, or efflux and greater dependence on mechanisms that regulate Ca2+ sensitivity. Consistent with this possibility, recent studies from our laboratory indicate that spermine NONOate reverses Ca2+ sensitization generated by the small G protein, RhoA, and Rho kinase (ROK) in permeabilized small pulmonary arteries from CH but not control animals (17). However, the mechanism by which CH impairs NO-dependent decreases in VSM [Ca2+]i remains to be investigated and is the focus of the current study.

NO-mediated stimulation of PKG elicits relaxation through several mechanisms that result in either a decrease in [Ca2+]i or a decrease in the sensitivity of the contractile apparatus to Ca2+ (2). Ca2+ entry into VSM cells is mediated by voltage-operated Ca2+ channels (30) as well as several types of Ca2+-permeable channels that are not voltage gated. These include channels mediating store-operated Ca2+ entry (SOCE), activated by depletion of Ca2+ from the sarcoplasmic reticulum (SR), and receptor-operated Ca2+ entry (ROCE), commonly activated by agonist stimulation of G protein-coupled receptors (23). We hypothesized that CH impairs NO-mediated decreases in pulmonary VSM [Ca2+]i by interfering with PKG-dependent inhibition of either SOCE or ROCE. To test this hypothesis, we measured changes in [Ca2+]i and inner diameter (ID) elicited by SOCE and ROCE in isolated, endothelium-denuded, pressurized small pulmonary arteries from control and CH rats. In addition, we examined effects of the NO donor spermine NONOate on SOCE and ROCE. Our findings suggest that CH is associated with diminished VSM SOCE and ROCE in small pulmonary arteries. Furthermore, the inhibitory effects of NO on SOCE and ROCE are impaired following CH.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All protocols and surgical procedures employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine (Albuquerque, NM).

Experimental groups. Male Sprague-Dawley rats (200–300 g body wt, Harlan Industries) were divided into two groups for each experiment. Animals designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ~380 mmHg for 4 wk. The chamber was opened three times per week to provide animals with fresh food, water, and clean bedding. On the day of experimentation, rats were removed from the hypobaric chamber and immediately placed in a Plexiglas chamber continuously flushed with a 12% O2-88% N2 gas mixture to reproduce inspired PO2 (~70 mmHg) within the hypobaric chamber. Control animals were housed at ambient barometric pressure (~630 mmHg). All animals were maintained on a 12:12-h light-dark cycle.

Endothelial disruption and cannulation of small pulmonary arteries for dimensional analysis. Rats were anesthetized with pentobarbital sodium (200 mg/kg ip), and the heart and lungs were exposed by midline thoracotomy. The left lung was removed and immediately placed in ice-cold physiological saline solution (PSS) containing (in mM) 129.8 NaCl, 5.4 KC1, 0.83 MgSO4, 19 NaHCO3, 1.8 CaC12, and 5.5 glucose. The lung was pinned out in iced PSS in a Silastic-coated dissection dish. A fourth or fifth order intrapulmonary artery (~100- to 300-µm ID) of ~1-mm length and without visible side branches was dissected free and transferred to a vessel chamber (Living Systems, CH-1) containing iced PSS. The proximal end of the artery was cannulated with a tapered glass pipette, secured in place with a single strand of silk ligature, and gently flushed to remove any blood from the lumen. The vessel was monitored during this time to ensure blood exited the distal end and not through small openings in the length of the vessel that may be indicative of side branches. Before the distal end of the artery was cannulated, the vessel lumen was rubbed with a strand of moose mane to disrupt the endothelium. The vessel was then stretched longitudinally to approximate its in situ length and pressurized with either a column or a servo-controlled peristaltic pump (Living Systems) to 12 mmHg. A transmural pressure of 12 mmHg was chosen for both control and CH vessels based on our earlier findings that NO-mediated vasodilation and associated VSM [Ca2+]i responses were similar between control and CH arteries whether studied at 12 or 35 mmHg (17). The absence of leaks was assessed by vessel distension upon this initial pressurization as well as the ability of the vessel to further distend to an additional small increase in pressure. For those vessels used to assess Mn2+ quenching of fura-2 fluorescence, pressurization was achieved using the servo-controlled peristaltic pump. In addition to the above criteria, these arteries were required to hold a steady pressure upon switching off the servo-control function to confirm the absence of a leak. Any vessels with apparent leaks were discarded. The vessel chamber was transferred to the stage of a Nikon Eclipse TS100 microscope where vessels from both groups were superfused with PSS equilibrated with a gas mixture containing 10% O2, 6% CO2, and balance N2. A vessel chamber cover was positioned to permit this same gas mixture to flow over the top of the chamber bath. Bright-field images of vessels were obtained with an IonOptix CCD100M camera, and dimensional analysis was performed by IonOptix Sarclen software to measure ID. The effectiveness of endothelial disruption was verified by the lack of a vasodilatory response to ACh (1 µM) in UTP (3–5 µM)-constricted vessels.

Measurement of VSM [Ca2+]i. Pressurized arteries were loaded abluminally with the cell-permeant, ratiometric, Ca2+-sensitive fluorescent indicator fura-2 AM (Molecular Probes). Immediately before being loaded, fura-2 AM (1 mM in anhydrous DMSO) was mixed 2:1 with a 20% solution of Pluronic acid in DMSO, and this mixture was diluted in PSS to yield a final concentration of 2 µM fura-2 AM and 0.05% Pluronic acid. Arteries were incubated in this solution for 45 min at room temperature in the dark. The diluted fura-2 AM solution was equilibrated with the 10% O2 gas mixture during this loading period. Vessels were rinsed for 20 min with aerated PSS (37°C) after the loading period to wash out excess dye and to allow for hydrolysis of AM groups by intracellular esterases. Fura-2-loaded vessels were alternately excited at 340 and 380 nm at a frequency of 10 Hz with an IonOptix Hyperswitch dual excitation light source, and the respective 510-nm emissions were collected with a photomultiplier tube. Background-subtracted 340/380 emission ratios were calculated with IonOptix Ion Wizard software and recorded continuously throughout the experiment, with simultaneous measurement of ID from red wavelength bright-field images as described above.

Isolated vessel experiments. Because the endothelium is a source of vasoactive factors that may influence VSM reactivity to NO, all experiments were conducted in endothelium-denuded arteries (16).

Effect of CH on SOCE. After fura-2 AM loading and the 20-min equilibration, pulmonary arteries from control and CH rats were superfused with Ca2+-free PSS containing 3 mM EGTA to chelate any residual Ca2+ and 50 µM diltiazem to prevent Ca2+ entry through L-type voltage-gated Ca2+ channels. The efficacy of diltiazem was verified in control arteries by blockade of the [Ca2+]i response to a depolarizing concentration of KCl (50 mM). In addition, arteries were incubated with the SR Ca2+-ATPase (SERCA) inhibitors cyclopiazonic acid (CPA; 10 µM) or thapsigargin (TG; 1 µM) to deplete intracellular Ca2+ stores and prevent Ca2+ reuptake. The changes in [Ca2+]i (SOCE) and ID were then determined upon restoration of extracellular Ca2+ (1.8 mM) in the continued presence of diltiazem and CPA or TG. Separate sets of arteries from each group were pretreated with the relatively nonselective inhibitor of Ca2+ entry, NiCl2 (10 mM) (40). Parallel experiments were performed in the presence of SKF-96365 (50 µM), which has been reported to selectively inhibit ROCE vs. SOCE (8, 12, 25).

Effect of NO on SOCE. Effects of the NO donor, spermine NONOate (1 µM), on CPA-induced SOCE and vasoconstriction were assessed in vessels from each group as above. Spermine NONOate was continually present in both the Ca2+-free and normal PSS. [Ca2+]i and ID responses to spermine NONOate (1 µM) were further determined in the presence of the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 50 µM) or the PKG inhibitor Rp-8-Br-PET-cGMPS (30 µM). Additional experiments examined responses to the PKG agonist 8-pCPT-cGMP (10 µM) in separate sets of control and CH vessels.

Effects of CH and NO on Mn2+ quenching of fura-2 fluorescence. Store-operated cation entry was quantified by the rate of quenching of fura-2 fluorescence with Mn2+, which enters the VSM as a Ca2+ surrogate and reduces fura-2 fluorescence on binding to the dye. Pulmonary arteries were loaded with fura-2, as previously described, and washed with aerated PSS for 20 min. Fura-2 was excited at 360 nm, and emission light was recorded at 510 nm. At the excitation wavelength of 360 nm, fura-2 fluorescence intensity is not influenced by [Ca2+]i changes; therefore, changes in fluorescence are assumed to be caused by Mn2+ alone. After stable baseline fluorescence was attained, store-operated channels were activated by superfusing the vessel with Ca2+-free PSS (without EGTA) containing diltiazem (50 µM) and CPA (10 µM) for 15 min. MnCl2 (500 µM) was then added to the superfusate for 10 min. In experiments examining the effect of NO on store-operated cation entry, spermine NONOate (1 µM) was continuously present in the superfusate.

Effects of CH and NO on ROCE. ROCE was induced with the G protein-coupled P2Y receptor agonist UTP (14). To assess ROCE independently of SOCE and to eliminate other UTP-mediated Ca2+ entry mechanisms, vessels were pretreated with diltiazem (50 µM) and CPA (10 µM), and a stable SOCE response was obtained (described above) before the addition of UTP (100 µM). Parallel protocols were performed in arteries from each group pretreated with NiCl2 (10 mM) and SKF-96365 (50 µM). Similar experiments were conducted in the presence of spermine NONOate (1 µM) to examine potential inhibitory effects of NO on UTP-induced Ca2+ entry through receptor-operated channels.

Calculations and statistics. Vasoconstrictor responses were calculated as a percent of baseline ID for SOCE experiments and as a percent of ID after SOCE for ROCE experiments. All data are expressed as means ± SE, and values of n refer to the number of animals in each group. A t-test or two-way ANOVA was used to make comparisons when appropriate. If differences were detected by ANOVA, individual groups were compared with the Student-Newman-Keuls test. P ≤ 0.05 was accepted as significant for all comparisons.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Efficacy of CPA, TG, and diltiazem. To assess influences of CH on SOCE, the SERCA inhibitors CPA and TG were used to deplete intracellular Ca2+ stores and thereby activate store-operated cation channels, whereas diltiazem was employed to eliminate any contribution of Ca2+ influx through L-type voltage-dependent Ca2+ channels. Representative traces illustrating the efficacy of these compounds in isolated control arteries are depicted in Fig. 1, A and B. UTP elicited a transient increase in VSM [Ca2+]i ({Delta} in F340/F380 = 0.08 ± 0.01; n = 3) associated with a transient constriction (40% ± 3%; n = 3) in Ca2+-free PSS, signifying Ca2+ release from intracellular stores (Fig. 1A, left). Repeating this protocol in the presence of either CPA (Fig. 1A, middle) or TG (Fig. 1A, right) largely inhibited UTP-mediated changes in [Ca2+]i ({Delta} in F340/F380 = –0.01 ± 0.01, n = 4 for CPA; and 0.00 ± 0.01, n = 3 for TG) and ID (7% ± 2%, n = 4 for CPA; and 3% ± 1%, n = 3 for TG), thus demonstrating effective depletion of intracellular Ca2+ stores by these inhibitors.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 1. A: representative traces of changes in vascular smooth muscle (VSM) free calcium concentration ([Ca2+]i) (F340/F380, top) and vessel inner diameter (µm, bottom) in endothelium-denuded control arteries demonstrating UTP (10 µM)-induced Ca2+ release from intracellular stores (left) and depletion of intracellular stores with cyclopiazonic acid (CPA; 10 µM; middle) and thapsigargin (TG; 1 µM; right). Experiments were performed in Ca2+-free physiological saline solution (PSS). B: representative traces of changes in VSM [Ca2+]i (F340/F380, top) and inner diameter (µm, bottom) in endothelium-denuded control arteries demonstrating the ability of diltiazem (50 µM; right) to inhibit increases in [Ca2+]i and vasoconstriction induced by the depolarizing stimulus, KCl (50 mM; left).

 
Figure 1B illustrates that KCl (50 mM)-induced increases in [Ca2+]i ({Delta} in F340/F380 = 0.38 ± 0.03, n = 3) were diminished by 50 µM diltiazem ({Delta} in F340/F380 = 0.02 ± 0.01, n = 3). Diltiazem similarly prevented KCl-induced vasoconstriction (76% ± 10%, n = 3 without diltiazem; and 3% ± 3%, n = 3 with diltiazem), indicating blockade of voltage-gated Ca2+ channels.

CH inhibits SOCE. Baseline ID and [Ca2+]i were similar between groups (Table 1). Figure 2A depicts traces of VSM [Ca2+]i and ID from individual control arteries, indicating SOCE (left) and blockade of SOCE and associated vasoconstriction by NiCl2 (middle) but not the putative ROCE inhibitor SKF-96365 (right).


View this table:
[in this window]
[in a new window]
  		Table 1. Baseline inner diameter and vascular smooth muscle [Ca2+]i of pulmonary arteries from control and chronically hypoxic rats

 

Figure 2
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2. Chronic hypoxia (CH) attenuates CPA-induced store-operated Ca2+ entry (SOCE). A: representative traces of changes in VSM [Ca2+]i (F340/F380, top) and vessel inner diameter (µm, bottom) in endothelium-denuded control arteries demonstrating SOCE and associated vasoconstriction (left) and effects of NiCl2 (middle) and SKF-96365 (right). Summary data are shown for store-operated change in VSM [Ca2+]i (F340/F380) (B) and SOCE-induced vasoconstriction (C; % baseline inner diameter) in endothelium-denuded control (n = 5/treatment) and CH (n = 5/treatment) small pulmonary arteries after restoration of Ca2+-containing PSS. Experiments were performed in the presence of CPA (10 µM) and diltiazem (50 µM) plus either vehicle (PSS), NiCl2 (10 mM), or SKF-96365 (50 µM). Values are means ± SE. *P ≤ 0.05 vs. corresponding control group. #P ≤ 0.05 vs. corresponding vehicle-treated group.

 
SOCE was significantly attenuated in vessels isolated from CH rats compared with controls after SERCA inhibition with CPA (Fig. 2B). Similar results were obtained for TG-induced SOCE (Table 2). Although the change in [Ca2+]i was diminished after CH, the percent changes in ID were not different between groups (Fig. 2C, Table 2). SOCE and associated vasoconstriction were blocked with NiCl2, but not SKF-96365, in each group (Fig. 2, Table 2). Furthermore, CH-induced SOCE remained attenuated in the presence of SKF-96365 (Fig. 2B).


View this table:
[in this window]
[in a new window]
  		Table 2. Chronic hypoxia inhibits thapsigargin-induced store-operated Ca2+ entry

 
CH impairs NO-dependent inhibition of SOCE. Treatment with either the NO donor spermine NONOate or the cGMP analog 8-pCPT-cGMP attenuated SOCE in control, but not CH, pulmonary arteries (Fig. 3A). Although there is a tendency for both spermine NONOate and 8-pCPT-cGMP to inhibit SOCE-induced vasoconstriction in control vessels, these differences did not achieve statistical significance (Fig. 3B). Furthermore, both the soluble guanylyl cyclase inhibitor ODQ and the PKG inhibitor Rp-8-Br-PET-cGMPS prevented spermine NONOate-induced attenuation of SOCE in vessels from control animals (Fig. 3A), suggesting NO inhibits SOCE through a cGMP/PKG-dependent mechanism. Rp-8-Br-PET-cGMPS was without effect on SOCE in CH vessels (Fig. 3A) but augmented vasoconstrictor responses to SOCE in this group (Fig. 3B).


Figure 3
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. Nitric oxide (NO), via protein kinase G (PKG), inhibits SOCE in control, but not CH, vessels. Store-operated change in VSM [Ca2+]i (F340/F380) (A) and SOCE-induced vasoconstriction (B; % baseline inner diameter) in endothelium-denuded control (n = 5/treatment) and CH (n = 5/treatment) small pulmonary arteries after restoration of Ca2+-containing PSS in the presence of CPA (10 µM) and diltiazem (50 µM) plus either vehicle (PSS), spermine NONOate (1 µM), 8-pCPT-cGMP (10 µM), spermine NONOate (1 µM) + 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (50 µM), or spermine NONOate (1 µM) + Rp-8-Br-PET-cGMPS (30 µM). Values are means ± SE. *P ≤ 0.05 vs. corresponding control group. #P ≤ 0.05 vs. corresponding vehicle-treated group. {tau}P < 0.05 vs. corresponding spermine NONOate-treated group.

 
CH attenuates both store-operated Mn2+ influx and NO-mediated inhibition of Mn2+ entry. Similar to effects of CH on SOCE, Mn2+ quenching of fluorescence was markedly decreased in arteries from CH rats vs. controls (Fig. 4). Spermine NONOate inhibited Mn2+ influx in control arteries but was without effect in vessels from CH rats, thus providing additional support for an effect of CH to impair NO-dependent inhibition of SOCE.


Figure 4
View larger version (22K):
[in this window]
[in a new window]
  		Fig. 4. Changes in fura-2 fluorescence (% of baseline) in response to CPA (10 µM)-induced Mn2+ entry in small pulmonary arteries from control (n = 4/treatment) and CH rats (n = 4/treatment) with or without spermine NONOate (1 µM). All experiments were performed in the presence of diltiazem (50 µM) and Ca2+-free PSS. Values are means ± SE. *P ≤ 0.05 for all groups vs. control.

 
CH inhibits ROCE. Figure 5A depicts traces for VSM [Ca2+]i and ID in individual control vessels, demonstrating UTP-induced ROCE after induction of stable SOCE. Whereas NiCl2 blocked SOCE, UTP-dependent increases in [Ca2+]i persisted in the presence of this inhibitor, suggesting specificity of NiCl2 for SOCE vs. ROCE. In contrast, the putative ROCE inhibitor SKF-96365 prevented UTP-dependent [Ca2+]i responses but was without effect on SOCE.


Figure 5
View larger version (29K):
[in this window]
[in a new window]
  		Fig. 5. CH attenuates receptor-operated Ca2+ entry (ROCE) and abolishes the inhibitory influence of NO on ROCE. A: representative traces of changes in VSM [Ca2+]i (F340/F380, top) and vessel inner diameter (µm, bottom) in endothelium-denuded control arteries demonstrating UTP-induced ROCE and associated vasoconstriction (left) and effects of NiCl2 (middle) and SKF-96365 (right). UTP (100 µM) was added subsequent to generation of stable SOCE. Summary data are shown for receptor-operated change in VSM [Ca2+]i (F340/F380) (B) and ROCE-induced vasoconstriction (C; % SOCE inner diameter) in endothelium-denuded control (n = 5/treatment) and CH (n = 5/treatment) small pulmonary arteries upon administration of UTP after a stable SOCE response. Experiments were conducted in the presence of CPA (10 µM) and diltiazem (50 µM) plus either vehicle (PSS), NiCl2 (10 mM), SKF-96365 (50 µM), or spermine NONOate (1 µM). Values are means ± SE. *P < 0.05 vs. corresponding control group. #P < 0.05 vs. corresponding vehicle-treated group.

 
Similar to effects of CH on SOCE, UTP-induced ROCE (Fig. 5B) was attenuated in CH vessels compared with controls. However, despite CH-induced inhibition of ROCE, vasoconstrictor responses to UTP were not different between groups (Fig. 5C). NiCl2 inhibited UTP-dependent changes in [Ca2+]i and vasoconstriction in control vessels but did not alter responses in CH arteries. In contrast, SKF-96365 abolished UTP-induced changes in [Ca2+]i and largely diminished vasoconstrictor responses in both groups. Spermine NONOate provided a modest but significant inhibition of ROCE in control arteries, although vasoconstrictor responses to UTP were unaltered by this NO donor. Whereas spermine NONOate was without effect on UTP-induced changes in [Ca2+]i in CH arteries, there was a significant decrease in UTP-mediated vasoconstriction, which may be a consequence of increased spermine NONOate-induced Ca2+ desensitization after CH (17).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Previous studies from our laboratory have demonstrated that long-term hypoxia impairs NO-mediated decreases in VSM [Ca2+]i in isolated small pulmonary arteries (16). The present study examined whether CH mediates this response by interfering with NO-dependent inhibition of SOCE and ROCE in pulmonary VSM. The major findings from this study are: 1) CH attenuates SOCE and ROCE; and 2) NO diminishes SOCE and ROCE in control arteries; however, these inhibitory effects of NO are absent after CH. These results, summarized in Fig. 6, suggest that hypoxic acclimation leads to diminished NO-dependent decreases in pulmonary VSM [Ca2+]i at least in part by interfering with NO-mediated inhibition of SOCE and ROCE.


Figure 6
View larger version (46K):
[in this window]
[in a new window]
  		Fig. 6. Summary of major findings. Ca2+ influx through store-operated channels (SOC) was induced by depletion of sarcoplasmic reticulum (SR) Ca2+ stores with the SR Ca2+-ATPase (SERCA) inhibitors CPA or TG. Ca2+ entry via receptor-operated channels (ROC) was elicited by the P2Y receptor agonist UTP after SOCE. A: control arteries. NO inhibited SOCE in pulmonary VSM via a cGMP/PKG pathway (solid line). NO provided a modest inhibition of ROCE, although the signaling pathway mediating this effect has not been defined (dashed line). NiCl2 prevented SOCE (solid line) and inhibited ROCE (dotted line), whereas SKF-96365 blocked only ROCE. B: CH arteries. CH attenuated both SOCE and ROCE. NO was without effect on either Ca2+ influx pathway. NiCl2 and SKF-96365 selectively inhibited SOCE and ROCE, respectively. These data support a shift in NO/PKG signaling from [Ca2+]i-lowering mechanisms to those involving Ca2+ desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway in CH arteries as previously reported (17).

 
Relaxation to NO/PKG can be elicited either by lowering [Ca2+]i or by decreasing the sensitivity of the contractile apparatus to Ca2+ (2). We have recently demonstrated that PKG-induced vasodilation is dependent on a reduction in VSM [Ca2+]i in pulmonary arteries from control rats, whereas PKG-mediated decreases in VSM [Ca2+]i are diminished after long-term hypoxia (16). This latter effect of CH to interfere with NO regulation of Ca2+ handling mechanisms in pulmonary VSM is compensated by an increase in PKG-dependent Ca2+ desensitization, which appears to be largely attributed to inhibition of RhoA/ROK (17). These recent findings have led us to examine CH-induced alterations to mechanisms of VSM Ca2+ handling in the pulmonary circulation.

Activation of many G protein-coupled receptors mediates Ca2+ influx in VSM through both receptor-operated and store-operated cation channels (27). Whereas receptor-operated channels can be stimulated via phospholipase C-derived diacylglycerol, store-operated channels are activated secondary to depletion of Ca2+ from the SR, a process termed capacitative Ca2+ entry or SOCE (23). SOCE has been demonstrated in isolated pulmonary arterial smooth muscle cells (7, 11, 26, 36, 39, 40) and in VSM from both conduit (22) and distal pulmonary arteries (34). Snetkov et al. (34) have shown that SOCE occurs in small arteries from different beds (mesenteric, renal, femoral, and pulmonary), although the coupling of SOCE to vasoconstriction was observed only in intrapulmonary arteries, demonstrating the potential significance of this Ca2+ entry mechanism in regulation of pulmonary vascular reactivity. Our present finding that capacitative Ca2+ entry mediates vasoconstriction in isolated small pulmonary arteries is in agreement with these earlier observations. We have further identified a novel effect of CH to attenuate both SOCE and ROCE. Interestingly, the vasoconstriction associated with each Ca2+ entry pathway was similar between groups, consistent with recent evidence from our laboratory for enhanced pulmonary VSM Ca2+ sensitivity after CH (17). However, neither the mechanisms by which CH inhibits SOCE and ROCE nor the physiological significance of these responses is understood.

The best candidates for store-operated and receptor-operated channels are several members of the canonical transient receptor potential (TRPC) family of cation channels (41). Although first characterized as light-activated cation channels in photoreceptor cells of Drosophila (13), mammalian TRPC ion channels have recently gained recognition for their involvement in SOCE and ROCE. Of the seven known TRPC channels, denoted TRPC1–7, expression of TRPC1–6 has been identified by RT-PCR (21, 22, 26, 38, 39) in rat pulmonary arterial smooth muscle cells. The role of TRPC channels in SOCE has been evaluated in cell culture by either overexpression of TRPC protein or interference with expression by genetic manipulations [antisense oligonucleotides, small interfering (si) RNA, or gene knockout]. In human pulmonary artery smooth muscle cells, SOCE and cationic current were significantly reduced when treated with TRPC1 antisense (36). Moreover, Lin et al. (21) demonstrated that siRNA knockdown of TRPC1 attenuates thapsigargin-induced Ca2+ entry, whereas knockdown of TRPC6 inhibits influx of Ca2+ mediated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol. Therefore, TRPC1 and TRPC6 appear to contribute to SOCE and ROCE, respectively, in pulmonary VSM.

It is possible that diminished SOCE and ROCE after CH observed in the current study is a result of decreased expression or activity of one or more TRPC channel proteins. At odds with this hypothesis, however, Lin and colleagues (21) have observed enhanced SOCE and ROCE in transiently cultured pulmonary myocytes from CH rats compared with controls, which correlates with an upregulation of both TRPC1 and TRPC6 expression. The reason for these apparent discrepancies between the current study and earlier work (21) is unclear but may be a consequence of the different preparations used. One possible explanation is that the enzymatic tissue digestion and cell culture procedures used previously (21) resulted in differential alterations in cellular phenotype between groups. A further possibility is that segmental heterogeneity exists in Ca2+ influx mechanisms or TRPC channel expression between the smaller vessels used in the present study (~200 µm) and those larger intrapulmonary arteries (~300–800 µm) from which VSM cells were isolated by Lin and colleagues (21). Finally, the current study was performed with rats that were exposed to hypobaric hypoxia rather than normobaric hypoxia used by Lin et al. (21). Recent evidence suggests that hypoxemia, hypocapnia, and arterial pH are greater and arterial O2 saturation is lower in acute hypobaric compared with normobaric hypoxia at a given inspired PO2 (31). Future studies will be necessary to examine the reason for such disparate influences of CH on Ca2+ signaling mechanisms between experimental preparations.

Although our present observation that basal fura-2 emission ratios were not different between control and CH vessels is in agreement with previous studies from our laboratory in both endothelium-intact and -denuded pulmonary arteries (16, 17), these results are in contrast to several previous reports that CH elevates basal [Ca2+]i in pulmonary arterial smooth muscle (1, 33). The explanation for these discrepant results is not apparent but again may be explained by segmental differences in VSM cell phenotype or differences in the preparations employed. Nevertheless, an effect of CH to elevate basal cytosolic free Ca2+ would not be predicted based on our present findings of diminished SOCE and ROCE after CH.

Despite the lack of NO-mediated inhibition of SOCE in CH vessels, there was a significant PKG-dependent inhibitory effect of NO on SOCE in arteries from control animals. NO can inhibit SOCE indirectly via inactivation of phospholamban, subsequent stimulation of SERCA, and increased filling of SR Ca2+ stores (3–5, 18, 24). In the current study, however, it is likely that NO/PKG attenuated SOCE independently of the SR Ca2+ load, given that SERCA was continually inhibited with CPA. A similar direct effect of NO to inhibit capacitative Ca2+ entry has been suggested in bovine endothelial cells (6). One possible explanation for these findings is that NO/PKG mediates a direct inhibitory effect on the channels involved in SOCE, presumably members of the TRPC family of cation channels (41). Consistent with this possibility is evidence that PKG can directly phosphorylate TRPC3 channels in HEK-293 cells leading to diminished SOCE (19). Although our findings support an effect of PKG to inhibit SOCE independently of functional coupling to the SR Ca2+ load in control arteries, they do not preclude a potential role for increased SERCA activity in mediating NO-induced pulmonary vasodilation in this preparation. However, neither the mechanism by which PKG stimulation leads to inhibition of capacitative Ca2+ entry in pulmonary VSM nor the mechanism by which long-term hypoxia attenuates this response is understood; both represent viable areas of future investigation.

Similar to effects of CH on NO-dependent regulation of SOCE, we observed a unique role for CH to prevent NO-mediated inhibition of UTP-induced ROCE. Together, these results suggest that the diminished effect of NO to reduce cytosolic [Ca2+] previously observed in CH pulmonary VSM (16, 17) is mediated by a lack of NO-dependent attenuation of both ROCE and SOCE. Although these results may be explained by a derangement in NO signaling following CH, it is alternatively possible that the inability of NO to inhibit Ca2+ entry in CH arteries is a function of already diminished SOCE and ROCE. Further study will be required to determine whether CH is having additional effects to alter other mechanisms of NO signaling involving regulation of Ca2+ sequestration, influx, and efflux.

It is noteworthy that SKF-96365 inhibited UTP-induced increases in [Ca2+]i, but not SOCE, in arteries from both control and CH rats, demonstrating a selectivity of SKF-96365 for ROCE in this preparation. These findings are consistent with evidence that SOCE is resistant to SKF-96365 in smooth muscle cells of rabbit pial arterioles (10) and in isolated, pressurized small pulmonary arteries from rats (12). SKF-96365 was additionally found to exhibit selectivity for ROCE vs. SOCE in A7r5 cells (25) and in nonpressurized rat renal interlobular arterioles (8). In contrast, other reports have demonstrated inhibition of SOCE by SKF-96365 in isolated pulmonary arterial myocytes (26, 39). The reason for these conflicting results in the literature is not presently clear. Interestingly, our results further suggest that NiCl2 exhibits a degree of selectivity for SOCE over ROCE in this preparation, as evidenced by attenuation of ROCE by NiCl2 only in arteries from control animals, but nearly complete blockade of SOCE in vessels from both groups of animals. Therefore, NiCl2 and SKF-96365 appear to be useful pharmacological tools to help discriminate between SOCE and ROCE pathways in VSM of small pulmonary arteries.

In conclusion, the present study demonstrates a novel effect of CH to attenuate both SOCE and ROCE in pulmonary VSM and to impair NO-dependent inhibition of these Ca2+ influx pathways in isolated small pulmonary arteries (Fig. 6). These data are consistent with recent observations from our laboratory that CH inhibits NO-mediated decreases in VSM [Ca2+]i (16) and support previous studies indicating vasoreactivity is largely independent of Ca2+ mobilization after CH (16, 17, 33). Understanding the mechanisms by which CH alters NO signaling in pulmonary VSM will require additional studies to evaluate potential influences of CH on the expression or activity of PKG targets, including TRPC channels. Considering that [Ca2+]i is a major stimulus for VSM contraction, gene expression, and proliferation, such a derangement in NO signaling may have important implications for regulation of not only pulmonary vascular tone but for arterial remodeling and resultant pulmonary hypertension associated with CH.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-07736, RR-16480, HL-77876, HL-58124, HL-63207, and P30-ES-012072.


   ACKNOWLEDGMENTS
 
The authors thank Pam Allgood and Minerva Murphy for technical assistance and Jessica Snow for editorial comments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. C. Resta, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, MSC 08-4750, 1 Univ. of New Mexico, Albuquerque, NM 87131-0001 (e-mail: TResta{at}salud.unm.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bonnet S, Hyvelin JM, Bonnet P, Marthan R, and Savineau JP. Chronic hypoxia-induced spontaneous and rhythmic contractions in the rat main pulmonary artery. Am J Physiol Lung Cell Mol Physiol 281: L183–L192, 2001.[Abstract/Free Full Text]
  2. Carvajal J, Germain A, Huidobro-Toro J, and Weiner C. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol 184: 409–420, 2000.[CrossRef][Web of Science][Medline]
  3. Cogolludo AL, Perez-Vizcaino F, Zaragoza-Arnaez F, Ibarra M, Lopez-Lopez G, Lopez-Miranda V, and Tamargo J. Mechanisms involved in SNP-induced relaxation and [Ca+]i reduction in piglet pulmonary and systemic arteries. Br J Pharmacol 132: 959–967, 2001.[CrossRef][Web of Science][Medline]
  4. Cohen RA, Weisbrod RM, Gericke M, Yaghoubi M, Bierl C, and Bolotina VM. Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx. Circ Res 84: 210–219, 1999.[Abstract/Free Full Text]
  5. Cornwell TL, Pryzwansky KB, Wyatt TA, and Lincoln TM. Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. Mol Pharmacol 40: 923–931, 1991.[Abstract]
  6. Dedkova EN and Blatter LA. Nitric oxide inhibits capacitative Ca2+ entry and enhances endoplasmic reticulum Ca2+ uptake in bovine vascular endothelial cells. J Physiol 539: 77–91, 2002.[Abstract/Free Full Text]
  7. Doi S, Damron DS, Horibe M, and Murray PA. Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 278: L118–L130, 2000.[Abstract/Free Full Text]
  8. Facemire CS and Arendshorst WJ. Calmodulin mediates norepinephrine-induced receptor-operated calcium entry in preglomerular resistance arteries. Am J Physiol Renal Physiol 289: F127–F136, 2005.[Abstract/Free Full Text]
  9. Fagan KA, Morrissey B, Fouty BW, Sato K, Harral JW, Morris KG Jr, Hoedt-Miller M, Vidmar S, McMurtry IF, and Rodman DM. Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension. Respir Res 2: 306–313, 2001.[CrossRef][Medline]
  10. Flemming R, Xu SZ, and Beech DJ. Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells. Br J Pharmacol 139: 955–965, 2003.[CrossRef][Web of Science][Medline]
  11. Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ, and Yuan JX. Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol 280: H746–H755, 2001.[Abstract/Free Full Text]
  12. Guibert C, Marthan R, and Savineau JP. 5-HT induces an arachidonic acid-sensitive calcium influx in rat small intrapulmonary artery. Am J Physiol Lung Cell Mol Physiol 286: L1228–L1236, 2004.[Abstract/Free Full Text]
  13. Harteneck C, Plant TD, and Schultz G. From worm to man: three subfamilies of TRP channels. Trends Neurosci 23: 159–166, 2000.[CrossRef][Web of Science][Medline]
  14. Hartley SA, Kato K, Salter KJ, and Kozlowski RZ. Functional evidence for a novel suramin-insensitive pyrimidine receptor in rat small pulmonary arteries. Circ Res 83: 940–946, 1998.[Abstract/Free Full Text]
  15. Jernigan NL, Walker BR, and Resta TC. Pulmonary PKG-1 is upregulated following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 285: L634–L642, 2003.[Abstract/Free Full Text]
  16. Jernigan NL, Walker BR, and Resta TC. Endothelium-derived reactive oxygen species and endothelin-1 attenuate NO-dependent pulmonary vasodilation following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 287: L801–L808, 2004.[Abstract/Free Full Text]
  17. Jernigan NL, Walker BR, and Resta TC. Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling. Am J Physiol Lung Cell Mol Physiol 287: L1220–L1229, 2004.[Abstract/Free Full Text]
  18. Karczewski P, Hendrischke T, Wolf WP, Morano I, Bartel S, and Schrader J. Phosphorylation of phospholamban correlates with relaxation of coronary artery induced by nitric oxide, adenosine, and prostacyclin in the pig. J Cell Biochem 70: 49–59, 1998.[CrossRef][Web of Science][Medline]
  19. Kwan HY, Huang Y, and Yao X. Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G. Proc Natl Acad Sci USA 101: 2625–2630, 2004.[Abstract/Free Full Text]
  20. Le Cras T, Xue C, Rengasamy A, and Johns R. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am J Physiol Lung Cell Mol Physiol 270: L164–L170, 1996.[Abstract/Free Full Text]
  21. Lin MJ, Leung GPH, Zhang WM, Yang XR, Yip KP, Tse CM, and Sham JSK. Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells: a novel mechanism of hypoxic pulmonary hypertension. Circ Res 95: 496–505, 2004.[Abstract/Free Full Text]
  22. McDaniel SS, Platoshyn O, Wang J, Yu Y, Sweeney M, Krick S, Rubin LJ, and Yuan JX. Capacitative Ca2+ entry in agonist-induced pulmonary vasoconstriction. Am J Physiol Lung Cell Mol Physiol 280: L870–L880, 2001.[Abstract/Free Full Text]
  23. McFadzean I and Gibson A. The developing relationship between receptor-operated and store-operated calcium channels in smooth muscle. Br J Pharmacol 135: 1–13, 2002.[CrossRef][Web of Science][Medline]
  24. Mingone CJ, Gupte SA, Iesaki T, and Wolin MS. Hypoxia enhances a cGMP-independent nitric oxide relaxing mechanism in pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 285: L296–L304, 2003.[Abstract/Free Full Text]
  25. Moneer Z, Dyer JL, and Taylor CW. Nitric oxide co-ordinates the activities of the capacitative and non-capacitative Ca2+-entry pathways regulated by vasopressin. Biochem J 370: 439–448, 2003.[CrossRef][Web of Science][Medline]
  26. Ng LC and Gurney AM. Store-operated channels mediate Ca2+ influx and contraction in rat pulmonary artery. Circ Res 89: 923–929, 2001.[Abstract/Free Full Text]
  27. Parekh AB and Putney JW Jr. Store-operated calcium channels. Physiol Rev 85: 757–810, 2005.[Abstract/Free Full Text]
  28. Resta TC, Chicoine LG, Omdahl JL, and Walker BR. Maintained upregulation of pulmonary eNOS gene and protein expression during recovery from chronic hypoxia. Am J Physiol Heart Circ Physiol 276: H699–H708, 1999.[Abstract/Free Full Text]
  29. Resta TC, Kanagy NL, and Walker BR. Estradiol-induced attenuation of pulmonary hypertension is not associated with altered eNOS expression. Am J Physiol Lung Cell Mol Physiol 280: L88–L97, 2001.[Abstract/Free Full Text]
  30. Sanders KM. Invited review: mechanisms of calcium handling in smooth muscles. J Appl Physiol 91: 1438–1449, 2001.[Abstract/Free Full Text]
  31. Savourey G, Launay JC, Besnard Y, Guinet AL, and Travers SP. Normo- and hypobaric hypoxia: are there any physiological differences? Eur J Appl Physiol 89: 122–126, 2003.[CrossRef][Medline]
  32. Shaul P, North A, Brannon T, Ujiie K, Wells L, Nisen P, Lowenstein C, Snyder S, and Star R. Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung. Am J Respir Cell Mol Biol 13: 167–174, 1995.[Abstract]
  33. Shimoda LA, Sham JS, Shimoda TH, and Sylvester JT. L-type Ca2+ channels, resting [Ca2+]i, and ET-1-induced responses in chronically hypoxic pulmonary myocytes. Am J Physiol Lung Cell Mol Physiol 279: L884–L894, 2000.[Abstract/Free Full Text]
  34. Snetkov VA, Aaronson PI, Ward JP, Knock GA, and Robertson TP. Capacitative calcium entry as a pulmonary specific vasoconstrictor mechanism in small muscular arteries of the rat. Br J Pharmacol 140: 97–106, 2003.[CrossRef][Web of Science][Medline]
  35. Steudel W, Scherrer-Crosbie M, Bloch KD, Weimann J, Huang PL, Jones RC, Picard MH, and Zapol WM. Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3. J Clin Invest 101: 2468–2477, 1998.[Web of Science][Medline]
  36. Sweeney M, Yu Y, Platoshyn O, Zhang S, McDaniel SS, and Yuan JX. Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol 283: L144–L155, 2002.[Abstract/Free Full Text]
  37. Tyler RC, Muramatsu M, Abman SH, Stelzner TJ, Rodman DM, Bloch KD, and McMurtry IF. Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 276: L297–L303, 1999.[Abstract/Free Full Text]
  38. Walker RL, Hume JR, and Horowitz B. Differential expression and alternative splicing of TRP channel genes in smooth muscles. Am J Physiol Cell Physiol 280: C1184–C1192, 2001.[Abstract/Free Full Text]
  39. Wang J, Shimoda LA, and Sylvester JT. Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 286: L848–L858, 2004.[Abstract/Free Full Text]
  40. Wilson SM, Mason HS, Smith GD, Nicholson N, Johnston L, Janiak R, and Hume JR. Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells. J Physiol 543: 917–931, 2002.[Abstract/Free Full Text]
  41. Xu SZ and Beech DJ. TrpC1 is a membrane-spanning subunit of store-operated Ca2+ channels in native vascular smooth muscle cells. Circ Res 88: 84–87, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. L. Jernigan, M. L. Paffett, B. R. Walker, and T. C. Resta
ASIC1 contributes to pulmonary vascular smooth muscle store-operated Ca2+ entry
Am J Physiol Lung Cell Mol Physiol, August 1, 2009; 297(2): L271 - L285.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. L. Jernigan, B. R. Walker, and T. C. Resta
Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia
Am J Physiol Lung Cell Mol Physiol, September 1, 2008; 295(3): L515 - L529.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
B. R. S. Broughton, B. R. Walker, and T. C. Resta
Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries
Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L797 - L806.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/3/L517    most recent
00308.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jernigan, N. L.
Right arrow Articles by Resta, T. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jernigan, N. L.
Right arrow Articles by Resta, T. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.