Postnatal development alters functional compartmentalization of myosin light chain kinase in ovine carotid arteries

Abstract

The rate-limiting enzyme for vascular contraction, myosin light chain kinase (MLCK), phosphorylates regulatory myosin light chain (MLC₂₀) at rates that appear faster despite lower MLCK abundance in fetal compared with adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC₂₀. Under optimal conditions, common carotid artery homogenates from nonpregnant adult female sheep and near-term fetuses exhibited similar values of V_max and K_m for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 s, calculation of in situ rates of MLC₂₀ phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC₂₀. The in situ rate of MLC₂₀ phosphorylation divided by total MLCK abundance averaged to values 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded, respectively, to 43 ± 10% and 31 ± 3% of the V_max values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC₂₀, and that MLCK activation was greater in periluminal than periadventitial regions over the time course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.

INTRODUCTION

The transition from fetal to postnatal life involves numerous major changes in vascular structure and reactivity. These include altered patterns of sarcolemmal receptor expression and calcium handling that continuously modulate pharmacomechanical coupling throughout fetal and postnatal maturation (1–3). An important component of these maturational adjustments is a gradual postnatal decrease in the magnitude of agonist-induced myofilament calcium sensitization (4–6) resulting from altered relations between cytosolic calcium concentration and the extent of myosin light chain phosphorylation, defined as thick filament reactivity (7, 8). In turn, this altered thick filament reactivity is attributable in large part to enhanced rates of myosin phosphorylation, in situ, despite a lower abundance of myosin light chain kinase (MLCK) in fetal relative to adult arteries (8).

Although multiple previous studies have documented that MLCK abundance and activity are often tightly correlated (9–12), other studies of fetal and postnatal development have demonstrated the opposite correlation; in immature arteries, the MLCK abundance is low but 20-kDa myosin light chain (MLC₂₀) phosphorylation rates, in situ, are high (13, 14). Our previous studies agree with this latter finding, and further suggest that it is not attributable to age-related differences in MLC₂₀ concentration (15). Other potential mechanisms for age-related differences in observed MLC₂₀ phosphorylation rates can be divided into two major categories. First, MLCK is activated by calcium calmodulin (Ca²⁺-CaM), so age-related differences in cytosolic calcium or calmodulin (CaM) concentrations and/or availability could be involved. Correspondingly, abundant evidence demonstrates that agonist-induced increases in cytosolic calcium vary considerably with fetal and postnatal age (5, 8, 16). Paradoxically, the MLC₂₀ phosphorylation rates, in situ, are greater in fetal arteries, which also have reduced rates and magnitudes of calcium mobilization (2, 5). Similarly, published evidence argues against the possibility that CaM is reduced in mature arteries (15, 17). Among the remaining alternatives, elevated abundances of endogenous inhibitors of MLCK activity, such as polyamines (18) or telokin (19), might explain reduced rates of MLC₂₀ phosphorylation in adult arteries. Finally, age-dependent differences in posttranslational modifications of MLCK resulting in altered catalytic activity remain possible; studies with protein kinase A (PKA), protein kinase C (PKC), and calcium-calmodulin kinase II (CaM-Kinase II) have demonstrated that each of these kinases can attenuate MLCK catalytic activity (20).

An important but untested explanation of why the catalytic activity of MLCK appears greater in fetal than adult arteries, arises from possible differences in contractile protein organization. A variety of recent studies demonstrate that contractile protein abundance and colocalization are independently regulated (21), and more importantly, that contractility is better correlated with the extent of colocalization among contractile proteins, than with their abundances (22–24). A critical characteristic of most contractile proteins, including MLCK, is that local cellular concentrations and resulting activities follow a heterogeneous distribution within and between cells (25–28). Our previous studies support this later notion that the expression of contractile proteins, such as MLCK and MLC₂₀, follows a gradient from the lumen to the adventitia in an age-dependent manner (29). In addition, the distribution of MLCK is known to be highly dynamic (30–34), especially during smooth muscle contraction (35). From this perspective, it is possible that contractile proteins, and in particular MLCK and MLC₂₀, are colocalized and distributed differently in fetal and adult arteries, which in turn may help to explain the greater apparent catalytic activity of MLCK in fetal arteries. The present study was designed to explore this hypothesis.

MATERIALS AND METHODS

The Loma Linda University Institutional Animal Care and Use Committee approved all techniques, protocols, and experimental procedures used in these studies. All procedures adhered to the policies and practices outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

General Preparation

These experiments utilized common carotid artery segments harvested from nonpregnant adult female sheep (18–24 mo old) and near-term (∼140 days gestation) fetuses. Full-term pregnant sheep (139–142 days gestation) anesthetized with 30 mg/kg pentobarbital underwent intubation then maintenance on 1.5%–2.0% isoflurane gas. Surgical removal through a midline incision provided access to each fetus, followed by cardiac excision and subsequent harvest of the fetal carotid arteries. Intravenous administration of 100 mg/kg pentobarbital to nonpregnant adult sheep produced a rapid euthanasia, with subsequent harvest of the adult carotid arteries. Incubation of all freshly harvested carotid arteries in cold Krebs buffer (in mM: 122 NaCl, 25.6 NaHCO₃, 5.17 KCl, 2.56 dextrose, 2.49 MgSO₄, 1.60 CaCl₂, and 0.027 EGTA) helped maintain artery viability. Artery preparation included removal of loose extracellular and adventitial tissues and endothelium denudation, as previously described (15, 36). Arteries not designated for immediate use in contractility experiments underwent freezing in liquid nitrogen and storage at −80°C.

Preparation of Carotid Artery Homogenates

The ice-cold buffer used to homogenize frozen carotid arteries included 20 mM imidazole, 1 mM cysteine, 60 mM KCl, 1 mM MgCl₂, 10 mM sodium azide, 0.25 mM PMSF, 1 mM DTT, 1% phosphatase inhibitor cocktail (Sigma-Aldrich, P2850), and 0.5% protease inhibitor cocktail (Sigma-Aldrich, P8340), at pH 7.5, as previously described (15). After tissue homogenization for 15 min at 4°C using a motor-driven glass-on-glass mortar and pestle, sample centrifugation at 6,000 g for 1 min produced an aliquot of supernatant used for protein determination. Final adjustments in protein concentration ensured similar amounts of MLCK in each homogenate. The utilization of purified chicken gizzard MLCK as absolute standards on each gel during electrophoresis allowed standardization of MLCK concentrations across all homogenates during Western blot analysis. Daily preparation of fresh homogenates ensured consistent measurements of enzyme activity.

Determination of MLCK and MLC₂₀ Abundances in Artery Homogenates

Western immunoblots involved addition of 15 µg of total protein from each artery homogenate to separate lanes of 8% SDS-PAGE gels, followed by electrophoresis then transfer to nitrocellulose membranes. Membranes underwent overnight blockade of nonspecific staining at 4°C with 5% nonfat dry milk in 20 mM Tris·HCl at pH 7.5, with 150 mM NaCl and 0.1% Tween-20, and then incubation with anti-MLCK primary antibody (Sigma-Aldrich, M7905) at 1:10,000 dilution or with anti-MLC₂₀ antibody (Sigma-Aldrich, M4401) at 1:300 dilution. Incubations with horseradish peroxidase (HRP)-conjugated goat antimouse secondary antibodies (Thermo Fisher Scientific, No. 31430 at 1:50,000 with Pierce Supersignal West Dura substrate no. 34076) allowed detection and visualization of primary antibody-antigen complexes through utilization of chemiluminescence direct photon capture with a charge-coupled device (CCD) camera (AlphaInnotech, ChemiImager). Each membrane carried lanes with serial dilutions of known masses of either purified chicken gizzard MLCK or MLC₂₀ or an ovine reference homogenate containing the detected protein to construct standard curves. Plots of standard intensities against their known masses allowed determination of the best-fit coefficients of the logistic equation, and rearrangement of this equation into its inverse form allowed direct calculation of the mass of target protein in each sample based on the band intensity it produced, as previously described (15, 22). Although all immunoblots were designed to produce bands that fell in the linear central portion of the standard curves, the practice of constructing standard curves using known masses of authentic standards to directly quantify protein abundances, largely eliminated variability due to slight run-to-run differences in transfer efficiency, antibody avidity, the efficiency of photon detection, and nonlinearities in the relations between mass and optical density.

Determination of Optimum Calcium and Calmodulin Concentrations for Activating MLCK

To retard the reaction rates enough to enable accurate measurements of product (phosphorylated MLC₂₀; pMLC₂₀) accumulation and also negate the influence of endogenous substrate concentration, the enzyme velocity assays involved dilution of all artery homogenates by 400-fold with subsequent adjustments as necessary to achieve 200 ng of MLCK in each assay tube at a final concentration of ≈33 nM. Additions of exogenous purified MLC₂₀ to the dilute homogenates raised MLC₂₀ concentration to 1.5 µM. All homogenates also contained Phosphatase Inhibitor Cocktail (Sigma-Aldrich, P2850) at 10 µL/mL, the concentration identified as optimum in our previous studies (15). After incubation of fetal and adult artery homogenates with 0, 0.3, 0.6, or 1 µM CaM for 20 min at 4°C and then for 10 min at 37°C, the reactions began by the addition of calcium at 0, 0.3, 1, 3, or 10 mM with 1 mM ATP. Addition of ice-cold 10 mM EDTA followed by transfer of the samples to dry ice, allowed precise termination of the reaction at exactly 0, 1, 2, and 3 s. Sample analysis was achieved via resolution on using 10% urea glycerol gels followed by transfer to nitrocellulose membranes and incubation with primary anti-MLC₂₀ antibody (MY-21, Sigma-Aldrich, M4401), as previously described (7). Incubations with goat antimouse HRP-conjugated secondary antibodies (Thermo Fisher Scientific, No. 31430 at 1:50,000 with Pierce Supersignal West Dura substrate no. 34076) enabled the use of a ChemiImager (AlphaInnotech) to detect and determine integrated optical density values of both phosphorylated and unphosphorylated MLC₂₀ bands. Inclusion of multiple known masses of purified MLC₂₀ on each gel allowed generation of a standard curve and determination of masses of phosphorylated and unphosphorylated MLC₂₀. Again, the use of standard curves constructed from known masses of authentic standards run side-by-side with the samples, largely eliminated variability due to slight run-to-run differences in transfer efficiency, antibody avidity, the efficiency of photon detection, and slight nonlinearities in the relations between mass and optical density. Calculation of the slope of the relation between ng pMLC₂₀ as a function of time of incubation provided a measurement of MLCK activity.

Effects of Protein Kinase Inhibitors on MLCK Activity

Suggestions from multiple previous studies that PKA (37), PKC (38), and CaM-Kinase II (39) can inhibit MLCK activity necessitated a series of experiments to verify that phosphorylation of MLCK by these kinases did not contribute to the observed age-related variations in MLCK activity. Conduct of these experiments followed the protocol described for Determination of Optimum Calcium and Calmodulin Concentrations for Activating MLCK with the exception that the 20 min incubation at 4°C included H-89 (PKA inhibitor) at 0, 0.05, 0.15, or 0.5 µM or calphostin-C (PKC Inhibitor) at 0, 0.05, 0.15, or 0.5 µM or KN-93 (CaM Kinase-II inhibitor) at 0, 1, 3, or 10 µM. The reactions began by addition of 3 mM calcium and 1 mM ATP, followed by precise termination after 0, 1, 2, and 3 s via addition of ice-cold 10 mM EDTA and transfer to dry ice. Sample analysis and calculation of reaction velocities followed the protocol detailed for Determination of Optimum Calcium and Calmodulin Concentrations for Activating MLCK.

Determination of V_max and K_m Values for MLCK

Incubation of artery homogenates with purified chicken gizzard MLC₂₀ at 1.5, 3, 5, 7.5, 10, 15, and 20 µM for 10 min at 37°C enabled determination of values of V_max and K_m for MLCK. Initiation and termination of MLCK activity followed the protocol described in Determination of Optimum Calcium and Calmodulin Concentrations for Activating MLCK. Calculation of the mass of pMLC₂₀ divided by reaction duration (600 s) normalized to the mass of MLCK in each tube (≈200 ng), at each MLC₂₀ concentration, provided a measurement of MLCK activity. Curve fitting using least-square error minimization routines for the relationships between activity and MLC₂₀ concentration allowed determinations of V_max and K_m. Nonlinear analysis of concentration-velocity relations relied on curve fits to rectangular hyperbolae.

Measurement of MLC₂₀ Phosphorylation in Situ

A custom-designed, computer-controlled apparatus enabled measurement of rates of MLC₂₀ phosphorylation in intact arteries, as previously described (15). Briefly, platinum foil electrodes delivered electrical field stimulation at optimal currents (≈100 mA at ≈21 volts direct current, 4 ms duration) to wire-mounted 3-mm artery segments equilibrated at optimal resting tension in Na⁺-Krebs solution containing optimal concentrations of a phosphatase inhibitor (10 nM Calyculin A), bubbled with 95% O₂-5% CO₂. At precisely determined durations of electrical stimulation (0, 1, 2, 3 s), servo-controlled valves permitted the high-speed exchange (<200 ms) of Krebs buffer for methacarn at −70°C (60% methanol, 30% chloroform, and 10% acetic acid) with 5 mM NaF. The use of supercooled methacarn allowed rapid termination of enzyme activities and fixation without generating cross linking that could limit extraction of myosin light chain (MLC₂₀) for phosphorylation determinations (40). Extraction of MLC₂₀ from methacarn fixed and fresh artery segments yielded equivalent abundances. Bisection of each electrically stimulated and frozen segment yielded half the segment for pMLC₂₀ determination (Fig. 3) and the other half for confocal imaging (Fig. 4) and analysis (Fig. 5). For each segment used for pMLC₂₀ determination and confocal imaging, an adjacent segment enabled measurements of MLCK abundance used to normalize rates of MLC₂₀ phosphorylation.

Figure 3.

Apparent myosin light chain kinase (MLCK) activity in intact fetal and adult common carotid arteries. As determined with a custom-built, servo-controlled cuvette capable of rapidly freezing contracting artery segments at precise 1-s intervals, electrical stimulation in the presence of phosphatase inhibitors increased the phosphorylation of 20-kDa myosin light chain (MLC₂₀). When the rates of in situ phosphorylation measured over 3 s were normalized relative to the abundance of MLCK measured via immunoblotting, the apparent catalytic activity of MLCK (in ng pMLC₂₀/ng MLCK/s) was 2.4-fold greater in fetal than adult arteries; this difference was statistically significant (Behrens–Fisher). Values shown indicate averages ± SE for 28 segments from 7 adult animals and 24 segments from 6 fetuses. Each animal contributed equally to each duration of stimulation employed.

Figure 4.

Estimation of catalytically active myosin light chain kinase (MLCK) using confocal microscopy. Paired adjacent artery segments were electrically stimulated at 1-s intervals. After stimulation and freezing, one-half of each segment was frozen for analysis of 20-kDa myosin light chain (MLC₂₀) phosphorylation, as shown in Fig. 3, and the other half was analyzed using confocal microscopy to quantify colocalization between MLCK (488 nm; green) and phosphorylated MLC₂₀ (633 nm; red). The confocal images shown represent each of the four different timepoints (0, 1, 2, and 3 s) for seven adult sheep (top) and seven fetal lambs (bottom). In both age groups, colocalization analysis of image samples from periluminal (Lum) and periadventitial (Adv) locations revealed how interactions between MLCK and MLC₂₀ differed among regions of the artery wall. Brightness and contrast were adjusted for all images simultaneously to optimize visual clarity. All measurements of colocalization were performed on raw images only.

Figure 5.

Colocalization-corrected myosin light chain kinase (MLCK) activity. In both fetal and adult common carotid segments, electrical stimulation increased colocalization between MLCK and phosphorylated 20-kDa myosin light chain (pMLC₂₀) in a time-dependent manner, as indicated by the Mander’s M1 coefficient (top left). Multiplication of the fraction of MLCK colocalized with pMLC₂₀ by the total abundance of MLCK in artery homogenates of adjacent segments enabled calculation of the catalytically active mass of MLCK as a fraction of total MLCK. The change in fractional activation of total MLCK was significantly greater (*; ANOVA) in fetal than adult arteries after 3 s of electrical stimulation (top right). The lower left (adult) and lower right (fetus) panels indicate stimulation-induced regional changes in periluminal and periadventitial MLCK colocalization with pMLC₂₀. Vertical error bars indicate SE for n = 7 artery segments from each of 7 animals for both age groups. Adv, periadventitial; Lum, periluminal.

Determination of In Situ MLCK Activity

The buffer used to homogenize methacarn-quenched arterial segments contained 8 M Urea, 500 mM NaCl, 20 mM Tris, 23 mM Glycine, 10 mM EGTA, 10 mM DTT, 5 mM NaF, and 10% glycerol at pH 8.6 with 0.5% protease inhibitor cocktail (Sigma-Aldrich, P8340) and 1% phosphatase inhibitor cocktail (Sigma-Aldrich, P0044). After tissue homogenization (motorized glass-on-glass mortar and pestle), sample centrifugation at 10,000 g for 15 min produced the supernates used for the determination of in situ MLCK activity. Western immunoblots involved addition of equal amounts of supernate protein, as determined by Bradford protein assay, to separate lanes of SDS-PAGE gels, followed by electrophoresis then transfer to nitrocellulose membranes using Towbin’s buffer (25 mM Tris, 192 mM glycine, and 20% methanol). Membranes underwent incubation with primary antibodies against MLCK (Sigma-Aldrich, M7905) or MLC₂₀ (Cell Signal, 8505) and pMLC₂₀ (Ser¹⁹) (Cell Signal, 3675). Incubations with DyLight-conjugated secondary antibodies (at 1:15,000, Thermo Fisher Scientific) allowed detection and determination of integrated optical density values using an infrared membrane imager (LI-COR Bioscience’s Odyssey). The use of standard curves constructed from known masses of authentic standards run side-by-side with the samples largely eliminated variability due to slight run-to-run differences in transfer efficiency, antibody avidity, the efficiency of photon detection, and slight nonlinearities in the relations between mass and optical density. Analysis of samples to calibrate the ratios of unphosphorylated and phosphorylated MLC₂₀ proceeded using 10% urea glycerol gels, as previously described (7).

Immunohistochemistry and Confocal Colocalization of MLCK and pMLC₂₀

Processing of methacarn-fixed, paraffin-embedded artery segments for immunohistochemistry began with microtome sectioning at 6 µm, followed by deparaffinization in Histoclear and rehydration. Immunostaining involved nonspecific blocking in 1X PBS buffer containing 1% BSA, 5% normal goat serum (NGS) and 0.1% Triton X-100 at room temperature for an hour, followed by overnight incubation at 4°C with primary antibodies against MLCK and pMLC₂₀ (Ser¹⁹) in blocking buffer containing 2% NGS. Incubation of sections with DyLight-conjugated secondary antibodies (at 1:200, Thermo Fisher Scientific), coverslipped using Slowfade Gold Antifade Reagent (Life Tech.) allowed imaging at 488 nm (MLCK) and 633 nm (pMLC₂₀) on an Olympus FV1000 confocal microscope. Sequential scanning at 10 µs/pixel with a Semi-Apochromat UPLFLN ×40 objective (NA = 1.3) produced images with an axial resolution of 1,099–1,397 nm and a lateral resolution of 207–263 nm, depending on the wavelength of excitation. Setting the confocal aperture for 1 airy unit captured images at 640 × 480 pixels with a pixel size of ≈0.57 µm. Images were collected as Z-stacks in the vertical plane with six sections per stack. Appropriate controls verified a lack of antibody cross talk, spectral crossover, and autofluorescence contributions to the images from immunolabeling and scanning conditions. Image deconvolution with either ImageJ (Fiji v2.1.0) or Bitplane Imaris (v9.3) routines demonstrated a modest ability to restore systematic losses in image contrast, but the overall patterns of statistical significance among colocalization values were not changed; we did not routinely employ deconvolution in the present study. Colocalizer Pro software (v2.6.1) provided Mander’s colocalization coefficients between MLCK and pMLC₂₀. Previous studies have compared multiple different measures of colocalization and determined that Manders’ M1 and M2 coefficients rely less on correlation and more on co-occurrence than other colocalization coefficients, making these coefficients particularly appropriate for studies of enzyme-substrate interactions (41–43). Our own validation studies have confirmed these interpretations (44), as has our use of Manders’ coefficients, in previous studies of contractile protein colocalization (45–48).

For quantitative analysis of confocal colocalization between MLCK and pMLC₂₀, we assigned the MLCK signal to channel 1 and this thus defined the M1 Mander’s coefficient (42, 49) as the number of pixels positive for both MLCK and pMLC₂₀ divided by the total number of pixels positive for MLCK [Σ (MLCK × pMLC₂₀)/Σ (MLCK)]. All pixels between the basal elastic lamina and the adventitial-medial border in each arterial section were included in the colocalization analysis to assure that the entire medial layer was represented in the colocalization results and thus would optimally correspond with the results from our in situ activity analyses (Fig. 3). The extent of colocalization between MLCK and pMLC₂₀ was determined in each optical slice in each Z-stack for each image, after which the M1 Mander’s values were averaged across all the slices in the Z-stack. Multiplication of the averaged Mander’s M1 coefficient by the mass of MLCK in the paired artery segment used for in situ velocity calculations produced time-dependent estimates of fractional activation. The capture of small images (≈10 µm by 60 µm) just medial to the basal elastic lamina (periluminal) and just medial to the external elastic lamina (periadventitial) enabled an analysis of time-dependent MLCK activation in different regions of the artery wall. These dimensions were selected to encompass the area of approximately one smooth muscle cell with enough pixels per optical slice (≈2,200) to produce a reliable measure of colocalization. Each small image was taken at a location that minimized areas of contact between adjacent cells to obtain images that represented as much as possible one smooth muscle cell. Given that the arteries imaged consisted of ≈8–12 concentric layers of smooth muscle cells, our approach could reliably detect significant differences between individual smooth muscle cells in the periluminal and periadventitial layers.

Calculations and Statistics

All curve-fitting procedures employed least-square error minimization routines. Nonlinear fits for standard curves (optical density vs. mass) relied on the logistic equation for rectangular hyperbolae. Coefficients of fit for the standard curves, prepared from known absolute masses of standards, allowed for derivation of inverse forms of the logistic equation that enabled direct calculation of sample masses from band optical densities. Division of estimates of homogenate V_max by the corresponding mass of MLCK measured in the same homogenate-yielded estimates of apparent specific activity.

Throughout the text, all values indicate the means ± SE for the number of animals indicated. Values of n refer to the numbers of animals and not the numbers of segments or experiments. For unpaired comparisons between two variables, statistical analysis relied on Behrens–Fisher comparisons with pooled variance. Analysis with SPSS v23 confirmed normal distributions for all data sets. Factorial ANOVA within SPSS v23 was used for all multigroup comparisons with a Fisher’s Protected Least Significant Difference (PLSD) for post hoc comparisons. Preliminary experiments provided the initial estimates of variance required to estimate the number of replicates needed to attain a statistical power ≥0.8 for all groups. In all cases, the null hypothesis was rejected when P < 0.05.

RESULTS

This study utilized a total of 31 fetal lambs and 31 adult sheep, which provided a total of 3.9 g and 2.8 g of carotid arterial tissues, respectively, for the preparation of all fetal and adult artery homogenates used in this study. These animals also provided the 14 paired segments of common carotid arteries from each group used for in situ measurements of MLCK activity and colocalization between MLCK and pMLC₂₀.

Optimum Concentrations of Calcium and Calmodulin

Optimization experiments revealed optimal MLCK activities (ng MLC₂₀ phosphorylated/s/ng MLCK) in artery homogenates containing 10 ng of MLCK in a volume of 40 µL, in the presence of 0.3 µM CaM and 3 mM calcium in both fetal (2.51 ± 0.12) and adult (1.93 ± 0.60) arteries (Fig. 1). Additions of exogenous CaM had no significant effect (2-factor ANOVA) at any calcium concentration. To assure that all homogenates had equivalent concentrations of exogenous CaM that far exceeded any unbound endogenous CaM, all subsequent measurements of MLCK velocity utilized concentrations of 0.3 µM exogenous CaM together with 3 mM calcium.

Figure 1.

Optimization of conditions for measurement of myosin light chain kinase (MLCK) activity in fetal and adult carotid homogenates. Addition of calcium-stimulated rates of 20-kDa myosin light chain (MLC₂₀) phosphorylation similarly in fetal and adult artery homogenates; rates were optimal at 3 mM in both age groups. Exogenous CaM had little effect on MLC₂₀ phosphorylation in either age group. Addition of phosphatase inhibitors at 10 µL/mL yielded maximal rates of MLC₂₀ phosphorylation in both age groups. The values shown represent the averages ± SE obtained from homogenates of 60 adult and 60 fetal artery segments, with n = 3 homogenates of 3 arteries from 3 animals for each average shown. Each animal contributed equally to each combination of calcium and calmodulin concentrations used.

Effect of Protein Kinase Inhibitors on MLCK Velocity

To assess the potential inhibitory influence of protein kinase-mediated phosphorylation on MLCK activity, the experimental design included measurements of MLCK activities in the presence of differing concentrations of kinase inhibitors. In fetal artery homogenates, MLCK activities (ng MLC₂₀ phosphorylated/s/ng MLCK) did not increase significantly (2-factor ANOVA) compared with control homogenates (2.23 ± 0.68), following treatment with 0.5 µM H-89 (1.22 ± 0.44), 0.5 µM calphostin-C (1.36 ± 0.28), or 10 µM KN-93 (0.91 ± 0.31). Similarly, MLCK activities did not increase significantly (2-factor ANOVA) in adult homogenates compared with control homogenates (2.02 ± 0.43), following treatment with 0.5 µM H-89 (1.08 ± 0.17), 0.5 µM calphostin-C (1.47 ± 0.26), or 10 µM KN-93 (0.80 ± 0.17) (Supplemental Table S1; see https://doi.org/10.6084/m9.figshare.14888235). Given that none of the kinase inhibitors tested increased MLCK velocities in either fetal or adult artery homogenates, and in some cases significantly inhibited MLCK activity, all subsequent assays excluded kinase inhibitors.

Age-Related Differences in V_max and K_m Values for MLCK

In artery homogenates containing ≈33 nM MLCK, MLCK activity (in ng MLC₂₀ phosphorylated/s/ng MLCK), following activation by rapid addition of calcium and ATP, exhibited classic hyperbolic substrate dependence in both fetal and adult homogenates (Fig. 2). With reaction times of exactly 10 min, fetal and adult values of MLCK activity did not differ significantly (2-factor ANOVA with Fisher's PLSD) at any MLC₂₀ concentration used. Correspondingly, estimates of V_max for MLCK obtained via nonlinear regression did not differ significantly (Behrens–Fisher) in fetal (7.3 ± 0.5) and adult (6.9 ± 0.5) homogenates. K_m values also did not differ significantly (Behrens–Fisher) in fetal (4.8 ± 1.2 µM) and adult (3.3 ± 1.0 µM) samples. These measurements involved 42 artery segments in each age group, distributed across seven concentrations of MLC₂₀, with six segments at each concentration; statistical power was ≥0.8 for all comparisons.

Figure 2.

Myosin light chain kinase (MLCK) substrate velocity in fetal and adult carotid homogenates. Artery homogenates containing equivalent amounts of MLCK were incubated for exactly 10 min with different concentrations (1.5, 3, 5, 7.5, 10, 15, or 20 µM) of purified chicken gizzard 20-kDa myosin light chain (MLC₂₀). MLCK velocities exhibited classic substrate dependence in both age groups. Estimates of kinetic parameters were obtained via nonlinear regression with a rectangular hyperbola; the solid and dotted lines shown indicate the curves of best fit. The values shown represent the averages ± SE obtained from homogenates of 42 adult and 42 fetal artery segments, with n = 6 homogenates of 6 arteries from 6 animals for each average shown. Each animal contributed equally to each concentration of MLC₂₀ used.

Age-Related Differences in Apparent in Situ MLCK Activity

Intact artery segments mounted in our rapid-freeze cuvette, incubated at optimum stretch in the presence of 10 mM Calyculin A, and stimulated at optimal currents, reached a maximum MLC₂₀ phosphorylation of 2.13 ± 0.18 ng pMLC₂₀/ng MLCK within 1 s of stimulation in adult segments but continued to increase through 3 s in fetal arteries, at which point MLC₂₀ phosphorylation averaged 3.16 ± 0.70 ng pMLC₂₀/ng MLCK (Fig. 3). The apparent catalytic activity of MLCK, estimated as the average rate of increase in MLC₂₀ phosphorylation over 3 s divided by MLCK abundance, averaged to values more than 2.4-fold greater (P < 0.05, Behrens–Fisher) in fetal arteries (1.06 ± 0.28 ng pMLC₂₀/s/ng MLCK), relative to adult arteries (0.43 ± 0.08 ng pMLC₂₀/s/ng MLCK). Division of the apparent rate of MLCK activity in situ by the values of V_max obtained in homogenates (Fig. 2) yielded estimates of fractional activation that averaged 43 ± 10% and 31 ± 3% in fetal and adult arteries, respectively; these values were significantly different at P < 0.05 (Behrens–Fisher).

Colocalization of MLCK with pMLC₂₀ and Age-Dependent Fractional Activation of MLCK

Immunostaining for MLCK and pMLC₂₀ in intact artery segments rapidly frozen after 1, 2, and 3 s of electrical stimulation enabled capture of confocal microscopy images (Fig. 4). Quantitative analysis of these confocal images revealed that the colocalization between pMLC₂₀ and MLCK, averaged across the entire artery wall between the basal elastic lamina and the adventitial-medial border, increased significantly as a function of time in both fetal and adult arteries, as indicated by the Mander’s M1 coefficient (Σ pMLC₂₀ × MLCK/Σ MLCK) (Fig. 5, top left). Consistent with the measurements of apparent in situ MLCK activity (Fig. 3), values of the M1 colocalization coefficient plateaued after 1 s of stimulation in adult arteries, but rose steadily throughout the 3 s period of stimulation in fetal arteries.

To estimate the mass of MLCK directly involved in phosphorylation of MLC₂₀, the calculations involved multiplication of the M1 coefficient values by the total MLCK abundance, as determined by calibrated immunoblot. In paired segments of the arteries used for confocal colocalization studies, MLCK abundances averaged 1.98 ± 0.09 and 1.03 ± 0.08 µg MLCK per mg total protein in seven adult and seven fetal arteries, respectively. The time-dependent change in colocalized MLCK mass, relative to the total content of MLCK, provided estimates of time-dependent fractional activation for the whole artery segment (Fig. 5, top right). At 3 s, these values averaged 20 ± 5% and 33 ± 6% and in adult and fetal arteries, respectively; these values were significantly different (Behrens–Fisher). When the colocalized fractions of MLCK were used to calculate MLCK activity in situ (as ng MLC₂₀/s/ng MLCK), the averaged fetal values (3.21 ± 0.08) were 49.3% greater (P < 0.05) than adult values (2.15 ± 0.04). However, before the correction for colocalization, the averaged fetal values (1.06 ± 0.28) were 147% greater than the corresponding adult values (0.43 ± 0.08); correction for the extent of colocalization between MLCK and MLC20 accounted for 68.6% of the original difference between fetal and adult estimates of MLCK activity, in situ (Fig. 5).

Image samples from the periluminal and periadventitial regions of the artery wall (Fig. 4) revealed that estimates of single cell activation of MLCK followed different time courses and age-dependent magnitudes of activation in different regions of the artery wall (Fig. 5, bottom). In fetal arteries, both periluminal and periadventitial regions exhibited significant increases in MLCK-pMLC₂₀ colocalization within 1 s of stimulation that differed from each other and persisted for 3 s at which time the changes in colocalization averaged 54 ± 5% and 19 ± 11% in the periluminal and periadventitial regions, respectively. In adult arteries, the local changes in MLCK-pMLC₂₀ colocalization exhibited magnitudes of change much smaller than observed in the fetal arteries and differed from each other only at 3 s, where changes in colocalization in the periluminal and periadventitial regions averaged 29 ± 11% and 1 ± 6%, respectively.

DISCUSSION

MLCK is a dedicated serine/threonine kinase that phosphorylates only T18 and S19 of MLC₂₀ to initiate smooth muscle contraction (50). When activated by Ca²⁺-CaM (51), it is a very fast enzyme that can fully phosphorylate MLC₂₀ in as little as 2 s following nerve stimulation in some preparations (52, 53). To a certain extent, MLCK may compete with other kinases (54), and integrin-linked kinase in particular (55) for regulating the phosphorylation of sites on MLC₂₀. In contrast to MLCK, however, the rates of phosphorylation of MLC₂₀ by these other kinases is relatively slow (56), which makes phosphorylation by MLCK the predominant rate-limiting step in the initiation of contraction. Correspondingly, the rate of contraction is generally tightly coupled to the smooth muscle abundance of MLCK (9–12). One major exception to this rule, however, is the contraction of certain immature smooth muscle preparations (13, 15).

Beginning in the 1990s and for many years thereafter, a primary motivation for studying contractility in immature smooth muscle preparations was better understanding of the mechanisms responsible for the pulmonary hypertension (57) and airway hyperreactivity (58) commonly encountered in critically ill neonates. Within this context, several different groups reported that MLCK abundance increased significantly after birth in guinea pig tracheal smooth muscle (59) and rat pulmonary artery (13). The Belik study further demonstrated that total MLCK activity was similar in homogenates of fetal and adult arteries, even though MLCK abundance was lower in fetal than adult homogenates. This latter finding suggested for the first time that the specific catalytic activity of MLCK was somehow greater in fetal than adult arteries (13). Subsequent studies have similarly reported that the apparent catalytic activity of MLCK was greater in fetal than adult ovine arteries even though MLCK abundance was less in the fetal arteries (8, 15). The mechanisms responsible for this apparent age dependency of MLCK catalytic activity remain unexplained.

Prior studies have demonstrated that age-related differences in substrate (MLC₂₀) concentration cannot explain why in situ MLCK activity is greater in fetal arteries, because the MLC₂₀ concentration greatly exceeded the K_m values for MLCK in both fetal and adult arteries (15). Differences in CaM availability also cannot explain why in situ catalytic activity of MLCK is greater in fetal than adult arteries because CaM was significantly more abundant in adult than in fetal ovine arteries (15). A remaining possibility is that the specific catalytic activity of MLCK is greater in fetal than adult arteries, as suggested by Belik (13).

To compare the catalytic activity of MLCK in fetal and adult arteries, the present studies employed an artery homogenate preparation in which MLCK concentration was diluted ∼400-fold, as previously described (15). This dilution offered multiple advantages. First, it eliminated any possible effects of endogenous inhibitors of MLCK (18, 19). Second, it essentially eliminated any possible effects of differences in endogenous substrate concentrations. Third, dilution of MLCK reduced the observed rates of MLC₂₀ phosphorylation and enabled more accurate measurements. Finally, it minimized any possible age-related differences in CaM concentration (15, 17). Using purified chicken gizzard MLC₂₀ as a substrate for MLCK, activation of MLCK in both fetal and adult artery homogenates yielded maximal rates of MLC₂₀ phosphorylation in the presence of 3 mM calcium and 0.3 µM CaM (Fig. 1). Our dilution method also effectively attenuated phosphatase activities (60) owing to the inclusion of optimal concentrations of inhibitors of cantharidin and microcystin to inhibit PP1c phosphatase (61), along with bromotetramisole to inhibit alkaline phosphatase, as previously described (15). Given that inclusion of inhibitors of PKA (37), PKC (38), and CaM-Kinase II (20, 39) were unable to increase MLCK activity (Supplemental Table S1), the present results suggest that the potential inhibitory activities of these kinases did not significantly influence measurements of MLCK reaction velocity under our reaction conditions. Together, these experiments validated our MLCK homogenate assay, which produced results that agreed well with other MLCK homogenate studies (11, 15). Correspondingly, all subsequent measurements of MLCK activity in homogenates included 3 mM calcium and 0.3 µM CaM with 10 µL/ml of the phosphatase inhibitor cocktail.

Under optimal conditions, substrate-velocity measurements revealed that MLCK velocity (ng MLC₂₀ phosphorylated/s/ng MLCK) measured 10 min after the rapid addition of calcium and ATP did not differ significantly between fetal and adult homogenates at any MLC₂₀ concentration used and exhibited similar V_max values in fetal (7.3 ± 0.5) and adult (6.9 ± 0.5) artery homogenates (Fig. 2). Estimates of substrate affinity (K_m) also did not differ significantly between fetal (4.8 ± 1.2 µM) and adult (3.3 ± 1.0 µM) homogenates. These results extend the findings of Belik obtained in rat pulmonary arteries (13) and suggest that age-related differences in the apparent whole tissue rates of MLC₂₀ phosphorylation probably cannot be explained by differences in the catalytic activity of MLCK. In turn, age-related differences in MLC₂₀ or CaM abundance also cannot explain why the rates of whole tissue MLC₂₀ phosphorylation appear greater in fetal arteries, because fetal arteries actually exhibit lower abundances of both MLC₂₀ and CaM (15).

In their early studies of MLCK function in multiple muscle types, Cavadore et al. suggested that the subcellular localization of MLCK may influence its function (62). Subsequent studies have clearly demonstrated that MLCK contains a specific actin-binding sequence (63, 64) and directly binds both actin and myosin (65). Detailed studies of intracellular MLCK distribution have further suggested that both its distribution and function depend on isoform, the type(s) of myosin(s) present and the pattern of actin organization (30). Correspondingly, a broad variety of studies have also implicated involvement of MLCK in numerous noncontractile plasmalemmal functions including ion channel function in gastric smooth muscle (66), translocation of secretory granules in pancreatic β cells (67), membrane transporter function in neurons (68), antigen internalization in monocytes (69), and vesicle endocytosis in nerve terminals (70). In light of this broad variety of noncontractile MLCK functions localized to the cell membrane, it seems reasonable to suspect that MLCK may also serve some noncontractile functions in vascular smooth muscle cells. If so, then not all MLCK expressed in vascular smooth muscle would be directly involved in contraction, and the fraction involved in contraction could vary, for example, with changes in smooth muscle cell phenotype and differentiation associated with postnatal age, exposure to hypoxia, and many other perturbations (23, 24, 71).

To test the hypothesis that not all MLCK expressed in our artery preparations were directly involved in contraction, we modified our rapid-freeze cuvette to inject −70°C methacarn fixative, which preserved ultrastructural detail (40). This modification enabled rapid freezing of arterial segments at precise short durations of electrical stimulation, followed by immunohistochemical analysis with confocal microscopy to determine the time course of colocalization between MLCK and pMLC₂₀. A paired approach using bisected arterial segments enabled measurements of changes in pMLC₂₀ in one-half of each segment, via standard immunoblotting techniques (Fig. 3) and MLCK/pMLC₂₀ colocalization (Fig. 4) in the other half of the segment. Under these conditions in mounted and optimally stretched artery segments, we assumed that MLCK and MLC₂₀ were not homogeneously distributed, in contrast to our activity measurements in homogenates (Fig. 2). This assumption was verified by the measurements of colocalization between MLCK and MLC₂₀. When we calculated the whole artery rate of pMLC₂₀ accumulation relative to the whole artery abundance of MLCK, we corroborated our previous findings (15) and found that the apparent catalytic activity for MLCK was 147% greater in fetal than adult arteries (Fig. 3). Basal levels of MLC₂₀ phosphorylation at time 0 were not significantly different in fetal and adult arteries (Fig. 3) and cannot help explain why the apparent rate of MLC₂₀ phosphorylation was so much greater in fetal than adult arteries. Importantly, both the phosphorylation and colocalization measurements produced similar time courses for MLC₂₀ phosphorylation with both methods of measurement. Multiplication of the fraction of MLCK colocalized with pMLC₂₀ quantified with the Mander’s M1 coefficient across the entire artery wall between the basal elastic lamina and the adventitial-medial border (Fig. 5, upper left), by total MLCK abundance measured by calibrated immunoblots in a paired artery segment, produced estimates of the mass of MLCK involved in MLC₂₀ phosphorylation as a function of time in both fetal and adult arteries. In turn, division of the mass of MLCK colocalized with pMLC₂₀ after 3 s of electrical stimulation, by the total MLCK mass, yielded estimates of fractional activation that averaged to values at least 1.6-fold greater in fetal (33%) than adult (20%) arteries (Fig. 5, upper right). Together, these results strongly suggest that not all vascular MLCK is involved in contraction, and thus total vascular MLCK cannot be used to reliably estimate its catalytic activity, in situ. In addition, these findings suggest that the reason that in situ MLCK catalytic activity has appeared to be greater in fetal than adult arteries was because the fraction of total MLCK mass directly involved in MLC₂₀ phosphorylation was markedly less in adult than fetal arteries.

Another advantage offered by time-dependent confocal analysis of MLCK-pMLC₂₀ colocalization includes the ability to resolve highly regional changes throughout the arterial wall. This approach revealed that in both fetal and adult arteries, the rate of MLCK activation was faster and more pronounced in smooth muscle near the lumen than near the adventitial border (Fig. 5, bottom). In the periluminal regions of both fetal and adult arteries, activation of MLCK increased over the entire period of stimulation. In contrast, MLCK activation in the periadventitial regions returned to baseline values by 3 s in adult arteries, but remained at constant but elevated levels in fetal arteries. Together, these findings reveal that not all smooth muscle cells in the artery wall react the same way in response to electrical stimulation, particularly in relation to age-dependent activation of MLCK. The results also support our “gradient hypothesis” (29), which posits that growth factors released from the endothelium (e.g., VEGF) promote smooth muscle differentiation most in the periluminal layers, and least in the periadventitial layers, which are farthest from the endothelium. Conversely, growth factors released from parenchymal tissues [e.g., brain-derived neurotrophic factor (BDNF), FGF] adjacent to the serosal surfaces of arteries promote different patterns of smooth muscle differentiation that are greatest in the periadventitial layers and least in the periluminal layers farthest from the serosal surface (72). The presence of these opposing gradients could help explain why different concentric layers of arterial smooth muscle exhibit different functional characteristics, as observed in the present study.

Perspectives and Significance

Overall, the present results emphasize that contractile protein colocalization and abundance can be independently regulated (21), and that for MLCK, not all vascular MLCK contributes to contraction. Indeed, estimates of fractional activation of MLCK from this and previous studies (15, 73) suggest that less than half of all MLCK contributes to active contraction, regardless of age. Through the use of a novel combination of rapid-freeze and imaging techniques to simultaneously quantify the rates of both MLC₂₀ phosphorylation and colocalization of MLCK with MLC₂₀ in whole arteries, our findings demonstrate that the portion of total MLCK involved in smooth muscle contraction is significantly greater in fetal than adult arteries. From a methodological perspective, the present study demonstrates the utility of confocal optical approaches to follow regional changes in MLCK activity. These confocal methods avoid the need to homogenize arterial samples and thereby preserve not only the intracellular distributions of MLCK but also the concentrations of all endogenous activator and inhibitor molecules that might influence MLCK activity. The use of confocal colocalization methods also uniquely enabled resolution of age-dependent transmural differences in the time course of MLCK activation, which appeared more robust in periluminal than in periadventitial smooth muscle cells in both age groups. Importantly, the overall time courses of MLCK activation revealed by our confocal colocalization methods in both fetal and adult arteries agreed well with those produced by a conventional homogenate approach, which cross-validated both approaches. Altogether, the novel working hypothesis arising from this study is that in immature arteries, the first priority location for MLCK is the contractile apparatus, but with progressing age comes greater MLCK abundance, and an accumulation of “extracontractile” MLCK. A key consequence of this developmental trajectory is that the use of total MLCK to normalize rates of MLC₂₀ phosphorylation, which is common in whole artery estimates of MLCK activity (13, 14), underestimates the true in situ catalytic activity of MLCK, and more so in adult than in fetal arteries.

The limitations constraining interpretation of the present results fall into at least three categories including spatial, temporal, and biochemical. Our confocal microscopy methods provided lateral resolutions of 207–263 nm for our sampling voxel, which were significantly larger than the 7–8 nm size reported for a single molecule of MLCK (74, 75), suggesting a possible overestimation of colocalization. The extent of this overestimation, however, was probably mitigated by the fact that pMLC₂₀ could be detected only following phosphorylation by MLCK. These spatial limitations could be overcome through future use of more resource intensive measurements of colocalization using superresolution confocal microscopy (76); indeed, the present results offer a strong premise to carry out such studies.

Temporal limitations of the present findings arise from the possible ability of MLCK to move during contraction (35), in which case it may have escaped detection at the 1-s freezing increments employed, leading to a possible underestimation of MLCK-pMLC₂₀ colocalization. In addition, our measurements spanned an interval of only 3 s, which may not have been sufficient to capture the entire MLCK response, and thus may have underestimated the extent of fractional activation, particularly in fetal arteries (Figs. 3 and 5).

Biochemically, the present methods cannot unambiguously quantify highly localized patterns of MLCK activation due to lack of information about precise regional MLCK concentrations. In addition, our methods cannot exclude possible age-related differences in the distribution and localization of Ca-CaM, which is prerequisite for activation of MLCK. The present data also do not exclude possible age-dependent involvement of kinases other than MLCK that may influence MLC₂₀ phosphorylation, including either direct (77, 2967818) or indirect (54, 78) effects of PKC or Integrin-Linked Kinase (55). Even so, the results raised numerous questions about MLCK function. For example, why is catalytically inactive MLCK bound to α-actin near the contractile apparatus (79, 80)? What are the main nonkinase functions of MLCK in smooth muscle, are nonkinase and catalytically active MLCK interchangeable, and what cellular factors and stimuli regulate the distribution and roles of MLCK (81)? More specifically, is the reported kinase-independent ability of MLCK to influence cytoskeletal organization through its actin-binding domain (82, 83) significant in whole arteries, and if so, are these effects equally important in fetal and adult arteries? Similarly, can kinase-independent binding of MLCK to myosin modulate myosin ATPase activity in whole arteries, as reported in other preparations (84–86), and if so, is this effect equally significant in fetal and adult arteries? Finally, how significant are the influences of nonenzymatic endogenous activators and inhibitors of MLCK (18, 19), and the abilities multiple kinases, including p21-activated kinase (87), MAPK (88), and CDK1 (89), to activate or inhibit local MLCK activity? Specifically, can these latter influences explain the 32% difference between fetal and adult estimates in MLCK, in situ, that remain following correction for MLCK colocalization with MLC₂₀?

From a more general perspective, the results from the present study represent the vascular biology only of term fetal and adult ovine carotid arteries; owing to long-established principles of structural and functional heterogeneity among arteries of differing sizes from different vascular beds (90, 91), it should be expected that the spatial and temporal differences identified between fetal and adult carotids might be even more pronounced, or of a completely different character altogether, in other vascular beds such as the cerebral, coronary, or mesentery. The above questions aside, the present results emphasize that the abundance, dynamics, and patterns of both intercellular and intracellular distribution and activation of MLCK differ markedly in fetal and adult arteries, which help explain why the characteristics of vascular contractility are so different in these two age groups.

SUPPLEMENTAL DATA

Supplemental Table S1: https://doi.org/10.6084/m9.figshare.14888235.

GRANTS

The work was supported by National Institutes of Health Grants HD-054920, HD-31266, HL-54120, HL-64867, and NS-076945 and the Loma Linda University School of Medicine. A portion of this research used resources in the Loma Linda University School of Medicine Advanced Imaging and Microscopy Core; the facility is supported in part by the National Science Foundation through the Major Research Instrumentation program of the Division of Biological Infrastructure Grant No. 0923559 and the Loma Linda University School of Medicine.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.M.W. and W.J.P. conceived and designed research; D.W.S., E.R.I., L.M-A., and J.M.W. performed experiments; D.W.S., E.R.I., J.M.W., and W.J.P. analyzed data; D.W.S., E.R.I., J.M.W., and W.J.P. interpreted results of experiments; D.W.S. and W.J.P. prepared figures; D.W.S. and W.J.P. drafted manuscript; D.W.S. and W.J.P. edited and revised manuscript; D.W.S., E.R.I., L.M-A., J.M.W., and W.J.P. approved final version of manuscript.