AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 284: H1321-H1331, 2003. First published December 27, 2002; doi:10.1152/ajpheart.00795.2002
0363-6135/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
284/4/H1321    most recent
00795.2002v1
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 ISI 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 ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Littler, C. M.
Right arrow Articles by Dempsey, E. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Littler, C. M.
Right arrow Articles by Dempsey, E. C.
Vol. 284, Issue 4, H1321-H1331, April 2003

Protein kinase C-epsilon -null mice have decreased hypoxic pulmonary vasoconstriction

Cassana M. Littler1, Kenneth G. Morris Jr.1, Karen A. Fagan1, Ivan F. McMurtry1, Robert O. Messing3, and Edward C. Dempsey1,2

1 Cardiovascular Pulmonary Research Laboratory, University of Colorado Health Sciences Center, and 2 Denver Veterans Administration Medical Center, Denver, Colorado 80262; and 3 Ernest Gallo Clinic and Research Center, Department of Neurology, University of California San Francisco, Emeryville, California 94608


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, -/-) mouse to test the hypothesis that PKC-epsilon is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-epsilon would decrease acute HPV in adult C57BL6×SV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% O2) was compared with responses to angiotensin II (ANG II) and KCl. PKC-epsilon -/- mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-L-arginine augmented HPV in PKC-epsilon +/+ but not -/- mice. Inhibition of Ca2+-gated K+ channels (KCa) with charybdotoxin and apamin did not enhance HPV in -/- mice relative to wild-type (+/+) controls. In contrast, the voltage-gated K+ channel (KV) antagonist 4-aminopyridine increased the response of -/- mice beyond that of +/+ mice. This suggested that increased KV channel expression could contribute to blunted HPV in PKC-epsilon -/- mice. Therefore, expression of the O2-sensitive KV channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and -/- mice. A subtle increase in Kv3.1b was detected in -/- vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in -/- vs. +/+ PASMC. Thus deletion of PKC-epsilon blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or KCa channel activity. Instead, the absence of PKC-epsilon allows increased expression of KV channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV.

murine knockout; isolated, perfused lung; pulmonary artery; smooth muscle cells; potassium channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR TONE IN THE LUNG circulation is regulated at least in part by hypoxic pulmonary vasoconstriction (HPV) (37). HPV serves as an adaptive response of the pulmonary circulation to regional differences in alveolar oxygen tension. Local vasoconstriction in response to alveolar hypoxia leads to redirection of blood flow to areas of the lung with higher oxygen tension. As a result, matching of ventilation and perfusion is preserved (44). However, when HPV is persistent, it initiates a cascade of cellular events that leads to vascular remodeling. The end result is fixed pulmonary hypertension (21, 47).

Basal vascular tone and HPV are regulated by a balance of vasodilators such as nitric oxide (NO) and PGI2 and vasoconstrictors such as endothelin-1 (ET-1). Contractile responses to these mediators are dependent on intracellular signaling cascades. One important signaling pathway is PKC (29, 35, 38). Cell-permeant PKC activators (phorbol esters) stimulate contraction and potentiate HPV (29). Various PKC inhibitors have been shown to decrease HPV in rats, rabbits, and dogs (2, 29, 45). PKC is also important in other early responses to hypoxia including cell proliferation (12, 13).

The PKC family includes at least 11 related intracellular kinases that are classified into four groups and share varying degrees of homology (14, 28). Studies that rely on broad PKC inhibitors have been complicated by the recent observation that individual isozymes of PKC may contribute to competing responses (6). Concerns have also been raised about the specificity of PKC inhibitors (10). Therefore, the role of individual isozymes of PKC in the control of pulmonary vascular reactivity has remained unclear. However, selected isozymes of PKC have been implicated in other pulmonary vascular cell responses (14). PKC-alpha , -beta , and -zeta are thought to be important in proliferation, PKC-beta has been implicated in permeability, and PKC-delta plays a key role in apoptosis (7, 25, 27, 34, 46). In the systemic circulation, PKC-epsilon has been implicated in contraction, although this remains controversial (19, 41). Activation of PKC-epsilon also protects rodent myocardium from ischemic or hypoxic injury (6, 15). Cell-permeant inhibitors of PKC-epsilon have not been available in sufficient quantities for use in perfused lung preparations. Thus the role of PKC-epsilon in the intact pulmonary circulation has remained unclear.

Recently, a mouse model with a body-wide deletion of PKC-epsilon has been characterized (18). The pulmonary vascular phenotype of these mice remains unknown. Organ-specific targeting for the study of a complex response like HPV and of an intracellular kinase like PKC-epsilon is not possible in the lung because of heterogeneity in cell populations important in pulmonary vascular reactivity. Thus studies of the pulmonary circulation require a whole body deletion of the PKC-epsilon gene. These knockout (null, -/-) mice therefore provide a novel in vivo approach for investigating the role of a specific PKC isozyme in the regulation of pulmonary vascular reactivity.

We took a knockout mouse approach to test the hypothesis that PKC-epsilon is an important determinant of the magnitude of acute HPV. An isolated, salt solution-perfused mouse lung preparation was used to test for differences in reactivity between PKC-epsilon +/+ and -/- mice. The HPV response of PKC-epsilon -null mice was blunted compared with +/+ controls, whereas ANG II and KCl responses were preserved. To begin to explore the mechanism for the blunted HPV response in PKC-epsilon -null mice, we tested whether derangements in either nitric oxide synthase (NOS) or K+ channel activity were involved. These experiments pointed to a key role for voltage-gated K+ (KV) channels. Expression of one potentially relevant O2-sensitive KV channel (Kv3.1b) was investigated in whole lung and isolated pulmonary artery (PA) smooth muscle cells (SMC). We found that increased expression of Kv3.1b in PASMC may contribute to decreased HPV in PKC-epsilon -/- mice.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Anti-PKC isozyme antibodies and corresponding blocking peptides were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PKC-gamma isozyme antibody was purchased from BD Transduction Laboratories (San Diego, CA). Anti-Kv3.1b K+ channel antibody was purchased from Alomone Labs (Jerusalem, Israel). Protease inhibitor cocktail, ANG II, KCl, nitro-L-arginine (L-NNA), charybdotoxin (ChTX), apamin, 4-aminopyridine (4-AP), and DMEM-F-12 cell culture medium were purchased from Sigma (St. Louis, MO).

Animals

Adult female C57BL6×SV129 PKC-epsilon wild-type (+/+), heterozygote (+/-), and knockout (null, -/-) mice were generated by homologous recombination as previously described (18, 22). Southern blot analysis was used for genotyping the resulting animals (22). F2 hybrid mice were generated at the Gallo Clinic and Research Center (Emeryville, CA; sea level altitude), sent to Denver altitude (5,280 ft) at 11-13 wk of age, and used for all studies. They were allowed to acclimate to Denver altitude for 10-12 additional weeks before study. The animal protocols used were approved by the University of Colorado Health Sciences Center Institutional Animal Care and Use Committee.

Measurement of Baseline Hemodynamics

Mice were anesthetized by intramuscular injection with ketamine-Rompun (100 mg/kg and 15 mg/kg; Fort Dodge and Miles Laboratories, respectively). Closed-chest measurements of right ventricular (RV) systolic pressure (RVSP) were then performed on three spontaneously breathing PKC-epsilon +/+ and -/- mice as previously described (16). After calibration of the pressure transducer (Statham), RV pressures were measured and recorded (Lockheed data recorder).

Comparison of Baseline Lung and Heart Histology

After baseline hemodynamic measurements, the chest was opened and 100 U of heparin was injected into the RV. The pulmonary circulation was gently perfused with PBS to remove blood. The left bronchus was ligated, and the left lung was then removed and quick-frozen in liquid nitrogen (LN). A slit was made in the trachea, and a 16-gauge tubing adapter was inserted and tied in place. A syringe filled with 1% warm agarose (GIBCO, Grand Island, NY) and 1% paraformaldehyde (Sigma) in PBS was attached to the adapter, and the right lung was slowly inflated. The trachea was tied off, and the lung was removed and placed in cold 2% paraformaldehyde-PBS for 2 h. Tissues were then cut, placed in cassettes in 10% buffered formalin (VWR, West Chester, PA), and embedded in paraffin. Trichrome and hematoxylin and eosin staining was performed on lung and heart sections from +/+ and -/- mice to screen for any baseline differences in lung or heart structure.

PKC Isozyme Expression in Mouse Whole Lung Homogenates

Lung tissue was removed from the thorax, quick-frozen in LN, and then stored at -80°C. Frozen lung tissue was weighed and homogenized on ice (PowerGen 700, Pittsburgh, PA) in 5 vols of homogenization buffer (20 mM Tris pH 7.5 containing 0.25 M sucrose, 3 mM EDTA, 3 mM EGTA, 1 mM benzamidine, 0.1% Triton X-100, and a protease inhibitor cocktail including 1.04 mM AEBSF, 0.8 µM aprotinin, 21 µM leupeptin, 15 µM pepstatin A, 36 µM bestatin, and 14 µM E-64). The homogenates were centrifuged at 1,500 relative centrifugal force for 20 min at 4°C (Sorvall Super T21 centrifuge, Newtown, CT). Supernatant aliquots were immediately frozen in LN and stored at -80°C. Protein concentrations were determined by micro-Bradford assay as previously described (11). Seventy-five micrograms of whole lung homogenate was loaded per lane of a 10% reduced SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane as previously described (12). Prestained high-molecular weight (MW) protein markers (Bio-Rad, Hercules, CA) were also loaded into the first lane of each gel. After a 1-h incubation at room temperature (RT) in 5% dry milk-PBS-0.05% Tween 20 to block nonspecific binding, the nitrocellulose was probed with a 1:1,000 dilution of primary PKC isozyme-specific antisera in 5% milk-PBS-0.05% Tween 20 overnight at 4°C. The nitrocellulose was then washed twice for 7 min with PBS-0.05% Tween 20. To detect bound primary antibody, blots were incubated for 1 h at RT with horseradish peroxidase-conjugated goat anti-rabbit IgG at a dilution of 1:5,000 in 5% milk-PBS-0.05% Tween 20. The nitrocellulose was washed again with PBS-0.05% Tween 20 three times for 5 min and then with PBS once for 5 min. Blots were developed with Kodak Biomax Light Film (Eastman Kodak, Rochester, NY) and an enhanced chemiluminescence detection kit (NEN, Boston, MA). Determination of MW for each isozyme detected was made by comparison with known MW markers. Band intensity was quantified with NIH Image Software (National Institutes of Health, Bethesda, MD). To confirm the specificity of binding between primary antibody and immunoreactive protein, Western blots were routinely performed in the presence and absence of isozyme-specific immunizing peptide. Isozyme-specific antisera were preincubated with the respective peptide antigen (1:10) used for immunization for 2 h at RT or overnight at 4°C and then diluted to the working concentration as described above. When no signal was detectable, a known positive control was used to verify antibody activity and specificity.

Modified Salt Solution-Perfused Mouse Lung Preparations

An intact-chest modified salt solution-perfused lung preparation was used as previously described (16) to determine the effect of PKC-epsilon deletion on acute pulmonary vascular reactivity to hypoxia. After mice were adequately anesthetized, the trachea was intubated and lungs were ventilated with a mixture of 21% O2-5% CO2-74% N2 (balance) at 60 breaths/min, a maximum inspiratory pressure of 9 cmH2O, and an expiratory pressure of 2.5 cmH2O. After placement of cannulas into the main PA and similar placement of a catheter in the left ventricle, the lung was perfused by a peristaltic pump at a constant flow of 0.04 ml · g body wt-1 · min-1. The perfusate was a physiological salt solution (16) containing Ficoll (4%) as a colloid and 3.1 mM sodium meclofenamate to inhibit synthesis of prostacyclin. Mean pulmonary arterial perfusion pressure (typically 8-12 mmHg at baseline) was continuously measured with a transducer at a constant flow and recorded with the Biopac system. A 20-min period of equilibration was allowed during ventilation with 21% O2 before vascular responses were measured.

Isolated, Perfused Lung Protocols

Comparison of HPV. After a baseline perfusion pressure was established, the lung was ventilated with three challenges of severe hypoxia (0% O2) for 5 min followed by 5 min of normoxic ventilation. To detect subtle differences in HPV the lungs were then ventilated once with a submaximal dose of hypoxia (3% O2) for 5 min. The absolute magnitude of each of the three acute hypoxic vasoconstrictor responses were averaged and compared between +/+ and -/- mice. This HPV technique was applied to all lungs before specific pharmacological protocols. Mice of different genotypes were studied serially in each experimental protocol.

Comparison of vasoconstriction in response to ANG II and KCl. After measurement of the hypoxic responses, the lungs of +/+ and -/- mice were ventilated with 21% O2 and 0.2 µg of ANG II was added to the perfusate. After the ANG II pressor response was measured, increasing concentrations of KCl were added stepwise to the perfusate (20-80 mM). Ventilation was maintained at 21% O2 throughout the KCl additions (3). To test the effect of gene dosing on HPV, ANG II, and KCl responses, PKC-epsilon +/- mice were also studied.

Contribution of NO and KCa channel activation to differences in HPV. The nonselective NOS inhibitor L-NNA was added at a concentration of 100 µM to the perfusate of +/+ and -/- lungs and circulated for 10 min after initial evaluation of HPV (16). After L-NNA lungs were ventilated with 0% O2. To test whether the blunted HPV in -/- lungs was due to increased KCa channel expression/activity, 50 nM ChTX and 50 nM apamin were added simultaneously to block big- and small-conductance KCa channels (3). The KCa channel blockers were circulated for 10 min, and the maximal (0% O2) challenge was repeated.

Contribution of KV rectifying channel activation to differences in HPV. To determine whether the blunted HPV in -/- lungs was due to KV channel activity, the selective blocker 4-AP was added at concentrations of 0.1 and 1.0 mM after the initial evaluation of HPV (17). 4-AP was circulated in the perfusate for 10 min after each addition, and a maximal (0% O2) challenge was performed.

Kv3.1b Protein Expression in +/+ and -/- Whole Lung Homogenates

To measure expression of the KV channel Kv3.1b, frozen lung tissue was homogenized in 0.3 ml of homogenization buffer containing 10% glycerol, 20 mM Tris · HCl pH 7.5, 150 mM NaCl, 0.5 M NaF, 0.02 M Na pyrophosphate, 100 µM Na orthovanadate, 0.5% Triton X-100, 1.0% NP-40, and the protease inhibitor cocktail. Homogenates were centrifuged at 10,000 rcf for 10 min at 4°C (Savant µSpeedFuge, Holbrook, NY) and stored as noted above. Eighty micrograms of whole lung homogenate were loaded per lane of an 8% reduced SDS-polyacrylamide gel and run at 100 V. After a 1-h incubation at RT in 10% dry nonfat milk-1% protease-free BSA-Tris-buffered saline (TBS)-0.05% Tween 20 to block nonspecific binding, the nitrocellulose was probed with a 1:200 dilution of primary Kv3.1b-specific antisera overnight at 4°C. The nitrocellulose was washed three times for 5 min with TBS-0.05% Tween 20. To detect bound primary antibody, blots were processed as described for PKC isozyme expression. Determination of MW was made by comparison with known MW markers and the anticipated MWs of the glycosylated (100 kDa) and core (70 kDa) forms of the Kv3.1b protein (8, 31). To confirm the specificity of binding between primary antibody and immunoreactive protein, Western blots were also performed in the presence of excess immunizing peptide. Mouse brain extract was used as a positive control for the 100-kDa form of the Kv3.1b peptide; the sample also yielded a faint 70-kDa signal. Antiserum was preincubated with the peptide antigen (1 µg:2 µg ratio) and diluted to the concentration described above.

Kv3.1b Protein Expression in +/+ and -/- Mouse PASMC Lysates

Heart and lung tissue blocks were removed from the thorax of +/+ and -/- mice and placed in MEM containing 200 U/ml penicillin and 0.2 mg/ml streptomycin (Sigma). The main PA was dissected away and placed in fresh MEM. After incubation in 100 µl of 10× trypsin, small pieces of PA were placed in digest medium (HBSS containing elastase, collagenase, albumin, and soybean trypsin inhibitor) and incubated on a rotator plate for 90 min at 37°C. The resulting solution was passed thru a 100-µm cell strainer, rinsed with 10% FBS and DMEM-F-12 medium, and centrifuged. The pelleted cells were resuspended in 10% FBS and DMEM-F-12 and pipetted into 2 wells of a 48-well plate. Digested cells were maintained in 10% FBS-DMEM-F-12 medium and incubated at 37°C. Medium was supplemented every other day for the first 7 days and then replaced twice a week thereafter. Cells were characterized by staining for alpha -smooth muscle (SM)-specific actin and SM-specific myosin. To harvest PASMC lysates, 435 × 103 cells from +/+ and -/- mice were plated in P-100 dishes and grown to confluence. The cells were exposed for 2 days to fresh DMEM-F-12 containing 0.1% FBS to induce a quiescent state. Cells were then harvested with 200 µl of cell lysis buffer (20 mM Tris, pH 7.5, containing 0.25 M sucrose, 3 mM EDTA, 3 mM EGTA, 50 mM mercaptoethanol, 50 µg/ml leupeptin, 50 µg/ml aprotinin, 1 mM PMSF, and 0.1% Triton X-100) per plate on ice. Resulting lysates were centrifuged, and the supernatant was carefully transferred to new tubes and stored at -80°C.

Fifteen micrograms of +/+ and -/- SMC lysate were loaded per lane of a 10% reduced SDS-polyacrylamide gel and run at 100 V for 90 min. The blot was then transferred to a nitrocellulose membrane overnight at 4°C. After a 1-h incubation at RT in 5% dry milk-TBS-0.05% Tween 20 to block nonspecific binding, the nitrocellulose was probed with a 1:200 dilution of primary Kv3.1b-specific antisera in 5% milk-TBS-0.05% Tween 20 overnight at 4°C. The nitrocellulose was washed three times for 15 min with TBS-0.05% Tween 20. The blots were processed as described above to detect the bound primary antibody.

Data Analysis

Routine review of lung and heart histology, initial measurement of baseline RVSP, and detection of PKC isozyme expression was performed on n = 3 mice per genotype. Otherwise, n = 3-6 individual mice per genotype were used for each experimental condition. Mechanistic studies were limited to +/+ and -/- mice. After each perfused lung study, tissue was saved to reconfirm the original genotype data. One- and two-way ANOVA, followed by the Student-Newman-Keuls multiple-comparison test were used for individual comparisons within and between groups of data points. Significance was defined as P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Initial Characterization of PKC-epsilon Mice

Baseline lung and heart structure was examined before the vascular reactivity studies were undertaken. Review of trichrome- and hematoxylin and eosin-stained tissue from +/+ and -/- mice (n = 3/group) showed no differences in the lung or heart structure. Figure 1 shows representative trichrome stains of proximal and distal vessels and adjacent lung parenchyma from +/+ and -/- mice. RVSP values were also initially measured in a subgroup of +/+ and -/- mice before the vascular reactivity studies were undertaken (n = 3/group). No difference was seen in baseline RVSP values in the two groups (+/+ mice, 25.8 ± 1.5 mmHg; -/- mice, 26.1 ± 1.6 mmHg).


View larger version (128K):
[in this window]
[in a new window]
  		Fig. 1.   Normal lung histology in adult PKC-epsilon wild-type and null mice. Trichrome staining was performed on formalin-fixed paraffin-embedded lung sections from +/+ and -/- mice to screen for any differences in lung structure (n = 3 mice/genotype). Representative proximal and distal (see arrows) vessels shown.

PKC Isozyme Expression in PKC-epsilon Mice

PKC-epsilon peptide was detected in +/+ mouse lungs as a doublet at 84 kDa. The absence of PKC-epsilon was also confirmed in PKC-epsilon -null mice. Because adaptive changes in the expression of other PKC isozymes may occur when one isoform is deleted (36, 43), expression levels of other PKC isozymes were measured in lung lysates from +/+ and -/- mice (Fig. 2). The same blot was stripped and reprobed for each PKC isozyme. No significant differences were seen in the expression of other isozymes detected (alpha , beta I, beta II, gamma , delta , eta , theta , zeta , µ) in the lungs of +/+ and -/- mice (n = 3; representative data shown). In addition to those isozymes shown, PKC-lambda was detected in +/+ and -/- mouse lung lysates and no significant differences were noted (data not shown). PKC-gamma expression was not detected in +/+ and -/- mouse lung lysates; antibody activity was verified with a positive control (mouse brain extract).


View larger version (55K):
[in this window]
[in a new window]
  		Fig. 2.   Expression of PKC isozymes in lungs from PKC-epsilon wild-type and null mice. Western blot analysis was completed on whole lung homogenates from PKC-epsilon +/+ and -/- mice. Lysates were subjected to SDS-PAGE and immunoblot analysis with PKC isozyme-specific antibodies (n = 3 mice/genotype; blots done in duplicate, representative blots shown).

Comparison of HPV in PKC-epsilon Mice

To evaluate the effect of the targeted deletion of PKC-epsilon on acute HPV, we compared the magnitudes of the hypoxic pressor responses in +/+ and -/- mice in response to three maximal and one submaximal hypoxic challenges (0% and 3% O2, respectively). Figure 3 illustrates representative examples of pressure tracings from isolated perfused +/+ and -/- lungs showing a blunting of HPV in the -/- mice. The magnitudes of the three challenges were averaged together for several additional mice and compared. The -/- mice had a significantly blunted response compared with the +/+ mice (Fig. 4). In response to the submaximal hypoxic challenge, the PKC-epsilon -/- mice again had significantly reduced HPV compared with their +/+ counterparts.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 3.   Representative pulmonary arterial pressure tracings from isolated, salt solution-perfused lungs from a PKC-epsilon wild-type mouse (A) and a null mouse (B). Three maximal hypoxic (0% O2) challenges followed by one submaximal hypoxic (3% O2) challenge were performed. HPV was blunted in PKC-epsilon -null compared with +/+ mice. A 20-min period of equilibration was established before vascular responses were measured. HPV was expressed as Delta mmHg above baseline.



View larger version (9K):
[in this window]
[in a new window]
  		Fig. 4.   PKC-epsilon inhibition decreases acute hypoxic pulmonary vasoconstriction (HPV). Mean acute HPV data from +/+ and PKC-epsilon -null mice (open bars, PKC-epsilon +/+; filled bars, PKC-epsilon -/-) demonstrating that HPV was blunted in the PKC-epsilon -null mice in response to 3 maximal hypoxic challenges (0% O2) as well as to a submaximal hypoxic challenge (3% O2). The 3 maximal hypoxic challenge values were averaged together to obtain a representative HPV response for each mouse. *P < 0.05 vs. +/+ (n = 4-6 mice).

Comparison of ANG II and KCl Responses in PKC-epsilon Wild-Type and Null Mice

To determine whether the blunted responses to HPV could be attributed to a general decrease in vasoreactivity, we measured the contractile response induced by ANG II and increasing concentrations of KCl (Table 1). In contrast to the blunting of reactivity to acute hypoxia observed in the absence of PKC-epsilon , no significant difference in responsiveness to ANG II was found. KCl was then incrementally increased in the perfusate to a final concentration of 80 mM. The cumulative effects of the increasing KCl concentration on the perfusion pressure were similar for each genotype. These observations suggested that the blunted HPV in the -/- mice could not be attributed to a generalized decrease in vasoreactivity.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Effect of ANG II and KCl on pressor response in isolated, perfused lungs from PKC-varepsilon +/+ and -/- mice

Effect of PKC-epsilon Gene Dosing on HPV

To assess the effect of PKC-epsilon gene dosing on the blunting of HPV, PKC-epsilon heterozygote (+/-) mice were evaluated. The relative amount of PKC-epsilon protein expressed in lungs from these mice was first determined and found to be 66.0% of +/+ levels (n = 3 mice). The reduced level of PKC-epsilon peptide expression was associated with an intermediate response to both maximal (1.4 ± 0.2 Delta mmHg; n = 3) and submaximal (0.5 ± 0.2 Delta mmHg; n = 3) hypoxia. Responses to ANG II and KCl, like those in null mice, were preserved (ANG II, 1.3 ± 0.3 Delta mmHg; KCl, 15.4 ± 5.1 Delta mmHg).

Impact of NOS and KCa Channel Inhibition on Genotype-Specific Differences in HPV

We questioned whether inhibition of NOS activity would restore the HPV in lungs from PKC-epsilon -null mice. Nonselective inhibition of NOS isoforms by L-NNA increased the HPV response in +/+ lungs, as others have observed (3), but did not reverse the attenuated HPV in -/- mice (Fig. 5). KCa channel activation has been implicated in the blunted HPV response observed in a rat model of hepatopulmonary syndrome (3). As seen in Table 1, extracellular K+ normalized the cumulative pressor response of +/+ and -/- lungs, suggesting involvement of K+ channels in the differential response. Therefore, to evaluate the role of KCa channel activation in the blunted HPV of the -/- mice, large- and small-conductance KCa channels were inhibited with ChTX and apamin. These agents were added after meclofenamate and L-NNA. The addition of these KCa channel blockers did not restore the HPV responses of the -/- mice (Fig. 5).


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of nitro-L-arginine (L-NNA) and the KCa channel blockers charybdotoxin (ChTX) and apamin on acute HPV in PKC-epsilon wild-type and null isolated, perfused mouse lungs. Baseline HPV data from +/+ and PKC-epsilon -null mice (open bars, PKC-epsilon +/+; closed bars, PKC-epsilon -/-) are shown. Three maximal hypoxic challenge values were averaged together to obtain a representative pressor response for each mouse. Maximal hypoxic pulmonary vasoreactivity was assessed after a 100 µM dose of the nonselective nitric oxide synthase (NOS) inhibitor L-NNA. After the addition of L-NNA to the perfusate, the KCa channel blockers ChTX and apamin were added to the perfusate simultaneously and the response to 0% O2 was assessed again. *P < 0.05 compared with the difference in the initial +/+ vs. -/- response; **P < 0.05 compared with the difference after L-NNA addition (n = 3-5 mice).

Impact of KV Channel Inhibition on Genotype-Specific Differences in HPV

Delayed-rectifying KV channels are thought to be important in the HPV response (17). 4-AP is an inhibitor of KV channels and causes hypoxia-independent vasoconstriction. Therefore, we added two concentrations of 4-AP to the perfusate and allowed it to circulate for 10 min after each addition to inhibit KV channels. Figure 6 shows that the addition of this KV channel blocker causes significant potentiation of responses in -/- mice compared with +/+ controls at 0.1 and 1.0 mM concentrations. This pharmacological maneuver also markedly enhanced the subsequent response of the -/- mice to 0% O2. The post-4-AP response of the -/- mice was increased to a level above that observed with +/+ controls.


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of the KV channel blocker 4-aminopyridine (4-AP) on acute HPV in PKC-epsilon wild-type and null isolated, perfused mouse lungs. Data from wild-type and PKC-epsilon -null mice (open bars, PKC-epsilon +/+; closed bars, PKC-epsilon -/-) are shown. Increasing doses (0.1 and 1.0 mM) of 4-AP were added to the perfusate. The response to 0% O2 was then assessed after 4-AP. *P < 0.05 vs. +/+ at the 0.1 mM concentration; **P < 0.05 vs. +/+ at the 1.0 mM concentration; ***P < 0.05 vs. +/+ response to 0% O2.

Differential Expression of O2-Sensitive Kv3.1b Channel in PKC-epsilon Mice

The studies with 4-AP suggested a key role for KV channels in the blunted HPV of the PKC-epsilon -null mouse. Specifically, the potentiation of HPV observed after 4-AP treatment suggested that one or more KV channels may be upregulated in PKC-epsilon -null mice. We focused on Kv3.1b because 1) it is O2 sensitive and thought to be important in HPV (30, 48); 2) it is expressed in the bovine pulmonary vasculature (8); and 3) it is susceptible to inhibition by low concentrations of 4-AP but not by ChTX. We compared the protein expression of the Kv3.1b K+ channel subunit in whole lung homogenates from +/+ and -/- mice. A 100-kDa band was detected in both +/+ and -/- mouse lung homogenates. A faint 70-kDa band was also detected in both groups of lungs (data not shown), as well as in a positive control (mouse brain extract). The signals were extinguished when the blots were treated with antibody in the presence of blocking peptide. Both Kv3.1b proteins were slightly upregulated (21%) in the PKC-epsilon -null mice (n = 4 mice), suggesting a possible role for the KV channel in the blunted HPV (Fig. 7A). To test whether greater differences might exist within the pulmonary vasculature, we also isolated PASMC from +/+ and -/- mice and again compared the protein expression of the Kv3.1b K+ channel subunit between the two groups (Fig. 7B). PASMC from +/+ mice had a faint 100-kDa signal; a faint 70-kDa peptide was also variably detected although not seen in Fig. 7. A more substantial increase in expression of both forms of the Kv3.1b protein was detected in -/- PASMC isolated at the same density. Quantitation of changes in the 100-kDa peptide showed a 125% increase over +/+ PASMC. These bands were absent when probed in the presence of excess immunizing peptide.


View larger version (34K):
[in this window]
[in a new window]
  		Fig. 7.   Expression of O2-sensitive KV channel Kv3.1b in whole lung and isolated pulmonary artery smooth muscle cells (PASMC) from PKC-epsilon wild type and null mice. A: comparison of Kv3.1b in whole lung homogenates. Western blot analysis was completed on whole lung homogenates from PKC-epsilon +/+ and null mice. Eighty micrograms of protein sample were applied per lane. Blots were probed with anti-Kv3.1b antibody. PKC-epsilon -null mice expressed slightly more (21%) Kv3.1b 100-kDa protein compared with +/+ mice. The same subtle increase was seen in the faint 70-kDa band (not shown). A mouse brain positive control was used to help localize both forms of the KV channel. The signals were extinguished by treatment with an excess of antigenic blocking peptide (BP) (n = 4 mice/genotype). B: comparison of Kv3.1b in PASMC lysates. Western blot analysis was completed on PASMC lysates from PKC-epsilon +/+ and null mice. Fifteen micrograms of protein sample were applied per lane. Blots were probed with anti-Kv3.1b antibody. The dominant band detected was the 100-kDa glycosylated form of the protein (open bars, PKC-epsilon +/+; closed bars, PKC-epsilon -/-). PASMC derived from null mice expressed more of the glycosylated form (100 kDa) of the Kv3.1b protein (122.3%) than cells from +/+ mice. (n = 3 replicate samples from 1 mouse/genotype shown; blots done in duplicate). The 70-kDa core protein was also increased in PKC-epsilon -/- cell lysates.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our studies showed that PKC-epsilon deletion decreases acute HPV in the mouse. We demonstrated that the blunted response of PKC-epsilon -null mice to maximal and submaximal challenges of hypoxia in the isolated, perfused lung was not due to a generalized decrease in vasoconstriction. This observation agrees with the histology that showed no obvious differences in airway or vascular structure. A selective difference in HPV was demonstrated by contrasting the hypoxic responses with those induced by ANG II and increasing concentrations of KCl. Both the ANG II and KCl responses were preserved in the null mice.

PKC was previously shown to be an important signaling pathway in contractile responses in the systemic and pulmonary circulation (29, 35, 37); however, the contribution of individual isozymes of PKC has been unclear. The PKC-epsilon isozyme has been implicated in several responses including migration (20), shear stress and ventricular stretch (40), Golgi trafficking (9), and growth and transformation (23, 42). PKC-epsilon has been linked to contraction in the systemic circulation (19), although there has been some controversy in this area (41). Horowitz et al. (19) showed that in ferrets a constitutively active form of PKC-epsilon applied to single aortic SMC induced contraction that could be reversed with an inhibitory peptide. Using a knockout mouse approach, we have now shown that PKC-epsilon is important in the regulation of HPV. Although it is very unlikely, we acknowledge that the blunted HPV in PKC-epsilon -null mice could be due to an as yet unidentified adaptive developmental response to the gene deletion rather than a direct consequence of the specific PKC-epsilon knockout. Against this possibility is the observation that expression levels of the other PKC isozymes were unchanged in the lungs of PKC-epsilon +/+ and null mice. Our results are similar in this regard to the findings of Khasar et al. (22) in brain tissue from the same mice. In future studies we plan to extend the characterization of the PKC isozymes to isolated cells from +/+ and -/- mice.

We have not explored how hypoxia activates PKC-epsilon , but several factors (phosphatidylinositol 3-kinase, 3-phosphoinositide-dependent kinase) are likely involved (5). PKC-epsilon is normally located in the nucleus and translocates to cross-striated structures and cell-cell contact regions after stimulation (26). Elaborate PKC-epsilon signaling complexes have recently been identified in the heart that suggest ways in which hypoxic sensing mechanisms, PKC-epsilon , and contractile proteins could interact (32).

Our findings suggest that deletion of PKC-epsilon may be protective against the development of pulmonary hypertension. In contrast to our results in the lung, several studies have shown that activation of PKC-epsilon is important in protection of cardiac myocytes from ischemic/hypoxic injury (6, 32). Thus the role of PKC-epsilon in adaptive responses to hypoxia may be tissue specific.

Our studies support the idea that K+ channels play a key role in the regulation of HPV. K+ channel activity was previously implicated in the regulation of HPV and vascular tone. Several types of K+ channels have been reported in PASMC that modulate vascular tone, including KCa and delayed-rectifier KV channels (2, 3, 8, 17). Post et al. (33) suggested that K+ channels are inhibited by acute hypoxia in isolated canine PASMC; this inhibition was unique to the pulmonary circulation, because a similar sensitivity to hypoxia could not be demonstrated in canine renal artery cells. Because extracellular K+ concentration restored the cumulative pressor response of the PKC-epsilon -null mice, it suggested that the blunted HPV in the null mice could involve K+ channel activation.

We demonstrated that blocking delayed-rectifier KV channels with 4-AP restored the HPV response of the PKC-epsilon -/- mice to beyond that of their +/+ counterparts. Delayed-rectifier KV channels have been shown to be important in the regulation of the HPV response. Sweeney and Yuan (39) suggest that acute hypoxia inhibits KV channels selectively and induces membrane depolarization in PASMC. Membrane depolarization causes voltage-dependent Ca2+ channels to open and allows Ca2+ influx that causes contraction in the SMC. Previous studies in other experimental models including rats and dogs reported that the delayed-rectifier KV channel blocker 4-AP causes vasoconstriction under normoxic conditions (2, 17). Barman (2) reported that in isolated canine lungs, pharmacological activation of PKC with phorbol myristate acetate and thymeleatoxin caused pulmonary vasoconstriction; the effect was potentiated by 4-AP.

We found that expression of the Kv3.1b channel was slightly increased in lungs from PKC-epsilon -/- mice compared with +/+ controls. We then isolated PASMC from PKC-epsilon +/+ and null mice and found that both the glycosylated (100 kDa) and core (70 kDa) forms of the protein were more substantially upregulated in the PKC-epsilon -null SMC. This suggested that the Kv3.1b channel may play a role in the blunted HPV response of the PKC-epsilon -null mice. Although we have not directly proven that activation of PKC-epsilon negatively regulates the expression of Kv3.1b, a link is strongly suggested. We focused on Kv3.1b because 1) it is O2 sensitive and thought to be important in HPV (30, 48); 2) it is expressed in the pulmonary vasculature (8), and 3) it is susceptible to inhibition by 4-AP but not by ChTX.

KV channels are tetramers made of two subunits. The alpha -subunits are the pore-forming portion, whereas the beta -subunits perform a regulatory function (39). There are many candidates that could form O2-sensitive channels (48). Osipenko et al. (30) suggest that there is a potential role for the Kv3.1b channel as an O2 sensor in PASMC. Hypoxia was shown to inhibit the Kv3.1b channel in PASMC but did not cause complete depolarization of the cells alone. Osipenko et al. suggested that the Kv3.1b channel amplifies a primary response to hypoxia. Archer et al. (1) showed that PASMC contain numerous types of Kv channels from the Kv1 and Kv2 families, and Kv1.5 and Kv2.1 may play a role in the HPV response as well. We have not yet studied the effects of chronic hypoxia on the expression of the Kv3.1b channel in +/+ or -/- mice; however, this is an interesting future direction to pursue.

We also tested the possibility that KCa channel activation may be involved in the blunted HPV response of the PKC-epsilon -null mice. Inhibition of the KCa channels did not reverse the response of the null mice. The KCa channel blockers appeared to both enhance the +/+ HPV response and further attenuate the response of the null mice. The significance of the slight further attenuation of HPV in the PKC-epsilon -null mice is not clear. These results are different than those reported by Carter et al. (3) in a rat model of hepatopulmonary syndrome with impaired arterial oxygenation. Their cirrhotic rats had a blunted HPV response that was not dependent solely on elevated NO and decreased ET-1 levels. They reported that the blockade of KCa channels with ChTX and apamin normalized the response between sham-injured and cirrhotic rats, suggesting an important role for KCa channels in their model.

NO plays a central role in the regulation of vascular tone. NOS is the enzyme responsible for the production of NO, and three isoforms have been described (endothelial, inducible, and neuronal). Expression of endothelial and inducible NOS isoforms is increased in chronic hypoxic pulmonary hypertension (24). These two isoforms have also been implicated in modulating pulmonary vascular tone. However, the attenuated HPV in PKC-epsilon -/- mice was not reversed by a nonselective NOS inhibitor in our model, suggesting that differences in NOS activity could not account for the result. L-NNA did augment the acute HPV response of the +/+ mice, confirming that the isolated +/+ mouse lungs behaved as others have described (16). Interestingly, macrophages from PKC-epsilon -null mice have an attenuated response to LPS and interferon with decreased generation of NO. Macrophages from these null mice failed to induce NOS expression (4). However, Fagan et al. (16) showed that endothelial NOS-derived NO is the major mediator of vasodilation in the murine pulmonary circulation.

In summary, we have found that the isolated lungs from PKC-epsilon -null mice have blunted acute HPV. The NOS inhibitor L-NNA and the KCa channel inhibitors ChTX and apamin did not reverse the blunted HPV of the PKC-epsilon -null mice. In contrast, the delayed rectifying KV channel blocker 4-AP potentiated the hypoxic response of the -/- mice compared with +/+ controls. This observation suggests that the blunted hypoxic pressor response of the -/- mice may be the result of increased KV channel expression and/or activity and membrane hyperpolarization. Our findings of increased Kv3.1b protein expression in PKC-epsilon -null PASMC supports this concept. Thus increased expression of KV channels likely contributes to the decreased HPV observed in the PKC-epsilon -/- mouse. Further investigation of KV channel subunit interactions is clearly needed, because Kv3.1b is only part of a complex sensing system that transduces hypoxic signals (48).


    ACKNOWLEDGEMENTS

The authors thank Cheryl Oliver-Pickett for assistance with animal protocols, Hannah Young for animal handling at the Ernest Gallo Clinic and Research Center, Sandra Walchak and Dustin Tallman for excellent technical assistance, and Dr. Ethan Carter and Dr. Vijaya Karoor for helpful discussions about this manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute PPG-HL-14985 and Veterans Administration Merit Review.

Preliminary results were presented at the 2002 Annual Meeting of the American Thoracic Society, Atlanta, GA, on May 22, 2002 (Am J Respir Crit Care Med 165: A749, 2002).

Address for reprint requests and other correspondence: E. C. Dempsey, Cardiovascular Pulmonary Research Laboratory; B-133, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: Edward.Dempsey{at}uchsc.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.

First published December 27, 2002;10.1152/ajpheart.00795.2002

Received 10 September 2002; accepted in final form 5 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Archer, SL, Weir EK, Reeve HL, and Michelakis E. Molecular identification of O2 sensors and O2-sensitive potassium channels in the pulmonary circulation. Adv Exp Med Biol 475: 219-240, 2000[ISI][Medline].

2.   Barman, SA. Potassium channels modulate canine pulmonary vasoreactivity to protein kinase C activation. Am J Physiol Lung Cell Mol Physiol 277: L558-L565, 1999[Abstract/Free Full Text].

3.   Carter, EP, Sato K, Morio Y, and McMurtry IF. Inhibition of KCa channels restores blunted hypoxic pulmonary vasoconstriction in rats with cirrhosis. Am J Physiol Lung Cell Mol Physiol 279: L903-L910, 2000[Abstract/Free Full Text].

4.   Castrillo, A, Pennington DJ, Otto F, Parker PJ, Owen MJ, and Bosca L. Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection. J Exp Med 194: 1231-1242, 2001[Abstract/Free Full Text].

5.   Cenni, V, Doppler H, Sonnenburg ED, Maraldi N, Newton AC, and Toker A. Regulation of novel protein kinase Cepsilon by phosphorylation. Biochem J 363: 537-545, 2002[ISI][Medline].

6.   Chen, L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Banci L, Guo Y, Bolli R, Dorn GW, 2nd, and Mochly-Rosen D. Opposing cardioprotective actions and parallel hypertrophic effects of delta  PKC and epsilon  PKC. Proc Natl Acad Sci USA 98: 11114-11119, 2001[Abstract/Free Full Text].

7.   Chou, MM, Hou W, Johnson J, Graham LK, Lee MH, Chen CS, Newton AC, Schaffhausen BS, and Toker A. Regulation of protein kinase Czeta by PI 3-kinase and PDK-1. Curr Biol 8: 1069-1077, 1998[ISI][Medline].

8.   Coppock, EA, and Tamkun MM. Differential expression of KV channel alpha - and beta -subunits in the bovine pulmonary arterial circulation. Am J Physiol Lung Cell Mol Physiol 281: L1350-L1360, 2001[Abstract/Free Full Text].

9.   Csukai, M, Chen CH, De Matteis MA, and Mochly-Rosen D. The coatomer protein beta '-COP, a selective binding protein (RACK) for protein kinase Cepsilon . J Biol Chem 272: 29200-29206, 1997[Abstract/Free Full Text].

10.   Davies, SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95-105, 2000[ISI][Medline].

11.   Dempsey, EC, Badesch DB, Dobyns EL, and Stenmark KR. Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: dependence on cell size, time from birth, insulin-like growth factor I, and auto-activation of protein kinase C. J Cell Physiol 160: 469-481, 1994[ISI][Medline].

12.   Dempsey, EC, Frid MG, Aldashev AA, Das M, and Stenmark KR. Heterogeneity in the proliferative response of bovine pulmonary artery smooth muscle cells to mitogens and hypoxia: importance of protein kinase C. Can J Physiol Pharmacol 75: 936-944, 1997[ISI][Medline].

13.   Dempsey, EC, McMurtry IF, and O'Brien RF. Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia. Am J Physiol Lung Cell Mol Physiol 260: L136-L145, 1991[Abstract/Free Full Text].

14.   Dempsey, EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, and Messing RO. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 279: L429-L438, 2000[Abstract/Free Full Text].

15.   Dorn, GW, 2nd, Souroujon MC, Liron T, Chen CH, Gray MO, Zhou HZ, Csukai M, Wu G, Lorenz JN, and Mochly-Rosen D. Sustained in vivo cardiac protection by a rationally designed peptide that causes epsilon  protein kinase C translocation. Proc Natl Acad Sci USA 96: 12798-12803, 1999[Abstract/Free Full Text].

16.   Fagan, KA, Tyler RC, Sato K, Fouty BW, Morris KG, Jr, Huang PL, McMurtry IF, and Rodman DM. Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation. Am J Physiol Lung Cell Mol Physiol 277: L472-L478, 1999[Abstract/Free Full Text].

17.   Hasunuma, K, Rodman DM, and McMurtry IF. Effects of K+ channel blockers on vascular tone in the perfused rat lung. Am Rev Respir Dis 144: 884-887, 1991[ISI][Medline].

18.   Hodge, CW, Mehmert KK, Kelley SP, McMahon T, Haywood A, Olive MF, Wang D, Sanchez-Perez AM, and Messing RO. Supersensitivity to allosteric GABAA receptor modulators and alcohol in mice lacking PKCepsilon . Nat Neurosci 2: 997-1002, 1999[ISI][Medline].

19.   Horowitz, A, Clement-Chomienne O, Walsh MP, and Morgan KG. epsilon -Isoenzyme of protein kinase C induces a Ca2+-independent contraction in vascular smooth muscle. Am J Physiol Cell Physiol 271: C589-C594, 1996[Abstract/Free Full Text].

20.   Ivaska, J, Whelan RD, Watson R, and Parker PJ. PKCepsilon controls the traffic of beta 1 integrins in motile cells. EMBO J 21: 3608-3619, 2002[ISI][Medline].

21.   Jeffery, TK, and Wanstall JC. Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension. Pharmacol Ther 92: 1-20, 2001[ISI][Medline].

22.   Khasar, SG, Lin YH, Martin A, Dadgar J, McMahon T, Wang D, Hundle B, Aley KO, Isenberg W, McCarter G, Green PG, Hodge CW, Levine JD, and Messing RO. A novel nociceptor signaling pathway revealed in protein kinase Cepsilon mutant mice. Neuron 24: 253-260, 1999[ISI][Medline].

23.   Kim, L, Lee T, Fu J, and Ritchie ME. Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo. Am J Physiol Heart Circ Physiol 277: H1808-H1816, 1999[Abstract/Free Full Text].

24.   Le Cras, TD, Xue C, Rengasamy A, and Johns RA. 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].

25.   Leitges, M, Mayr M, Braun U, Mayr U, Li C, Pfister G, Ghaffari-Tabrizi N, Baier G, Hu Y, and Xu Q. Exacerbated vein graft arteriosclerosis in protein kinase Cdelta -null mice. J Clin Invest 108: 1505-1512, 2001[ISI][Medline].

26.   Mochly-Rosen, D, and Gordon AS. Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J 12: 35-42, 1998[Abstract/Free Full Text].

27.   Murray, NR, Davidson LA, Chapkin RS, Clay Gustafson W, Schattenberg DG, and Fields AP. Overexpression of protein kinase C beta II induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J Cell Biol 145: 699-711, 1999[Abstract/Free Full Text].

28.   Nishizuka, Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258: 607-614, 1992[Abstract/Free Full Text].

29.   Orton, EC, Raffestin B, and McMurtry IF. Protein kinase C influences rat pulmonary vascular reactivity. Am Rev Respir Dis 141: 654-658, 1990[ISI][Medline].

30.   Osipenko, ON, Tate RJ, and Gurney AM. Potential role for kv3.1b channels as oxygen sensors. Circ Res 86: 534-540, 2000[Abstract/Free Full Text].

31.   Ozaita, A, Martone ME, Ellisman MH, and Rudy B. Differential subcellular localization of the two alternatively spliced isoforms of the kv3.1 potassium channel subunit in brain. J Neurophysiol 88: 394-408, 2002[Abstract/Free Full Text].

32.   Ping, P, Zhang J, Pierce WM, Jr, and Bolli R. Functional proteomic analysis of protein kinase Cepsilon signaling complexes in the normal heart and during cardioprotection. Circ Res 88: 59-62, 2001[Abstract/Free Full Text].

33.   Post, JM, Hume JR, Archer SL, and Weir EK. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol Cell Physiol 262: C882-C890, 1992[Abstract/Free Full Text].

34.   Rahman, A, Bando M, Kefer J, Anwar KN, and Malik AB. Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene transcription. Mol Pharmacol 55: 575-583, 1999[Abstract/Free Full Text].

35.   Rasmussen, H, Takuwa Y, and Park S. Protein kinase C in the regulation of smooth muscle contraction. FASEB J 1: 177-185, 1987[Abstract].

36.   Romanova, LY, Alexandrov IA, Nordan RP, Blagosklonny MV, and Mushinski JF. Cross-talk between protein kinase C-alpha (PKC-alpha ) and -delta (PKC-delta ): PKC-alpha elevates the PKC-delta protein level, altering its mRNA transcription and degradation. Biochemistry 37: 5558-5565, 1998[Medline].

37.   Shimoda, LA, Sham JS, and Sylvester JT. Altered pulmonary vasoreactivity in the chronically hypoxic lung. Physiol Res 49: 549-560, 2000[ISI][Medline].

38.   Shimoda, LA, Sylvester JT, Booth GM, Shimoda TH, Meeker S, Undem BJ, and Sham JS. Inhibition of voltage-gated K+ currents by endothelin-1 in human pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 281: L1115-L1122, 2001[Abstract/Free Full Text].

39.   Sweeney, M, and Yuan JX. Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels. Respir Res 1: 40-48, 2000[Medline].

40.   Traub, O, Monia BP, Dean NM, and Berk BC. PKC-epsilon is required for mechano-sensitive activation of ERK1/2 in endothelial cells. J Biol Chem 272: 31251-31257, 1997[Abstract/Free Full Text].

41.   Walker, LA, Gailly P, Jensen PE, Somlyo AV, and Somlyo AP. The unimportance of being (protein kinase C) epsilon . FASEB J 12: 813-821, 1998