Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Ca+/calmodulin-dependent protein kinase II (CaM kinase II) has been implicated in the regulation of smooth muscle contractility. The goals of this study were to determine: 1) to what extent CaM kinase II is activated by contractile stimuli in intact arterial smooth muscle, and 2) the effect of a CaM kinase II inhibitor (KN-93) on CaM kinase II activation, phosphorylation of myosin regulatory light chains (MLC20), and force. Both histamine (1 µM) and KCl depolarization activated CaM kinase II with a time course preceding maximal force development, and suprabasal CaM kinase II activation was sustained during tonic contractions. CaM kinase II activation was inhibited by KN-93 pretreatment (IC50 ~1 µM). KN-93 inhibited histamine-induced tonic force maintenance, whereas early force development and MLC20 phosphorylation responses during the entire time course were unaffected. Both force development and maintenance in response to KCl were inhibited by KN-93. Rapid increases in KCl-induced MLC20 phosphorylation were also inhibited by KN-93, whereas steady-state MLC20 phosphorylation responses were unaffected. In contrast, phorbol 12,13-dibutyrate (PDBu) did not activate CaM kinase II and PDBu-stimulated force development was unaffected by KN-93. Thus KN-93 appears to target a step(s) essential for force maintenance in response to physiological stimuli, suggesting a role for CaM kinase II in regulating tonic contractile responses in arterial smooth muscle. Pharmacological activation of protein kinase C bypasses the KN-93 sensitive step.
smooth muscle contractility; myosin light-chain phosphorylation; calcium/calmodulin-dependent protein kinase II; phorbol esters; protein kinase C
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INTRODUCTION |
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IT IS WIDELY ACCEPTED that the primary regulatory
pathway for smooth muscle contraction is through the Ca2+
and calmodulin-dependent, reversible phosphorylation of the 20,000-Da myosin light chain (MLC20) (19). However, a number of
studies suggest that additional modulatory pathways exist that may
modify the activities of regulatory enzymes and/or the properties of regulatory proteins involved in the development or maintenance of
contractile force. For example, the Ca2+ sensitivity of
MLC20 phosphorylation and force can be altered by the
regulation of myosin light-chain kinase (MLCK) (29, 33) and/or
MLC20 phosphatase activities (13). Also, during tonic force
maintenance, the relationship between force and MLC20
phosphorylation levels can be modified or even dissociated (4, 8, 17). Thus secondary regulatory pathways are likely to be functionally important in the control of smooth muscle contractility.
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II)
is a ubiquitous multifunctional serine/threonine kinase that has been
implicated in the regulation of diverse cellular proteins or functions
including synaptic transmission, gene transcription and cell growth,
ion channels and transporters, and hormone production (3). CaM kinase
II is expressed in vascular smooth muscle as multimers of - and/or
-subunits (24, 28, 36). Whereas much of the kinase activity in
differentiated smooth muscle can be readily extracted and is expected
to be cytosolic (27), a substantial fraction is resistant to extraction
and is associated with a particulate fraction enriched in myofibrils
(11, 25, 27) where several potential substrates, such as
MLC20 (6), MLCK (11, 33), calponin (35), and caldesmon (12,
25) are found.
Phosphorylation of MLCK by CaM kinase II in vitro (11), as well as in cultured tracheal smooth muscle cells (32, 33), has been shown to reduce its sensitivity to calmodulin. Conversely, selective CaM kinase II inhibitors have been shown to increase the Ca2+ sensitivity of MLC20 phosphorylation and potentiate MLC20 phosphorylation in cultured tracheal smooth muscle cells (32, 33). Phosphorylation of MLCK has been documented following activation of intact swine carotid smooth muscle (34). However, the role of CaM kinase II or the physiological consequences of this phosphorylation event were not clear in that study.
Therefore, the first goal of this study was to determine if CaM kinase II was activated in intact arterial smooth muscle in response to physiological contractile stimuli. The second goal was to determine whether CaM kinase II activation could be correlated with modulation of contractile responses. For example, if CaM kinase II is activated and phosphorylates MLCK in response to contractile stimuli, then this should result in a feedback decrease in the Ca2+ sensitivity of MLCK and attenuation of MLC20 phosphorylation and force development. If so, inhibition of CaM kinase II should lead to enhanced force development through an increase in MLC20 phosphorylation. To test this, we used KN-93, a selective CaM kinase II inhibitor that acts through inhibition of calmodulin binding to the kinase (30).
The results of this study indicate that CaM kinase II undergoes rapid activation in arterial smooth muscle, the magnitude of which is dependent on the physiological stimulus applied. A low suprabasal activation of the kinase is maintained during tonic contraction. KN-93 pretreatment inhibited CaM kinase II activation. Unexpectedly, the CaM kinase II inhibitor KN-93 inhibited contractile force maintenance in response to physiological stimuli, whereas phorbol 12,13-dibutyrate (PDBu)-induced contractile responses were unaffected. The inhibitory effect of KN-93 on tonic force maintenance could not be explained by nonspecific inhibition of MLCK activity or by decreased phosphorylation of MLC20. These data are consistent with a predominantly positive role for CaM kinase II in the regulation of tonic force maintenance in response to physiological stimuli in arterial smooth muscle.
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METHODS |
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Tissue preparation. Swine carotid arteries were obtained from a local abattoir and transported to the laboratory in ice-cold physiological salt solution (PSS: 140 mM NaCl, 4.7 mM KCl, 1.2 mM Na2HPO4, 2.0 mM MOPS, pH 7.0, 0.02 mM Na2EDTA, 1.6 mM CaCl2, 1.2 mM MgSO4, 1 g/l dextrose). After removing adventitial and intimal layers, circumferential medial strips (~2 mm wide, 6-9 mm long, ~0.2 mm thick, 1-2 mg dry wt) were dissected.
Force measurement. Tissues were mounted between plastic clips (Harvard Apparatus) in water jacketed tissue baths in oxygenated PSS at 37°C. Force development was recorded isometrically using Grass FT03 force-displacement transducers, Gould model 13-4615-50 transducer amplifiers, and an eight-channel Gould polygraph, or more recently, using Harvard Apparatus isometric force transducers coupled to an eight-channel Biopac computerized data acquisition system with AcqKnowledge 3.5 software. Forces are normalized to the force induced by 110 mM KCl at the optimal length for force development (Lo) for each tissue. After equilibration and determination of Lo, tissues were stimulated and frozen either with liquid N2-cooled tongs (for measurement of CaM kinase II activity), or in a 6% TCA/acetone/dry ice slurry (for the measurement of CaM kinase II autophosphorylation and MLC phosphorylation).
CaM kinase II activity assay.
The basis and specificity of the assay for CaM kinase II have been
previously documented (1, 10). Activation of the kinase is inferred
through an increase in the tissue extracts of
Ca2+/calmodulin-independent (or "autonomous")
activity assayed with a specific peptide substrate (autocamtide 2;
KKALRRQETVDAL) of CaM kinase II. This increase in autonomous activity
is dependent on a specific autophosphorylation event (threonine-287 in
- and
-subunits) that occurs between Ca2+/calmodulin
(Ca2+/CaM)-activated subunits within the multimeric
holoenzyme (3, 23) and is preserved during extraction of the kinase
from intact tissues by inclusion of phosphatase inhibitors.
Myosin light-chain phosphorylation measurements. Frozen tissues were thawed at room temperature, air-dried, and then homogenized on ice in glass-to-glass homogenizers (Radnoti, Monrovia, CA) in a buffer consisting of 10% glycerol, 1% SDS and 1 mM DTT at 6 mg dry wt/ml. Homogenates were subjected to isoelectric focusing (pH 4.5-5.4), followed by SDS-PAGE in the second dimension (26). Gels were stained with Coomassie brilliant blue R-250 and analyzed using a Molecular Dynamics laser densitometer. Data are expressed as a ratio of phosphorylated MLC over total (phosphorylated plus unphosphorylated) MLC (only smooth muscle specific light-chain isoforms were evaluated).
Chemicals.
Microcystin LR, KN-92, KN-93, and KN-62 were purchased from Calbiochem
(La Jolla, CA), or from Seikagaku America (Rockville, MD), PDBu from
Alexis (San Diego, CA), and [-32P]adenosine
trisphosphate from Amersham (Arlington Heights, IL). All other
chemicals were of reagent grade and were obtained from standard
suppliers. KN-93 was dissolved in deionized H2O (Seikagaku) or in DMSO (Calbiochem). Microcystin LR, KN-92, KN-62, and PDBu were
dissolved in DMSO. Stock solutions were diluted into PSS so that the concentration of DMSO did not exceed 0.3% (with the exception of KN-62, where to achieve a 30 µM concentration in the
working salt solutions, as much as 10% DMSO was required), and control
specimens were incubated with identical concentrations of the vehicle.
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RESULTS |
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Ca2+/calmodulin-independent or autonomous CaM kinase II
activity depends on the magnitude of autophosphorylation (on
threonine-286) in response to activation by
Ca2+/calmodulin. Autonomous activity can be preserved and
measured in cell or tissue extracts containing phosphatase inhibitors, providing an index of CaM kinase II activation (1, 10). As shown in the
left panel of Fig. 1,
autonomous CaM kinase II activity, expressed as a percentage of total
CaM kinase II activity, was low (3.7 ± 0.9%, n = 15) in
unstimulated arterial strips, consistent with low levels of free
intracellular Ca2+ in the resting muscles. Physiological
contractile stimuli (1 µM histamine and membrane depolarization with
110 mM KCl) triggered rapid increases in autonomous CaM kinase II
activity in tissue extracts indicative of CaM kinase II activation in
situ. The early time course and magnitude of CaM kinase II activation
was stimulus specific. Depolarization-induced autonomous CaM kinase II
activity levels were only slightly elevated after 10 s and peaked at 20 s, whereas histamine-induced autonomous CaM kinase II activity was
maximal by 10 s. Autonomous activity stimulated by 110 mM KCl rarely
exceeded 30% of total activity (28.8 ± 7.1%), whereas autonomous
activity generated in response to histamine averaged 73.7 ± 13.4% of
the total activity. CaM kinase II activation in response to either
stimulus declined to suprabasal levels by 60 s (26.6 ± 7.9% and 24.7 ± 5.2% for histamine and KCl stimulation, respectively) and remained
significantly elevated even after 70 min during tonic contractions
(Fig. 1, right).
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To infer whether CaM kinase II was involved in regulating contractile
activity, we tested the effect of KN-93, a selective CaM kinase II
inhibitor in vitro (2, 30), on activation of CaM kinase II and force
development in the intact tissues. Treatment of tissues with KN-93
inhibited KCl- and histamine-induced CaM kinase II activation and
generation of autonomous activity with an IC50 of ~1 µM
(Fig. 2), comparable with IC50
values reported using purified enzymes (30). These results are similar
to the effects of KN-93 on CaM kinase II activation in cultured
vascular smooth muscle cells (1). Preincubation for 30 min with
1-100 µM KN-93 inhibited KCl depolarization-induced force
development and maintenance in a dose-dependent manner (Fig.
3, A and C). Early force
development was significantly attenuated and tonic force maintenance
(after 70 min) was completely inhibited in the presence of 30 µM
KN-93 (Fig. 3, A and C). In contrast, the early phase
of histamine-induced force development was virtually unaffected by as
much as 30 µM KN-93 (Fig. 3, B and D), whereas again
tonic force maintenance after 70 min was nearly eliminated. The
inhibitory effect of KN-93 on tonic force could not be readily reversed
by repeated washes of either KCl- or histamine-stimulated tissues, although phasic force responses could still be elicited (data not
shown). The IC50 for KN-93 inhibition of tonic KCl-induced (Fig. 4A) or histamine-induced
(Fig. 4B) tonic force response was ~3-5 µM. Addition
of 30 µM KN-93 after maximal force had developed in response to
either KCl depolarization or histamine also resulted in a slow
relaxation of the tissues with similar kinetics for each stimulus (Fig.
5).
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In control experiments, preincubation with KN-92, which is an inactive analog of KN-93 with respect to inhibition of CaM kinase II activation (2, 30), had only a weak inhibitory effect on tonic KCl- or histamine-induced force maintenance over the same range of KN-93 concentrations that maximally relaxed the tissues (Fig. 4). Adding it to precontracted tissues also had no effect (not shown). A second CaM kinase II inhibitor, KN-62 (33), was evaluated in this system. However, addition of as much as 30 µM KN-62 had no significant inhibitory effect on either contractile stimulus-induced CaM kinase II activation or force development (not shown). Concentrations of KN-62 in excess of 30 µM in PSS resulted in obvious precipitation unless the concentration of the vehicle, DMSO, was raised to ~6-10%, a concentration of DMSO that is in itself deleterious to these tissues. It appears that the lack of a significant effect of KN-62 on CaM kinase II is due to poor solubility and/or permeability in these relatively thick arterial preparations.
Because it is well established that MLC20 phosphorylation
is required for and is the primary determinant of force development in
smooth muscle in response to physiological stimuli, we evaluated the
effects of KN-93 on this parameter. Figure
6A shows that 30 µM KN-93
inhibited peak MLC20 phosphorylation at 1 min of KCl depolarization (50.7 ± 7.8% control vs. 21.3 ± 6.0% in the
presence of KN-93), consistent with its inhibitory effect on rapid
force development with this stimulus (Fig. 3, A and C).
However, by 5 min there was no significant difference between
MLC20 phosphorylation values from control and KN-93-treated
tissues (21.6 ± 4.4% vs. 22.4 ± 2.0%) while force was inhibited
by ~70% at this time (Fig. 3C). MLC20
phosphorylation values during tonic KCl-induced contractions (70 min)
were unaffected by 30 µM KN-93. In histamine-stimulated tissues, 30 µM KN-93 had no inhibitory effect on MLC20
phosphorylation at early or late time points (Fig. 6B).
Overall, these data indicate that the inhibition of tonic force
maintenance in either KCl-depolarized or histamine-stimulated tissues
by KN-93 cannot be simply explained by nonspecific inhibition of MLCK
catalyzed MLC20 phosphorylation.
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Phorbol ester activators of protein kinase C stimulate contraction in
carotid arterial smooth muscle by an unknown mechanism that appears to
be largely or totally independent of MLC20 phosphorylation (7, 26) on those residues (serine-19 or threonine-18) whose phosphorylation is otherwise considered both necessary and sufficient for actin-activated myosin ATPase (reviewed in Ref. 19). Interestingly, force development or maintenance in response to 1 µM PDBu was unaffected by pretreatment with up to 100 µM KN-93 (Fig.
7), concentrations of KN-93 that completely
inhibited tonic force in response to the physiological activators. This
observation confirms the unique properties of a phorbol ester-induced
contractile response and suggests that pharmacological activation of
protein kinase C can circumvent the requirement for physiological
activation of CaM kinase II during activation of arterial smooth
muscle.
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DISCUSSION |
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Our current understanding of CaM kinase II function in smooth muscle is limited and mostly based on data from smooth muscle cell culture models where it has been linked to regulation of cell motility (21), modulation of MLCK activity (32, 33), and activation of the mitogen-activated protein kinase signaling pathway (2, 20). In the present study, we have documented CaM kinase II activation in an intact smooth muscle preparation in response to prototypical contractile stimuli, and based on a pharmacological approach, provided evidence that CaM kinase II may be involved in regulating smooth muscle contractility.
The assay for in situ CaM kinase II activation used in this study
measures accumulation of the Ca2+-independent or autonomous
form of CaM kinase II that results from autophosphorylation of a
threonine residue conserved in all CaM kinase II subunits
(threonine-287 in CaM kinase II - and
-subunits) (3). Although
this autophosphorylation event is not required for
Ca2+/calmodulin-dependent activity of the kinases (10, 23),
it provides the kinase with unique functional properties that may be of
physiological importance. For example, calmodulin binding affinity is
increased by almost three orders of magnitude in the autophosphorylated
kinase (18). These autoregulatory properties may result in a
prolongation of kinase activity following a transient Ca2+
signal and provide the kinase with the property of responding to
Ca2+ transients in a frequency-dependent manner (5).
Irrespective of the functional importance of the autophosphorylated
form of the kinase, its appearance in a tissue provides an index of the state of CaM kinase II activation because the autophosphorylation only
occurs between Ca2+/calmodulin-bound (activated) subunits
within the holoenzyme (10, 23). However, there are at least two factors
that impinge on the fidelity of the assay from the standpoint of
estimating Ca2+/calmodulin-dependent CaM kinase II
activation in situ. One is that the amount of autonomous activity
measured in the assay reflects net autophosphorylation on threonine-287
and is therefore also dependent on in situ phosphatase activities. The
other factor is that the autophosphorylation reaction requires at least
two activated subunits per holoenzyme and it is cooperative (1, 23).
Thus the assay would be predicted to underestimate the extent of
Ca2+/calmodulin-dependent CaM kinase II activation in situ,
particularly at submaximal [Ca2+]i
levels and/or when phosphatase activities are high. The low levels of
autonomous activity that were measured during tonic contraction in
response to either histamine or KCl are consistent with a predicted
low, but suprabasal, free intracellular [Ca2+]
and these assay limitations.
Although smooth muscle CaM kinase II has a lower affinity for calmodulin (~10 nM) (27; unpublished observations) compared with MLCK (~1 nM) (29), our data from cultured vascular smooth muscle cells (1) and intact arterial tissues (this study) strongly argue that, under physiological conditions, neither [Ca2+]i nor free calmodulin was a limiting factor in the activation of the kinase. The observed histamine-stimulated increase in autonomous activity, which reached 73% of the total CaM kinase II activity within 10 s, is one of the highest values of autonomous CaM kinase II reported in any intact cell or tissue. Because autonomous CaM kinase II activity rarely exceeds 80% of total activity in vitro, even under optimal autophosphorylation conditions (28), it can be inferred that 1 µM histamine results in a nearly complete activation of CaM kinase II in the intact tissues.
Based on previous reports using this tissue and Ca2+ indicators to determine concentrations of free intracellular Ca2+, 1 µM histamine and 110 mM KCl would have been predicted to stimulate comparable increases in [Ca2+] (9). In fact, the kinetics of MLC20 phosphorylation and force generation in response to these stimuli were found in the present study to be similar, consistent with this prediction. In contrast to these similarities, KCl depolarization resulted in a significantly slower and smaller increase in autonomous CaM kinase II activity compared with 1 µM histamine. Recent developments in our understanding of the spatial distribution of [Ca2+]i (22), as well as data suggesting that calmodulin is compartmentalized and liberated during stimulation (15, 32), may help explain the high level of CaM kinase II activation we observed in response to histamine in arterial smooth muscle. It appears that in both the carotid artery and cultured vascular smooth muscle cells (1), agents that release Ca2+ from internal stores very efficiently activate CaM kinase II, consistent with a kinase pool located in the proximity of the sarco/endoplasmic reticulum where locally high concentrations of free Ca2+ would be expected.
In previous studies reporting effects of the CaM kinase II inhibitor, KN-62, in cultured tracheal smooth muscle cells, it was concluded that CaM kinase II phosphorylation of MLCK decreases MLCK sensitivity to Ca2+/calmodulin and attenuates stimulus-induced MLC20 phosphorylation responses (32, 33). In the present study, we reasoned that if this mechanism was functionally significant in intact carotid artery, then inhibition of CaM kinase II with KN-93, a more soluble analog of KN-62, should potentiate MLC20 phosphorylation and force development in response to contractile stimuli. In contrast to the prediction, there was no potentiation of MLC20 phosphorylation or force by KN-93. This result, coupled with the lack of correlation between CaM kinase II activation levels and parameters of MLC20 phosphorylation and force in histamine- and KCl-stimulated tissues, suggests that CaM kinase II is not an important regulator of force development in intact carotid arterial smooth muscle. Our results are also consistent with the previously observed lack of correlation between MLCK phosphorylation levels and net MLC20 phosphorylation and force in the same tissue (34).
The predominant effect of KN-93 on contractile force responses in carotid artery is during sustained force maintenance and the effect is inhibitory, suggesting a positive role for CaM kinase II during this period. The fact that addition of KN-93 to tonically contracted tissues results in relaxation (Fig. 5) indicates that the early phase of CaM kinase II activation is not related to its apparent role during force maintenance. We considered the possibility that the effects of KN-93 were due to nonspecific inhibition of MLCK activity, either directly by inhibiting Ca2+/calmodulin binding or indirectly by interfering with availability of free intracellular Ca2+. The major arguments against this are: 1) KN-93 is not an effective inhibitor of smooth muscle MLCK in vitro (30), 2) KN-93 potencies for inhibiting histamine- and KCl-stimulated increases in autonomous CaM kinase II activity and tonic force maintenance are similar, and 3) neither the transient increases in histamine-stimulated force and MLC20 phosphorylation nor sustained MLC20 phosphorylation in response to KCl or histamine were inhibited by KN-93. Because KN-93 had no effect on PDBu-induced contractile responses, a similar argument can be made that it did not nonspecifically inhibit protein kinase C. However, in the case of KCl-depolarization, KN-93 did attenuate early increases in MLC20 phosphorylation and force. These data could be interpreted to indicate that KN-93 has a nonspecific inhibitory effect on Ca2+ entry through plasmalemmal Ca2+ channels, or, alternatively, that KN-93 is acting specifically on Ca2+ entry through such channels. There are reports in the literature that indicate a role for CaM kinase II in mediating Ca2+-dependent facilitation of Ca2+ entry through voltage-dependent Ca2+ channels in smooth muscle (16) and in heart (14).
Although we do not yet fully understand the underlying mechanism(s), the data reported here indicate a KN-93-induced dissociation of MLC20 phosphorylation and force in tonically contracted arterial smooth muscle. The results are qualitatively similar to other studies that also demonstrated inhibition of force with sustained suprabasal levels of MLC20 phosphorylation in smooth muscle following addition of okadaic acid (31), nitroprusside (17), high Mg2+ concentrations (4), or by a manipulation of extracellular Ca2+ concentrations during agonist stimulation (8). In a general sense, the maintenance of disproportionately high MLC20 phosphorylation levels in the face of decreasing force is consistent with the idea that tonic maintenance of smooth muscle force, i.e., the "latch" state (19), involves regulatory mechanisms in addition to those affecting MLC20 phosphorylation.
Assuming that KN-93 specifically inhibits CaM kinase II, our data are consistent with the existence of an unidentified CaM kinase II-dependent phosphorylation event that is required for tonic force maintenance in arterial smooth muscle. If this model is correct, then regulation of both force development through MLCK activation and force maintenance through CaM kinase II would be primarily dependent on intracellular free [Ca2+]. Based on the PDBu-induced contractile responses that are both MLC20 phosphorylation independent (7, 26) and KN-93 insensitive, it appears that pharmacological activation of protein kinase C can circumvent the physiological requirements for MLCK and CaM kinase II activation. To the extent that protein kinase C is activated physiologically, it could function in parallel with or in place of CaM kinase II to support force maintenance. It is reasonable to speculate that both CaM kinase II and protein kinase C, as multifunctional protein kinases, could share a common substrate or set of substrates. Potentially relevant substrates for the kinases from the standpoint of regulating smooth muscle contraction include thin- filament-associated regulatory proteins such as caldesmon or calponin, proteins required for transmission of force through actomyosin/cytoskeleton attachment sites, or a downstream mediator or signaling molecule that ultimately impinges on such proteins.
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ACKNOWLEDGEMENTS |
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We thank Clark Brothers (Shamokin, PA), Economy Locker Storage (Pennsdale, PA), and Hilltown Pork (Canaan, NY) for providing swine carotid arteries. We are indebted to David Cooney and Holly Benscoter for help in procuring arteries and for advice on the measurement of CaM kinase II activity, respectively. Drs. Dee A. Van Riper, S. Thomas Abraham, and Charles M. Schworer are acknowledged for helpful discussions.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-49426 (H. A. Singer) and by the Henry Hood, M. D. Research Program at the Weis Center for Research.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Rokolya, Center for Cardiovascular Sciences, M/C 8, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: rokolya{at}mail.amc.edu).
Received 2 September 1999; accepted in final form 30 September 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abraham, S. T.,
H. A. Benscoter,
C. M. Schworer,
and
H. A. Singer.
In situ Ca2+ dependence for activation of Ca2+/calmodulin-dependent protein kinase II in vascular smooth muscle.
J. Biol. Chem.
271:
2506-2513,
1996
2.
Abraham, S. T.,
H. A. Benscoter,
C. M. Schworer,
and
H. A. Singer.
A role for Ca2+/calmodulin-dependent protein kinase II in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells.
Circ. Res.
81:
575-584,
1997
3.
Braun, A. P.,
and
H. Schulman.
The multifunctional calcium/calmodulin-dependent protein kinase: from form to function.
Annu. Rev. Physiol.
57:
417-445,
1995[ISI][Medline].
4.
D'Angelo, E. K.,
H. A. Singer,
and
C. M. Rembold.
Magnesium relaxes arterial smooth muscle by decreasing intracellular Ca2+ without changing intracellular Mg2+.
J. Clin. Invest.
89:
1988-1994,
1992[ISI][Medline].
5.
De Koninck, P.,
and
H. Schulman.
Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations.
Science
279:
227-230,
1998
6.
Edelman, A. M.,
W. H. Lin,
D. J. Osterhout,
M. K. Bennett,
M. B. Kennedy,
and
E. G. Krebs.
Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase.
Mol. Cell. Biochem.
97:
87-98,
1990[ISI][Medline].
7.
Fulginiti, J., III,
H. A. Singer,
and
R. S. Moreland.
Phorbol-ester-induced contractions of swine carotid artery are supported by slowly cycling crossbridges which are not dependent on calcium or myosin light chain phosphorylation.
J. Vasc. Res.
30:
315-322,
1993[ISI][Medline].
8.
Gerthoffer, W. T.
Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle.
J. Pharmacol. Exp. Ther.
240:
8-15,
1987[Abstract].
9.
Gilbert, E. K.,
B. A. Weaver,
and
C. M. Rembold.
Depolarization decreases the [Ca2+]i sensitivity of myosin light-chain kinase in arterial smooth muscle: comparison of aequorin and fura-2 [Ca2+]i estimates.
FASEB J.
5:
2593-2599,
1991
10.
Hanson, P. I.,
M. S. Kapiloff,
L. L. Lou,
M. G. Rosenfeld,
and
H. Schulman.
Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation.
Neuron
3:
59-70,
1989[ISI][Medline].
11.
Ikebe, M.,
and
S. Reardon.
Phosphorylation of smooth muscle myosin light chain kinase by smooth muscle Ca2+/calmodulin-dependent multifunctional protein kinase.
J. Biol. Chem.
265:
8975-8978,
1990
12.
Ikebe, M.,
S. Reardon,
G. C. Scott-Woo,
Z. Zhou,
and
Y. Koda.
Purification and characterization calmodulin-dependent protein kinase from smooth muscle: isolation of caldesmon kinase.
Biochemistry
29:
11242-11248,
1990[ISI][Medline].
13.
Lee, M. R.,
L. Li,
and
T. Kitazawa.
Cyclic GMP causes Ca2+ desensitization in vascular smooth muscle by activating the myosin light chain phosphatase.
J. Biol. Chem.
272:
5063-5068,
1997
14.
Li, L.,
H. Satoh,
K. S. Ginsburg,
and
D. M. Bers.
The effect of Ca2+-calmodulin-dependent protein kinase II on cardiac excitation-contraction coupling in ferret ventricular myocytes.
J. Physiol. (Lond.)
501:
17-31,
1997[Abstract].
15.
Luby-Phelps, K.,
M. Hori,
J. M. Phelps,
and
D. Won.
Ca2+-regulated dynamic compartmentalization of calmodulin in living smooth muscle cells.
J. Biol. Chem.
270:
21532-21538,
1995
16.
McCarron, J. G.,
J. G. McGeown,
S. Reardon,
M. Ikebe,
F. S. Fay,
and
J. V. Walsh.
Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II.
Nature
357:
74-77,
1992[ISI][Medline].
17.
McDaniel, N. L.,
X. L. Chen,
H. A. Singer,
R. A. Murphy,
and
C. M. Rembold.
Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i and uncoupling stress from myosin phosphorylation.
Am. J. Physiol. Cell Physiol.
263:
C461-C467,
1992
18.
Meyer, T.,
P. I. Hanson,
L. Stryer,
and
H. Schulman.
Calmodulin trapping by calcium-calmodulin-dependent protein kinase.
Science
256:
1199-1202,
1992[ISI][Medline].
19.
Murphy, R. A.
What is special about smooth muscle? The significance of covalent crossbridge regulation.
FASEB J.
8:
311-318,
1994
20.
Muthalif, M. M.,
I. F. Benter,
M. R. Uddin,
and
K. U. Malik.
Calcium/calmodulin-dependent protein kinase II mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells.
J. Biol. Chem.
271:
30149-30157,
1996
21.
Pauly, R. R.,
C. Bilato,
S. J. Sollott,
R. Monticone,
P. T. Kelly,
E. G. Lakatta,
and
M. T. Crow.
Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration.
Circulation
91:
1107-1115,
1995
22.
Rembold, C. M.,
D. A. Van Riper,
and
X. L. Chen.
Focal [Ca2+]i increases detected by aequorin but not by fura-2 in histamine- and caffeine-stimulated swine carotid artery.
J. Physiol. (Lond.).
488:
549-564,
1995[Abstract].
23.
Rich, R. C.,
and
H. Schulman.
Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.
J. Biol. Chem.
273:
28424-28429,
1998
24.
Schworer, C. M.,
L. I. Rothblum,
T. J. Thekkumkara,
and
H. A. Singer.
Identification of novel isoforms of the -subunit of Ca2+/calmodulin-dependent protein kinase II. Differential expression in rat brain and aorta.
J. Biol. Chem.
268:
14443-14449,
1993
25.
Scott-Woo, G. C.,
C. Sutherland,
and
M. P. Walsh.
Kinase activity associated with caldesmon is Ca2+/calmodulin-dependent kinase II.
Biochem. J.
268:
367-370,
1990[ISI][Medline].
26.
Singer, H. A.
Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle.
Am. J. Physiol. Cell Physiol.
259:
C631-C639,
1990
27.
Singer, H. A.,
S. T. Abraham,
and
C. M. Schworer.
Calcium/calmodulin-dependent protein kinase II.
In: Biochemistry of Smooth Muscle Contraction, edited by M. Barany. San Diego: Academic, 1996, p. 143-153.
28.
Singer, H. A.,
H. A. Benscoter,
and
C. M. Schworer.
Novel Ca2+/calmodulin-dependent protein kinase II -subunit variants expressed in vascular smooth muscle, brain, and cardiomyocytes.
J. Biol. Chem.
272:
9393-9400,
1997
29.
Stull, J. T.,
L. C. Hsu,
M. G. Tansey,
and
K. E. Kamm.
Myosin light chain kinase phosphorylation in tracheal smooth muscle.
J. Biol. Chem.
265:
16683-16690,
1990
30.
Sumi, M.,
K. Kiuchi,
T. Ishikawa,
A. Ishii,
M. Hagiwara,
T. Nagatsu,
and
H. Hidaka.
The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells.
Biochem. Biophys. Res. Commun.
181:
968-975,
1991[ISI][Medline].
31.
Tansey, M. G.,
M. Hori,
H. Karaki,
K. E. Kamm,
and
J. T. Stull.
Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle.
FEBS Lett.
270:
210-221,
1990.
32.
Tansey, M. G.,
K. Luby-Phelps,
K. E. Kamm,
and
J. T. Stull.
Ca2+-dependent phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain phosphorylation within smooth muscle cells.
J. Biol. Chem.
269:
9912-9920,
1994
33.
Tansey, M. G.,
R. A. Word,
H. Hidaka,
H. A. Singer,
C. M. Schworer,
K. E. Kamm,
and
J. T. Stull.
Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.
J. Biol. Chem.
267:
12511-12516,
1992
34.
Van Riper, D. A.,
B. A. Weaver,
J. T. Stull,
and
C. M. Rembold.
Myosin light chain kinase phosphorylation in swine carotid artery contraction and relaxation.
Am. J. Physiol. Heart Circ. Physiol.
268:
H2466-H2475,
1995
35.
Winder, S. J.,
and
M. P. Walsh.
Calponin: thin filament-based regulation of smooth muscle contraction.
Cell. Signal.
5:
677-686,
1993[ISI][Medline].
36.
Zhuo, Z. L.,
and
M. Ikebe.
New isoforms of Ca2+/calmodulin-dependent protein kinase II in smooth muscle.
Biochem. J.
299:
489-495,
1994[ISI][Medline].