Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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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 (,
,
,
,
/
, and
µ) of PKC were identified by Western blot analysis.
Immunolocalization of 5 isoforms (
,
,
,
/
, and
µ) revealed a unique pattern of staining for each individual isoform.
ANG II caused translocation of PKC-
from the cytosol to the nuclear
envelope and of PKC-
to the myofilaments. In contrast, cytosolic
PKC-
did not translocate, but nuclear PKC-
was upregulated.
Translocation of PKC-
and PKC-
and upregulation of PKC-
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
and
isoforms may act to regulate nuclear events, whereas PKC-
may be
involved in modulating contraction via actions on the myofilaments.
protein kinase C isoforms; angiotensin II
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INTRODUCTION |
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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 (,
I,
II, and
) that are
Ca2+ dependent,
2) novel group
B PKCs (
,
,
/L, and
) that are Ca2+ independent,
3) atypical group
C PKCs (
and
/
) 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 (
,
,
,
, and
) 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-
in contraction, whereas PKC-
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.
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MATERIALS AND METHODS |
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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 -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(-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 (
,
,
,
,
,
/
, and
) or Jurkat cell (µ and
) 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-
antibody (polyclonal; 1:500 dilution) was used to confirm
the absence of PKC-
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
(107 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 (
,
,
,
,
,
,
,
/
, and µ) were used for the immunofluorescence
protocols. An additional anti-PKC-
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- ![]() |
RESULTS |
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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 (,
,
,
,
,
/
, and
) or
Jurkat cell lysate (µ and
). 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-
and anti-PKC-
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-
,
72 kDa) molecular mass.
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Western blots of the PASMC homogenates are shown in Fig.
1B. This Western blot demonstrates the
presence of the µ, ,
,
,
,
/
, and
isoforms in PASMCs. Immunodetection of PKC-µ and PKC-
was faint,
and PKC-
and PKC-
were not detected. Similar to the positive
controls, anti-PKC-
and anti-PKC-
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, ,
,
and
), the antigen-antibody complex failed to recognize any protein
in the PASMC homogenate.
Because the affinity of the anti-PKC- (monoclonal) for PKC-
in
the brain lysate was weak (Fig.
1A) and no PKC-
was detected in
the PASMC homogenate (Fig. 1B), we
tested the ability of another anti-PKC-
to detect PKC-
in rat
brain lysate and PASMC homogenate (Fig. 2).
Anti-PKC-
(polyclonal) recognized a single band with high affinity
in the rat brain lysate but failed to recognize any PKC-
in the
PASMC homogenate regardless of the PASMC protein concentration on the
nitrocellulose membrane.
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Immunofluorescence localization of PKC isoforms in
PASMCs. Initially, the antibodies that gave a positive
result on the Western blot (,
,
,
,
/
, and
µ) were utilized for immunocytochemistry, with the exception of
anti-PKC-
. Anti-PKC-
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
isoform of PKC. Anti-PKC-
and anti-PKC-
provided no
immunodetectable fluorescence in PASMCs and were not useful for further
immunocytochemical studies. Therefore, anti-PKC-
was used for
immunolocalization of PKC-
/
in PASMCs. The other isoforms
(
,
,
,
/
, 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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Effects of ANG II on the cellular distribution of PKC
isoforms. Exposure of PASMCs to ANG II
(107 M) revealed spatial
translocation of two isoforms (
and
) and upregulation of one
isoform (
) compared with untreated cells (Figs. 3-5). All
treatments were for 10 min.
Immunolocalization of PKC-. In
untreated cells, PKC-
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-
immunoreactivity being more
pronounced on the nuclear envelope (Fig.
3B). Treatment with DOG mimicked ANG
II-induced translocation of PKC-
to the nuclear envelope (Fig.
3C). The competitive
AT1-receptor agonist-antagonist saralasin also induced translocation of PKC-
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-
compared with
untreated cells (Fig. 3E). However,
pretreatment of PASMCs with losartan before ANG II treatment abolished
the ANG II-induced translocation of PKC-
(Fig.
3F).
Immunolocalization of PKC-. In
untreated cells, PKC-
was detected in both the nucleus and the
cytoplasm (Fig. 4A). The nuclear immunoreactivity of PKC-
included numerous, intensely bright, discrete spots, whereas the cytoplasmic immunoreactivity was amorphous and diffuse. Exposure to ANG II resulted in cytoplasmic PKC-
becoming more localized onto myofilaments, whereas the nuclear fluorescence was similar to that in untreated cells (Fig.
4B). In some cells, staining of
PKC-
on the myofilaments was very strong, resembling staining for
-smooth muscle actin. Treatment with DOG (Fig.
4C) or saralasin (Fig.
4D) mimicked the ANG II translocation of PKC-
to the myofilaments. Losartan alone did not
alter the subcellular distribution of PKC-
compared with untreated
cells (Fig. 4E), whereas
pretreatment with losartan abolished the ANG II-induced translocation
of PKC-
(Fig. 4F).
Immunolocalization of PKC-. In
untreated cells, PKC-
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-
, as reflected by the appearance of several
bright intranuclear coarse fluorescent granules (Fig.
4B). No redistribution of PKC-
was observed in cells exposed to DOG (Fig.
5C). Saralasin upregulated PKC-
similar to that observed with ANG II (Fig.
5D). Losartan alone (Fig.
5E) did not alter the subcellular
distribution of PKC-
but abolished the ANG II-induced upregulation
of PKC-
(Fig. 5F).
Immunolocalization of PKC-/
and
PKC-µ. PKC-
/
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-
/
or PKC-µ in ANG
II-treated cells.
Intranuclear localization of PKC- and
PKC-µ. PKC-
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-
during ANG II treatment was also confirmed (Fig.
7C).
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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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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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DISCUSSION |
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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 (/
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-
to the nuclear envelope and
PKC-
to the myofilaments, whereas intranuclear PKC-
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 (,
,
,
,
,
,
, and
µ). In addition, an appropriate molecular-mass correlation was
demonstrated for each. Anti-PKC-
and anti-PKC-
each recognized
two proteins. Detection of a lower molecular mass protein with
anti-PKC-
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-
is not known but
was not observed when we used anti-PKC-
, which should recognize the
same protein.
Western blots of cultured PASMCs.
Western blot analysis of cultured PASMCs revealed the presence of seven
isoforms (µ, ,
,
,
,
/
,
). However, the
presence of PKC-
on the immunoblot is likely due to anti-PKC-
cross-reacting with PKC-
because PKC-
appears to be restricted
primarily to the brain (44). Cross-reactivity of anti-PKC-
with
PKC-
has been documented by the commerical supplier. However, two
different anti-PKC-
antibodies did not recognize PKC-
in PASMCs.
This result suggests that anti-PKC-
is not detecting the
isoform
of PKC. Anti-PKC-
and anti-PKC-
(mouse variant) each identified
the same protein in PASMC homogenates. However, because anti-PKC-
labeled two bands and anti-PKC-
detected a single protein, this
result could suggest that there are some differences in specificity.
Anti-PKC-
appeared to be the more specific probe for
PKC-
/
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 /
. Therefore, it appears that PASMCs express six
different isoforms of PKC (
,
,
,
, µ, and
/
). 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
/
and µ isoforms of PKC in any type of vascular
smooth muscle.
Intracellular translocation of PKC- to the nuclear
envelope by ANG II. PKC-
underwent
translocation to the nuclear envelope in response to ANG II.
Translocation of PKC-
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-
along
the myofilaments, followed by translocation to the nucleus (14). In
contrast, PKC-
immunoreactivity was at the surface membrane in
hypertrophied cells from the rat aorta (25). In addition, translocation
of PKC-
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-
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- to
myofilaments by ANG II. Translocation of PKC-
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-
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-
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-
may colocalize with the cytoskeletal protein
talin, as suggested for PKC-
(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- by ANG
II. In contrast to PKC-
and PKC-
, intranuclear
PKC-
was upregulated in response to ANG II. Translocation of PKC-
into the nucleus has been observed in ferret aorta exposed to
phenylephrine (21). However, in rat aorta, neither phenylephrine nor
phorbol esters mobilized PKC-
, although increased nuclear levels of
PKC-
were observed in hypertrophied rat aortic cells (25).
Immunoreactivity of PKC-
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- 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-
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- and PKC-µ
isoforms. One unique finding of the present study was
the differential nuclear localization of PKC-
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-
and PKC-µ
were not affected by ANG II stimulation, suggesting that these isoforms
may be involved in steady-state nuclear functions. PKC-
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- 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- ![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Dr. Hiroshi Hamada, Dr. Sumihiko Seki, and Elizabeth Ghoubrial for technical assistance and Ronnie Sanders for preparation of this manuscript.
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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.
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