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Departments of 1Neurology, 3Pediatrics, 4Medicine, and 5Anesthesiology, 2The Medical College of Wisconsin and Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin
Submitted 10 September 2007 ; accepted in final form 26 March 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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kinase-related protein; cerebral artery
The contractile state of a vascular smooth muscle cell depends heavily on the interaction of myosin light chain (MLC) with two enzymes, myosin light chain kinase (MLCK) and myosin phosphatase. In the presence of Ca2+ and calmodulin, MLCK phosphorylates MLC, and the cell contracts. When myosin phosphatase dephosphorylates MLC, the cell relaxes. Activator or repressor proteins, whose activities are determined by their own phosphorylation status, can modify myosin phosphatase. One such protein is telokin, also known as kinase-related protein (KRP). Telokin is a 17-kDa acidic protein with a sequence identical to the COOH-terminal domain of MLCK (6, 9). However, rather than being simply a proteolytic fragment of the kinase, telokin is expressed independently of MLCK (6). Telokin expression seems to be restricted to smooth muscle (7, 8) where it is generally more prevalent in phasic than tonic smooth muscle (17). Substantial amounts of telokin have been found in the phasic smooth muscle of the uterus, ovary, vas deferens, bladder, colon, kidney, ureter, trachea (8), and ileum (3). Within the vasculature, telokin has been found in the phasic muscle of the portal vein (3) and to a lesser extent in the tonic smooth muscle of umbilical and renal arteries (8), abdominal and thoracic aorta (3, 8), and femoral artery (3, 22).
In vitro studies have shown that telokin binding to the S1/S2 region of unphosphorylated myosin stabilizes myosin filaments, enhances myosin phosphatase activity (3), and induces Ca2+ desensitization (9) to initiate or maintain smooth muscle relaxation (7, 9, 18, 22). Current evidence suggests that telokin exerts these effects when it is phosphorylated. In both phasic and tonic smooth muscle cells, Ca2+ desensitization and relaxation induced by cyclic GMP (PKG) and/or cyclic AMP-dependent (PKA) protein kinases occurs concomitantly with the telokin phosphorylation (11, 22). Telokin phosphorylation is enhanced in the presence of forskolin and 8-bromo-cGMP, which suggests that telokin may have a functional role in smooth muscle contraction by regulating the level of MLC phosphorylation (22).
While telokin has been found in several different artery types, there have been no reports as to whether telokin expression or content differs within the same vascular bed. In preliminary studies we found greater telokin expression in feline SPA and their smooth muscle cells than in the LPA and their smooth muscle cells (15). Such differential expression might be correlated with the presence and operation of distinct regulatory mechanisms within the different regions of the pulmonary vasculature. We hypothesized that telokin expression and content would be graded in different sizes of pulmonary arteries. We also hypothesized that if telokin were greater in SPA than LPA, then during acute hypoxia, telokin phosphorylation in SPA smooth muscle cells would change concomitantly with, and in the opposite direction to changes in MLC phosphorylation, whereas in LPA smooth muscle cells there would be little or no change of either. This study was designed to determine the pattern of telokin expression in SPA and LPA and their smooth muscle cells, and also in cerebral artery smooth muscle cells, which relax to hypoxia (16). Changes in the phosphorylation status of both telokin and MLC were measured in response to acute hypoxia in SPA and LPA smooth muscle cells and to phenylephrine (PE) in LPA smooth muscle cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Smooth Muscle Cell Isolation
Immediately upon dissection, the arteries were placed into cold Pucks saline solution containing penicillin-streptomycin (Sigma- Aldrich, St. Louis, MO) at pH 7.4 and 4°C. The arteries were then minced and transferred to 1 ml of an enzyme solution (described below) and incubated for 80 min at 37°C to isolate smooth muscle cells. Enzyme digestion was stopped by adding an equal volume of M231 medium (Cascade Biologics, Portland, OR) + 10% fetal bovine serum. The solution was filtered through two different sizes of mesh filters onto 60-mm culture plates to remove connective tissue and cell debris. The supernatant was collected and centrifuged at 325 g at 4°C for 10 min. The pellet was suspended in 1 ml of M231 medium + 10% fetal bovine serum. The smooth muscle cells were grown in T-25 flasks containing M231 medium + 10% fetal bovine serum. At confluence, the cells were split 1:5 and replated in T-25 flasks for continued growth or onto 60-mm culture dishes and grown to confluence for the experimental protocols described below.
Northern Blot
Northern hybridization was performed using standard methods. Six micrograms of total RNA isolated with TRIzol reagent (Synovis Life Technologies, St. Paul, MN) from cultured smooth muscle cells were loaded on each line. Cloned full-length cat telokin cDNA (GenBank acc. no. AY831775) was used as a probe. Labeling was done with the Random Primer Labeling kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's protocol. GAPDH cDNA (gift from D. Levy, NYU Medical Center) was used to probe for GAPDH mRNA, which was used as a control for RNA loading of the gel.
Immunohistochemistry
Arteries. Immunoperoxidase staining for telokin was performed on formalin-fixed, paraffin-embedded sections of SPA using no. 6091 anti-human KRP (telokin) IgG (gift from D. M. Watterson, Northwestern Univ.).
Smooth muscle cells. Smooth muscle cells from pulmonary arteries were grown on 22-mm coverslips coated with poly-D-lysine (0.1 mg/ml, Sigma-Aldrich). The cells were then fixed for 10 min with formaldehyde, washed three times in PBS (Sigma-Aldrich), incubated for 10 min with 0.1% Triton X-100 in PBS, washed three times in PBS, and then blocked for 30 min with 2% BSA in PBS. The cells were subsequently incubated for 1 h at room temperature with a 1:200 dilution of the primary antibody for MLC [monoclonal antimyosin (light chains 20K) clone MY-21, Sigma-Aldrich], a 1:100 dilution of the telokin antibody, and washed three times in PBS. This was followed by a 1-h incubation in 1:1,000 dilutions of Alexa Fluor 568 goat anti-mouse IgG for MLC, Alexa Fluor 488 goat anti-rabbit IgG for telokin, and washing in PBS. The cell nuclei were stained with TO-PRO-3 iodide (all from Molecular Probes, Eugene, OR) and washed three times with PBS. Negative control experiments were done as above in the absence of both primary and secondary antibodies to verify the absence of autofluorescence. The coverslips were mounted on glass slides using Vectashield mounting media (Vector Laboratories, Burlingame, CA) and sealed. Images were acquired using a laser fluorescence imaging system and a confocal microscope (Nikon). The cells were visualized with a x40 objective, which resulted in a x400 magnification. A krypton-argon laser was used for excitation, and emitted fluorescence was determined after long-pass filtering.
Immunoprecipitation
SPA smooth muscle cells were grown to confluence on two 100-mm dishes. The cells from each dish were lysed with the buffer described below and collected into two eppendorf tubes. The tubes were then centrifuged for 15 min at 14,000 rpm. This and all subsequent centrifugations were done at 4°C. The supernatant (lysate) from each tube was collected, and the protein concentration was determined using a Coomassie blue-based assay (Biorad). Each tube was loaded with 500 µg of total protein, which was precleared to remove IgG antibodies by adding 20 µl of protein A-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). After 1 h of rotation at 4°C, the tubes were centrifuged and the lysate removed. The lysate was incubated overnight with 2 µl of the MLC antibody at 4°C with rotation. Fifty microliters of the protein A-agarose beads were added to this lysate-antibody mixture and incubated for 2 h with rotation. After 5 min of centrifugation at 2,400 rpm, the supernatant was removed, and the beads, which now contained the protein and antibody, were resuspended in PBS. After 2 min of centrifugation at 2,400 rpm, the supernatant was discarded, and 50 µl of loading buffer was added to the beads, which were then heated at 95°C for 5 min. A one dimensional Western blot was then performed as described below. The nitrocellulose membranes were then probed separately with the MLC and telokin antibodies. Data were collected and analyzed using the Kodak image station and software as described below.
One-Dimensional Western Blots
Smooth muscle cells grown to confluence in 60-mm culture dishes were placed on ice and lysed using a 1.5 ml 1% Triton X-100 Lysis Buffer (described below). They were then treated with 30 µl of protease inhibitor cocktail and 1.5 µl of 100x PMSF and centrifuged at 22,600 g and 4°C for 15 min. The supernatant was collected and loaded onto a gel at uniform protein concentration. A Bis-Tris 4–12% 1.5-mm precast gel (NuPAGE; Invitrogen, Carlsbad, CA) was run with a NuPAGE MES SDS Running Buffer and SeeBlue prestained molecular weight standard (both from Invitrogen) at 200 V for 
45 min and then transferred to a 0.22-µm nitrocellulose membrane (BioRad, Hercules, CA) and stained with Ponceau S for total protein load. The membrane was blocked for 30 min with 2% BSA in TBS-T buffer and probed overnight at 4°C with the primary MLC antibody and then for 1 h with the secondary antibody, goat-anti mouse HRP-conjugated antibody (Pierce Biotechnology, Rockford, IL), in 2% dry milk/TBS-T solution. Enhanced chemiluminescent agent (Pierce Biotechnology) was applied, and the membrane was developed on CL-X Posure film (Pierce Biotechnology). The membrane was reprobed with the primary polyclonal telokin antibody and then the secondary antibody, goat-anti rabbit HRP (Pierce Biotechnology). The smooth muscle 
-actin internal standard was probed with the monoclonal anti-actin antibody, clone C4 (MP Biomedicals, Irvine, CA).
Two-Dimensional Western Blots
Cells collected at each time point of an experiment were kept on ice, and total proteins were precipitated using 10% trichloroacetic acid and 50 mM DTT (Sigma-Aldrich). The precipitate was collected into separate eppendorf tubes and centrifuged at 5,600 g at 4°C for 10 min. The resulting pellet was then washed once with 4°C acetone, dried for 10–15 min, and stored at –80°C. The pellet was treated with a buffer containing 100 µg/ml DNase I and 25 µg/ml RNase A (both from Sigma-Aldrich) for 30 min at room temperature. All treatments were done in the presence of a protease inhibitor cocktail for mammalian tissues and phosphatase inhibitor cocktail I (both from Sigma-Aldrich). SDS (BioRad) 0.4% was added and adjusted to 0.1% with a stock rehydration buffer composed of 2.4 M urea, 8.4 M thiourea, and 4.8% CHAPS (UTC rehydration buffer). After 30 min, samples were cup loaded onto immobilized pH gradient (IPG) strips (BioRad) for isoelectric focusing. The strips were presoaked overnight with UTC rehydration buffer supplemented with IPG buffer, pH 3–10, containing 50 mM DTT. The samples were focused at 3,000 V/h for a total of 20,000 V/h. Second-dimension Criterion Bis Tris 4–12% 1.0-mm precast gels (BioRad) were loaded with the focused IPG strips. Before loading, the strips were soaked in an equilibration buffer (described below) for 15 min and then sealed on the top of the gel with 0.5% agarose in a 1:20 dilution of XT MES Running Buffer (BioRad). The gel was run with XT MES Running Buffer and transferred to the nitrocellulose membranes, which were treated as described above for the one-dimensional Western blots. After application of the chemiluminescent solution, the membranes were analyzed on a Kodak imaging station as described below.
To verify that phosphorylated MLC protein was being detected, pulmonary artery smooth muscle cells were grown to 95% confluence in 60-mm dishes and loaded with 800 µl of the macromolecule Chariot complex (Active-Motif, Carlsbad, CA) plus 1 µl of alkaline phosphatase (Amersham Biosciences, Pittsburgh, PA). The dishes were incubated for 2 h at 37°C, lysed, and focused onto two-dimensional strips for Western analysis as described above.
Solutions
The physiological saline solution (PSS) was (in mM): 141 Na+, 4.7 K+, 2.5 Ca2+, 0.72 Mg2+, 124 Cl, 1.7 H2PO4–, 22.5 HCO3–, and 11 glucose.
The Pucks saline solution was (in mM): 0.1 CaCl2·2H2O, 4.7 KCl, 1.18 KH2PO4, 1.19 MgSO4·7H2O, 120 NaCl, 0.12 Na2HPO4·7H2O, 5.5 D-glucose, and 0.013 phenol red (pH 7.34).
For the enzyme solution, the following ingredients were added to 10 ml of PSS: 157 U/ml collagenase (type II), 50 U/ml elastase, 5 U/ml DNase, 1.5% wt/vol BSA, 4 mM ATP, 0.1% wt/vol, soybean trypsin inhibitor, and 1 µM isoproterenol. The solution was maintained at pH 7.4 by gassing with 95% O2-5% CO2. All enzymes were from Worthington Biomedical (Newark, NJ), and all other chemicals were from Sigma-Aldrich.
Triton X-100 lysis buffer (1x) solution included: 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 5% wt/vol glycerol, 1%wt/vol Triton X-100 diluted up to 50 ml with nanopure deionized H2O.
Lysis buffer for immunoprecipitation included: 20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 40 mM β-glycerophosphate, phosphatase inhibitor (Sigma-Aldrich), and protease inhibitor (Roche, Indianapolis, IN).
Equilibration buffer for two-dimensional electrophoresis included: 6 M urea, 30% glycerol; 10X MES, and bromothymal blue.
Gases
Weather balloons were filled with calibrated gas mixtures. The gas was pumped from the balloon through dispersion stones into a PSS-filled reservoir at 37°C that held the culture dishes containing the smooth muscle cells. The gas mixtures were as follows: normoxia (control; PO2 140 Torr, PCO2 37 Torr), hypoxia (PO2 50 Torr, PCO2 37 Torr). The PO2, PCO2, and pH of the PSS were measured with a Radiometer ABL 500 blood gas analyzer.
Experimental Protocols
Cultured SPA and LPA smooth muscle cells grown to confluence in 60-mm-diameter dishes were placed in PSS at 37°C, pH 7.37, and exposed to hypoxia (PO2 <50 mmHg) or PE (1 µM) for 1, 3, and 10 min.
Data Analysis
The nitrocellulose membranes were scanned using a Kodak 2,000 mmT Imaging Station. The images were analyzed and the values quantified using Kodak 1D analysis software. Some images were developed on film, which were scanned using a Hewlett Packard ScanJet ADF scanner and saved as JPEG files. The files were opened in Adobe Photoshop 7.0 and saved in grayscale format. Densitometry was done using the UN-SCAN-IT version 5.1 (Silk Scientific, Orem, UT) program, and the values were used for statistical analysis and graphing. Quantitative data are expressed as means ± SE. Statistical significances were determined by ANOVA with appropriate post hoc tests or Student's t-test where applicable.
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RESULTS |
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Northern blots were performed using total RNA purified from smooth muscle cells of SPA with diameters <500 µm and a mixture of LPA, whose diameters ranged from 600 µm to >1,000 µm. The blots were probed with full-size telokin cDNA, stripped, and subsequently hybridized with a purified GAPDH cDNA probe. As shown in Fig. 1, the SPA smooth muscle cells showed strong expression of RNA for telokin, whereas SPA cells showed only minimal expression. The average pixel intensity of the RNA measured in the SPA cells was eight times greater than that in the LPA. Cerebral artery smooth muscle cells did not express any RNA for telokin (data not shown). Immunoperoxidase staining with and without the monoclonal antibody against recombinant telokin was done in cross sections of SPA and LPA. The SPA showed greater staining for telokin (Fig. 2A) than the LPA (Fig. 2C).
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Figure 3A shows a one-dimensional Western blot of telokin in smooth muscle cells from four sizes of cat pulmonary arteries: SPA (<500 µm), LPAsm (600–800 µm), LPAmed (800–1,000 µm), and LPAlg (>1,000 µm). Figure 3B shows a histogram of these data expressed as the ratio of the pixel intensity of telokin to the pixel intensity of the -actin standard. The SPA cells expressed significantly more telokin than both LPAmed and LPAlg cells (P < 0.05). Although the telokin expression in SPA was higher than in LPAsm cells, the difference was not statistically significant. Among the LPA cells, both LPAsm and LPAmed expressed significantly more telokin than LPAlg (P < 0.05). In keeping with the absence of RNA expression for telokin, the cerebral artery smooth muscle cells were devoid of telokin protein (Fig. 3A).
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As shown in Fig. 7A, exposing the SPA smooth muscle cells to acute hypoxia resulted in a shift of telokin from the phosphorylated state [isoelectric point (PI) of 4.1 at time 0] to the dephosphorylated state (PI of 4.3 at 1 min and thereafter). At the same time, MLC became increasingly phosphorylated. The distance measured between unphosphorylated myosin, which remained constant throughout the experiment, and telokin decreased significantly between time 0 and 10 min of hypoxia (P < 0.01; n = 6). The quantified values for phosphorylated MLC divided by the sum total of phosphorylated and unphosphorylated MLC increased from 0.83 ± 0.05 under normoxic conditions to 0.92 ± 0.04 after 10 min of hypoxia (P < 0.05; n = 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In previous studies, the telokin content of arteries, which are composed of tonic smooth muscle, was generally lower than that measured in phasic smooth muscle. However, these studies used large-diameter vessels such as aorta (9) and femoral artery (12, 22). Our finding of a low telokin content in the LPA mirrors these findings. The higher telokin levels in the SPA suggests that not all arterial smooth muscle has a low telokin content and that within the same vascular bed, telokin can be differentially expressed. This finding is not entirely unexpected since smooth muscle is derived from diverse embryological sources (4), and many different and distinct nuclear factors regulate vascular smooth muscle development and differentiation. Indeed, diverse phenotypes and functions among vascular smooth muscle cells (1), and in particular within pulmonary artery smooth muscle cells (2, 5, 19), have been documented. The present study did not examine any characteristics of the pulmonary artery smooth muscle cells other than the size of their vessel of origin, but it is reasonable to expect that differences, particularly in signaling mechanisms, could occur and that they might contribute to the differences in telokin expression and/or function.
At this point, the functional consequence of the differential expression of telokin in the cat pulmonary vasculature is not clear. However, a teleological argument could be made that under normal, unstimulated conditions the SPA would be more likely to remain relatively relaxed, due perhaps in part to phosphorylated telokin, and blood flow to the alveoli would be optimized. As mentioned previously, the lower telokin content of the LPA is similar to the relatively low amounts found in other large-diameter vessels. How a lower telokin content relates to tone and/or function in large-diameter vessels is unknown. In the case of the cat cerebral arteries, the total absence of telokin mRNA and telokin protein agrees with the finding of Wu et al. (22) in rabbit cerebral arteries. Whether the absence of telokin in cerebral arteries is related to the necessity of this vasculature to change diameter instantaneously in response to changes in pressure, blood flow, and brain metabolism also remains to be determined.
Immunostaining for telokin in unstimulated SPA smooth muscle cells showed that it was uniformly distributed throughout the cell and what appeared to be colocalization with MLC (Fig. 5). However, subsequent coimmunoprecipitation experiments showed that the two were not associated (Fig. 6). This is consistent with a previous report that while telokin and myosin appeared to be colocalized within a cell, binding activity between the two was weak (10). The effect of contractile agonists on telokin localization in tonic smooth muscle has not been determined. However, in tracheal muscle, which is phasic, sodium nitroprusside activation of the cGMP pathway resulted in telokin being translocated to the near plasma membrane (10). Thus the cellular location of telokin may depend on the presence or absence of relaxing or contracting agonists.
It is generally conceded that telokin exerts its effect on both phasic and tonic smooth muscle through activation of myosin phosphatase (3), but how differences in intracellular signaling mechanisms modulate telokin activity remains to be determined. To date, most studies have studied telokin with respect to its role in smooth muscle cell relaxation. Increasing telokin phosphorylation through activation of cyclic nucleotides and the involvement of PKA and PKG protein kinases results in smooth muscle cell relaxation. In this study, we looked at whether telokin dephosphorylated in pulmonary artery smooth muscle cells when they were exposed to a contractile agonist. During hypoxia, which evokes contraction of SPA smooth muscle cells (16), telokin phosphorylation did decrease and MLC phosphorylation increased. What triggered the dephosphorylation is not known, but it would seem reasonable to speculate that decreases in cAMP and/or cGMP levels in the SPA cells as well as possible additional contributions from other pathways involved in hypoxic pulmonary vasoconstriction could be involved. In contrast, in LPAmed smooth muscle cells, exposure to hypoxia did not affect the phosphorylation status of either telokin or MLC. The absence of MLC phosphorylation during hypoxia in this work is consistent with our previous studies showing neither contraction nor MLC phosphorylation during hypoxia in cat LPA or their smooth muscle cells (13, 16). When the LPA smooth muscle cells were exposed to PE, MLC phosphorylation increased while the phosphorylation status of telokin remained unchanged. There are several reasons why this may have occurred. First, the small amount of telokin present in LPA smooth muscle cells may be insufficient to be effective. Second, telokin cannot be considered an all-or-none inhibitor. Rather, by reducing the level of MLC phosphorylation, it functions as a smooth muscle relaxing factor (21). Third, telokin is neither a phosphatase itself nor a direct inhibitor of MLCK. It enhances myosin phosphatase activity (9). A strong agonist like PE produces a large, transient increase in intracellular Ca2+. This results in full activation of MLCK, MLC phosphorylation, and contraction, even in the presence of telokin. Aortas from wild-type mice, which contain little telokin, and aortas from telokin-deficient animals showed no difference in their Ca2+-force relationships (9). In the comparatively telokin-deficient LPA smooth muscle cells then, the increased intracellular Ca2+ evoked by PE would be more than sufficient to result in contraction. Once contraction to an agonist has occurred, the transient increase in intracellular Ca2+ declines and myosin phosphatase activity increases. In the presence of sufficient telokin, as in the case of the SPA smooth muscle cells, MLC phosphorylation would be retarded and its dephosphorylation accelerated. Different influences on smooth muscle contraction emphasize the fact that while telokin may help to maintain the SPA in a relaxed state under unstimulated conditions, during hypoxia, other contractile processes may dominate.
It should be noted that until the data obtained in this study are verified in the pulmonary vasculature of other species, it could be that they are peculiar to the cat. In the rat, both SPA and LPA have been shown to constrict to hypoxia (23). Mechanisms that help regulate contractility in the cat pulmonary arteries may differ from those in the rat and/or other species as well. Finally, the mechanisms underlying how contractile agonists affect telokin in smooth muscle cells deserve further study since regulation of telokin content and/or function may offer possible therapeutic opportunities to treat diseases involving the vasculature.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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