K+ depolarization induces RhoA kinase translocation to caveolae and Ca2+ sensitization of arterial muscle

Nicole H. Urban, Krystina M. Berg, and Paul H. Ratz

Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23501

Submitted 29 October 2002 ; accepted in final form 22 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
KCl causes smooth muscle contraction by elevating intracellular free Ca2+, whereas receptor stimulation activates an additional mechanism, termed Ca2+ sensitization, that can involve activation of RhoA-associated kinase (ROK) and PKC. However, recent studies support the hypothesis that KCl may also increase Ca2+ sensitivity. Our data showed that the PKC inhibitor GF-109203X did not, whereas the ROK inhibitor Y-27632 did, inhibit KCl-induced tonic (5 min) force and myosin light chain (MLC) phosphorylation in rabbit artery. Y-27632 also inhibited BAY K 8644- and ionomycin-induced MLC phosphorylation and force but did not inhibit KCl-induced Ca2+ entry or peak (~15 s) force. Moreover, KCl and BAY K 8644 nearly doubled the amount of ROK colocalized to caveolae at 30 s, a time that preceded inhibition of force by Y-27632. Colocalization was not inhibited by Y-27632 but was abolished by nifedipine and the calmodulin blocker trifluoperazine. These data support the hypothesis that KCl caused Ca2+ sensitization via ROK activation. We discuss a novel model for ROK activation involving translocation to caveolae that is dependent on Ca2+ entry and involves Ca2+-calmodulin activation.

vascular smooth muscle; signal transduction; caveolin; Y-27632; confocal microscopy


TO ACHIEVE SMOOTH MUSCLE CONTRACTION, many stimuli activate subcellular signaling systems that mobilize Ca2+ from extracellular and intracellular stores, resulting in an elevation in intracellular free Ca2+ concentration ([Ca2+]i) (for review see Ref. 14). An elevation in [Ca2+]i increases myosin light chain (MLC) kinase activity, MLC phosphorylation, and cross-bridge cycling, resulting in the production of contractile force (for review see Ref. 26). However, the regulation of contraction cannot be fully explained by Ca2+ activation of MLC kinase in all smooth muscle types (11, 24; for review see Ref. 37). A second smooth muscle regulatory system that is activated by plasma membrane G protein-coupled receptor stimulation and involves sensitization of contractile proteins to Ca2+ is known to be responsible for an elevation in the degree of contractile force without further increases in [Ca2+]i (8, 19, 27).

Ca2+ sensitization of smooth muscle produced on receptor stimulation involves a decrease in MLC phosphatase activity mediated through the actions of protein kinase C (PKC) and RhoA-associated kinase (ROK) (for review see Ref. 39). ROK and PKC inhibit MLC phosphatase by phosphorylating MLC phosphatase targeting subunit (MYPT1) and CPI-17, respectively, resulting in an increase in MLC phosphorylation and, thus, contractile force (6, 7, 48).

K+ depolarization (KCl) of smooth muscle causes an increase in [Ca2+]i and muscle contraction. For this reason, KCl is often used to induce contractile protein activation independently of plasma membrane receptor activation, and the degree of Ca2+ sensitization produced by stimulation of a particular receptor is based on the steepness of the resulting force-[Ca2+]i relation compared with that produced on stimulation with KCl (for review see Ref. 17). However, Yanagisawa and Okada (50) proposed that KCl also increases the Ca2+ sensitivity of arteries. This hypothesis is supported by work from our laboratory showing that KCl-induced contraction can be desensitized (30, 32) and that the degree of Ca2+ sensitivity for KCl and a receptor agonist, as determined by the steepness of each force-[Ca2+]i relation, is dependent on the history of receptor activation (31). Additionally, a very recent study using the ROK inhibitor Y-27632 supports the proposal that KCl activates ROK to induce an increase in Ca2+ sensitivity in rat caudal artery (22).

Ca2+ sensitization involves the coordination of complex signal transduction events at the plasma membrane and contractile apparatus (for review see Ref. 9). Caveolae, invaginations of the plasma membrane found in many cell types including smooth muscle cells, are proposed to integrate complex cell signaling systems (for review see Refs. 2 and 36). Muscarinic receptor stimulation has been shown to cause the translocation of RhoA, its effector, ROK, and PKC to the smooth muscle cell periphery, and introduction of a peptide corresponding to the scaffolding domain of caveolin-1 inhibits this translocation (44, 45). These studies suggest that caveolae play a role in coordinating Ca2+ sensitization elicited by receptor agonists in smooth muscle. Although KCl appears to cause Ca2+ sensitization, whether a component of KCl-induced contraction is dependent on the recruitment of signaling molecules to caveolae at the plasma membrane of smooth muscle cells remains to be determined.

We tested the hypothesis that KCl induces Ca2+ sensitization via the activation of ROK and that ROK plays a prominent role in KCl-induced force maintenance. We also tested the hypothesis that Ca2+, acting downstream from K+ depolarization, causes translocation of ROK to caveolae as a critical step in the signaling pathway leading from K+ depolarization to contraction.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue preparation. Tissues were prepared as previously described (29). Femoral and renal arteries from adult New Zealand White rabbits were cleaned of adhering tissue and stored in cold (0-4°C) physiological saline solution (PSS) [in mM: 140 NaCl, 4.7 KCl, 1.2 MgSO4, 1.6 CaCl2, 1.2 NaHPO4, 2.0 MOPS (adjusted to pH 7.4), 0.02 Na2EDTA (to chelate heavy metals), and 5.6 D-glucose]. To produce K+ depolarization, KCl (110 mM) was substituted isosmotically for NaCl. High-purity water (17 M{Omega}), distilled and deionized, was used throughout the study. The endothelium of each artery was removed by gentle rubbing of the intimal surface with a metal rod. Tissues were cut into 3- to 4-mm-wide artery rings.

Each muscle ring was secured in a tissue bath (Radnoti Glass Technology, Monrovia, CA) between stainless steel wire stirrups attached to a micrometer and isometric force transducer for length adjustments and isometric force measurements, respectively (model 52, Harvard Apparatus, South Natick, MA).

Isometric contractions were measured as previously described (29). Voltage signals from force transducers were digitized (CIO-DAS16F, Computer Boards, Middleboro, MA), and visualized on a computer screen as force (in g). Data acquisition and analyses were accomplished using DasyLab (DasyTec, Amherst, NH) and Microsoft Excel (Microsoft).

Isometric force. Contractile force (F) was measured as previously described (29). Tissues were allowed to equilibrate for 1 h at 37°C in PSS with aeration. The muscle length for which active force was maximum (Lo) was determined for each tissue using an abbreviated length-tension curve and 110 mM KCl (substituted isosmotically for NaCl) as the stimulus (12, 33). For each preloaded tissue, the degree of steady-state force (F) produced at Lo by incubation in KCl for 5-10 min was equal to the optimal force for muscle contraction (Fo), and subsequent contractions were calculated as F/Fo. BAY K 8644 (0.56-1 µM), a nifedipine analog that increases the open-state probability of L-type voltage-operated Ca2+ channels, was utilized as a contractile agent in some experiments (15, 41). To produce contraction with BAY K 8644, it was necessary to preincubate arteries with 7.5 mM KCl, a concentration of KCl that, alone, did not cause contraction. Ionomycin (10 µM), a Ca2+ ionophore, was also used as a contractile agent in some experiments. No further length changes were imposed once Lo was established. All tissues were incubated with 1 µM phentolamine to block potential {alpha}-adrenergic receptor activation caused by the release of norepinephrine from periarterial nerves that may occur on stimulation with KCl.

MLC phosphorylation. The degree of MLC phosphorylation was measured as previously described (29). Arteries were quick-frozen in an acetone-dry ice slurry, slowly warmed to room temperature, dried, weighed, and homogenized in 8 M urea, 2% Triton X-100, and 20 mM dithiothreitol. Isoelectric variants of the 20-kDa MLCs were separated by two-dimensional (isoelectrical focusing-SDS) PAGE followed by Western blot with visualization using colloidal gold staining. The relative amounts of phosphorylated and nonphosphorylated MLCs were quantified by digital image analysis (Scion Image, NIH).

[Ca2+]i. [Ca2+]i was measured in renal arteries as previously described (29). Arterial rings positioned inside a quartz cuvette housed in a fluorometer (Photon Technology International, Lawrenceville, NJ) and maintained at 37°C were loaded for 2.5 h with ~7.5 mM fura 2-PE3-AM and 0.01% (wt/vol) Pluronic F-127 (TefLabs, Austin, TX) to enhance solubility. Tissues were then washed for 30 min in three changes of PSS. The fluorescence emission at a wavelength of 510 nm was collected for alternating excitations at 340 and 380 nm. The [Ca2+]i signals produced during KCl-induced steady-state contraction in the absence and presence of Y-27632 were quantified as the fraction of the steady-state value produced during the tonic phase of the KCl contraction used for the Lo determination and the minimum signal produced in a Ca2+-free solution containing 5 mM EGTA plus 10 µM ionomycin, corrected for background fluorescence. The background fluorescence was recorded by incubating tissue in a 4 mM MnCl2 quench solution containing 10 µM ionomycin.

Tissue and slide preparation for ROK and caveolin colocalization experiments. The extent of ROK colocalization with caveolin at the plasma membrane of femoral artery smooth muscle cells was investigated. Femoral artery rings (3-4mm long) were treated as described above (see Tissue preparation). No difference in ROK colocalization to caveolin was identified in stretched (Lo) vs. unstretched (Lz) tissues under basal conditions (24 ± 2 and 18 ± 2% for Lo and Lz, respectively, n = 3, P > 0.05) and when stimulated with KCl for 30 s (34 ± 2 and 34 ± 2% for Lo and Lz, respectively, n = 3, P > 0.05). Unstretched artery rings, which were technically easier to fix than stretched tissues, were used for all subsequent histological studies. Tissues stimulated with KCl were fixed at 30 s and 5 min. Colocalization of ROK with caveolin in KCl-stimulated tissues was compared with that in tissues not contracted with KCl (basal). Tissues were fixed in 100% methanol at -20°C for 5 min and then placed in a solution containing 5% sucrose for 1 h. Tissues were rapidly frozen in optimal cutting temperature frozen tissue embedding medium (OCT, VWR Scientific) using a low-temperature freezing bath (Histobath2, Thermo Shandon, Pittsburgh, PA). Frozen tissues were cut into ~18-µm-thick sections on a cryostat (Microm HM505E, Richard Allan Scientific, Kalamazoo, MI), and each section was placed onto charged slides (SuperFrost Plus, Fisher Scientific). Slides were dried for >=1 h before storage at -80°C.

Immunohistochemistry. Slides were thawed at room temperature for 30 min and washed twice in PSS. Small wells were created on each slide using an ImmEdge Pen (Vector Laboratories, Burlingame, CA). Tissues were permeabilized and blocked in one step using a solution of 0.1% Triton X-100 and 3% normal goat serum (Vector Laboratories) in PSS. The slides were washed twice in PSS and incubated with primary antibodies to ROK (anti-ROK-2, goat polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) and caveolin (anticaveolin, rabbit polyclonal; BD Biosciences) for 1 h at a dilution of 1:50. The anticaveolin antibody used in this study does not discriminate among the three isoforms of caveolin. Slides were washed twice in PSS and incubated with fluorophore-conjugated secondary antibodies (Alexa Fluor 488 donkey anti-goat to label anti-ROK and Alexa Fluor 594 chicken anti-rabbit to label anticaveolin; Molecular Probes, Eugene, OR) for 1 h at a dilution of 1:200. Tissue sections were washed a final time, and coverslips were applied with an antifading medium (VectaShield, Vector Laboratories). Slides were viewed and images were recorded using a laser scanning confocal microscope (model LSM 510, Carl Zeiss). Each tissue section was excited with two lasers, argon (488 nm) and HeNe (543 nm) with use of a rapid line-scanning protocol for excitation, and emission signals were collected through 505- to 550-nm band-pass and 560-nm long-pass emission filters, respectively, and quantified using a photomultiplier tube and proprietary software. For display purposes, a composite image was constructed in which ROK was green, caveolin was red, and signal overlap (colocalization) was yellow. Approximately 11 images were recorded in the z dimension, which constituted one z series. Images were taken 0.5 µm apart with a zoom of x2 on a x100 oil immersion objective.

One slide was randomly selected for viewing and analyses from each sectioned artery ring. A minimum of two z series were recorded for each slide from different areas of the tissue. Images were exported in an uncompressed tagged-image format file, and image analysis was conducted using MetaMorph software (version 4.6r9, Universal Imaging).

Image analysis and data collection for confocal images. Five consecutive images selected from 2 different z series per slide permitted 10 images to be averaged for each artery ring. MetaMorph software separated each composite image into two images, one for each laser. The software compared the images, pixel-by-pixel, and determined the percent overlap of the fluorescent signals. In particular, our aim was to test the hypothesis that KCl increased the degree of ROK translocation to caveolae at the plasma membrane, so colocalization of the two fluorophores was determined as the percent overlap of the ROK signal onto the caveolin signal. Colocalization measured for all 10 images was averaged using a spreadsheet (Excel, Microsoft) and considered an n of 1.

Drugs. Nifedipine and trifluoperazine (TFP) were obtained from Sigma; BAY K 8644, Y-27632, and ionomycin from Calbiochem; and HA-1077 and GF-109203X from Alexis. Nifedipine, BAY K 8644, and ionomycin were dissolved in ethanol. Y-27632 was dissolved in water. Ethanol was added at a final concentration <=0.1%, which had no effect on KCl- or BAY K 8644-induced contractions or on the degree of ROK colocalization with caveolin.

Statistics. The null hypothesis was examined using Students' t-test (when 2 groups were compared) or a one-way ANOVA. To determine differences between groups after ANOVA, the Student-Newman-Keuls post hoc test was used. In all cases, the null hypothesis was rejected at P < 0.05. For each study, n was the number of rabbits from which arteries were taken.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of ROK, PKC, and L-type Ca2+ channel inhibitors on KCl-induced force. A KCl-induced contraction (110 mM KCl substituted isosmotically for NaCl) of rabbit femoral and renal artery consisted of a rapid rise in force that reached a maximum value within ~15 s (peak; Fig. 1A) followed by a slower rise to a sustained level within ~3-5 min (tonic; Fig. 1A). To determine the effect of ROK inhibition on KCl-induced contraction, tissues were exposed to two different ROK inhibitors, Y-27632 (1 and 3 µM) and HA-1077 (10 µM), and then contracted with KCl. Although peak force was not affected (Fig. 1B), Y-27632 and HA-1077 strongly inhibited tonic force by >50% (Fig. 1C). PKC has been shown to play a role in receptor agonist-induced Ca2+ sensitization, so we also investigated the role of PKC in KCl-induced contraction. GF-109203X, when used at 1 µM, inhibits conventional and novel isoforms of PKC (PKCc,n) (6, 10, 21). GF-109203X did not inhibit KCl-induced peak or tonic force (Fig. 1, B and C). These data suggest that ROK does, but PKCc,n does not, play a role in the maintenance of KCl-induced tonic force in rabbit large arteries.



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Fig. 1. Effects of the RhoA-associated kinase (ROK) inhibitors Y-27632 [1 and 3 µM (1Y and 3Y, respectively)] and HA-1077 (HA; 10 µM) and conventional and novel PKC inhibitor GF-109203X (GF; 1 µM) on peak (B) and tonic (C) KCl-induced force. Control (Con) tissues were not exposed to a drug. A: typical force traces. Values in B and C are means ± SE; n = 3-5. F, steady-state force; Fo, optimal force for muscle contraction. *P < 0.05 compared with control.

 

Effect of Y-27632 and nifedipine on KCl-induced force and [Ca2+]i. Inhibition of KCl-induced tonic force by Y-27632 could be caused by inhibition of ROK or by inhibition of Ca2+ entry caused by a nonspecific effect of the drug on Ca2+ channels. We therefore measured the effect of Y-27632 on steady-state increases in force and [Ca2+]i produced by KCl. Tissues were loaded with the Ca2+ indicator fura 2 (see METHODS) and contracted with KCl. Y-27632 (1 µM) was added, and force and [Ca2+]i were allowed to reach a new steady state. The L-type Ca2+ channel inhibitor nifedipine (10 nM) was subsequently added to completely relax tissues by causing Ca2+ channel blockade (Fig. 2, A and C). Y-27632 reduced tonic KCl-induced force by ~40% (Fig. 2, A and B) but did not alter tonic [Ca2+]i (Fig. 2, C and D). Nifedipine further reduced tonic force to the basal level (Fig. 2, A and B) and nearly abolished [Ca2+]i (Fig. 2, C and D). These data indicate that 1 µM Y-27632 does not block KCl-induced Ca2+ entry and suggest that KCl induces Ca2+ sensitization via activation of ROK.



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Fig. 2. Effect of 1 µM Y-27632 and 10 nM nifedipine on KCl-induced force (A and B) and intracellular Ca2+ concentration ([Ca2+]i; C and D). A and C: typical force and Ca2+ traces, respectively. Values in B and D are means ± SE; n = 3. F340/F380, ratio of fluorescence at 340 nm to fluorescence at 380 nm. *P < 0.05 compared with KCl-induced responses.

 

Effect of Y-27632 on KCl-, BAY K 8644-, and ionomycin-induced MLC phosphorylation and force. The level of MLC phosphorylation basally was 9 ± 1%, and KCl-induced contraction resulted in an increase in MLC phosphorylation to ~37%. Y-27632 (1 µM) nearly abolished this increase (Fig. 3A). We also investigated the effect of Y-27632 on responses produced by an agent that selectively activates L-type Ca2+ channels to cause Ca2+ entry and contraction. BAY K 8644 (0.56 µM), a nifedipine analog but Ca2+ channel agonist, produced a slow rise in force that reached a plateau in ~20 min. Y-27632 (1 µM) nearly abolished BAY K 8644-induced steady-state MLC phosphorylation (Fig. 3A) and reduced steady-state force by >70% (Fig. 3B). We next examined the ability of Y-27632 to inhibit responses produced by the Ca2+ ionophore ionomycin, an agent that can bypass L-type Ca2+ channels to produce contraction by Ca2+ entry or release of intra-cellular Ca2+. Ionomycin (10 µM) produced a rise in force that reached a plateau in ~6.5 min. Y-27632 (3 µM) nearly abolished ionomycin-induced MLC phosphorylation (Fig. 3A) and force (Fig. 3C). The ionophore-dependent transfer of Ca2+ into smooth muscle cells elicited by ionomycin has been shown to cause membrane depolarization and, thus, force development (3). However, we found that 1 µM nifedipine did not inhibit 10 µM ionomycin-induced contraction in rabbit femoral artery (Fig. 3C).



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Fig. 3. Effect of the ROK inhibitor Y-27632 (1 µM) on KCl-, BAY K 8644 (0.56 µM)-, and ionomycin (10 µM)-induced myosin light chain (MLC) phosphorylation (A) and BAY K 8644- and ionomycin-induced force (B and C, respectively). C: effect of 1 µM nifedipine on ionomycin-induced force. Control tissues were not exposed to a drug. Values are means ± SE; n = 3-5. MLC20, 20-kDa MLC; MLC20-P, phosphorylated MLC20. *P < 0.05 compared with control.

 

Time-dependent effects of Y-27632 and nifedipine on KCl-induced force and MLC phosphorylation. Basal force and MLC phosphorylation were not affected by 1 µM Y-27632 or 1 µM nifedipine (Fig. 4). Whereas nifedipine nearly abolished the KCl-induced increase in force and phosphorylation at 10 s and 1 and 5 min of contraction, Y-27632 did not decrease KCl-induced force and phosphorylation at 10 s but did inhibit force and phosphorylation at 1 and 5 min of contraction (Fig. 4).



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Fig. 4. Effect of 1 µM Y-27632 and 1 µM nifedipine compared with control (no drug addition) on KCl-induced increases in force (A) and MLC phosphorylation (B). Values are means ± SE; n = 3-6. *P < 0.05 compared with control.

 

Effect of KCl and BAY K 8644 on ROK colocalization with caveolin at the cell periphery. In a x40 unstained fluorescence image of femoral artery obtained using a laser scanning confocal microscope (see METHODS), autofluorescence was evident in the adventitia and intima (Fig. 5A). The media, however, displayed very little autofluorescence, although elastic lamina between layers of smooth muscle cells could be seen (Fig. 5A). Images at x200 used for fluorescence colocalization measurements in this study were taken from areas of the media between elastic lamina rich in vascular smooth muscle cells and nearly devoid of autofluorescence.



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Fig. 5. Laser scanning confocal images of rabbit femoral artery. A: unstained image showing autofluorescence in adventitia (top left) and intima (bottom right) but no autofluorescence between elastic lamina within the media (middle). Magnification x40. B: image from an area within the media between elastic lamina labeled with Alexa Fluor 488 donkey anti-goat and Alexa Fluor 594 chicken anti-rabbit secondary antibodies showing minimal nonspecific labeling. Magnification x200. C-E: image of an artery stimulated for 30 s with 110 mM KCl taken from an area within the media between elastic lamina, double-labeled with anti-ROK-{alpha} goat polyclonal antibody (plus Alexa Fluor 488 donkey anti-goat secondary antibody) and anticaveolin rabbit polyclonal antibody (plus Alexa Fluor 594 chicken anti-rabbit secondary antibody). Magnification x200. E: composite of C [showing caveolin (red) staining only] and D [showing ROK (green) staining only]. Yellow-orange color in E represents ROK colocalization with caveolin.

 

The secondary antibodies used in this study (see METHODS), when applied alone without primary antibodies, displayed minimal fluorescence when a section of rabbit femoral artery media was viewed at x200 between layers of elastic lamina (Fig. 5B). However, vascular smooth muscle cells were clearly evident in fluorescence confocal images taken from tissues labeled with ROK and caveolin primary antibodies, such that caveolin appeared red (Fig. 5C) and ROK appeared green (Fig. 5D). Caveolin was localized at the cell periphery, as expected (Fig. 5C), whereas ROK was distributed throughout the cytosol and at the cell periphery (Fig. 5D).

In uncontracted (basal) tissues, ~18% of ROK was colocalized with caveolin at the cell periphery (quantified using MetaMorph software and visualized graphically as yellow in Fig. 5E). The amount of ROK colocalized with caveolin at the cell periphery nearly doubled at 30 s of KCl stimulation (Fig. 6A). At 5 min of a KCl stimulus, the amount of ROK colocalized with caveolin at the cell periphery had returned to the basal level (Fig. 6A). Thus ~16% of ROK translocated to caveolae at the smooth muscle cell membrane early during development of a KCl-induced contraction (30 s) and before development of the slower component of a KCl-induced contraction (tonic phase) that was inhibited by the ROK inhibitor Y-27632 (Figs. 1 and 4). Moreover, an equal amount of ROK (~16%) returned to the cytosol during the tonic phase of contraction. These data together indicate that ROK translocation to the periphery of the cell preceded the tonic phase of a KCl-induced contraction but that sustained ROK translocation was not necessary for the maintenance of tonic contraction.



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Fig. 6. Percent colocalization of ROK with caveolin at the cell periphery in rabbit femoral artery at 30 s (30'') and 5 min (5') of stimulation with KCl (A) and 0.56 µM BAY K 8644 (B). Tissues stimulated with BAY K 8644 were exposed to 7.5 mM KCl, a concentration that alone did not cause contraction. Basal tissues were not stimulated. Values are means ± SE; n = 3. *P < 0.05 compared with basal.

 

The Ca2+ channel agonist BAY K 8644, like KCl, produced a similar movement of ROK to the periphery of vascular smooth muscle cells. Addition of 7.5 mM KCl 10 min before addition of BAY K 8644, which was needed for BAY K 8644-induced contraction in rabbit femoral and renal arteries, did not produce a contraction, nor did it increase ROK translocation from the cytosol to the periphery [basal (Fig. 6B) compared with basal in the absence of 7.5 mM KCl (Fig. 6A)]. However, at 30 s of stimulation with BAY K 8644, ~31% of ROK was colocalized with caveolin at the cell periphery (Fig. 6B). At 5 min, the percentage of ROK colocalized with caveolin returned to the basal level (Fig. 6B).

Effect of nifedipine and TFP on the ability of KCl, BAY K 8644, and ionomycin to increase ROK colocalization with caveolin at the cell periphery. Our data show that stimuli that increase [Ca2+]i also increase the degree of ROK translocation to the cell periphery, suggesting that increased [Ca2+]i causes ROK translocation to caveolin. Thus we examined whether inhibition of L-type voltage-operated Ca2+ channels and Ca2+-calmodulin would inhibit KCl-induced ROK translocation to the cell periphery. Nifedipine (Fig. 7A) abolished the increase in colocalization of ROK with caveolin at the cell periphery at 30 s of stimulation with KCl. TFP, a Ca2+-calmodulin inhibitor (Fig. 7B), also abolished the increase in colocalization of ROK with caveolin at 30 s of stimulation with KCl. Y-27632 did not inhibit ROK translocation (Fig. 7C), suggesting that ROK activation was not necessary for KCl-induced ROK translocation to the cell periphery.



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Fig. 7. Effect of 1 µM nifedipine (A), 100 µM trifluoperazine (TFP, B), and 1 µM Y-27632 (C) on percent colocalization of ROK with caveolin at the cell periphery in rabbit femoral artery stimulated for 30 s and 5 min with KCl. Basal tissues were not stimulated. Values are means ± SE; n = 3. *P < 0.05 compared with basal.

 

Although 1 µM nifedipine inhibited KCl-induced (Fig. 7A) and BAY K 8644-induced (Fig. 8) increases in ROK translocation to caveolin at 30 s, nifedipine did not inhibit ionomycin-induced ROK translocation (Fig. 8). These data, along with our finding that nifedipine did not inhibit ionomycin-induced early force development (Fig. 3C), suggest that nifedipine-insensitive Ca2+ translocation or release of intracellular Ca2+ by ionomycin caused ROK translocation to caveolin. TFP inhibited BAY K 8644- and ionomycin-induced ROK translocation to caveolin (Fig. 8), supporting a role for Ca2+-calmodulin in regulation of ROK translocation.



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Fig. 8. Effect of 1 µM nifedipine and 100 µM TFP on ability of 1 µM BAY K 8644 and 10 µM ionomycin to increase ROK colocalization to caveolae at 30 s of stimulation. Values are means ± SE; n = 3. *P < 0.05 compared with basal.

 

Nifedipine concentration-dependent inhibition of ROK translocation to caveolin. If 1 µM nifedipine inhibited KCl-induced ROK translocation to caveolin because of inhibition of Ca2+ entry, then lower nifedipine concentrations that partially inhibit Ca2+ entry should cause partial inhibition of ROK translocation. This was found to be the case. Whereas 1 µM nifedipine abolished the increase in ROK translocation produced by KCl at 30 s (Figs. 7A and 9), lower nifedipine concentrations known to cause partial inhibition of Ca2+ entry and contraction (41) caused only partial inhibition of KCl-induced ROK translocation to caveolin (Fig. 9).



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Fig. 9. Concentration-dependent inhibition of ROK translocation to caveolae by the Ca2+ channel blocker nifedipine. Values are means ± SE; n = 4. *P < 0.05 compared with basal. KCl data at 30 s in the absence of nifedipine are from Fig. 6A.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has long been known that K+ depolarization (KCl) contracts smooth muscle by elevating [Ca2+]i and MLC phosphorylation and that KCl-induced increases in force and MLC phosphorylation are abolished by Ca2+ channel inhibitors and Ca2+-free solutions (1, 33, 40, 47). Receptor agonists cause smooth muscle contraction by increasing [Ca2+]i and by causing Ca2+ sensitization (for review see Ref. 17), and several studies indicate that an increase in ROK activity acts as the primary Ca2+-sensitizing mechanism (for review see Ref. 39). To induce Ca2+ sensitization, ROK must interact with the plasma membrane (for review see Ref. 38). Small plasma membrane invaginations, named caveolae and first described in 1955 (49), are thought to be major centers for coordination of cellular signal transduction, because they contain multiple receptors, signal transduction molecules, voltage-gated ion channels, and numerous effectors (for review see Refs. 2, 28, 36, and 43). Caveolins act as scaffolds for assembly and interaction of signaling molecules at caveolae (for review see Ref. 28). That caveolae are loci for receptor agonist-induced Ca2+ sensitization was recently proposed by Taggart (43), who showed that receptor agonist stimulation of smooth muscle causes a redistribution of RhoA, ROK, and PKC-{alpha} from the cytosol to the cell periphery (45). The present study extends this model to include K+ depolarization as a stimulus that also causes ROK translocation to caveolae at the plasma membrane of rabbit arterial smooth muscle cells. The significance of this finding is the conclusion that Ca2+ can induce Ca2+ sensitization by causing Ca2+-dependent ROK translocation to caveolae.

Our laboratory previously showed that KCl-induced tonic force can be desensitized by pretreatment of arteries with receptor agonists (30, 32, 34). That is, the degree of force produced for a given KCl-induced increase in [Ca2+]i can be substantially altered by the history of receptor stimulation (31). This is also true for receptor-induced Ca2+ sensitivity (31). Thus receptor-and KCl-induced contractile mechanisms share at least one feature; namely, the degree of Ca2+ sensitivity induced on muscle stimulation is dependent on the history of receptor stimulation. We propose that this shared feature reflects a common subcellular mechanism regulating Ca2+ sensitivity.

Y-27632 is known to be a highly selective inhibitor of ROK. The concentrations of Y-27632 required for 50% inhibition of phenylephrine-contracted aorta and 50% inhibition of isolated ROK are 0.7 µM and ~0.1-0.8 µM, respectively (4, 7, 16, 46); 2-11 µM HA-1077 is required for 50% inhibition of isolated ROK. Neither Y-27632 nor HA-1077 significantly inhibits MLC kinase or PKC-{alpha} activities at the concentrations used in the present study (4, 42). We found that tonic force produced by KCl in rabbit arteries was inhibited ~50% by 1 µM Y-27632 and >60% by 10 µM HA-1077, whereas early peak force was not affected by either ROK inhibitor. Likewise, early peak MLC phosphorylation was unaffected by 1 µM Y-27632, whereas 1 µM nifedipine nearly abolished peak and tonic force and MLC phosphorylation. Given the high selectivity of Y-27632 and HA-1077 for ROK inhibition, these data suggest that ROK did not participate in early force development but played an essential role in the maintenance of KCl-induced steady-state force. Moreover, KCl-induced increases in [Ca2+]i were not affected by 1 µM Y-27632, but MLC phosphorylation was affected, indicating that, at this concentration, Y-27632 did not act as a Ca2+ channel blocker to reduce MLC phosphorylation and force. The Ca2+ channel blocker nifedipine inhibited peak and tonic MLC phosphorylation and force, supporting a role for Ca2+ in early force development and delayed activation of ROK, leading to tonic force maintenance.

Although Y-27632 is highly selective for inhibition of ROK, a recent study revealed that 1 µM Y-27632 can also inhibit PKC-{delta}, a novel PKC isotype (6). However, PKC-{delta} is also strongly inhibited by 1 µM GF-109203X, a staurosporine analog that is used to concentration dependently inhibit PKCc,n (6, 10, 21). In the present study, 1 µM GF-109203X had no effect on KCl-induced contraction (Fig. 1, B and C). Together, these data support the hypothesis that a common mechanism shared by KCl and receptor agonists is activation of ROK.

Precisely which subcellular signal activated ROK during KCl-induced contraction was not discerned in the present study. However, receptor agonists and KCl increase [Ca2+]i, and our data support the hypothesis that Ca2+ plays an essential role in KCl-induced ROK translocation. This is because the Ca2+ channel blocker nifedipine and the calmodulin blocker TFP, when used at concentrations that are known to inhibit contraction (23), prevented KCl-induced ROK translocation to caveolae. The fact that nifedipine concentration dependently inhibited KCl-induced ROK translocation further supports a role for Ca2+ in the regulation of ROK activation and concomitant force maintenance. Moreover, 1 µM Y-27632 inhibited BAY K 8644- and Ca2+ ionophore (ionomycin)-induced MLC phosphorylation and force, whereas nifedipine did not inhibit ionomycin-induced increases in ROK translocation and force. These data suggest that increases in [Ca2+]i, rather than strong membrane depolarization, per se, caused translocation of ROK to caveolae. Y-27632 did not prevent KCl-induced ROK translocation at 30 s but did inhibit KCl-induced tonic (i.e., 5 min) contraction, suggesting that ROK activation may not be required for ROK translocation. Moreover, KCl-induced ROK translocation to the plasma membrane preceded the ability of Y-27632 to inhibit force. These data together support our working model that increases in [Ca2+]i caused increased ROK translocation to caveolae by a calmodulin-dependent mechanism, permitting ROK activation at the plasma membrane, resulting in KCl-induced long-term force maintenance.

In summary, this study presents evidence that rapid increases in [Ca2+]i in smooth muscle (13, 25, 35) induced by the stimulus, KCl, activates two distinct, temporally separated events: 1) the well-established activation of MLC kinase causing rapid, strong increases in MLC phosphorylation and cross-bridge cycling, producing a peak contraction (5, 13, 33), and 2) Ca2+-calmodulin-activated increases in ROK translocation to caveolae, leading to maintenance of tonic force (shown here). In conclusion, these data support the hypothesis that any stimulus that increases [Ca2+]i in arterial muscle will cause increased ROK translocation to caveolae at the cell periphery, where additional signaling events, such as activation of ZIP-like kinase (20), that lead to sustained MLC phosphorylation and force in the face of temporally falling [Ca2+]i may be coordinated. Receptor agonists share this mechanism with KCl but, additionally, produce a greater increase in Ca2+ sensitivity (31), presumably by causing stronger increases in ROK activity through activation of RhoA (for review see Ref. 38) and by activating additional Ca2+-sensitizing mechanisms involving PKC (6, 45) that are independent of increases in [Ca2+]i (18).


    DISCLOSURES
 
This study was supported by National Heart, Lung, and Blood Institute Grant R01-HL-61320.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. H. Ratz, Dept. of Biochemistry and Pediatrics, School of Medicine, Virginia Commonwealth University, 1101 East Marshall St., PO Box 980614, Richmond, VA 23298-0614 (E-mail: phratz{at}vcu.edu).

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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