Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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Our objectives were to
identify the relative contributions of intracellular free
Ca2+ concentration ([Ca2+]i) and
myofilament Ca2+ sensitivity in the pulmonary artery smooth
muscle (PASM) contractile response to the -adrenoreceptor agonist
phenylephrine (PE) and to assess the role of PKC, tyrosine kinases
(TK), and Rho kinase (ROK) in that response. Our hypothesis was that
multiple signaling pathways are involved in the regulation of
[Ca2+]i, myofilament Ca2+
sensitization, and vasomotor tone in response to
-adrenoreceptor stimulation of PASM. Simultaneous measurement of
[Ca2+]i and isometric tension was performed
in isolated canine pulmonary arterial strips loaded with fura 2-AM.
PE-induced tension development was due to sarcolemmal Ca2+
influx, Ca2+ release from inositol
1,4,5-trisphosphate-dependent sarcoplasmic reticulum Ca2+
stores, and myofilament Ca2+ sensitization. Inhibition of
either PKC or TK partially attenuated the sarcolemmal Ca2+
influx component and the myofilament Ca2+ sensitizing
effect of PE. Combined inhibition of PKC and TK did not have an
additive attenuating effect on PE-induced Ca2+
sensitization. ROK inhibition slightly decreased
[Ca2+]i but completely inhibited myofilament
Ca2+ sensitization. These results indicate that PKC and TK
activation positively regulate sarcolemmal Ca2+ influx in
response to
-adrenoreceptor stimulation in PASM but have relatively
minor effects on myofilament Ca2+ sensitivity. ROK is the
predominant pathway mediating PE-induced myofilament Ca2+ sensitization.
intracellular calcium ion; myofilament calcium ion sensitivity; phenylephrine; vascular smooth muscle; pulmonary artery smooth muscle; protein kinase C
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INTRODUCTION |
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CATECHOLAMINE-INDUCED
ACTIVATION of -adrenoreceptors on vascular smooth muscle cells
results in an increase in vasomotor tone. Vasomotor tone is regulated
by intracellular free Ca2+ concentration
([Ca2+]i) and myofilament Ca2+
sensitivity. The initial increase in tone is mediated by an increase in
[Ca2+]i, which triggers activation of myosin
light chain kinase, myosin light chain phosphorylation, and an
increase in tension. Maintenance of vascular smooth muscle contraction
is more complex and likely involves several signaling pathways.
Sensitization of the contractile apparatus to Ca2+
appears to be one important mechanism of pharmacomechanical
coupling (12). Activation of PKC (4, 5, 11,
22), tyrosine kinases (TK) (7, 18, 19, 42), and/or
Rho kinase (ROK) (18, 46) have been suggested to play
important roles in the signal transduction events associated with
vascular smooth muscle contraction. However, the relative roles of PKC,
TK, and ROK in regulating [Ca2+]i,
myofilament Ca2+ sensitivity, and tension in response to
-adrenoreceptor stimulation have not been fully elucidated.
Although the cellular mechanisms involved in
-adrenoreceptor-mediated vasoconstriction have been studied in a
variety of systemic vascular smooth muscle types (14, 40, 45,
49), comparatively less is known about the cellular mechanisms
mediating
-adrenoreceptor activation in the pulmonary circulation.
Moreover, differential responses to hypoxia in the systemic
(vasodilation) vs. the pulmonary (vasoconstriction) circulation
underscore the difficulty in extrapolating results obtained in systemic
vascular smooth muscle to cellular mechanisms that regulate pulmonary
vasomotor tone. In the pulmonary circulation, the extent to which PKC,
TK, and ROK mediate
-adrenoreceptor pulmonary vasoconstriction is controversial (5, 18, 19, 22). In endothelium-intact canine pulmonary artery smooth muscle (PASM) rings,
norepinephrine-induced contractions were insensitive to PKC inhibitors
but were abolished by TK inhibition and ROK inhibition
(18). In contrast, others demonstrated that the
vasoconstrictor response to norepinephrine in the feline pulmonary
vascular bed was attenuated by PKC inhibitors (22) and
likely mediated by Ca2+-independent PKC-
isozyme/calmodulin-dependent kinase III (5). Others
suggested that the contractile response to norepinephrine in isolated
rat pulmonary artery is largely mediated by TK activation (42). In none of these studies were
[Ca2+]i and tension simultaneously measured
in the same tissue so that the role of PKC, TK, or ROK in
[Ca2+]i and myofilament Ca2+
sensitivity in response to
-adrenoreceptor activation could be
adequately assessed. Only one study assessed the role of sarcolemmal Ca2+ influx, as well as release of Ca2+ from
the sarcoplasmic reticulum, in mediating the actions of
-adrenoreceptor activation in PASM (17). However, that
study did not directly measure [Ca2+]i nor
did it assess the role(s) of protein kinase activation in mediating
increases in tension in response to
-adrenoreceptor activation
(17). The present study is unique in that our goal was to
simultaneously measure [Ca2+]i and tension in
PASM strips and to identify the extent to which PKC, TK, and ROK
modulate [Ca2+]i and myofilament
Ca2+ sensitivity in response to
-adrenoreceptor
stimulation with phenylephrine (PE).
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MATERIALS AND METHODS |
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All surgical procedures and experimental protocols were approved by the Cleveland Clinic Foundation Institutional Animal Care and Use Committee (Cleveland, OH).
Preparation of pulmonary arterial strips.
Heartworm-negative mongrel dogs (20-30 kg) were anesthetized with
intravenous pentobarbital sodium (30 mg/kg) and fentanyl citrate (15 µg/kg) and placed on positive-pressure ventilation. A catheter was
inserted into the right femoral artery, and the dogs were exsanguinated
by controlled hemorrhage. A left lateral thoracotomy was performed
through the fifth intercostal space, and the heart was arrested with
electrically induced ventricular fibrillation. The heart and lungs were
removed from the thorax en bloc. Right and left intralobar pulmonary
arteries [2- to 4-mm internal diameter (ID)] were dissected free and
immersed in cold modified Krebs-Ringer bicarbonate (KRB) solution
composed of (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25 NaHCO3, 0.016 Ca-EDTA, and 11.1 glucose. The pulmonary
arteries were cleaned of fat and connective tissue and cut into strips (2 × 8 mm) or rings (2- to 4-mm ID). The endothelium was denuded by gently rubbing the intimal surface with a cotton swab. The absence
of an intact endothelium was later verified by assessing the
vasorelaxant response to 106 M acetylcholine.
Simultaneous measurement of
[Ca2+]i and tension.
As described previously (37), pulmonary arterial strips
without endothelium were loaded with 5 µM fura 2-AM at room
temperature (22-24°C). A noncytotoxic detergent, 0.05%
cremophor EL, was added to solubilize the fura 2-AM in the solution and
act as a vehicle for penetrating the tissue. After fura 2 loading, the
muscle strips were washed with KRB buffer for 30-60 min to remove
unhydrolyzed fura 2-AM and then placed between two stainless steel
hooks in a temperature-controlled (37°C) cuvette (volume 3 ml), which
was continuously perfused (12 ml/min) with KRB solution bubbled with 95% air-5% CO2 (pH 7.4). One hook was anchored, and the
other was connected to a strain gauge transducer (Grass model FTO3; Quincy, MA) to measure isometric force. The resting tension was adjusted to 4 g, which was determined in preliminary studies to be
optimal for achieving a maximum contractile response to 20 mM KCl.
Fluorescence measurements were performed with a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, Lawrenceville, NJ) at excitation wavelengths of 340 and 380 nm and an
emission wavelength of 510 nm. Fura 2 fluorescence signals (340 nm, 380 nm, and 340 nm-to-380 nm ratio) were continuously monitored at a
sampling frequency of 2.5 Hz and collected with a software package from
Photon Technology International. The 340-to-380 fluorescence ratio was
used as an indicator of [Ca2+]i. In control
experiments, the baseline fluorescence intensities, as well as the
signal ratios measured in the absence of dye, were not altered by any
of the experimental interventions. After changes in tension and
[Ca2+]i in response to 60 mM KCl were
measured, the strips were washed with fresh KRB for 30 min. All strips
were pretreated with propranolol (5 µM, 30 min) to inhibit the
-agonist effect of PE. A high dose of PE (10 µM) was used
throughout the study to achieve a significant increase in tension in
protocols in which extracellular Ca2+ was absent. The
response to PE (10 µM) was assessed in the presence (10 min) or
absence of the PKC inhibitor (48) bisindolylmaleimide I
(Bis I; 3 µM), the TK inhibitor (26) tyrphostin (Tyr)
A47 (10 µM), the ROK inhibitor (15) Y-27632 (10 µM),
or the inositol 1,4,5-trisphosphate (IP3) receptor
inhibitor (2) 2-aminoethoxydiphenyl borate (2-APB; 100 µM). Structurally different inhibitors of PKC (calphostin C; 0.1, 0.3, and 1 µM) and TK (genistein; 100 µM) were also investigated.
Structurally similar inactive analogs of Bis (Bis V; 3 µM), Tyr A47
(Tyr A1; 10 µM) and genistein (daidzein, 100 µM) were also
assessed. Nonspecific effects of the inhibitors on voltage-gated
Ca2+ channels and myosin light chain kinase activity were
assessed in strips contracted with KCl. Bis I, Bis V, calphostin C, Tyr A47, Tyr A1, genistein, daidzein, and 2-APB had no effect on
KCl-induced contraction, whereas Y-27632 inhibited the increase in
tension by 20 ± 9%. Early [Ca2+]i and
tension values represent the peak increases in each parameter in the 2 min after administration of PE. Late [Ca2+]i
and tension values represent measurements for each parameter 15 min
after administration of PE. Summarized data for the changes in
[Ca2+]i and tension after an intervention are
expressed as the percent change from the previous response measured in
that particular protocol.
Materials. Phenylephrine HCl, propranolol HCl, Tyr A47, Tyr A1, genistein, daidzein, Bis I, and Bis V were purchased from Sigma (St. Louis, MO). 2-APB and calphostin C were obtained from Calbiochem (La Jolla, CA). Y-27632 was obtained from Biomol (Plymouth Meeting, PA).
Statistical analysis and data presentation. Results are expressed as means ± SE. The sample size, n, represents the number of dogs from which strips or rings were studied. Statistical comparisons used ANOVA and the Bonferroni-Dunn post hoc test. Student's t-test for paired comparisons was used when appropriate. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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Effect of KCl and PE on
[Ca2+]i and tension.
Figure 1A illustrates
simultaneous recordings of [Ca2+]i and
tension during exposure to 60 mM KCl and 10 µM PE in a PASM strip. Addition of KCl resulted in rapid, early increases in
[Ca2+]i and tension, followed by late,
sustained increases near peak levels until washout. PE caused early
peak increases in [Ca2+]i and tension
followed by late, sustained increases at lower values. PE increased
tension to a similar degree as KCl, whereas the increase in
[Ca2+]i was only about half of that observed
with KCl. During washout of PE, tension remained elevated even when
[Ca2+]i had returned to baseline (Fig.
1A). PE caused a leftward shift in the continuous
[Ca2+]i-tension relationship (Fig.
1B). The reproducibility of the PE response was assessed by
administering PE three successive times to PASM strips. As summarized
in Fig. 2, increases in
[Ca2+]i and tension in response to the second
and third applications of PE were similar although somewhat greater
then the first response to PE. We next investigated the effects of the
various interventions on the early (defined as the peak changes
occurring within 2 min of PE application) and late (measured 15 min
after administration of PE) changes in
[Ca2+]i and tension in response to PE in PASM
strips.
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Effect of removing extracellular
Ca2+.
To test the hypothesis that influx of extracellular Ca2+ is
required for PE-induced contraction, we measured
[Ca2+]i and tension in response to PE in the
presence or absence of extracellular Ca2+. As illustrated
in Fig. 3, removal of extracellular
Ca2+ decreased the early increase in
[Ca2+]i and tension by 32 ± 6% and
55 ± 5%, respectively. In the absence of extracellular
Ca2+ the late phase of the PE-induced increase in
[Ca2+]i was abolished, whereas the late phase
of tension development was reduced by 65 ± 2%.
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Effect of IP3 receptor inhibition in absence of
extracellular Ca2+.
Our next goal was to test the hypothesis that IP3-mediated
release of Ca2+ from intracellular stores is involved in
the PE-induced contractile response. We pretreated the strips with the
IP3 receptor blocker 2-APB before PE stimulation in the
absence of extracellular Ca2+ (Fig.
4). Inhibition of IP3
receptors with 2-APB (100 µM) abolished the early increase in
[Ca2+]i in response to PE and reduced the
early increase in tension by 43 ± 5% compared with that observed
in the absence of extracellular Ca2+. 2-APB had no effect
on the late PE-induced increase in tension.
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Effect of PKC inhibition.
To test the hypothesis that PKC activation plays a role in PE-induced
increases in [Ca2+]i and tension, PASM strips
were pretreated with the PKC inhibitor Bis I before PE stimulation. In
the presence of extracellular Ca2+, pretreatment with Bis I
(3 µM) had no effect on the early increase in
[Ca2+]i but reduced the late increase in
[Ca2+]i by 60 ± 3% (Fig.
5). Bis I reduced both the early (20 ± 5%) and late (33 ± 4%) increases in tension in response to
PE. The inactive analog of Bis I (Bis V) had no effect on PE-induced
increases in [Ca2+]i or tension. To further
confirm a role for PKC in PE-induced contraction,
concentration-response curves for KCl and PE were obtained in PASM
rings in the presence or absence of another specific PKC inhibitor,
calphostin C. Because of the light-sensitive nature of calphostin C,
[Ca2+]i could not be measured in these
studies. Pretreatment with calphostin C (1 µM) for 60 min had no
effect on resting tension or KCl-induced contraction (Fig.
6). However, PE-induced contraction was
inhibited by calphostin C, resulting in a 43 ± 5% decrease in
maximal tension.
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Effect of PKC inhibition in absence of extracellular
Ca2+.
We next tested the hypothesis that PKC activation is involved in
regulating the PE-induced increase in tension observed in the absence
of extracellular Ca2+. We pretreated strips with Bis I in
the absence of extracellular Ca2+ before stimulation with
PE. In the absence of extracellular Ca2+, Bis I (3 µM)
had no effect on the early increase in
[Ca2+]i induced by PE but reduced the early
increase in tension by 25 ± 6% (Fig.
7). Bis I had no significant effect on
the late PE-induced increase in tension.
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Effect of PKC inhibition in absence of extracellular
Ca2+ and IP3-mediated SR
Ca2+ release.
We tested the hypothesis that the increase in tension observed in
the absence of any increase in [Ca2+]i is
mediated by activation of PKC. PASM strips were pretreated with Bis I
in the absence of extracellular Ca2+ and in the presence of
2-APB before stimulation with PE. Under these conditions, PE-induced
increases in tension were not associated with concomitant increases in
[Ca2+]i. As summarized in Fig.
8A, Bis I reduced both the
PE-induced early (20 ± 5%) and late (18 ± 6%) increases
in tension.
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Effect of TK inhibition.
To test the hypothesis that activation of TK plays a role in PE-induced
increases in [Ca2+]i and tension, we
pretreated PASM strips with the TK inhibitor Tyr A47 before PE
stimulation. Pretreatment with Tyr A47 (10 µM) had no effect on the
early increase in [Ca2+]i but,
surprisingly, enhanced the late increase in
[Ca2+]i by 79 ± 20% (Fig.
9). Tyr A47 had no effect on the early
increase in tension but enhanced the late increase in tension by
20 ± 8%. The inactive analog of Tyr A47 (Tyr A1; 10 µM) did
not alter PE-induced increases in [Ca2+]i or
tension. In contrast to Tyr A47, pretreatment with genistein (100 µM)
reduced the early increase in [Ca2+]i and
tension by 55 ± 6% and 38 ± 12%, respectively. Genistein also reduced the late increases in [Ca2+]i
and tension by 60 ± 8% and 20 ± 8%, respectively. The
inactive analog of genistein (daidzein; 100 µM) had no effect on
PE-induced increases in [Ca2+]i or tension.
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Effect of TK inhibition in absence of extracellular
Ca2+.
We tested the hypothesis that activation of TK is involved in
regulating the PE-induced increase in tension observed in the absence
of extracellular Ca2+. PASM strips were pretreated with Tyr
A47 in the absence of extracellular Ca2+ before stimulation
with PE. Similar to Bis I, Tyr A47 (10 µM) had no effect on the early
increase in [Ca2+]i in response to PE but
reduced the early increase in tension by 22 ± 6% (Fig.
10). Tyr A47 had no effect on late
changes in tension. Pretreatment with genistein (100 µM) reduced the
early PE-induced increase in [Ca2+]i
and tension by 68 ± 12% and 60 ± 16%, respectively.
Genistein reduced the late phase of PE-induced contraction by 55 ± 15%.
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Effect of TK inhibition in absence of extracellular Ca2+ and IP3-mediated SR Ca2+ release. To test the hypothesis that the increase in tension observed in the absence of any increase in [Ca2+]i is mediated by activation of TK, we pretreated strips with Tyr A47 in the absence of extracellular Ca2+ and in the presence of 2-APB before stimulation with PE. As noted above, under these conditions PE-induced increases in tension were not associated with concomitant increases in [Ca2+]i. As summarized in Fig. 8B, Tyr A47 reduced the early (34 ± 3%) and late (30 ± 5%) increases in tension in response to PE.
Effect of combined TK and PKC inhibition in absence of extracellular Ca2+ and IP3-mediated SR Ca2+ release. We also tested the hypothesis that the effects of PKC and TK on myofilament Ca2+ sensitization were additive (i.e., the signaling pathways are in parallel). PASM strips were pretreated with both Bis I and Tyr A47 in the absence of extracellular Ca2+ and in the presence of 2-APB before stimulation with PE. As summarized in Fig. 8C, the effect of combined PKC and TK inhibition on PE-induced contraction was no greater than that observed with Bis I or Tyr A47 alone.
Effect of ROK inhibition.
To test the hypothesis that ROK plays a role in PE-induced increases in
[Ca2+]i and tension, PASM strips were
pretreated with the ROK inhibitor Y-27632 before PE stimulation. In the
presence of extracellular Ca2+, pretreatment with Y-27632
(10 µM) reduced the early (21 ± 7%) but not the late increase
in [Ca2+]i in response to PE (Fig.
11). However, pretreatment with Y-27632 reduced both the early and late PE-induced increases in tension by
52 ± 7% and 54 ± 8%, respectively.
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Effect of ROK inhibition in absence of extracellular Ca2+ and IP3-mediated SR Ca2+ release. Finally, to test the hypothesis that the increase in tension observed in the absence of any increase in [Ca2+]i is mediated by activation of ROK, we pretreated strips with Y-27632 in the absence of extracellular Ca2+ and in the presence of 2-APB before stimulation with PE. Under these conditions, inhibition of ROK with Y-27632 (10 µM) abolished PE-induced contraction (Fig. 8D).
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DISCUSSION |
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To our knowledge, this is the first study to simultaneously measure PE-induced changes in [Ca2+]i and tension in PASM in the presence and absence of extracellular Ca2+ before and after inhibition of PKC, TK, ROK, and IP3 receptors. Our results indicate the following. Transsarcolemmal Ca2+ influx and myofilament Ca2+ sensitization contribute to both the early and late increases in tension. IP3 receptor-mediated release of Ca2+ from the sarcoplasmic reticulum contributes to the increase in early tension but not the late phase of tension. PKC and TK activation positively regulate both PE-induced Ca2+ influx and myofilament Ca2+ sensitization. The effects of PKC and TK on myofilament Ca2+ sensitivity were comparatively minor and not additive. Finally, ROK is the predominant pathway mediating PE-induced myofilament Ca2+ sensitization. However, it should be noted that the relative roles of these pathways may vary depending on the stimulus intensity and/or degree of resting tension. In addition, cross talk (30, 39) likely occurs between these pathways and may account for some of the differences observed in other studies (18, 42).
[Ca2+]i-tension
relationship.
Our first goal was to determine the relationship between
[Ca2+]i and tension in response to membrane
depolarization with KCl (electromechanical coupling), in which no
phospholipid-derived second messengers are liberated, compared with
that observed with -adrenoreceptor stimulation with PE
(pharmacomechanical coupling). KCl essentially caused monotonic
increases in [Ca2+]i and tension. In
contrast, PE caused a transient early increase in
[Ca2+]i and tension, followed by a late phase
of tension and [Ca2+]i that remained elevated
above baseline but below peak values. Because the PE-induced increase
in tension was similar to that of KCl, whereas the increase in
[Ca2+]i was only about half of that observed
with KCl, our results suggest that PE increases myofilament
Ca2+ sensitivity. A PE-induced increase in myofilament
Ca2+ sensitivity is also suggested by the continued
contraction during the washout period when
[Ca2+]i had returned to baseline and by the
leftward shift in the continuous [Ca2+]i-tension relationship compared with
KCl. A PE-induced leftward shift in the
[Ca2+]i-tension relationship was also
observed in rabbit PASM (12). We then investigated the
roles of transsarcolemmal Ca2+ influx and
IP3-mediated Ca2+ release in mediating the
PE-induced increases in [Ca2+]i and tension.
Ca2+ influx and
Ca2+ release.
Removal of extracellular Ca2+ abolished the late
increase in [Ca2+]i in response to PE,
indicating that it is entirely dependent on transsarcolemmal
Ca2+ influx. The early increase in
[Ca2+]i was also modestly reduced, likely
reflecting a small depletion in the Ca2+ content of the
sarcoplasmic reticulum in response to removal of extracellular
Ca2+. Both the early and late increases in tension were
reduced when extracellular Ca2+ was removed, indicating
that transsarcolemmal Ca2+ influx plays a major role in
PE-induced contraction in PASM. For reasons that remain unclear, our
results appear to be in direct contrast to a recent study that reported
that removal of extracellular Ca2+ or administration of
voltage-gated Ca2+ channel blockers had no effect on
tension development in response to -adrenoreceptor stimulation in
endothelium-intact PASM (18). However, in a separate
study, PE-induced contractions were found to be reduced by >75% in a
nominally Ca2+-free buffer and involved a
nisoldipine-insensitive, SK&F-96365-sensitive Ca2+ influx
pathway (17). In that same study, depletion of
IP3-sensitive Ca2+ stores, but not
caffeine-sensitive Ca2+ stores, nearly abolished the PE
response. This study by Jabr and coworkers (17) suggests
that a nisoldipine-insensitive Ca2+ entry pathway is
normally involved in filling IP3-sensitive Ca2+
stores and that IP3-sensitive and caffeine-sensitive
Ca2+ stores are functionally separate and independent
entities in PASM. In our study, Ca2+ influx could be
mediated via activation of receptor-operated Ca2+ channels
(9), by activation of voltage-gated Ca2+
channels in response to PE-induced membrane depolarization
(35), or by capacitative Ca2+ entry in
response to depletion of Ca2+ stores in the sarcoplasmic
reticulum (8). In the absence of extracellular
Ca2+, IP3 receptor inhibition abolished the
early increase in [Ca2+]i, thereby indicating
that Ca2+ release from IP3-sensitive
Ca2+ stores in the sarcoplasmic reticulum was the mechanism
responsible for the early increase in [Ca2+]i
in response to PE. Our data are consistent with the findings of Jabr et
al. (17) that both sarcolemmal Ca2+ influx and
Ca2+ release from intracellular stores (IP3
sensitive) are involved in PE-induced contraction.
Myofilament Ca2+ sensitization. After removal of extracellular Ca2+ and inhibition of IP3 receptor-mediated Ca2+ release, PE increased tension without a concomitant increase in [Ca2+]i. The most likely explanation for this result is a PE-induced increase in myofilament Ca2+ sensitivity. An alternative explanation could be that there is a Ca2+-independent mechanism involved, rather than an increase in myofilament Ca2+ sensitivity. However, these two possibilities are not mutually exclusive and may exist in parallel or be linked to each other (i.e., Ca2+-independent pathway triggering an increase in myofilament Ca2+ sensitivity). This could be achieved through activation of PKC, TK, ROK, or a yet unidentified pathway. Thus we next investigated the roles of PKC, TK, and ROK in mediating this effect, as well as PE-induced changes in Ca2+ influx.
PKC.
PKC activation is concomitant with IP3 production in
response to agonist stimulation of phospholipase C (50).
Alternatively, agonist activation of phospholipase D (1,
34) or phospholipase A2 (10, 38) can
result in PKC activation in the absence of IP3 production.
PKC has been reported to play a role in -adrenoreceptor-mediated pulmonary vasoconstriction in isolated cat and rat lung (5, 22). In contrast, a recent report indicated that PKC is not involved in
-adrenoreceptor-mediated Ca2+ influx or
contraction in endothelium-intact canine pulmonary artery
(18). To resolve this controversy, we assessed the extent to which PKC mediates increases in [Ca2+]i
and tension simultaneously in response to PE. PKC inhibition with Bis I
attenuated the PE-induced late increases in
[Ca2+]i and tension, which suggests that a
component of the late increase in tension is mediated by PKC-dependent
sarcolemmal Ca2+ influx. Bis I had no effect on KCl-induced
increases in [Ca2+]i or tension, indicating
that its effect was not due to some nonspecific action on myosin light
chain kinase. Moreover, calphostin C, a structurally different PKC
inhibitor, also attenuated PE-induced, but not KCl-induced,
contraction. These data are consistent with other studies that
demonstrated that PKC activation by phorbol esters enhances L-type
channel-mediated Ca2+ influx in some smooth muscle cells
(28) as well as
-adrenoreceptor-stimulated Ca2+ influx through voltage-gated Ca2+ channels
in rat portal vein and tail artery (25). Our results clearly indicate that PKC activation is involved in
-adrenoreceptor-mediated contraction of canine PASM.
TK.
Because TK activation has also been implicated in the contractile
response to -adrenoreceptor activation in the pulmonary circulation
(8, 18, 42), we assessed the extent to which inhibition of
TK regulates [Ca2+]i and tension in response
to PE. Much to our surprise, pretreatment with Tyr A47 appeared to
enhance the late increase in [Ca2+]i and
tension in response to PE when extracellular Ca2+ was
present (Fig. 9). Tyr A47 was shown to exert no effect on arginine
vasopressin-induced increases in Ca2+ in A7r5 aortic smooth
muscle cells (21). In contrast, other studies demonstrated
that inhibition of TK with various Tyr derivatives results in decreased
Ca2+ entry in vascular smooth muscle cells (21, 43,
51, 52). It should be noted that repetitive exposure to PE alone
was associated with slightly potentiated late increases in
[Ca2+]i and tension (Fig. 2). This effect may
explain, at least in part, the late PE-induced increases in
[Ca2+]i and tension after Tyr A47.
Alternatively, the effects of Tyr A47 could potentially be explained by
the finding that some TK inhibitors, including Tyr A47, actually
enhance tyrosine phosphorylation (6, 21). To further
investigate this unexpected finding, we assessed the effects of a more
widely used and structurally different TK inhibitor, genistein. In
contrast to Tyr A47, genistein attenuated both the early and late
increases in [Ca2+]i and tension in response
to PE. This effect was observed in the presence and absence of
extracellular Ca2+. These results suggest that a component
of PE-induced contraction is due to Ca2+ release and
transsarcolemmal Ca2+ influx. Our results with genistein
are consistent with our previous finding (8) that TK
inhibition caused a rightward shift in the PE-induced
concentration-response relationship in pulmonary arterial rings and
provide additional support for the idea that TK plays a role in
regulating
-adrenoreceptor-mediated increases in
[Ca2+]i and tension in PASM strips.
Combined TK and PKC inhibition. Controversy surrounding the relative roles of PKC and TK in mediating the overall Ca2+-sensitizing effect of PE in the pulmonary vasculature (5, 18) led us to assess the extent to which combined inhibition of PKC and TK attenuated PE-induced Ca2+ sensitization. The effect of combined inhibition of PKC and TK on the PE-induced increase in tension in the absence of extracellular Ca2+ and in the presence of 2-APB was not additive, suggesting that PKC and TK were acting via a final common pathway. Inhibition of myosin light chain phosphatase is a possible candidate (46).
ROK. Recent evidence in a variety of smooth muscle preparations (16, 36), including PASM (18, 46), suggests that activation of ROK by the active GTPase RhoA may be a key mediator of Ca2+ sensitization in response to G protein-coupled receptor activation. ROK phosphorylates the regulatory subunit of myosin light chain phosphatase and inhibits its catalytic activity, thus resulting in increased myosin light chain phosphorylation, Ca2+ sensitization, and increased tension (46). In our study, inhibition of ROK with Y-27632 attenuated KCl-induced tension by 20%, with no concomitant effect on [Ca2+]i, indicating that Y-27632 may have some nonspecific inhibitory effect on myosin light chain kinase activity. When extracellular Ca2+ was present and intracellular Ca2+ stores were preserved, inhibition of ROK resulted in a slight reduction in the PE-induced increase in early [Ca2+]i but no effect on late [Ca2+]i. However, both early and late increases in tension in response to PE were attenuated. These data suggest that ROK positively regulates IP3-dependent SR Ca2+ release while having no significant effect on Ca2+ influx (16). The reduction in both PE-induced early and late tension is likely explained by a modest reduction in Ca2+ availability but primarily by a decrease in the sensitizing component of the late contraction. ROK inhibition abolished the contractile response to PE in the absence of extracellular Ca2+ and after IP3 receptor block, indicating that ROK activation is the predominant pathway mediating PE-induced Ca2+ sensitization (Fig. 8D). Activation of ROK by PE may be due to the GTPase RhoA (46) or the release of arachidonic acid in response to PE (37, 46).
Relative roles of signaling pathways. As noted above, the inhibitory effects of PKC and TK inhibition (alone and in combination) on PE-induced contraction in the absence of changes in [Ca2+]i were relatively modest and not additive, whereas ROK inhibition abolished PE-induced contraction under these conditions. ROK inhibition also blocked about half of the PE-induced contraction when Ca2+ was available. These results suggest that PKC and TK play a relatively minor role in mediating PE-induced late tension and that they act via a final common pathway. TK-dependent activation of ROK (18, 41) or PKC-dependent activation of ROK (32, 44) may explain these findings. PKC activation may serve dual roles in the PE response by positively regulating the L-type Ca2+ current (47, 53) and enhancing myofilament Ca2+ sensitivity, possibly via interactions with CPI-17 (27). TK activation also appears to positively regulate sarcolemmal Ca2+ influx in PASM, which is consistent with studies in other vascular beds (24, 29). TK positively regulates myofilament Ca2+ sensitivity, perhaps via direct phosphorylation of tyrosine residues on myosin light chain phosphatase (30, 39, 41). RhoA activation of ROK could exert a direct effect on myosin light chain phosphatase activity to enhance myofilament Ca2+ sensitivity (46). Phosphorylation of calponin by ROK (20), PKC (33), or TK (31) is another potentially important pathway. Further studies will be required to fully elucidate these interactions.
In summary, our results suggest that PE activates multiple signaling pathways that mediate ![]() |
ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-38291 and HL-40361.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. A. Murray, Center for Anesthesiology Research, FF-40, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195 (E-mail: murrayp{at}ccf.org).
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.
July 12, 2002;10.1152/ajplung.00345.2001
Received 27 August 2001; accepted in final form 8 July 2002.
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