Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II

Derek S. Damron, Hany S. Nadim, Sung Jin Hong, Ahmad Darvish, and Paul A. Murray

Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our goals were to identify the isoforms of protein kinase C (PKC) present in primary cultures of canine pulmonary artery smooth muscle cells (PASMCs) and to determine whether angiotensin II (ANG II) triggers translocation of specific PKC isoforms to discreet intracellular locations. Isoform-specific antibodies and Western blot analysis were utilized to identify the isoforms of PKC in PASMCs. Indirect immunofluorescence and confocal microscopy were used to examine the subcellular distribution of PKC isoforms. Inositol phosphate production was used to assess phospholipase C activation, and fura 2 was utilized to monitor intracellular Ca2+ concentration in response to ANG II. Six isoforms (alpha , delta , epsilon , zeta , iota /lambda , and µ) of PKC were identified by Western blot analysis. Immunolocalization of 5 isoforms (alpha , delta , zeta , iota /lambda , and µ) revealed a unique pattern of staining for each individual isoform. ANG II caused translocation of PKC-alpha from the cytosol to the nuclear envelope and of PKC-delta to the myofilaments. In contrast, cytosolic PKC-zeta did not translocate, but nuclear PKC-zeta was upregulated. Translocation of PKC-alpha and PKC-delta and upregulation of PKC-zeta in response to ANG II were blocked by the ANG II type 1-receptor antagonist losartan. In addition, ANG II stimulated inositol phosphate production and intracellular Ca2+ concentration oscillations, which were blocked by losartan. Thus activation of ANG II type 1 receptors triggers the phosphoinositide signaling cascade, resulting in translocation or upregulation of specific PKC isoforms at discreet intracellular sites. The alpha  and zeta  isoforms may act to regulate nuclear events, whereas PKC-delta may be involved in modulating contraction via actions on the myofilaments.

protein kinase C isoforms; angiotensin II

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PROLIFERATION AND HYPERTROPHY of pulmonary artery smooth muscle cells (PASMCs) are early pathological features of pulmonary hypertension (1). This disease is also characterized by activation of the renin-angiotensin system and an increase in pulmonary vascular resistance (17). Recent studies have focused primarily on the effects of angiotensin II (ANG II) in the systemic circulation and have demonstrated vasoconstrictor (12) as well as growth-promoting effects of ANG II on smooth muscle (3) via activation of the ANG II type 1 (AT1) receptor (39). The pressor and growth-promoting effects are thought to be mediated, at least in part, via common signaling pathways involving the activation of protein kinase C (PKC) (6). Phosphorylation of calponin and/or caldesmon may be involved in mediating contractile events (16), whereas phosphorylation of nuclear elements that alter gene expression (35), DNA synthesis (37), mitogenesis (19), and/or hypertrophy (25) may be involved in growth responses. However, the isoforms of PKC involved and their sites and mechanism of action that mediate these divergent cellular responses remain unclear.

PKC represents a family of 11 closely related serine-threonine kinases that can be subdivided into four groups (41): 1) classic group A PKCs (alpha , beta I, beta II, and gamma ) that are Ca2+ dependent, 2) novel group B PKCs (delta , epsilon , eta /L, and theta ) that are Ca2+ independent, 3) atypical group C PKCs (zeta  and iota /lambda ) that are Ca2+ independent and diacylglycerol insensitive, and 4) group D PKC (µ) that is similar to group C but contains a putative signal peptide and transmembrane domain. Several isozymes (alpha , beta , delta , epsilon , and zeta ) coexist in systemic vascular smooth muscle cells (21, 25). Expression patterns can vary depending on the species, the type or size of vessel, or the state of differentiation of the smooth muscle cells (13, 21, 32). Specific functions for individual isoforms are supported by findings in the ferret aorta that demonstrate the involvement of PKC-epsilon in contraction, whereas PKC-zeta is involved in cell growth (21, 25).

In the pulmonary circulation, ANG II increases vascular resistance in a number of species including human (26), rat (31), cat (20), and dog (8). ANG II appears to play a role in hypoxic pulmonary vasoconstriction (HPV) because angiotensin-converting enzyme inhibition and AT1-receptor antagonism attenuate the vasomotor and vascular remodeling effects of hypoxia (28). Conversely, ANG II has also been shown to exert a vasodilator influence mediated by cyclooxygenase metabolites and to prevent HPV and increased muscularization of the pulmonary arterial wall, suggesting an antimitogenic effect (33). The pressor response to ANG II is mediated, in part, by activation of PKC (20), which is thought to play an important role in HPV (18). In addition, the growth-promoting actions of other agonists (platelet-derived growth factor, thrombin) coupled to GTP binding proteins and phosphoinositide metabolism are dependent on the activation of PKC and are associated with activation of nuclear response elements (c-fos, c-jun, egr-1) in PASMCs (35). At the cellular level, ANG II stimulates production of inositol phosphates in cultured PASMCs (4) and Ca2+ oscillations in freshly dispersed PASMCs (11).

The goals of this study were to identify the isoforms of PKC expressed in cultured PASMCs and to investigate their subcellular localization before and after stimulation with ANG II. The final site of translocation of an individual isoform should provide insight concerning its potential role in regulating a specific cellular event.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. Pulmonary arteries were isolated from adult male mongrel dogs. The technique of euthanasia was approved by the Institutional Animal Care and Use Committee. Under general anesthesia with endotracheal intubation and positive-pressure ventilation, the dogs were euthanized by removing the mobilizable blood volume and administering saturated KCl. With the use of sterile surgical technique, a left thoracotomy was performed through the fifth intercostal space. The heart and lungs were removed en bloc and subsequently dissected in a laminar flow hood under aseptic conditions.

Cell culture. Primary cultures of PASMCs were obtained from segmental and subsegmental branches of the intralobar pulmonary artery (the third and fourth generations of branches from the main pulmonary artery), with diameters of ~3 mm. Explant cultures were prepared according to the method of Ross (34). Briefly, the endothelium was removed by rubbing gently with a sterile cotton swab. The tunica adventitia was carefully removed together with the most superficial part of the tunica media. The remaining portion of the media was cut into 1-mm2 pieces that were explanted on precleaned 22-mm2 no. 1 glass coverslips placed individually in six-well culture plates for immunofluorescence studies or alternatively into 60-mm culture plates to generate large numbers of cells for the Western blots and inositol phosphate assays. The explants were nourished by Dulbecco's modified Eagle's medium-F-12 medium containing 10% fetal bovine serum (FBS) and protected against infection with a 1% antibiotic-antimycotic mixture (100 units/ml of penicillin, 10,000 µg/ml of streptomycin, and 0.25 µg/ml of amphotericin B). Cultures were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. Cells began to migrate out of the explants within 1 wk and were used for experiments at subconfluence after 2 wk in culture. The cells were never passed. Cells were >90% pure as assessed by the filamentous staining pattern achieved with alpha -smooth muscle actin.

Western blot analysis. Confluent PASMCs grown from explants were harvested and suspended in ice-cold sucrose buffer (0.29 M) containing 3 mM sodium azide (NaN3), 1 mM dithiothreitol, 10 mM (N-morpholino)propanesulfonic acid, 2 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and the protease inhibitors leupeptin (0.5 µM), pepstatin (0.5 µM), and phenylmethylsulfonyl fluoride (0.2 mM), pH 7.4. The suspension of PASMCs was pelleted in a microfuge for 30 s at 700 revolutions/min (rpm), and the supernatant was removed. The pellet was homogenized with a hand-held motorized pestle, and the PASMC homogenates were resuspended in sucrose buffer containing 10% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at 4°C for 1 h. Gel electrophoresis was performed with 10% polyacrylamide gels to separate the solubilized PASMC proteins (22). In addition, positive controls for all of the isoform- specific antibodies were performed with either rat brain (alpha , beta , gamma , delta , epsilon , iota /lambda , and zeta ) or Jurkat cell (µ and theta ) lysate. After transfer of the proteins from the gel to a nitrocellulose membrane, the remaining protein binding sites were blocked with 5% gelatin. The membranes were subsequently incubated with the monoclonal isoform-specific PKC antibody (1:200 dilution) or antigen-antibody complex for 4 h at room temperature. The nitrocellulose membranes were washed in tris(hydroxymethyl)aminomethane (Tris)-buffered saline (3 times) and incubated with anti-mouse conjugated horseradish peroxidase (HRP) for 3 h at room temperature. After several washes in Tris-buffered saline (3 × 30 min), the membranes were incubated with HRP color-development buffer. In some experiments, an additional anti-PKC-beta antibody (polyclonal; 1:500 dilution) was used to confirm the absence of PKC-beta in the PASMC homogenates. For these experiments, goat anti-rabbit conjugated HRP was used as the secondary antibody.

Experimental design. The primary cultures of PASMCs were divided into seven experimental groups. All groups were rendered quiescent with medium lacking FBS for 24 h before experimentation. The first group of cells was untreated. The second group was treated with ANG II (10-7 M). This dose was selected because it is optimal for diacylglycerol production and induction of vascular smooth muscle cell hypertrophy (9). The third group was exposed to the membrane-permeant PKC activator dioctanoylglycerol (DOG; 50 µM). The fourth group was exposed to saralasin (1 µM), an AT1-receptor antagonist known also to have agonist activity. The fifth group was exposed to the specific AT1-receptor antagonist losartan (DuP-753; 1 µM). A sixth group was exposed to ANG II after pretreatment with losartan. Cells in the seventh group (vehicle control) were treated with buffer containing either ethanol (vehicle for DOG) or distilled water (vehicle for ANG II, losartan, and saralasin). All treatments were for 10 min. The vehicles had no effect on the cellular distribution of PKC isoforms. The experiments were reproduced in cells from at least four individual dogs on 3 separate days. For groups 1-6, micrographs were taken from cells of the same population exposed simultaneously to different agents.

Immunofluorescence labeling of PKC isoforms. The indirect immunofluorescence technique was used to localize the cellular distribution of PKC isoforms before and after the addition of ANG II. Immediately after each treatment, the reaction was stopped by placing the coverslips in 1:1 (vol/vol) acetone-methanol at -20°C for 10 min to simultaneously fix the cells and permeabilize their plasma membranes. Fixed cells were then washed with 0.1 M phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 10 min and subsequently incubated with monoclonal, isoform-specific PKC antibodies at a dilution of 10 µg/ml in PBS-BSA for 24 h at 4°C in a humidified chamber. Monoclonal antibodies to nine individual PKC isoforms (alpha , beta , gamma , delta , epsilon , theta , zeta , iota /lambda , and µ) were used for the immunofluorescence protocols. An additional anti-PKC-beta antibody (polyclonal) obtained from another commercial vendor was used in some experiments. After incubation with the primary antibody, the coverslips were thoroughly washed in PBS-BSA and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or goat anti-rabbit immunoglobulin G (secondary antibody) diluted 1:100 in PBS-BSA for 30 min at 37°C. An immunocytochemical control for antibody specificity was performed by incubating the cells with the secondary antibody only. After a thorough washing in PBS-BSA, the coverslips were mounted on microscope slides with Aquamount. The specimens were viewed and photographed with a Reichert epifluorescent microscope equipped with an Olympus PM-30 photomicrographic system with Kodak T-Max 400 film.

Confocal microscopy. To confirm the intranuclear localization of some isoforms of PKC, examination of thin (0.25-µm) optical sections in a Z series throughout the whole cell thickness was done with a Leica confocal laser scanning microscope. Persistence of immunoreactivity in the region of the nucleus throughout the whole series of cell sections was interpreted as the presence of the isoform inside the nucleus.

Fura 2 loading procedure. Twenty-four hours before experimentation, the culture medium containing 10% FBS was substituted with serum-free medium to arrest cell growth, allow for establishment of steady-state cellular events independent of cell division, and prevent a false estimate of intracellular Ca2+ concentration ([Ca2+]i) due to the binding of available dye by a high serum protein concentration in the medium (36). PASMCs were washed two times in loading buffer (LB), which contained (in mM) 125 NaCl, 5 KCl, 1.2 MgSO4, 11 glucose, 1.8 CaCl2, and 25 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) plus 0.2% BSA at pH 7.40 adjusted with NaOH. PASMCs were then incubated in LB containing 2 µM fura 2-AM, the acetoxymethyl ester derivative of fura 2, at ambient temperature for 30 min. After the 30-min loading period, the cells were washed two times in LB and incubated at ambient temperature for an additional 20 min before the study. This provided sufficient time to wash away any extracellular fura 2-AM and for intracellular esterases to cleave fura 2-AM into the active fura 2 (7).

Determination of [Ca2+]i. Culture dishes containing fura 2-loaded PASMCs were placed in a temperature-regulated (37°C) chamber (Bioptechs) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope. Fluorescence measurements were performed on individual PASMCs in a culture monolayer with a dual-wavelength spectrofluorometer (Deltascan RFK6002, Photon Technology International) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The volume of the chamber was 1.5 ml. The cells were superfused continuously at 1 ml/min with Krebs-Ringer buffer, which contained (in mM) 125 NaCl, 5 KCl, 1.2 MgSO4, 11 glucose, 2.5 CaCl2, and 25 HEPES at pH 7.40 adjusted with NaOH. The temperature of all solutions was maintained at 37°C in a water bath. Solution changes were accomplished rapidly by aspirating the buffer in the dish and transiently increasing the flow rate to 10 ml/min. Just before data acquisition, background fluorescence (i.e., fluorescence between cells) was obtained and subtracted automatically from the subsequent experimental measurement. Fura 2 fluorescence signals (340 nm, 380 nm, and 340- to 380-nm ratio) originating from individual PASMCs were continuously monitored at a sampling frequency of 25 Hz and were collected and stored with a software package (Felix, Photon Technology International).

Fura 2 titration. Estimates of [Ca2+]i were achieved by comparing the cellular fluorescence ratio with the fluorescence ratio acquired with fura 2 (free acid) in buffers containing known Ca2+ concentrations. [Ca2+]i was calculated as described by Grynkiewicz et al. (10).

Measurement of [3H]inositol phosphates. Primary cultures of PASMCs grown in 60-mm culture dishes were labeled with [3H]inositol (5 µCi/ml) for 24 h at 37°C. After the labeling period, the medium was aspirated, and the cells were washed two times with Krebs-Ringer buffer. The cells were then preincubated in Krebs-Ringer buffer containing LiCl (20 mM) in the presence or absence of losartan (1 µM) for 30 min at 37°C. Then, ANG II (100 nM) was added to the dishes, and the incubation was carried out for an additional 10 min. The reactions were terminated by addition of ice-cold perchloric acid (10%). Cell lysis after perchloric acid treatment was achieved by freezing the cells on dry ice and then allowing them to thaw. The cells were scraped from the dish, transferred in a 0.5 volume of 0.72 N KOH-0.6 M KHCO3 to centrifuge tubes, and sedimented at 2,000 rpm. The resulting supernatant was applied to 1-ml packed AG 1-X8 columns (100-200 mesh, formate form), and inositol phosphates were eluted with 1 M ammonium formate plus 0.1 M formic acid after the column was first washed with 16 ml of 0.1 M formic acid. Radioactivity, in fractions corresponding to inositol phosphate, inositol bisphosphate, and inositol trisphosphate, was quantified by liquid scintillation counting.

Materials. Dulbecco's modified Eagle's medium-F-12 medium, the antibiotic-antimycotic mixture, and BSA (fraction V) were from GIBCO (Grand Island, NY). FBS, FITC-labeled monoclonal anti-alpha -smooth muscle actin, and PBS were from Sigma (St. Louis, MO). ANG II and DOG were from Research Biochemicals (Natick, MA). Saralasin ([Sar1,Ile8]ANG II) was from Bachem (Torrance, CA). Losartan was from DuPont Merck Pharmaceutical (Wilmington, DE). Isoform-specific monoclonal antibodies to PKC and positive control cell lysates were from Transduction Laboratories (Lexington, KY). Anti-PKC-beta was obtained from Research & Diagnostic Antibodies (Berkeley, CA). FITC-labeled goat anti-mouse and HRP-labeled goat anti-mouse and goat anti-rabbit immunoglobulin G were from Jackson Immunoresearch Laboratories (West Grove, PA). Aquamount was purchased from Lerner (Pittsburgh, PA). Fura 2-AM was from Molecular Probes (Eugene, OR).

Cross-reactivity of PKC antibodies. Information concerning the cross-reactivity of the monoclonal PKC antibodies has recently been provided by the commercial supplier (39a). Competitive Western blot analysis was performed with the specific antigens that the antibodies were raised against. Only two cases of cross-reactivity were identified. Anti-PKC-alpha was shown to cross-react slightly with the beta  isoform of PKC, and anti-PKC-gamma cross-reacted with the alpha  isoform of PKC. Because PKC-iota and PKC-lambda are the same protein in different species, both anti-PKC-iota and anti-PKC-lambda recognize the same isoform and are referred to as PKC-iota /lambda .

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Western blots of PKC isoforms. Experiments were performed to assess the specificity of the antibodies. Positive controls for all the antibodies were performed with either rat brain lysate (alpha , beta , gamma , delta , epsilon , iota /lambda , and zeta ) or Jurkat cell lysate (µ and theta ). The results are shown in Fig. 1A. Immunoblotting of the lysates resulted in each antibody recognizing a single protein, with the exception that anti-PKC-iota and anti-PKC-zeta each recognized two proteins. Figure 1A also demonstrates the molecular mass correlation for the various isoforms, which are presented from the highest (PKC-µ, 115 kDa) to the lowest (PKC-zeta , 72 kDa) molecular mass.


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Fig. 1.   A: Western blot analysis of protein kinase C (PKC) isoforms using rat brain (alpha , beta , gamma , delta , epsilon , iota /lambda , zeta ) or Jurkat cell (µ, theta ) lysate as positive controls for antibody specificity. B: immunoblot of pulmonary artery smooth muscle cell (PASMC) homogenates. C: demonstration that preadsorption of a specific immunizing peptide [antigen (Ag)] to the antibody prevents recognition of isoform in PASMC homogenates. Nos. on left, molecular-mass indicators (M) in kDa. PKC isoforms are presented from highest (PKC-µ, 115 kDa) to lowest (PKC-zeta , 72 kDa) molecular mass.

Western blots of the PASMC homogenates are shown in Fig. 1B. This Western blot demonstrates the presence of the µ, epsilon , alpha , gamma , delta , iota /lambda , and zeta  isoforms in PASMCs. Immunodetection of PKC-µ and PKC-epsilon was faint, and PKC-beta and PKC-theta were not detected. Similar to the positive controls, anti-PKC-iota and anti-PKC-zeta recognized two proteins.

Immunoblots for some isoforms were performed on homogenates of PASMCs, probing with either antibody alone or an antigen-antibody complex provided by the commercial supplier (Fig. 1C). In all cases (µ, lambda , delta , and alpha ), the antigen-antibody complex failed to recognize any protein in the PASMC homogenate.

Because the affinity of the anti-PKC-beta (monoclonal) for PKC-beta in the brain lysate was weak (Fig. 1A) and no PKC-beta was detected in the PASMC homogenate (Fig. 1B), we tested the ability of another anti-PKC-beta to detect PKC-beta in rat brain lysate and PASMC homogenate (Fig. 2). Anti-PKC-beta (polyclonal) recognized a single band with high affinity in the rat brain lysate but failed to recognize any PKC-beta in the PASMC homogenate regardless of the PASMC protein concentration on the nitrocellulose membrane.


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Fig. 2.   Western blot analysis of PASMC homogenates with anti-PKC-beta from Research & Diagnostic Antibodies. Lanes A, D, and G, molecular-mass markers. Lanes B, E, and H, rat brain lysate (50 µg). PASMC homogenate was loaded in lanes C (50 µg), F (100 µg), and I (150 µg). Nos. on left, molecular-mass indicators in kDa.

Immunofluorescence localization of PKC isoforms in PASMCs. Initially, the antibodies that gave a positive result on the Western blot (alpha , delta , epsilon , zeta , iota /lambda , and µ) were utilized for immunocytochemistry, with the exception of anti-PKC-gamma . Anti-PKC-gamma was not used for immunolocalization because this isoform is specific to the nervous system (44), and its presence in PASMCs is likely due to the known cross-reactivity of this antibody with the alpha  isoform of PKC. Anti-PKC-epsilon and anti-PKC-iota provided no immunodetectable fluorescence in PASMCs and were not useful for further immunocytochemical studies. Therefore, anti-PKC-lambda was used for immunolocalization of PKC-iota /lambda in PASMCs. The other isoforms (alpha , delta , zeta , iota /lambda , and µ) exhibited a unique pattern of subcellular distribution, which can be observed in Figs. 3A, 4A, 5A, and 6A and are summarized in Table 1.


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Fig. 3.   Immunofluorescence of PKC-alpha in primary explant cultures of PASMCs. Untreated cells (a) show a cytosolic immunoreactivity. Increased immunoreactivity in nuclear envelope occurs in cells exposed to 100 nM ANG II (b), 50 µM dioctanoylglycerol (DOG; c), or 1 µM saralasin (d). Losartan (1 µM) alone has no effect on PKC-alpha immunoreactivity (e). Translocation of PKC-alpha to nuclear envelope in response to ANG II is blocked by pretreatment with losartan (f). All treatments were for 10 min.


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Fig. 4.   Immunofluorescence of PKC-delta in primary explant cultures of PASMCs. Untreated cells (a) show both a punctate nuclear and amorphous cytoplasmic distribution of isoform. Appearance of immunoreactivity on cytoplasmic filaments is evident in cells exposed to ANG II (b), DOG (c), or saralasin (d). Losartan alone has no effect on PKC-delta immunoreactivity (e). Translocation of PKC-delta to myofilaments in response to ANG II is blocked by pretreatment with losartan (f). All treatments were for 10 min.


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Fig. 5.   PKC-zeta immunofluorescence is entirely cytoplasmic in untreated PASMCs (a). Exposure of cells to ANG II (b) or saralasin (d) results in PKC-zeta immunoreactivity in nucleus. Exposure to DOG (c), pretreatment with losartan alone (e), or response to ANG II after losartan pretreatment (f) does not result in translocation of PKC-zeta . All treatments were for 10 min.


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Fig. 6.   PKC-iota /lambda in untreated cells (a) is localized on irregular cytoplasmic filamentous structures. PKC-µ immunoreactivity is both cytoplasmic (finely filamentous) and nuclear (b).

                              
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Table 1.   Pulmonary artery smooth muscle cell PKC isoforms

Effects of ANG II on the cellular distribution of PKC isoforms. Exposure of PASMCs to ANG II (10-7 M) revealed spatial translocation of two isoforms (alpha  and delta ) and upregulation of one isoform (zeta ) compared with untreated cells (Figs. 3-5). All treatments were for 10 min.

Immunolocalization of PKC-alpha . In untreated cells, PKC-alpha immunoreactivity was entirely absent from the nucleus, whereas in the cytoplasm, it showed a diffuse cytosolic disposition (Fig. 3A). Exposure of PASMCs to ANG II resulted in PKC-alpha immunoreactivity being more pronounced on the nuclear envelope (Fig. 3B). Treatment with DOG mimicked ANG II-induced translocation of PKC-alpha to the nuclear envelope (Fig. 3C). The competitive AT1-receptor agonist-antagonist saralasin also induced translocation of PKC-alpha similar to that observed with ANG II (Fig. 3D). In contrast, the non-peptide-specific AT1-receptor antagonist losartan did not alter the subcellular distribution of PKC-alpha compared with untreated cells (Fig. 3E). However, pretreatment of PASMCs with losartan before ANG II treatment abolished the ANG II-induced translocation of PKC-alpha (Fig. 3F).

Immunolocalization of PKC-delta . In untreated cells, PKC-delta was detected in both the nucleus and the cytoplasm (Fig. 4A). The nuclear immunoreactivity of PKC-delta included numerous, intensely bright, discrete spots, whereas the cytoplasmic immunoreactivity was amorphous and diffuse. Exposure to ANG II resulted in cytoplasmic PKC-delta becoming more localized onto myofilaments, whereas the nuclear fluorescence was similar to that in untreated cells (Fig. 4B). In some cells, staining of PKC-delta on the myofilaments was very strong, resembling staining for alpha -smooth muscle actin. Treatment with DOG (Fig. 4C) or saralasin (Fig. 4D) mimicked the ANG II translocation of PKC-delta to the myofilaments. Losartan alone did not alter the subcellular distribution of PKC-delta compared with untreated cells (Fig. 4E), whereas pretreatment with losartan abolished the ANG II-induced translocation of PKC-delta (Fig. 4F).

Immunolocalization of PKC-zeta . In untreated cells, PKC-zeta exhibited a bright granular cytoplasmic fluorescent pattern, which was especially abundant in the perinuclear zones, with some faint focal spots in the nucleus (Fig. 5A). In ANG II-treated cells, the cytoplasmic staining pattern was similar to that of untreated cells (Fig. 5B), whereas ANG II upregulated nuclear PKC-zeta , as reflected by the appearance of several bright intranuclear coarse fluorescent granules (Fig. 4B). No redistribution of PKC-zeta was observed in cells exposed to DOG (Fig. 5C). Saralasin upregulated PKC-zeta similar to that observed with ANG II (Fig. 5D). Losartan alone (Fig. 5E) did not alter the subcellular distribution of PKC-zeta but abolished the ANG II-induced upregulation of PKC-zeta (Fig. 5F).

Immunolocalization of PKC-iota /lambda and PKC-µ. PKC-iota /lambda immunoreactivity (Fig. 6A) exhibited faint, thin, interrupted filamentous arrays in the cytoplasm, with no nuclear staining. PKC-µ immunoreactivity (Fig. 6B) was detected in both the nuclear and cytoplasmic compartments. In the nucleus, one to three large fluorescent spots were observed. The cytoplasmic pattern of PKC-µ was diffuse, amorphous, and/or finely filamentous. There was no change in the staining pattern for PKC-iota /lambda or PKC-µ in ANG II-treated cells.

Intranuclear localization of PKC-delta and PKC-µ. PKC-delta and PKC-µ revealed positive fluorescent staining in the region of the nucleus. Intranuclear localization of these isoforms was confirmed by their persistent immunoreactivity in confocal optical sections through the entire thickness of the cell (Fig. 7, A and B). The intranuclear localization of PKC-zeta during ANG II treatment was also confirmed (Fig. 7C).


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Fig. 7.   Series of confocal optical sections in PASMCs at various depths (nos. in top right corner) from top surface of cells showing persistence of PKC-delta (a), PKC-µ (b), and PKC-zeta (c) immunoreactivity within nucleus throughout cell thickness.

Inositol phosphate production by ANG II. ANG II stimulated a 1.2-fold increase in total [3H]inositol phosphate production [basal, 406 ± 18 counts/min (cpm); ANG II, 897 ± 31 cpm]. In contrast, pretreatment of the PASMCs with losartan (1 µM) abolished production of inositol phosphates by ANG II (421 ± 22 cpm). The results are summarized in Fig. 8 (n = 3 experiments).


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Fig. 8.   Effect of exposure of PASMCs (n = 3 experiments) to ANG II (100 nM) in absence or presence of losartan (1 µM) on total inositol phosphate production. All treatments were for 10 min. Results are means ± SE. * P < 0.05 compared with basal level.

Intracellular Ca2+ signaling in response to ANG II in individual PASMCs. Baseline [Ca2+]i was 70 ± 17 nM. Superfusion of the PASMCs with ANG II (100 nM) stimulated repetitive [Ca2+]i oscillations, which were blocked by 1 µM losartan (Fig. 9). During exposure to ANG II, [Ca2+]i reached a peak value of 310 ± 27 nM and an oscillation frequency of 1.07 ± 0.18 transients/min (n = 7 cells).


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Fig. 9.   Effect of ANG II on intracellular Ca2+ concentration in individual PASMCs (n = 7), demonstrating that ANG II (100 nM) stimulates persistent oscillations in intracellular Ca2+ concentration, which are blocked by addition of ANG II type 1-receptor antagonist losartan (1 µM). 340/380, Ratio of fluorescence signals at 340 and 380 nm.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The main finding of this study is the identification of six different isoforms of PKC in PASMCs with diverse subcellular distribution. Two of these isoforms (iota /lambda and µ) have not been previously identified in systemic vascular smooth muscle, so they could be unique to the pulmonary circulation. Moreover, we have demonstrated differential translocation of PKC-alpha to the nuclear envelope and PKC-delta to the myofilaments, whereas intranuclear PKC-zeta was upregulated in response to ANG II. These translocation patterns suggest that these isoforms may be involved in mediating specific cellular responses to ANG II. Because the two major effects of ANG II on vascular smooth muscle are contraction and cell growth, the cellular function regulated by each isoform may be reflected by its final intracellular site of translocation.

Positive controls for Western blots. The results from the Western blot experiments with positive control cell lysates support the likelihood that the antibodies used in this study are recognizing specific PKC isoforms. Single bands were observed for most of the antibodies (alpha , beta , gamma , delta , epsilon , lambda , theta , and µ). In addition, an appropriate molecular-mass correlation was demonstrated for each. Anti-PKC-zeta and anti-PKC-iota each recognized two proteins. Detection of a lower molecular mass protein with anti-PKC-zeta is consistent with another report (43) and is likely due to rapid proteolysis of this isoform. The identity of the high-molecular-mass band identified by anti-PKC-iota is not known but was not observed when we used anti-PKC-lambda , which should recognize the same protein.

Western blots of cultured PASMCs. Western blot analysis of cultured PASMCs revealed the presence of seven isoforms (µ, epsilon , alpha , gamma , delta , iota /lambda , zeta ). However, the presence of PKC-gamma on the immunoblot is likely due to anti-PKC-gamma cross-reacting with PKC-alpha because PKC-gamma appears to be restricted primarily to the brain (44). Cross-reactivity of anti-PKC-alpha with PKC-beta has been documented by the commerical supplier. However, two different anti-PKC-beta antibodies did not recognize PKC-beta in PASMCs. This result suggests that anti-PKC-alpha is not detecting the beta  isoform of PKC. Anti-PKC-iota and anti-PKC-lambda (mouse variant) each identified the same protein in PASMC homogenates. However, because anti-PKC-iota labeled two bands and anti-PKC-lambda detected a single protein, this result could suggest that there are some differences in specificity. Anti-PKC-lambda appeared to be the more specific probe for PKC-iota /lambda and was used for the immunolocalization studies.

Expression of PKC isoforms is known to be tissue and cell-type specific. Moreover, variation in PKC isoform expression has been reported at different states of differentiation. Although commercial availability of the antigen-antibody complex was limited, antibody susceptibility to antigen binding in the PASMC homogenates was demonstrated for four of the isoforms, including the two novel isoforms µ and iota /lambda . Therefore, it appears that PASMCs express six different isoforms of PKC (alpha , delta , epsilon , zeta , µ, and iota /lambda ). To our knowledge, the present study is the first to describe PKC isoform distribution in cells derived from the pulmonary circulation. Also, this is the first study to report the presence of the iota /lambda and µ isoforms of PKC in any type of vascular smooth muscle.

Intracellular translocation of PKC-alpha to the nuclear envelope by ANG II. PKC-alpha underwent translocation to the nuclear envelope in response to ANG II. Translocation of PKC-alpha to the cell nucleus has been observed in NIH/3T3 cells in response to phorbol esters (24). In rat aortic smooth muscle cells, ANG II caused a transient localization of PKC-alpha along the myofilaments, followed by translocation to the nucleus (14). In contrast, PKC-alpha immunoreactivity was at the surface membrane in hypertrophied cells from the rat aorta (25). In addition, translocation of PKC-alpha from the cytosol to the surface membrane by phenylephrine or phorbol esters has been demonstrated in normal rat aortic smooth muscle cells (25). In this study, translocation of PKC-alpha to the nuclear envelope suggests that this isoform may be involved in regulating nuclear events. PKC substrates are known to exist on the nuclear envelope (lamin B) and are phosphorylated in response to ANG II in smooth muscle cells (40).

Intracellular translocation of PKC-delta to myofilaments by ANG II. Translocation of PKC-delta to the myofilaments in response to ANG II points toward its possible involvement in contractile events. Activation of PKC has been proposed to modulate the contractile activity of vascular smooth muscle either by activation of transsarcolemmal Ca2+ influx or by phosphorylation of myofilament regulatory proteins such as caldesmon or calponin (2). In rat aortic smooth muscle cells, Liou and Morgan (25) have described a redistribution of PKC-delta within the cytoplasmic compartment on agonist activation with phenylephrine as well as with hypertrophy of the aorta. Because we did not observe translocation of any PKC isoform to the sarcolemma but rather translocation of PKC-delta to the myofilaments, we speculate that phosphorylation of the myofilament substrate (e.g., caldesmon or calponin) may be a potential mechanism by which ANG II modulates the contractile activity of PASMCs. Alternatively, PKC-delta may colocalize with the cytoskeletal protein talin, as suggested for PKC-alpha (14). In addition to being a direct vasoconstrictor, ANG II also potentiates the vasoconstrictor response to other vasoactive substances (15). This potentiation is mediated by PKC and occurs without increasing Ca2+ influx (15). This further supports the possibility that ANG II enhances contractile activity via a PKC-mediated change in myofilament properties.

Upregulation of nuclear PKC-zeta by ANG II. In contrast to PKC-alpha and PKC-delta , intranuclear PKC-zeta was upregulated in response to ANG II. Translocation of PKC-zeta into the nucleus has been observed in ferret aorta exposed to phenylephrine (21). However, in rat aorta, neither phenylephrine nor phorbol esters mobilized PKC-zeta , although increased nuclear levels of PKC-zeta were observed in hypertrophied rat aortic cells (25). Immunoreactivity of PKC-zeta in PASMCs resembled discrete coarse intranuclear aggregates, suggesting an association with nucleoli and a possible role in modulating the synthesis of rRNA and protein biosynthesis. PKC substrates are known to exist in the nucleus (e.g., DNA polymerase, DNA topoisomerase II, histones, matrix proteins), and several studies (5, 30) have suggested that PKC has intranuclear functions and may play a role in transcriptional regulation.

Upregulation of intranuclear PKC-zeta in response to ANG II, but not to DOG, suggests that ANG II receptors may be coupled to additional signaling pathways independent of the inositol-specific phospholipase C pathway that produces diacylglycerol. PKC-zeta can be activated by unsaturated fatty acids via activation of phospholipase A2 as well as via a ceramide pathway (27, 29). ANG II has also been shown to stimulate phospholipase D-mediated hydrolysis of phosphatidylcholine in systemic vascular smooth muscle, which can result in the release of free fatty acids (23).

Nuclear localization of PKC-delta and PKC-µ isoforms. One unique finding of the present study was the differential nuclear localization of PKC-delta and PKC-µ. Both isoforms were found in the nucleus of untreated cells, but each isoform had its own unique pattern of staining. Nuclear PKC-delta and PKC-µ were not affected by ANG II stimulation, suggesting that these isoforms may be involved in steady-state nuclear functions. PKC-delta has been proposed to be involved in cell cycle regulation (42).

Effect of DOG on translocation of PKC isoforms. The finding that DOG caused translocation similar to that caused by ANG II indicates that the primary antibodies recognized subcellular redistribution of PKC because DOG is a specific activator of PKC. Also, the inability of DOG to induce translocation or upregulation of PKC-zeta is evidence to support the specificity of the antibodies for immunostaining because this isoform lacks a diacylglycerol-binding site and should not be activated by DOG.

Upregulation and translocation of PKC isoforms are mediated by activation of AT1 receptors. ANG II has been reported to stimulate contraction of the pulmonary artery through an AT1-receptor mechanism (38). Inhibiting the ANG II-mediated upregulation and/or translocation of PKC isoforms by pretreatment with losartan indicates that these effects are mediated specifically by AT1 receptors. This is also supported by the ability of saralasin, a competitive AT1-receptor agonist-antagonist, to mimic ANG II-induced effects.

Activation of the phosphoinositide cascade by ANG II. Activation of the phosphoinositide cascade, giving rise to inositol phosphates (inositol 1,4,5-trisphosphate) and diacylglycerol, is a primary signaling mechanism through which ANG II, via activation of AT1 receptors, exerts its effects on vascular smooth muscle (9). Activation of the phospholipase C pathway in PASMCs should result in changes in [Ca2+]i and activation of PKC. In this study, the addition of ANG II to PASMCs resulted in inositol phosphate production and oscillations in [Ca2+]i, which were blocked by the AT1-receptor antagonist losartan. These data are consistent with our findings of ANG II-mediated, losartan-sensitive translocation and upregulation of PKC-alpha , PKC-delta , and PKC-zeta , respectively. These results are also consistent with the production of inositol phosphates observed by others in cultured rat PASMCs (4) and with [Ca2+]i oscillations in freshly dispersed rat PASMCs (11). The [Ca2+]i signal is likely responsible for initiating the contractile response, whereas PKC activation maintains the contraction over time when inositol phosphate levels have returned to normal. Therefore, it appears that ANG II activates AT1 receptors coupled to the phosphoinositide cascade in PASMCs.

In summary, ANG II is known to cause PASMC contraction and has been postulated to stimulate PASMC growth, although this latter effect has not been conclusively documented. ANG II signaling via AT1 receptors diverges at the level of PKC, with activation of at least three different isoforms. PKC-alpha translocates to the nucleus and PKC-delta translocates to the myofilaments. Nuclear PKC-zeta is upregulated. The challenge of future studies will be to delineate specific roles for individual isoforms of PKC in regulating and/or modulating PASMC function. This can be achieved by developing specific antagonists to individual PKC isoforms. Selective inhibition of PASMC growth by antagonizing specific PKC isoform(s) may have a clinical application in preventing vascular remodeling associated with chronic pulmonary hypertension (33) while preserving homeostatic vasomotor regulation.

    ACKNOWLEDGEMENTS

The authors thank Dr. Hiroshi Hamada, Dr. Sumihiko Seki, and Elizabeth Ghoubrial for technical assistance and Ronnie Sanders for preparation of this manuscript.

    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-38291 and HL-40361.

Address for reprint requests: P. A. Murray, Center for Anesthesiology Research-FF4, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.

Received 15 April 1996; accepted in final form 21 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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