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Am J Physiol Renal Physiol 294: F1487-F1492, 2008. First published April 9, 2008; doi:10.1152/ajprenal.00060.2008
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INNOVATIVE METHODOLOGY

A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles

Kosuke Takeya,1,2 Kathy Loutzenhiser,1 Mitsuya Shiraishi,2 Rodger Loutzenhiser,1 and Michael P. Walsh2

Departments of 1Pharmacology and Therapeutics and 2Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Smooth Muscle Research Group, Calgary, Alberta, Canada

Submitted 4 February 2008 ; accepted in final form 2 April 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Phosphorylation of the 20-kDa myosin regulatory light chains (LC20) plays a key role in the regulation of smooth muscle contraction. The level of LC20 phosphorylation is governed by the relative activities of myosin light chain kinase and phosphatase pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of LC20 phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure LC20 phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound Mn2+-phosphate-binding tag and enhanced Western blot analysis, we were able to detect LC20 phosphorylation using as little as 5 pg (250 amol) of isolated LC20. Phosphorylated and unphosphorylated LC20 were detected in single isolated afferent arterioles, and LC20 phosphorylation levels could be accurately quantified in pooled samples of three arterioles (<300 cells). The phosphorylation level of LC20 in the afferent arteriole was 6.8 ± 1.7% under basal conditions and increased to 34.7 ± 5.1% and 44.6 ± 6.6% in response to 30 mM KCl and 10–8 M angiotensin II, respectively. The application of this technique will enable investigations of the different determinants of LC20 phosphorylation in afferent and efferent arterioles and provide insights into the signaling pathways that regulate LC20 phosphorylation in the renal microvasculature under physiological and pathophysiological conditions.

afferent arteriole; Phos-tag SDS-PAGE

THE RESISTANCE VESSELS IN the kidney, the interlobular artery, and the afferent and efferent arterioles exhibit a remarkable level of heterogeneity regarding the mechanisms regulating vascular tone. Knowledge of these differences is critical regarding developing a more complete understanding of renal hemodynamic control and insights into the impact of pathophysiological processes on renal microvascular function. Phosphorylation of the 20-kDa myosin regulatory light chain (LC20) is the primary biochemical signal initiating cross-bridge cycling in smooth muscle, and this key reaction plays a pivotal role in the regulation of arterial tone, at least in the larger vessels (4, 16). However, little is known about LC20 phosphorylation in smaller vessels, such as renal arterioles, because of the lack of a sufficiently sensitive method to quantify LC20 phosphorylation in minute samples. The isolated afferent and efferent arterioles, consisting of < 100 cells each, are approximately one-tenth the size of a human eyelash (10–20 µm in diameter) and one afferent arteriole contains ~50 pg (2.5 fmol) of LC20, well below the sensitivity of conventional assays (~0.3 ng or 15 fmol) (13).

Several techniques have been developed to detect and quantify LC20 phosphorylation, including urea/glycerol-PAGE (15, 20), isoelectric focusing (5, 14), and 2D-gel electrophoresis (1, 7), combined with Coomassie blue, colloidal gold, or silver staining, Western blot analysis, or radioisotope (32P) labeling. Although these techniques are suitable when a large amount of sample is available, as in the case of mid- to large-diameter arteries, their relatively low sensitivity precludes their utility in analyzing minute samples, as in the case of the renal microvasculature. We recently reported a novel technique using capillary isoelectric focusing and laser-induced fluorescence to separate, detect, and quantify phosphorylated and nonphosphorylated LC20 (13). Although highly sensitive (~1-pg detection limit), the application of this approach to assays of arteriolar LC20 proved problematic in that the LC20 must be isolated and concentrated prior to loading on the isoelectric focusing capillary (see below).

In the present study, we developed a new approach, based on SDS-PAGE with polyacrylamide-bound Mn2+-phosphate-binding tag (Phos-tag SDS-PAGE) (6), to separate LC20 species on the basis of their phosphorylation state, followed by detection of the separated LC20 species by Western blot analysis. A number of modifications were introduced to optimize sample retention during Western blot analysis and to improve detection and quantification using enhanced chemiluminescence. Using this new approach, LC20 phosphorylation could be reliably detected and accurately quantified in a small number of pooled, isolated afferent arterioles.


   METHODS
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 ABSTRACT
 METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of afferent arterioles. Renal afferent arterioles were isolated from Wistar rats (200–225 g) as described previously (12). The protocol for this study was approved by the University of Calgary Animal Care Committee. In brief, rats were anesthetized with halothane. The left kidney was perfused in vivo with warmed Ca2+-free atmospheric MEM (ATM MEM) composed of (in mM) 102.7 NaCl, 5.4 KCl, 1.7 NaH2PO4, 0.8 MgSO4, 5.6 HEPES, 5.6 glucose, 1.0 sodium pyruvate, 4.2 NaHCO3, and 30 sodium isethionic acid, and 1x MEM vitamin solution (Sigma), 1x MEM nonessential amino acid solution (Sigma), and 1x MEM amino acid solution (Sigma) containing 10–5 M diltiazem. The kidney was perfused with 1.5% Seaprep agarose (Cambrex) in ATM MEM (37°C), excised, and chilled (4 °C) to solidify the agarose. Cortical slices (~400 µm) were treated with 400 U/ml collagenase IV (Sigma), 0.5 U/ml dispase II (Roche), 18 µg/ml DNase I (Roche), and 0.1 mg/ml trypsin inhibitor I-S (Sigma) in ATM MEM at 37°C for 30 min to separate microvessels from tubules. Individual vessels were isolated using a dual-pipette system. Since Cl– current plays an important role in renal vascular contractions (17), isethionate was included as a counterion to Na+ to maintain the Cl– concentration within the physiological range.

Stimulation of isolated afferent arterioles. Isolated vessels were incubated in 100 µl of modified MEM containing (in mM) 1.5 CaCl2, 102.7 NaCl, 5.4 KCl, 1.7 NaH2PO4, 0.8 MgSO4, 5.6 HEPES, 5.6 glucose, 1.0 sodium pyruvate, 26.2 NaHCO3, 8 sodium isethionic acid, and 1x MEM vitamin solution, 1x MEM nonessential amino acid solution and 1x MEM amino acid solution. Ibuprofen (10–5 M) was added to eliminate the potential effect of prostaglandin E2 on LC20 phosphorylation (19). Vessels were treated with 1 U/ml agarase (Sigma) in modified MEM at 37°C for 10 min while gassing with 5% CO2-95% air prior to stimulation. Vessels were stimulated by adding 1 µl of 10–6 M angiotensin II (Sigma, 10–8 M final concentration) or 2.5 M KCl (30 mM final concentration), incubated at 37°C for 5 min while gassing with 5% CO2, 95% air and quick frozen by adding 300 µl of dry ice cold 10% TCA, 10 mM DTT in acetone. One microliter of 0.1 µg/µl bovine serum albumin was added to coprecipitate denatured proteins. Samples were placed at –20°C overnight and then centrifuged at 13,000 g for 10 min at 4°C. After the supernatant was removed, the pellets were washed 2–3 times with 400 µl of cold acetone (–20°C) containing 10 mM DTT and air dried.

Stimulation of skinned rat tail arterial smooth muscle strips. Rat tail artery was dissected and smooth muscle strips were prepared as described previously (22). Triton X-100 skinned strips were treated with 10–9 M Ca2+ (pCa 9), 10–4.5 M Ca2+ (pCa 4.5), or 1 µM microcystin LR (Alexis Biochemical) at pCa 9. The pCa 9 solution contained (in mM) 20 TES, 4 K2EGTA, 5.83 MgCl2, 7.56 potassium propionate, 1 NaN3, 3.9 Na2ATP, 0.5 dithioerythritol, and 16.2 phosphocreatine, and 15 U/ml creatine kinase, pH 6.9. The pCa 4.5 solution contained (in mM) 20 TES, 4 CaEGTA, 5.66 MgCl2, 7.53 potassium propionate, 1 NaN3, 3.9 Na2ATP, 0.5 dithioerythritol, and 16.2 phosphocreatine, and 15 U/ml creatine kinase, pH 6.9. Tissue samples were quick frozen in dry ice cold 10% TCA and 10 mM DTT in acetone at the plateau of the contractile response. Strips were washed with dry ice cold acetone containing 10 mM DTT and lyophilized overnight.

Measurement of LC20 phosphorylation. LC20 was extracted from afferent arteriole and tail artery in 20 or 200 µl, respectively, of SDS gel sample buffer [4% (wt/vol) SDS, 100 mM DTT, 5% glycerol, 0.04% (wt/vol) Bromphenol Blue, 65 mM Tris·HCl, pH 6.8] by constant shaking in a microcentrifuge tube for 2 h at room temperature and sonication for 10 min. Samples were heated to 95°C for 5 min prior to electrophoresis. Unphosphorylated, monophosphorylated, and diphosphorylated LC20 were separated by Phos-tag SDS-PAGE (6) with some modifications. The stacking gel was composed of 4.35% (wt/vol) acrylamide, 0.15% (wt/vol) N,N'-methylenebisacrylamide, 0.1% (wt/vol) SDS, 125 mM Tris·HCl, pH 6.8, 0.1% (wt/vol) ammonium persulfate, and 0.17% N,N,N',N'-tetramethylethylenediamine. The resolving gel was composed of 9.7% (wt/vol) acrylamide, 0.34% (wt/vol) N,N'-methylenebisacrylamide, 30 µM Phos-tag acrylamide (NARD Institute), 60 µM MnCl2, 0.1% (wt/vol) SDS, 375 mM Tris·HCl, pH 8.8, 0.05% (wt/vol) ammonium persulfate, and 0.07% N,N,N',N'-tetramethylethylenediamine. Electrophoresis was performed in 0.1% (wt/vol) SDS, 25 mM Tris, and 192 mM glycine at 20 mA until Bromphenol Blue ran off the gel. After electrophoresis, the gel was soaked in 2 mM EDTA, 25 mM Tris, 192 mM glycine for 15 min, and then in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) for 15 min. Proteins were transferred to PVDF membrane (0.45-µm pore size, Roche) overnight at 27 V and 4 °C.

The following steps were performed at room temperature unless otherwise indicated. Blotted membranes were washed in PBS for 5 min. LC20 was cross-linked and fixed on the membrane by soaking in 0.5% glutaraldehyde in PBS for 45 min. After being washed in 150 mM NaCl and 25 mM Tris·HCl, pH 7.5 (TBS), the membrane was blocked with 0.3% (wt/vol) I-Block (Tropix) in TBS containing 0.05% Tween-20 (TBST) for 1.5 h. All forms of LC20 were detected using rabbit anti-LC20 (Santa Cruz Biotechnology, Santa Cruz, CA) and phosphorylated forms of LC20 using phospho-specific anti-LC20 (Rockland). In two-step Western blot analysis, the primary (rabbit anti-LC20) and secondary antibodies [horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG; Pierce] were diluted in 1% normal goat serum in TBST at 200- to 1,000- and 10,000-fold, respectively. In three-step Western blot analysis, the primary (rabbit anti-LC20) and secondary antibodies (biotin-conjugated goat anti-rabbit IgG; Chemicon) and HRP-conjugated streptavidin (Pierce) were diluted in 1% normal goat serum in TBST at 200- to 1,000-, 40,000-, and 200,000-fold, respectively. Incubation with the primary antibody was overnight at 4°C, with the secondary antibodies was for 1 h at room temperature, and with HRP-streptavidin was for 30 min at room temperature. Membranes were washed for 4 x 5 min with TBST (0.02%) after these incubations. HRP was detected with the SuperSignal West Femto reagent (Pierce), and the emitted light was detected and quantified with a chemiluminescence imaging analyzer (LAS3000mini; Fujifilm). Obtained images were analyzed with Multi-Gauge version 3.0 software (Fujifilm).

Fixation of LC20 on PVDF membrane. Purified LC20 (1 ng) was loaded per lane of a 12.5% acrylamide SDS minislab gel and electrophoresed at 200 V for 45 min. Proteins were transferred to PVDF membrane at 27 V overnight at 4°C and washed (2 x 5 min) in distilled, deionized water. Individual lanes were cut out of the membrane and incubated in: 1) water, 2) 0.1% Ponceau S in 5% acetic acid for 5 min, 3) 0.5% formaldehyde for 45 min, or 4) 0.25% glutaraldehyde for 45 min. Membranes were then washed (2 x 5 min) in water and blocked by incubation in 0.5% I-Block in TBST for 1 h at room temperature prior to incubation with the primary antibody (anti-LC20 at 1:3,000 dilution in 0.5% I-Block in TBST) for 1 h at room temperature. After washing (6 x 5 min) with TBST, membranes were incubated with secondary antibody (goat anti-rabbit IgG-HRP conjugate (Chemicon) diluted 1:40,000 in 0.5% I-Block in TBST) for 1 h at room temperature prior to washing (6 x 5 min) with TBST. LC20 bands were detected by enhanced chemiluminescence using the Super Signal West Femto reagent (Pierce) and exposure to X-Omat Blue XB-1 film (Kodak) for 2 min.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Measurement of LC20 phosphorylation in smooth muscle tissues by capillary isoelectric focusing with laser-induced fluorescence detection. We previously reported the development of a capillary isoelectric focusing technique utilizing laser-induced fluorescence to quantify phosphorylation levels in very small samples of LC20 (13). This approach enabled measurement of phosphorylation with as little as 1 pg (50 amol) of purified LC20. We then applied this technique to measure LC20 phosphorylation in intact tissue samples. While this approach proved to be reliable when using large tissue samples, such as rat tail artery (data not shown), the method failed to detect LC20 in small arterioles due to high background signals. The background noise appeared to be introduced during the concentration step. Since our efforts to eliminate this problem were unsuccessful, we shifted our attention to enhance the sensitivity of electrophoretic techniques coupled with enhanced chemiluminescence detection.

Measurement of LC20 phosphorylation by Phos-tag SDS-PAGE. One of the reasons for the low sensitivity of the current methods for quantification of LC20 phosphorylation (urea/glycerol-PAGE, 2D-gel electrophoresis, and isoelectric focusing) appears to be that LC20, particularly at low concentrations, tends to aggregate in the presence of urea and fails to enter the gel. To overcome this problem, we exploited the recently described Phos-tag SDS-PAGE (6), which utilizes SDS as the denaturing agent. Thus the solubility and extraction efficiency of LC20 and other proteins of interest are expected to increase. Furthermore, SDS prevents nonspecific interactions of LC20 with acrylamide gel, which causes smearing of LC20 during urea/glycerol-PAGE.

To verify the ability of Phos-tag SDS-PAGE to separate phosphorylated and unphosphorylated forms of LC20 from tissue samples, we first examined LC20 phosphorylation in rat tail artery. Skinned rat tail arterial strips were maintained at pCa 9 (relaxed) or contracted by Ca2+ (pCa 4.5) or the phosphatase inhibitor microcystin (at pCa 9) (see METHODS). Tissue proteins were extracted with SDS containing sample buffer and subjected to Phos-tag SDS-PAGE and Western blot analysis. Up to three bands were detected with anti-LC20, which recognizes phosphorylated and unphosphorylated forms of the protein (Fig. 1A). Considering that a phosphorylated protein migrates more slowly than its unphosphorylated counterpart on a Phos-tag gel (6) and the unphosphorylated LC20 was predominant in pCa 9-treated rat tail artery (20–22), the fastest-migrating band is concluded to be unphosphorylated LC20 (Fig. 1A, lane 1). The intensity of the signal corresponding to the middle band increased in response to an increase in [Ca2+] to pCa 4.5, suggesting that this band corresponds to monophosphorylated LC20 (Fig. 1A, lane 2). It has been shown that LC20 is phosphorylated at Ser19 and Thr18 in the presence of the phosphatase inhibitor, microcystin (20, 21). Therefore, the slowest-migrating band observed in microcystin-treated rat tail artery (Fig. 1A, lane 3) can be identified as diphosphorylated LC20. These conclusions were confirmed by Western blot analysis with phospho-specific anti-LC20, which recognizes LC20 only when phosphorylated at Ser19. The phospho-specific antibody detected only the upper two bands, confirming that the lowest band corresponds to unphosphorylated LC20 and the upper two correspond to mono- and diphosphorylated species, each containing phosphoserine-19 (Fig. 1B). Western blotting with the phospho-specific antibody gave stronger signals (Fig. 1B, lanes 2 and 3) than did the pan-LC20 antibody (Fig. 1A, lanes 2 and 3), indicating a greater avidity of the phospho-specific antibody. It is also apparent that the phospho-specific anti-LC20 has a higher avidity for diphosphorylated than for monophosphorylated LC20.


Figure 1
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  		Fig. 1. Separation of unphosphorylated (0P), monophosphorylated (1P), and diphosphorylated (2P) 20-kDa myosin regulatory light chain (LC20) by Phos-tag SDS-PAGE. Skinned rat tail arterial strips were treated with 10–9 M Ca2+ (pCa 9), 10–4.5 M Ca2+ (pCa 4.5), or 1 µM microcystin at pCa 9 and analyzed by Phos-tag SDS-PAGE and Western blot analysis with anti-LC20 (A) or phospho-specific anti-LC20 (B).

 
To analyze LC20 phosphorylation in a minute amount of sample, such as an afferent arteriole, we improved the sensitivity of Western blot analysis by optimizing the conditions as follows. Since small proteins can be easily removed from a PVDF membrane in the presence of Tween-20, LC20 was fixed on the membrane by cross-linking with glutaraldehyde (3, 18), and the Tween-20 concentration in the washing solution (TBST) was reduced to 0.02% (Fig. 2). Incubating the membrane with primary and secondary antibodies in the presence of 1–5% normal serum further increased the signal intensity (data not shown).


Figure 2
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  		Fig. 2. Fixation of LC20 to PVDF membrane markedly improves its retention. LC20 was subjected to SDS-PAGE and Western blot analysis as described in METHODS. Following protein transfer to PVDF, the membrane was cut into lanes that, prior to blocking, were either untreated (lane 1), treated with Ponceau S in acetic acid for 5 min (lane 2), treated with 0.5% formaldehyde in PBS for 45 min (lane 3), or treated with 0.25% glutaraldehyde in PBS for 45 min (lane 4).

 
Figure 3 establishes the sensitivity of this Western blot analysis procedure using isolated unphosphorylated and phosphorylated LC20s. A dilution series of a mixture of unphosphorylated and phosphorylated LC20s (5–160 pg of each) were subjected to Phos-tag SDS-PAGE and detected by standard two-step (Fig. 3A) and the more sensitive three-step (Fig. 3B) Western blot analysis, which incorporates a streptavidin-biotin amplification step. Increasing the contrast of the image enhanced the LC20 bands (Fig. 3C), but did not match the accuracy of quantification achieved with the three-step procedure (Fig. 3B) in which as little as 5 pg of LC20 was detected, as shown by quantitative analysis of the data (Fig. 3, D and E). The images captured by the cooled charge-coupled device camera were analyzed, and the intensity of LC20 bands was quantified (Fig. 3, D and E). In two-step Western blot analysis, the percent phosphorylation deviated from the expected value at low loading levels because of the inhomogeneous background noise (Fig. 3D, bottom). Three-step Western blot analysis (Fig. 3E) increased the signal-to-noise ratio, resulting in a linear relationship even at the lowest loading levels. The detection limit for LC20 by enhanced Western blot analysis with Phos-tag SDS-PAGE (~5 pg) was significantly lower than with standard SDS-PAGE/Western blot analysis (100–200 pg) (13) or urea/glycerol-PAGE (~500 pg).


Figure 3
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  		Fig. 3. Detection sensitivity of enhanced Western blot analysis with Phos-tag SDS-PAGE: Isolated LC20. A dilution series of a mixture of unphosphorylated and phosphorylated LC20s (5–160 pg of each), prepared as previously described (20), was separated by Phos-tag SDS-PAGE and detected by enhanced 2-step (A and C) or 3-step (B) Western blot analysis. The images were captured by an LAS-3000mini (Fujifilm) with "super" sensitivity (A and C) or "high" sensitivity (B). The contrast of the image in C was enhanced compared with that in A. Charge-coupled device (CCD)-captured images were analyzed, and the LC20 signal intensity was measured using Multi-Gauge version 3.0 software (D and E). The signal intensity of unphosphorylated (bullet) and phosphorylated LC20 ({circ}) was plotted against the loaded amount (D and E). The value of %phosphorylation ({triangleup}) was calculated as follows: %phosphorylation = phosphorylated LC20/(unphosphorylated LC20 + phosphorylated LC20) x 100. In 2-step Western blot analysis (D), the %phosphorylation value deviated from the expected value at the lowest loading levels. Three-step Western blot analysis provided more consistent measures of phosphorylation stoichiometry (E).

 
We next used Phos-tag SDS-PAGE with enhanced Western blot analysis to quantify LC20 phosphorylation in afferent arterioles (Fig. 4). A dilution series of pooled angiotensin II-treated afferent arterioles (equivalent to 1–6 vessels) was loaded onto a Phos-tag gel and examined by three-step Western blot analysis (Fig. 4A, lanes 1–4). The unphosphorylated and monophosphorylated LC20 bands were detected in all samples down to one vessel. It should be noted, however, that the percent phosphorylation (Fig. 4B, bottom) deviated from the actual value at low loading levels. We conclude that at least three pooled vessels are required for reliable quantitative analysis (Fig. 4A, lanes 5 and 6). Western blotting with phospho-specific anti-LC20 confirmed that the upper band corresponded to monophosphorylated LC20 (data not shown).


Figure 4
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  		Fig. 4. Detection sensitivity of enhanced Western blot analysis with Phos-tag SDS-PAGE: Afferent arterioles. A dilution series of 10–8 M ANG II-treated afferent arterioles (equivalent to 1–6 vessels; lanes 1–4) was subjected to Phos-tag SDS-PAGE, and LC20 was detected by enhanced 3-step Western blot analysis (A). In addition, pooled samples of 3 afferent arterioles (untreated; lane 6) or treated with 10–8 M ANG II (lane 5) were similarly analyzed. CCD-captured images were analyzed, and the LC20 signal intensity was measured using Multi-Gauge version 3.0 software. The signal intensity of unphosphorylated (bullet) and phosphorylated LC20 ({circ}) was plotted against the loaded amount (B). Even though the LC20 bands were detected in as little as 1 vessel, the %phosphorylation values ({triangleup}) deviated from the actual value at the lowest loading levels.

 
Considering that the detection limit of the three-step Western blot analysis procedure was ~5 pg of LC20, and the estimated amount of LC20 in an isolated afferent arteriole is 50 pg (13), one vessel should be enough to quantify LC20 phosphorylation reliably. However, as indicated above, three vessels are required for accurate quantitative analysis. This may be because some of the LC20 is lost during TCA/acetone precipitation or is not fully recovered from the pellet. There will also be losses during electrophoresis and transfer to PVDF membrane. If the recovery of LC20 can be increased, it should be possible to quantify LC20 phosphorylation in a single vessel.

Finally, LC20 phosphorylation was measured under resting conditions and in response to two contractile stimuli: 30 mM KCl and 10–8 M angiotensin II (Fig. 5). The phosphorylation level in resting vessels was 6.8 ± 1.7%. This was increased after stimulation with 30 mM KCl or 10–8 M angiotensin II to 34.7 ± 5.1% and 44.6 ± 6.6%, respectively. Since these stimuli cause constriction of afferent arterioles (10, 11), the observed increases in LC20 phosphorylation level support a key role for myosin phosphorylation in the regulation of afferent arteriolar vasoconstriction, as is well established for larger vessels. Diphosphorylated LC20 was not detected, indicating that LC20 phosphorylation occurred exclusively at Ser19 in response to either KCl or angiotensin II.


Figure 5
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  		Fig. 5. LC20 phosphorylation in afferent arterioles in response to contractile stimuli. Isolated afferent arterioles (3–5 vessels per condition) were stimulated with 30 mM KCl or 10–8 M ANG II for 5 min, and LC20 phosphorylation was measured by Phos-tag SDS-PAGE and enhanced Western blot analysis (3-step). In the absence of any stimulus (basal), the phosphorylation level was 6.8 ± 1.7% (n = 4). The phosphorylation level increased to 34.7 ± 5.1% (n = 5) following treatment with 30 mM KCl (KCl) and to 44.6 ± 6.6% (n = 3) following treatment with 10–8 M ANG II. Data are expressed as the means ± SE. *Statistically significant differences from the basal value with P < 0.05 (Student's t-test).

 
In summary, we developed a novel technique that combines Phos-tag SDS-PAGE with high-sensitivity Western blot analysis to quantify myosin LC20 phosphorylation in tissue samples. Following separation of tissue proteins by Phos-tag SDS-PAGE and transfer to PVDF, they are cross-linked to the membrane with 0.5% glutaraldehyde. The concentration of Tween-20 in washing solutions was 0.02% and 1% normal serum was included in primary and secondary antibody solutions. A three-step Western blot analysis procedure that incorporated a streptavidin-biotin amplification step provided the greatest sensitivity of detection of phosphorylated and unphosphorylated LC20 species. This technique is applicable to a variety of vascular and other smooth muscles from microvessels to large arteries. It should also be suitable for analyzing LC20 phosphorylation in nonmuscle cell systems where myosin expression levels are much lower than they are in smooth muscles. LC20 phosphorylation has been implicated in nonmuscle motile events, such as cytokinesis and cell migration (4). Furthermore, this technique could be adapted for analysis of other phosphoproteins, such as PKC-potentiated inhibitor protein-17 (2, 8, 9) and calponin (23, 24), in the same smooth muscle tissue samples. Finally, this approach has the advantages of being considerably cheaper and easier to perform than capillary isoelectric focusing with laser-induced fluorescence detection.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Canadian Institutes of Health Research Grants MT14075 (to R. Loutzenhiser) and MT13101 (to M. P. Walsh).


   ACKNOWLEDGMENTS
 
M. Shiraishi was a recipient of Fellowships from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR). R. Loutzenhiser and M. P. Walsh are AHFMR Scientists and M. P. Walsh is holder of a Canada Research Chair (Tier 1) in Vascular Smooth Muscle Research. We thank Cindy Sutherland for expert technical assistance.

Present address of M. Shiraishi: Department of Veterinary Pharmacology, Faculty of Agriculture, Kagoshima University, Kagoshima. 890-065, Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Takeya, Univ. of Calgary Faculty of Medicine, Smooth Muscle Research Group, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada (e-mail: ktakeya{at}ucalgary.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 

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