Asthma Research Group, Firestone Institute for Respiratory Health, and Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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We investigated the mechanisms that underlie the responses to norepinephrine (NE) and thromboxane (Tx) A2 (TxA2) in the canine pulmonary vasculature with fura 2 fluorimetric, intracellular microelectrode, and force transduction techniques. KCl, caffeine, and cyclopiazonic acid elevated intracellular Ca2+ concentration levels and tone, indicating that Ca2+ mobilization is sufficient to produce contraction. However, contractions evoked by NE or the TxA2 mimetic U-46619 were unaffected by nifedipine or by omitting external Ca2+ and were reduced only partially by depleting the internal Ca2+ store; furthermore, NE-evoked depolarization was subthreshold for voltage-dependent Ca2+ currents. Agonist-evoked contractions were insensitive to inhibitors of protein kinase C (calphostin C and chelerythrine), mitogen-activated protein kinase kinase (PD-98059), and p38 kinase (SB-203580) but were abolished by the tyrosine kinase inhibitor genistein and the Rho kinase inhibitor Y-27632. We conclude that, although Ca2+ influx and Ca2+ release are sufficient for contraction, they are not necessary for adrenergic or TxA2 contractions. Instead, excitation-contraction coupling involves the activation of tyrosine kinase and Rho kinase, leading to enhanced Ca2+ sensitivity of the contractile apparatus.
mitogen-activated protein kinase; norepinephrine; thromboxane A2; intracellular calcium; protein kinase C; myosin light chain kinase; myosin light chain phosphatase
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INTRODUCTION |
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THE SIGNALING PATHWAYS THAT UNDERLIE contraction in smooth muscle have been the focus of intense study, and several mechanisms have been identified. Ultimately, contraction in smooth muscle is triggered by phosphorylation at Ser19 of the regulatory light chain of myosin (13). Generally, this is mediated by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), which, in turn, is activated by the influx of Ca2+ across the plasmalemma or the release of Ca2+ from an internal store. More recently, it has come to be appreciated that excitation-contraction (E-C) coupling can also involve increased Ca2+ sensitivity of the contractile apparatus such that even basal levels of intracellular Ca2+ concentration ([Ca2+]i) are sufficient to trigger contraction (14). Some studies (13) found that this latter phenomenon involves protein kinase C (after agonist-mediated activation of membrane receptors, cytosolic G proteins, and phospholipase C, with subsequent generation of diacylglycerol). Others (14) found that increased Ca2+ sensitivity involved tyrosine kinases and mitogen-activated protein (MAP) kinases, which, in turn, act on a variety of intracellular targets. For example, Rho kinase seems to phosphorylate one of the subunits of myosin light chain phosphatase (MLCP), thereby suppressing the activity of the latter and leading to a net increase in myosin light chain phosphorylation and contraction (14). Extracellular signal-regulated kinase MAP kinases, on the other hand, phosphorylate caldesmon, thereby removing its inhibitory effect on actin- and thin filament-mediated regulation of actomyosin ATPase activity (4).
Although E-C coupling has been investigated in many smooth muscles,
there still remains a general paucity in our understanding of the
mechanisms that operate in pulmonary vascular smooth muscles, particularly those in the vein (almost all studies of pulmonary vascular function use pulmonary arteries, even though there are many
documented differences between these and pulmonary veins). Neurogenic
regulation of the pulmonary vasculature is mediated almost exclusively
by adrenergic innervation acting on adrenoceptors on the smooth muscle
(8, 9). Also, many arachidonic acid metabolites excite the
pulmonary vasculature via action on thromboxane-selective prostanoid receptors including thromboxane A2
(TxA2) itself, PGF2 and PGD2
(10), and isoprostanes (15); levels of these
autacoids are markedly elevated under pathophysiological conditions
such as hypertension, inflammation, acute lung injury, and oxidative stress (2, 16).
In this study, we sought to examine the E-C coupling mechanisms that underlie responses to the adrenergic agonist norepinephrine (NE) and the TxA2 mimetic U-46619 in canine pulmonary arteries and veins.
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METHODS |
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Preparation of tissues. Whole lobes of lung were obtained from dogs that had been euthanized with pentobarbital sodium (100 mg/kg); these were pinned out in physiological saline, and ring segments (3- to 4-mm long) of pulmonary artery and pulmonary vein (outer diameter 2-8 mm; third- to fifth-division vessels) were excised. Tissues were either used immediately or stored at 4°C for use the next day; we found no functional differences in tissues that were studied immediately compared with those used after 24 h of refrigeration.
Muscle bath technique.
Ring segments were mounted in 3-ml muscle baths with stainless steel
hooks inserted into the lumen; care was taken not to damage the
endothelium while the hooks were inserted. One hook was fastened to a
Grass FT03 force transducer with silk thread (Ethicon 4-0); the other
was attached to a Plexiglas rod that served as an anchor. Tissues were
bathed in Krebs-Ringer buffer (for composition, see Solutions and
chemicals) containing 10 µM indomethacin, bubbled with
95% O2-5% CO2, and maintained at 37°C; preload tension was 1 g (determined to allow maximal responses). Isometric changes in tension were digitized (2 samples/s) and recorded
with an on-line program (DigiMed System Integrator; MicroMed, Louisville, KY). Tissues were equilibrated for 2 h and then
exposed to pharmacological inhibitors or experimental conditions (e.g., nominally Ca2+-free bathing medium) for 20-30 min
before the experiments were begun. Some tissues were dried and weighed
to express the contractile responses as grams of tension developed per
milligram of dry tissue weight.
Fura 2 fluorimetry. Ring segments (0.5-1.0 g wet wt) of the pulmonary vasculature were minced and transferred to dissociation buffer (for composition, see Solutions and chemicals) containing 2.7 U/ml of collagenase (type IV), 12.5 U/ml of elastase (type IV), and 1 mg/ml of BSA, then were either used immediately or stored at 4°C for use the next day. We have previously found that cells used immediately and those used after refrigeration exhibit similar functional responses (7). To liberate single cells, tissues in enzyme-containing solution were incubated at 37°C for 60-120 min and then gently triturated. Cells were studied with a filter-based photometer-driven system (DeltaScan, Photon Technology International, South Brunswick, NJ). After being settled on a glass coverslip mounted on a Nikon TMD inverted microscope, the cells were loaded with the membrane-permeant form of fura 2 (fura 2-AM; 2 µM for 30 min at 37°C) and then were superfused continuously with Ringer buffer (2-3 ml/min). The cells were alternately illuminated (0.5 Hz) at the excitation wavelengths and the emitted fluorescence (measured at 510 nm) induced by 340-nm excitation and that induced by 380-nm excitation were measured with a photomultiplier tube assembly. The fluorescence ratio was converted to [Ca2+]i with previously published methods (3). The fluorescence ratio values under saturating and Ca2+-free conditions (Rmax and Rmin, respectively) were obtained previously; the Ca2+-fura 2 dissociation constant (Kd) was assumed to be 224 nM (3). Agonists were applied by pressure ejection from a puffer pipette (Picospritzer II, General Valve, Fairfield, NJ).
Intracellular microelectrodes.
Intact tissues were carefully pinned out in a chamber with a bath
volume of 10 ml; Krebs-Ringer buffer (for composition, see
Solutions and chemicals) was bubbled with 95%
O2-5% CO2, heated to 37°C, and superfused
over the tissues at a rate of
3 ml/min. Conventional microelectrodes
(with a tip resistance of 30-80 M
when filled with 3 M KCl)
were pulled from borosilicate capillary tubes and used to impale single
smooth muscle cells. Membrane potential changes were observed on a
dual-beam oscilloscope (Tektronix D13; 5A22N differential amplifier;
5B12 dual-time base) and recorded on 0.25-inch magnetic tape with a
Hewlett-Packard instrumentation recorder. Portions of the recorded data
were played back, digitized (Digidata 1200), sampled at 500 Hz with
pCLAMP 6 software (Axon Instruments, La Jolla, CA), and then fitted
with the use of pCLAMP 6 and/or exported to SigmaPlot (Jandel, Corte
Madera, CA) for graphic presentation.
Solutions and chemicals. Dissociation buffer contained (in mM) 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 0.25 EDTA, 10 D-glucose, and 10 L-taurine, pH 7.0. Single cells were studied in Ringer buffer containing (in mM) 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose, pH 7.4. Intact tissues were studied with Krebs-Ringer buffer containing (in mM) 116 NaCl, 4.2 KCl, 2.5 CaCl2, 1.6 NaH2PO4, 1.2 MgSO4, 22 NaHCO3, and 11 D-glucose, bubbled to maintain pH at 7.4. Indomethacin (10 µM) was also added to the latter to prevent generation of cyclooxygenase metabolites of arachidonic acid.
Chemicals were obtained from Sigma, with the exception of Y-27632 (kindly provided by A. Yoshimura, Welfide, Osaka, Japan). Stock solutions (10Data analysis. Responses are reported as means ± SE; n refers to the number of animals. Statistical comparisons were made with Student's t-test or ANOVA (followed by Student-Newman-Keuls test for pairwise comparisons) as appropriate. P < 0.05 was considered significant.
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RESULTS |
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Mechanical responses to NE and to a Tx mimetic.
NE evoked contractions in a dose-dependent fashion in both pulmonary
arterial smooth muscle (PASM) and pulmonary venous smooth muscle (PVSM; Fig. 1). PVSM was
significantly more sensitive than PASM (log ED50 values
were 7.5 ± 0.1 and
6.7 ± 0.1 µM, respectively), but
it was not significantly more responsive (when normalized for tissue
dry weight, peak responses were 2.2 ± 0.4 and 1.6 ± 0.2 g/mg, respectively; n = 5 dogs). These two vascular
tissues also differed in their sensitivity to the Tx mimetic U-46619, with PASM being essentially unresponsive, whereas PVSM exhibited large
dose-dependent contractions even at submicromolar concentrations of
U-46619 (Fig. 1). The log ED50 value for U-46619 was
8.2 ± 0.2.
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Role of electromechanical coupling mechanisms in contraction of the
pulmonary vasculature.
KCl (60 mM) elevated [Ca2+]i and evoked
dose-dependent contractions in both PASM and PVSM (Fig.
2). The KCl-evoked change in [Ca2+]i was slower and smaller in magnitude
than that evoked by caffeine (32.6 ± 14.9%; 17 cells;
n = 5 dogs). In addition, this change was generally
sustained, although "oscillations" in
[Ca2+]i were seen in one cell (Fig.
2A, right). These findings indicate that membrane
depolarization and voltage-dependent Ca2+ influx alone are
sufficient to trigger contraction and might mediate the responses to NE
and U-46619.
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Release of internal Ca2+ is sufficient to evoke
contraction.
Caffeine (5 mM) evoked large Ca2+ transients in PASM and
PVSM (Fig. 5A). These reached
a mean peak magnitude of 364 ± 24 nM (n = 26 dogs) within 1-2 s after the application of caffeine and then
decayed almost as quickly to a plateau value of 96 ± 81 nM, a
value that was relatively sustained. When the application of caffeine
ended, [Ca2+]i fell back to
prestimulation levels; in some cases, [Ca2+]i
even briefly exceeded prestimulation levels, creating an
"undershoot."
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Agonist-evoked contractions are mediated in part by release of
internal Ca2+.
In single PASM and PVSM cells, NE (105 M) evoked a
transient elevation of [Ca2+]i, with a mean
magnitude (301 ± 32 nM; n = 18 dogs) and time course that were similar to those of the caffeine-evoked
responses (Fig. 6A). In
Role of electromechanical coupling mechanisms in contraction of
the pulmonary vasculature, we showed that these responses were
accompanied by contraction (Fig. 1). It is worth pointing out, however,
that although the fluorimetric responses to NE and caffeine appeared
somewhat similar with respect to time course (both rising and falling
within minutes; compare Figs. 5A and 6A), the
mechanical responses to NE were sustained (Fig. 1), which was in stark
contrast to the transient contractions triggered by caffeine (Fig. 5).
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Roles of various kinases in agonist-evoked contractions.
We examined the effects of several protein kinase inhibitors on the
contractile responses evoked by the -adrenergic agonist phenylephrine (PE) and by U-46619; mean dose-response relationships are
given in Fig. 7, and statistical analysis
of the effects of these antagonists on peak contractions is given in
Table 1. Calphostin C (1 µM) and
chelerythrine (1 µM), inhibitors of protein kinase C with differing
mechanisms of action, reduced PE contractions slightly but had no
effect on U-46619-evoked contractions. The tyrosine kinase inhibitor
genistein (100 µM), on the other hand, abolished adrenergic responses
and markedly attenuated responses to U-46619. Daidzen (the less active
analog of genistein; 100 µM) exerted only a fraction of the
inhibitory effect of genistein. To ascertain which MAP kinases might be
mediating the tyrosine kinase-sensitive contractions, we tested the
effects of the p38 kinase inhibitor SB-203580 (25 µM), the MAP kinase
kinase inhibitor PD-98059 (50 µM), and the Rho kinase inhibitor
Y-27632 (10 and 100 µM). The inhibitory effects of genistein on PE
and U-46619 contractions were mimicked by Y-27632, whereas SB-203580
and PD-98059 were largely ineffective.
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DISCUSSION |
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In this study, we sought to characterize the intracellular pathways that underlie E-C coupling in pulmonary arteries and pulmonary veins, particularly those involved in the responses to adrenergic and TxA2 stimulation.
Substantial contractions could be evoked in these tissues by KCl, caffeine, and CPA. These agents act by mobilizing Ca2+ in a G protein-independent fashion, suggesting that elevation of [Ca2+]i alone, as a result of either increased Ca2+ influx (KCl) or release from the sarcoplasmic reticulum (caffeine, CPA), is sufficient for contraction. Despite this, we obtained several lines of evidence that suggest that electromechanical coupling and Ca2+ mobilization are not necessary for mechanical responses to adrenergic or TxA2 stimulation.
First, although NE triggers a Ca2+ response, the contractions produced were at best marginally affected by the blocking of Ca2+ influx with nifedipine or omitting external Ca2+ and were reduced only partially by depletion of the internal Ca2+ store with CPA. This had previously been shown for pulmonary arteries (6) but not for pulmonary veins. Likewise, U-46619 did not evoke a change in [Ca2+]i, and the mechanical response that it evoked was unaffected by nifedipine, Ca2+ removal, or CPA.
Second, there was no correlation between the fluorimetric and mechanical responses evoked by these agonists. Caffeine and NE evoked Ca2+ transients with a similar magnitude and time course, yet the caffeine contractions were very small and transient in the pulmonary arteries but relatively large and transient in the pulmonary veins, whereas the NE contractions in both tissues were large and sustained. Likewise, U-46619 evoked little or no Ca2+ response but did evoke large and sustained contractions. Previous studies comparing U-46619-evoked responses to NE responses in rabbit pulmonary smooth muscle (5) or to carbachol responses in porcine epicardial coronary arteries (1) also found that the adrenergic and cholinergic responses were accompanied by a substantial Ca2+ transient, whereas U-46619 acted without elevating [Ca2+]i.
Third, the membrane depolarization evoked by nerve-released and
exogenously added NE rarely exceeded 40 mV and never exceeded
20
mV; voltage-dependent Ca2+ currents are not discernible in
this voltage range (11).
Collectively, these data indicate that electromechanical coupling and Ca2+ mobilization are sufficient for contraction in general but are not necessary for contractions evoked by these two physiologically relevant agonists.
Instead, the data suggest that NE and the Tx mimetic act primarily through enhanced Ca2+ sensitivity of the contractile apparatus. Other groups have investigated this phenomenon in various smooth muscle preparations and found it to involve protein kinase C or tyrosine kinase-triggered MAP kinases (14). We found that the protein kinase C inhibitors calphostin C and chelerythrine had no effect against U-46619 contractions and reduced NE contractions only partially, whereas genistein abolished the response to both agonists. We went on to study which MAP kinase(s) might be involved, finding adrenergic and U-46619 contractions to be essentially insensitive to specific inhibitors of MAP kinase kinase (PD-98509) or of p38 kinase (SB-203580), whereas the Rho kinase inhibitor Y-27632 reproduced the near total inhibitory effect of genistein. Thus E-C coupling in the pulmonary vasculature appears to be mediated largely by agonist-induced activation of tyrosine kinase, which, in turn, activates Rho kinase; the target of the latter is not clear from these data, but Somlyo and Somlyo (14) found that Rho kinase inhibits MLCP activity, leading to a net increase in myosin light chain phosphorylation and, hence, contraction. The contractions that remain in the presence of these tyrosine and Rho kinase inhibitors may be a product of Ca2+ mobilization.
The interpretation that these agonists act primarily by increasing the Ca2+ sensitivity of the contractile apparatus does not eliminate the importance of Ca2+ mobilization in E-C coupling. That is, suppression of MLCP activity alone will not lead to contraction; there must also be some ongoing level of kinase activity to lead to a net increase in the phosphorylation state of myosin. Thus agonists such as NE act in part by increasing MLCK activity while simultaneously suppressing MLCP activity. This would explain the slower rate of rise of U-46619-evoked contractions compared with those evoked by NE (Fig. 1). Although both act to suppress MLCP activity, NE (but not U-46619) also triggers a substantial Ca2+ transient that would greatly increase MLCK activity.
These findings are important from a clinical viewpoint; knowing that the physiologically relevant agonists tested here act in the pulmonary vascular bed primarily via a mechanism that does not involve Ca2+ mobilization, it should not be surprising that only a small percentage of patients with pulmonary hypertension respond favorably to Ca2+ channel blockers (12). Our data suggest that it would be much more effective to somehow target tyrosine and/or Rho kinases. Delivery via inhalation of candidate therapeutic agents may minimize the potential of undesirable side effects related to affecting such kinases in other tissues. Prostacyclin, which is currently used to treat primary pulmonary hypertension, may act through functional antagonism of the signaling pathway, which we describe in this study.
In this study, as in a previous one by our laboratory (7), we obtained further evidence for important functional differences between PASM and PVSM. For example, these two tissues differ substantially with respect to the mechanical responses evoked by NE, U-46619, and caffeine. Previously, we found a marked difference in their sensitivity to electric field stimulation (which evokes an adrenergic response) and to the nitric oxide donor S-nitroso-N-acetylpenicillamine. In ongoing studies, we have also found that they differ in the manner in which they regulate [Ca2+]i. Collectively, our findings emphasize that conclusions made with the use of pulmonary arteries (which are the tissues usually studied) cannot be extrapolated to apply to pulmonary veins and that a thorough understanding of pulmonary vascular physiology requires further studies with pulmonary veins. Moreover, our finding that PVSM is much more sensitive than PASM to several spasmogens is important from the point of view of edema formation, given that the latter is in part determined by the transcapillary bed pressure gradient.
We conclude that although Ca2+ mobilization (via voltage-dependent Ca2+ influx and/or release of internally sequestered Ca2+) is sufficient to trigger contraction in the pulmonary vasculature, it is not strictly necessary for the mechanical responses evoked by NE and TxA2. Instead, these spasmogens act primarily through tyrosine kinase and Rho kinase, which likely suppress MLCP activity and thereby increase the Ca2+ sensitivity of the contractile apparatus.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the gift of Y-27632 from Dr. A. Yoshimura (Welfide, Osaka, Japan) and the technical assistance of Matt Ostrowski and Kai Mardi, who performed the intracellular microelectrode recordings.
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FOOTNOTES |
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These studies were supported by a grant from the Medical Research Council of Canada, a Career Award (to L. J. Janssen) from the Pharmaceutical Manufacturers Association of Canada, and the Medical Research Council of Canada.
Address for reprint requests and other correspondence: L. J. Janssen, L-314, St. Joseph's Hospital, 40 Charlton Ave. East, Hamilton, Ontario, Canada L8N 4A6 (E-mail: janssenl{at}mcmaster.ca).
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.
Received 22 June 2000; accepted in final form 25 October 2000.
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