Inhibition of CaM kinase II activation and force maintenance by KN-93 in arterial smooth muscle

Aniko Rokolya and Harold A. Singer

Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma - and/or delta -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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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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 delta - and gamma -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.

Frozen tissues were stored in liquid N2, then pulverized in liquid N2-cooled stainless steel vials (Crescent Dental) in the following solution: 50 mM MOPS, 1% Nonidet P-40, 100 mM sodium pyrophosphate, 100 mM NaF, 250 mM NaCl, 3 mM EGTA, 1 mM 1,4-dithiothreitol (DTT), 0.23 U/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2 µg/ml trypsin inhibitor, and 2 µM microcystin LR. Homogenates were thawed on ice and centrifuged at 17,000 g for 15 min at 4°C. Autocamtide 2 kinase activity was assayed in a total volume of 25 µl containing 50 mM HEPES (pH 7.4), 10 mM magnesium-acetate, 0.2 mM [gamma -32P]ATP (specific activity 440 cpm/pmol), 20 µM autocamtide 2, plus 1 mM EGTA (for assay of autonomous, Ca2+/CaM-independent activity) or 600 nM calmodulin and 0.8 mM CaCl2 (for assay of total activity). Assays were carried out at 30°C, started by the addition of lysate (2-4 µg total protein), and stopped at 2 min by spotting aliquots onto P-81 phosphocellulose paper squares. P-81 papers were washed exhaustively in 75 mM phosphoric acid, followed by a wash in ethanol, then dried. Bound radioactivity was counted in a liquid scintillation counter. Protein concentrations were determined using the Lowry method. Total and autonomous CaM kinase II activities were calculated in nanomoles of Pi incorporated in autocamtide 2 per minute per milligram lysate protein. Autonomous activity, a measure of the activation state of CaM kinase II, was reported as a percentage of total activity measured in the same extract. Under these conditions, kinase activity assays were linear up to 7 min and over a range of 0-6 µg total protein/assay. To ensure that the reactions were enzyme limited, the concentration of autocamtide 2 was set at a sixfold excess based on the highest measured kinase activity.

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 [gamma -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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activation in intact carotid artery. Left: early time course of CaM kinase II activation in response to 110 mM KCl depolarization (black-triangle) and 1 µM histamine (open circle ). CaM kinase II was assayed in extracts from contracted strips, using autocamtide 2 as a specific peptide substrate. Autonomous (Ca2+/calmodulin-independent) CaM kinase II activity is expressed as a percentage of total activity. Values are means ± SE with n = 4-10 for each point. Right: autonomous CaM kinase II activities measured at 70 min in resting and KCl- and histamine (His)-stimulated intact arterial strips. Assays and calculations were performed as above. Values are means ± SE of 5-7 samples. Statistically significant increases in autonomous activity over resting values are indicated. * P < 0.05, ** P < 0.01, *** P < 0.001.

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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Fig. 2.   Inhibition of CaM kinase II by KN-93. Medial strips were preincubated with the indicated concentrations of KN-93 for 30 min, then stimulated with 110 mM KCl or 1 µM histamine for 25 s, frozen, and assayed for CaM kinase II activity as described in Fig. 1. Values are means ± SE with n = 3-5 for each point. KN-93 inhibited CaM kinase II activation with an IC50 of ~1 µM. Statistically significant decreases in autonomous activity are indicated. * P < 0.05, ** P < 0.01.



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Fig. 3.   Inhibition of contractile force by KN-93. Strips of swine carotid media were exposed to 0-100 µM KN-93 for 30 min, then stimulated with 110 mM K+-physiological salt solution (PSS; A and C) or 1 µM histamine (B and D) and isometric forces measured over 90 min. A and B show responses from representative experiments where individual strips from same artery were incubated with concentrations of KN-93 as indicated by legend to right. C and D show mean responses from 3-4 independent experiments using 30 µM KN-93.



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Fig. 4.   Effects of KN-93 and its inactive analog, KN-92, on tonic force maintenance. Medial strips from carotid artery were pretreated with 0-100 µM KN-93 (solid symbols) or 1-30 µM KN-92 (open symbols) for 30 min and then stimulated with 110 mM K+-PSS (A) or 1 µM histamine-PSS (B). Force development was measured after 70 additional min. Forces are normalized to a control response generated by addition of 110 mM K+-PSS at optimal length determined prior to experiment for each tissue (METHODS). Data are means ± SE, n = 3.



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Fig. 5.   Relaxation of precontracted arterial strips with KN-93. Medial strips of carotid artery were contracted with 110 mM K+-PSS (triangles) or 1 µM histamine (circles). After 40 min, KN-93 was added to a final concentration of 30 µM. KN-93 produced a slow relaxation of tissues contracted with either stimulus. Open and closed symbols represent KN-93-treated and vehicle-treated control tissues, respectively. Values are means ± SE from 3 independent experiments.

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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Fig. 6.   Effect of KN-93 on myosin light-chain (MLC) phosphorylation. A: KCl depolarization-induced MLC phosphorylation in presence () and absence (open circle ) of 30 µM KN-93. Carotid artery strips were exposed to 30 µM KN-93 or vehicle for 30 min and contracted with 110 mM K+-PSS for up to 70 min. Tissues were frozen and MLC isoelectric variants were quantified as described in METHODS. MLC monophosphorylation levels (MLC-P) are expressed as a percentage of total smooth muscle MLC (unphosphorylated + monophosphorylated). Data are means ± SE of 3-6 individual experiments. KN-93 inhibited initial, transient increase in MLC-P in response to KCl depolarization, but had no significant effect on MLC-P levels during sustained contraction (5-70 min). Statistically significant decreases in MLC-P levels are indicated. * P < 0.05. B: histamine-induced MLC phosphorylation in presence () and absence (open circle ) of 30 µM KN-93. Carotid artery strips were exposed to 30 µM KN-93 or vehicle for 30 min and contracted with 1 µM histamine for up to 70 min. Tissues were frozen and MLC isoelectric variants were quantified as described in METHODS and above. Data are means ± SE of 3-5 individual experiments. KN-93 had no significant effect on histamine-induced MLC phosphorylation either at early time points or during sustained contractions.

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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Fig. 7.   Effect of KN-93 on phorbol ester-induced force development. Carotid artery strips were preincubated with 1-100 µM KN-93 or vehicle for 30 min. Phorbol 12,13-dibutyrate (PDBu) was added directly to bathing solution to achieve a final concentration of 1 µM, and isometric force development was measured. KN-93 did not affect phorbol ester-induced force development. Data depicted in this figure are representative of several qualitatively similar experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 delta - and gamma -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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