Published online before print December 7, 2006, doi: 10.1161/01.RES.0000253902.90489.df
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© 2007 American Heart Association, Inc.
Cellular Biology |
Ca2+-Dependent Rapid Ca2+ Sensitization of Contraction in Arterial Smooth Muscle
From the Boston Biomedical Research Institute (G.J.D., S.S., K.K., T.K.), Watertown, Mass; and Department of Physiology (M.E.), Jefferson Medical College, Philadelphia, Pa.
Correspondence to Toshio Kitazawa, Boston Biomedical Research Institute, 64 Grove St, Watertown, MA 02472. E-mail Kitazawa{at}bbri.org
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Abstract |
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Ca2+ ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca2+ with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca2+ transient, we compared Ca2+ signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during
1 agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38±0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca2+ rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca2+ release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca2+-dependent protein kinase C. This was followed by a slow Ca2+-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29±0.09 at rest to the peak of 0.68±0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca2+ sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca2+ rise induces a rapid inhibition of MLC phosphatase coincident with the Ca2+-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.
Key Words: CPI-17 • MYPT1 • PKC • Rho-kinase
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Introduction |
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A wide range of excitatory agonists including
1-adrenergic agonist activates both heterotrimeric Gq and G12/13 G proteins in smooth muscle following their bindings to G protein-coupled receptors.1 The former G protein further activates phospholipase Cß to hydrolyze plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and concurrently generates 2 signaling messengers: a water-soluble inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The former messenger diffuses to the cytoplasm and binds to the IP3 receptor channel in the sarcoplasmic reticulum (SR) membrane to induce Ca2+ release from the lumen. This Ca2+ triggers the rapid phasic component of contraction and myosin light chain (MLC) phosphorylation through activation of the Ca2+/calmodulin-dependent MLC kinase (MLCK). Although Ca2+ influx is subsequently increased through opening of surface membrane Ca2+ channels, [Ca2+]i is not kept constant but rather transient and/or oscillates during the agonist stimulation.2 The average MLC phosphorylation is also transient; however, the tonic component of the phosphorylation after the peak is maintained at higher levels than expected from the Ca2+ signal, as compared with that of high K+ stimulation, suggesting an increase in the Ca2+ sensitivity of MLC phosphorylation via the inhibition of MLC phosphatase (MLCP).1,2 As a result, a relatively high level of MLC phosphorylation and contraction is achieved even at low MLCK activity under low [Ca2+]i during agonist-induced contraction.3 The inhibition of MLCP thus appears to play as critical a role as the Ca2+-dependent activation of MLCK in the maintenance of tonic contraction.
MLCP is a holoenzyme composed of 3 subunits: a 38-kDa catalytic subunit (
isoform of type 1 protein phosphatase, PP1c), a large 110- to 130-kDa regulatory subunit (MYPT1), and a small 20-kDa subunit.4 MYPT1 is responsible for binding to and activation of PP1c and for targeting myosin. Two major signaling pathways have been proposed for the in situ inhibition of MLCP. One is the phosphorylation of MYPT1 at Thr696 and Thr853 via G12/13/RhoA/Rho-kinase pathway.1 Recently, Hartshorne and colleagues5 demonstrated these results, although conflicting with their previous finding6 that the phosphorylation at not only Thr696 but also Thr853 equally suppresses the phosphatase activity. Several other kinases such as integrin-linked kinase (ILK), zipper-interacting protein kinase (ZIPK), p21-activated kinase (PAK), and dystrophia myotonica protein kinase (DMPK) can also phosphorylate the Thr696 site.1 However, the in situ phosphorylation at Thr696 in smooth muscle tissues and cultured cells is only minimally increased on G protein activation and not decreased by the Rho-kinase inhibitor Y-27632, which can significantly suppress agonist-induced Ca2+ sensitization of contraction and MLC phosphorylation.7,8 On the other hand, Thr853 is a Rho-kinase-specific site, and, in fact, the in situ phosphorylation is significantly increased in response to agonist stimulation via Rho-kinase pathway in smooth muscle tissues and cultured cells.5,7
The second mechanism of MLCP inhibition is through phosphorylation of smooth muscle-specific MLCP inhibitor protein CPI-17.9 Although there are multiple sites to be phosphorylated, the phosphorylation at only Thr38 increases the inhibitory effect of CPI-17 on MLCP by 1000-fold.10 A major kinase for agonist-induced CPI-17 phosphorylation at Thr38 in smooth muscle tissues is protein kinase C (PKC),9,11 which is activated by phospholipase Cß generation of DAG. Therefore, a potential signaling pathway through CPI-17 phosphorylation toward inhibition of MLCP is Gq/phospholipase Cß/DAG/PKC/CPI-17/MLCP. Another possible pathway is through Rho-kinase-induced phosphorylation of CPI-17. This is based on the evidence that the Rho-kinase inhibitor reduces agonist-induced CPI-17 phosphorylation.7,8,11 Furthermore, overexpression of constitutively active RhoA in cultured arterial smooth muscle cells increases CPI-17 phosphorylation.12 Thus, multiple signaling pathways mediate the agonist-induced inhibition of MLCP via the phosphorylation of MYPT1 and CPI-17. Furthermore, expression ratios of CPI-17 relative to MYPT1 (ie, MLCP) largely vary, depending on the type of smooth muscle.13 Thus, the Ca2+- sensitizing signal transduction through phosphorylation of MYPT1 and CPI-17 depends on the tissue type. However, the kinetics of 2 phosphorylations, CPI-17 at Thr38 and MYPT1 at Thr853, on agonist stimulation have not been investigated in detail.
We hypothesized that these 2 phosphorylations are activated in a specific time course on agonist stimulation. We therefore examined the temporal relationship among Ca2+, MLC phosphorylation, CPI-17 (Thr38) phosphorylation, MYPT1 (Thr853) phosphorylation, and contraction in response to the 1 agonist phenylephrine (PE) in rabbit femoral artery tissues and, furthermore, determined the effects of Ca2+ blockers, PKC inhibitors, and Rho-kinase inhibitors on these parameters. We also determined the stoichiometric amount of the in situ phosphorylation of CPI-17 and MYPT1. Our results reveal that, in addition to known Ca2+-independent phosphorylation of CPI-17 and MYPT1, CPI-17 is Ca2+-dependently phosphorylated by PKC as rapidly as MLCs are phosphorylated. The source of Ca2+ for the rapid phosphorylation is agonist-induced Ca2+ release from the sarcoplasmic reticulum (SR) but not Ca2+ influx from the extracellular space.
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Materials and Methods |
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Tissue Preparation and Force Measurement
All animal procedures were approved by the Animal Care and Use Committee of the Boston Biomedical Research Institute. Smooth muscle strips of rabbit femoral artery were prepared and mounted for force measurements and quick-freezing using liquid nitrogen-cooled propane, as described previously in detail.14 For more details, see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
Cytoplasmic Ca2+ Measurements
The method for intracellular Ca2+ ([Ca2+]i) measurement in the fura-2 loaded artery was essentially similar to that of Himpens et al.15 See the online data supplement for more details.
Measurement of MLC Phosphorylation
In situ phosphorylation of MLC in muscle strips was measured using the 2D electrophoresis, as described previously.14 See the online data supplement for more details.
Antibodies and Western Blotting
The antibodies used and Western blotting experiments have been described previously.7,11 See the online data supplement for more details.
Statistics
Results are expressed as the means±SEM of n experiments. Statistical significance was evaluated using ANOVA analysis. A level of P<0.05 was considered statistically significant.
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Results |
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Time Course of PE-Induced Contraction and Phosphorylation of MLC, CPI-17, and MYPT1 in Rabbit Femoral Artery
Figure 1A illustrates an example of the simultaneous measurements of Fura-2 ratio signal and isometric contraction in response to 50 µmol/L PE, with a clear indication of the Ca2+ rise in advance of force development. During the prolonged stimulation with PE, the Ca2+ level was partially decreased to 42±8% (n=5) of the transient peak. Figure 1B confirms that the increase in MLC phosphorylation precedes the development of contraction.16 At 7 seconds, MLC was already phosphorylated to 90% of the peak level (0.63±0.04 mol of Pi/mol of MLC at 15 seconds; n=6), whereas the force at the 7-second time point was developed to only 30% of the peak level at 5 minutes.
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Figure 1C and 1D illustrates a representative immunoblotting image and average extent of phosphorylated CPI-17 at Thr38 or MYPT1 at Thr853 in the PE-stimulated arterial tissues at various time points. CPI-17 was rapidly phosphorylated from a negligible value at rest (0 second in C and D) to a peak at 7 seconds similar to the rate of MLC phosphorylation but much faster than MYPT1 Thr853 phosphorylation and force development. The stoichiometry of CPI-17 phosphorylation was <0.01±0.00 (n=13) at rest and 0.38±0.04 mol of Pi/mol (n=4) of CPI-17 at 7 seconds after PE stimulation. In contrast, MYPT1 Thr853 at resting state was already phosphorylated to a considerable level (43±7% of value at 60 seconds). The phosphorylation was slowly increased similar to the rate at which the contractile force was developed (Figure 1B and 1D). In contrast to MYPT1 Thr853, the phosphorylation of MYPT1 at Thr696 was detected at rest and was not significantly increased at 60 seconds (not shown), confirming the previous results.8 The stoichiometry of MYPT1 phosphorylation at Thr853 was estimated as 0.29±0.09 mol of Pi/mol (n=13) of MYPT1 at rest and reached 0.68±0.14 mol/mol (n=4) at 60 seconds after PE stimulation. Assuming that the protein content of the typical mammalian cell is 18% of the total cell weight, the total MYPT1 concentration, ie, MLCP concentration, was 0.8±0.1 µmol/L (n=6) in rabbit femoral artery. Total expression level of CPI-17 in rabbit femoral artery is previously estimated 6±1 µmol/L13; thereby the cellular concentration of phosphorylated CPI-17 is increased to 2.3±0.2 µmol/L at 7 seconds.
After 15 seconds of PE stimulation, on the other hand, phosphorylation levels of MLC began to significantly but partially decline from 0.63±0.04 (n=6) to 0.47±0.03 mol of Pi/mol of MLC (n=4) at 60 seconds and then to 0.44±0.05 mol/mol (n=4) at 5 minutes. The phosphorylation level at 5 minutes was still much higher than that at rest, whereas average contraction level was maintained up to 5 minutes (Figure 1B) and thereafter started to decline during PE stimulation in many cases. The phosphorylated CPI-17 level also tended to decline slightly but not significantly to 1.7±0.18 µmol/L (n=5) at 5 minutes (Figure 1D), whereas MYPT1 phosphorylation level was not decreased and was maintained up to 5 minutes (Figure 1D; 0.67±0.07 mol/mol; n=7).
Effect of Inhibition of Ca2+ Rise on PE-Induced Phosphorylation and Contraction
A mixture of 2 Ca2+ blockers (2 µmol/L ryanodine,17 to open-lock the SR Ca2+ release channel plus 1 µmol/L nicardipine,18 to inhibit the voltage-dependent L-type Ca2+ channel) was applied to eliminate the [Ca2+]i increase by PE (Figure 2). The blocking of both the SR Ca2+ release and the voltage-dependent Ca2+ influx totally abolished an increase in cytoplasmic Ca2+ in response to PE (Figure 2A). The lack of PE-induced Ca2+ increase strongly inhibited the initial fast-rising phase and also the sustained phase of PE-induced contraction (Figure 2B), and MLC phosphorylation at 7 and 15 second-time points (Figure 2C). However, these Ca2+ blockers had no significant effect on MLC phosphorylation at 5 minutes after PE stimulation (Figure 2C) and did not prevent the slow development of contraction (Figure 2B). The basal levels of Ca2+ was slightly elevated possibly because of the leakage of Ca2+ through the ryanodine receptors17 or an increase in the Ca2+ influx by the depletion of Ca2+ stores.19 This may cause a slight increase in the resting MLC phosphorylation (0 second in Figure 2C) and force (not shown). The inhibition of the Ca2+ rise almost abolished the rapid increase in PE-induced phosphorylation of CPI-17 at 7 seconds (Figure 2D), whereas the phosphorylation was thereafter significantly increased at 15 seconds and 5 minutes by 0.39±0.13 and 0.41±0.08 µmol/L, respectively. In contrast, the MYPT1 phosphorylation in the presence of Ca2+ blockers increased similar to the control in the absence (Figure 2E).
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To further evaluate the effects of Ca2+ blockers on the initial rapid rising and sustained phases of PE-induced contraction, arterial strips were subjected to an individual blocker, nicardipine or ryanodine alone (Figure 3). Pretreatment with 1 µmol/L nicardipine for 10 minutes primarily inhibited the sustained but not initial rapid phase of PE-induced contraction (Figure 3A). Longer treatment with the Ca2+ entry blocker for 30 to 40 minutes caused a gradual suppression of both initial and sustained phases of contraction (not shown), possibly because of a depletion of the SR of Ca2+. Ryanodine, in contrast, suppressed the initial rapid-rising phase of PE-induced contraction but had no effect on the sustained phase of the contraction (Figure 3A). The ryanodine treatment diminished the phasic component of Ca2+ rise in response to PE, but the sustained phase of Ca2+ was higher than the control without the treatment (the red trace compared with the black in Figure 3B). The phosphorylation of CPI-17 in response to PE at 7 seconds was completely prevented by the ryanodine treatment (0±0% in Figure 3D) but partially increased at 15 seconds (25±8% for ryanodine treatment versus 122±12% for control; n=3). At 5 minutes, CPI-17 became phosphorylated to 58±18% (n=3; Figure 3E), which was not significantly different from the control at 5 minutes. The value was significantly higher than that of the channel blocker combination (Figure 3D). The ryanodine treatment had no significant effect on the PE-induced MYPT1 phosphorylation at 7 seconds and 5 minutes (Figure 3F and 3G). Furthermore, we tested the effects of another 2 Ca2+ blockers on contraction: thapsigargin, an inhibitor for the SR Ca2+ ATPase,20 and 2-aminoethoxydiphenyl borate (2-APB), an IP3 receptor antagonist.21 We confirmed that treatment with 10 µmol/L thapsigargin but not with 30 µmol/L 2-APB strongly suppressed 20 mmol/L caffeine-induced transient contraction in the absence of Ca2+. Both thapsigargin and 2-APB, like ryanodine, delayed the initial onset of PE-induced contraction (not shown), whereas the latter compound, in contrast to ryanodine and thapsigargin, strongly suppressed the sustained tonic level of the contraction to 24±2% (n=4) of control.
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The high K+-induced membrane depolarization of smooth muscle tissues is known to rapidly increase [Ca2+]i through direct opening of the voltage-dependent Ca2+ channels, MLC phosphorylation, and a contraction.15,16 We examined the effect of high (124 mmol/L) K+ on the Ca2+ signal and CPI-17 and MYPT1 phosphorylation in the ryanodine-treated strips. Both initial and maintained levels of Ca2+ rise were higher during the high K+ stimulation than that of PE in rabbit femoral artery (Figure 3C). The high K+ stimulation of the untreated strips, although producing a rapid increase in contraction, did not have a significant effect on phosphorylation of CPI-17 nor MYPT1 at 7 seconds, as compared with the respective resting value (Figure 3D and 3F). Five minutes of stimulation with high K+, however, significantly elevated MYPT1 phosphorylation (Figure 3G), whereas CPI-17 phosphorylation was still not significantly increased (Figure 3E). Simultaneous stimulation with high K+ and PE of the ryanodine-treated strips for 7 seconds and 5 minutes raised CPI-17 phosphorylation to a high level (Figure 3D and 3E). In contrast, the PE-induced phosphorylation of MYPT1 in the ryanodine-treated strips was significantly decreased by the addition of high K+ at 5 minutes (Figure 3G).
Effects of PKC Inhibitors on PE-Induced Phosphorylation and Contraction
Three modes of PKC inhibitors, GF-109203X (binding to the catalytic domain of both conventional and novel PKCs),22 calphostin C (binding to the regulatory domain of both conventional and novel PKCs),23 and Gö6976 (binding to the catalytic domain of conventional PKC)22 were used to examine the role of PKC in the PE-induced contraction. GF-109203X at 3 µmol/L significantly, but slightly, decreased the rate of initial rise in [Ca2+]i by PE (Figure 4A) but did not reduce the sustained level of [Ca2+]i. The GF compound also inhibited both the initial rising phase and the sustained phase of the PE-induced contraction, with a small delay in the onset (Figure 4C). The delay was much shorter than that caused by the Ca2+ channel blocker combination (Figure 4C versus Figure 2B). Calphostin C (1 µmol/L) had a similar inhibitory effect on both initial rising and sustained phases of PE-induced contraction (Figure 4D), but without obvious delay in the Ca2+ signals (Figure 4B). Gö6976 (10 µmol/L) had a similar inhibitory effect on the initial rising phase but less effect on the sustained level of the contraction, as compared with those of conventional PKC (cPKC)/novel PKC (nPKC) inhibitors (Figure 4E). GF-109203X significantly inhibited the PE-induced increase in MLC phosphorylation at all 3 time points but had no effect at rest (hatched bar at 0 second in Figure 4E). The CPI-17 phosphorylation at 7 seconds after stimulation with PE was almost completely blocked by the presence of GF-109203X, and thereafter slightly increased at 15 seconds and 5 minutes (Figure 4F) similar to the effect of the Ca2+ blocker combination (Figure 4D). Calphostin C at 1 µmol/L also significantly inhibited PE-induced CPI-17 phosphorylation to 21±1% (n=3) at 7 seconds, but less effective than 3 µmol/L GF-109203X. In contrast, the MYPT1 phosphorylation was not significantly decreased by either GF-109203X (Figure 4G) or calphostin C (62±10% at 7 seconds and 118±6% at 5 minutes; n=3).
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Effects of Rho-Kinase Inhibitors on PE-Induced Phosphorylation and Contraction
We examined the effect of Rho-kinase inhibitors (Y-2763224 and H-115225) on PE-induced contraction and phosphorylation. Y-27632 (10 µmol/L) had a slight inhibitory effect on the initial rising phase of [Ca2+]i increase by 50 µmol/L PE but not in the sustained phase (Figure 5A), similar to the effect of GF-109203X. Both Y-27632 and H-1152 (3 µmol/L) inhibited the sustained phase of PE-induced contraction, whereas the inhibitors (even 30 µmol/L Y-27632) had almost no effect on the initial rising phase of contraction (Figure 5B and 5C). The effect of the 3 types of inhibitors (Ca2+ blockers, GF-109203X, and Y-27632) on PE-induced contraction were additive in any combination of 2, and the pretreatment with all 3 types of inhibitors totally abolished the development of PE-induced contraction (not shown).
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Y-27632 (10 µmol/L) had no significant effect on phosphorylation of MLC and CPI-17 by the 7-second time point after 50 µmol/L PE stimulation, and, thereafter, phosphorylation of both proteins were significantly but partially inhibited (Figure 5D and 5E). H-1152 (3 µmol/L) also significantly reduced CPI-17 phosphorylation to a level similar to Y-27632 (n=3; 30±5% versus 42±7% in Y-27632) at 5 minutes. The MYPT1 phosphorylation at every time point, including rest, was almost abolished by Y-27632 (Figure 5F). H-1152 had a similar inhibitory effect on MYPT1 phosphorylation at 5 minutes (n=3; 12±2% versus 15±3% in Y-27632).
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Discussion |
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This study demonstrates unique roles of the phosphorylation of CPI-17 at Thr38 and MYPT1 at Thr853, respectively, in PE-induced contraction of artery. The rapid phosphorylation of CPI-17 in physiological conditions is totally reliant on Ca2+ release from the SR and on Ca2+-dependent PKC activity, and concomitant with a rapid rise in Ca2+ and an initial rapid development of MLC phosphorylation. On the other hand, the Ca2+-independent phosphorylation of both CPI-17 and MYPT1 by Rho-kinase occurs in the following sustained phase, in parallel with a force generation. These results indicate the existence of 2 distinct Ca2+-sensitizing signal transduction pathways, leading to inhibition of MLCP: rapid and slow mechanisms mainly driven by PKC and Rho-kinase, respectively (Figure 6). We propose a hypothesis that the rapid-signaling messenger Ca2+ synchronously increases and decreases MLCK and MLCP, respectively, toward the same target protein to synergistically increase a rapid phosphorylation of MLC and to initiate a rapid development of contraction in artery (left side of signal transduction pathways in Figure 6).
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Mechanism for Rapid Agonist-Induced Ca2+ Sensitization
The initial rapid phosphorylation of CPI-17 was Ca2+ dependent. Treatment of arterial smooth muscle with ryanodine in the presence of caffeine (see the expanded Materials and Methods section in the online data supplement) depleted the SR of Ca2+, and thus abolished the transient component of Ca2+ increase and the initial rapid but not slow increase in phosphorylation of CPI-17 and contraction in response to PE (Figure 3). Total inhibition of Ca2+ rise in response to PE with the mixture of ryanodine and nicardipine abolished the initial rapid and also inhibited the sustained slow components of CPI-17 phosphorylation (Figure 2). Together, these results suggest that the SR Ca2+ release is responsible for the initial rapid phase of CPI-17 phosphorylation and contraction, and the voltage-dependent Ca2+ influx plays a crucial role in the late sustained phase (Figure 6). The membrane depolarization by high K+ evokes Ca2+ rise, MLC phosphorylation, and smooth muscle contraction, whereas the Ca2+ rise did not trigger the CPI-17 phosphorylation, even though the [Ca2+]i level during high K+-induced contraction was higher than that of PE (Figure 3). In fact, increase in the [Ca2+]i to 1 µmol/L using the Ca2+/EGTA buffer in toxin-permeabilized strips did not significantly increase CPI-17 phosphorylation.11 These results, together, suggest that Ca2+ is required but not sufficient for triggering CPI-17 phosphorylation. The rapid CPI-17 phosphorylation by PE was also eliminated by a PKC inhibitor, either GF-109203X or calphostin C, but not by the Rho-kinase inhibitor Y-27632 (Figure 4G versus Figure 5E). These results, in conjunction with the fact that both PKC and PKCß but not PKC
are expressed as major Ca2+-dependent PKC isoforms in rabbit femoral artery (G. Dimopoulos, T. Kitazawa, unpublished results, 2005), suggest that both IP3-induced SR Ca2+ release and DAG-induced activation of Ca2+-dependent PKC and/or -ß isoforms are required for the CPI-17 phosphorylation (Figure 6). Removal of rapid CPI-17 phosphorylation by ryanodine treatment (Figure 3D) raises a possibility that Ca2+ release from the SR is specific for the rapid activation of PKC and thus the CPI-17 phosphorylation. However, even after destruction of the SR Ca2+ release by ryanodine treatment, PE with Ca2+ influx by high K+ was able to induce a rapid phosphorylation of CPI-17 to a level equivalent to control without ryanodine treatment (Figure 3D and 3E). We presume that, under the physiological conditions, PE-induced Ca2+ release but not Ca2+ influx is the mediator for the rapid CPI-17 phosphorylation, possibly because of coordinated timing between Ca2+ release and DAG production. The Ca2+ influx induced by PE appears to be too slow for activation of Ca2+-dependent cPKC (Figure 3B). This Ca2+-dependent phosphorylation of CPI-17, together with the activation of MLCK, initiates a rapid increase in MLC phosphorylation and contraction before the slow RhoA/Rho-kinase signaling pathway (Figure 6). Recently, we have demonstrated that chicken smooth muscle tissues lack both CPI-17 expression and 4-ß-phorbol-12,13-dibutyrate (PDBu)-induced contraction, unlike pigeon or other mammals.26 We found that the rise of PE-induced contraction in CPI-17–deficient chicken mesenteric artery was much slower than those of CPI-17–rich rabbit and pigeon arteries (also see Kitazawa et al26). These data support the hypothesis that CPI-17 is necessary for the rapid contraction induced on agonist stimulation in artery.
Rapid Phosphorylation of CPI-17 and Classical Ca2+ Sensitization
In toxin-permeabilized smooth muscle, PDBu, GTPS, and histamine rather slowly increase phosphorylation of CPI-17 and MLC, as well as contraction at resting or near resting Ca2+.1,7,8,14 The steady-state CPI-17 phosphorylation in permeabilized tissues is more strongly inhibited by GF-109203X than Gö6976 or Y-27632, suggesting that a major kinase is Ca2+-independent nPKC.9 In contrast, this study clearly shows that Ca2+ increase has a crucial role in the rapid phosphorylation of CPI-17 by Ca2+-dependent cPKC in intact tissues (Figures 3 and 6
). This rapid Ca2+-dependent CPI-17 phosphorylation cannot be seen in permeabilized smooth muscle tissues, where the SR is depleted of Ca2+ and/or Ca2+ is clamped with a high Ca2+/EGTA buffer. Any treatments modulating the Ca2+ release and/or Ca2+ loading of SR must affect the rapid CPI-17 phosphorylation and, thus, the rapid Ca2+ sensitization of contraction in intact smooth muscle.
The Ca2+-dependent translocation of PKC isoform from the cytosol to the cell surface has been shown in response to PE in smooth muscle cells isolated from ferret portal vein.27 If the translocation of Ca2+-dependent PKC is required for the enzymatic activation, CPI-17 should be translocated to the surface membrane. However, the PKC translocation appears a slow process with a half-time of approximately 3 minutes and dependent on a steady-state Ca2+ level but not on an initial large transient of Ca2+,27 suggesting that the phosphorylation of CPI-17 occurs in advance of the translocation of PKC. The issue, however, should be reevaluated in the fresh smooth muscle tissues or cells under conditions in which CPI-17 is rapidly phosphorylated by PKC in response to agonists.
Mechanisms for Slow Phase of Ca2+ Sensitization
The sustained tonic level of PE-induced contraction was more strongly inhibited by GF-109203X and calphostin C than Gö6976 (Figure 4E), indicating that Ca2+-independent nPKC takes the place of Ca2+-dependent cPKC in terms of CPI-17 phosphorylation with time. Phosphorylation of CPI-17 at the slow and sustained phase also contains a small but significant component insensitive to the Ca2+ blockers and PKC inhibitors, as compared with that of the initial phase (Figures 2D and 4G), suggesting that Ca2+-independent protein kinase(s) rather than PKC is involved in the slow phosphorylation of CPI-17 and the sustained tonic component of contraction. Furthermore, the MLC phosphorylation in the sustained tonic phase of contraction was also insensitive to the Ca2+ blockers or PKC inhibitors, compared with that of the initial phase (Figures 2C and 4F), suggesting that a mechanism(s) other than Ca2+-dependent PKC/CPI-17 phosphorylation is slowly developed at the late phase.
MYPT1 phosphorylation at Thr853 in response to either PE or high K+ is rather slowly augmented (Figures 1C and 3G) compared with the rapid phosphorylation of MLC and CPI-17. Both Y-27632 and H-1152 almost completely abolished this MYPT1 phosphorylation, whereas they did not attenuate the early phase of phosphorylation of MLC and CPI-17 and contraction (Figure 5). Therefore, the regulation of MLCP activity through Rho-kinase is involved in rather late sustained phase but not the early phase of PE-induced MLC phosphorylation and contraction. This is consistent with the observation that noradrenalin stimulation induces a slow increase in the amount of active GTP-bound form of RhoA in aorta28 and that photolysis of caged GTP-G14V RhoA/GDI complex in permeabilized portal vein induces a slow onset of contraction,1 similar to the time course of the MYPT1 Thr853 phosphorylation (Figure 1). On the other hand, when both Ca2+ release and Ca2+ influx were blocked with Ca2+ blockers, MLC phosphorylation and contraction were still significantly and slowly increased under the conditions in which MLCK was supposedly not increased (Figure 2). This contraction was partially inhibited by either GF-109203X or Y-27632 and completely inhibited by a mixture of 2 inhibitors (not shown). These results suggest that this slow development of MLC phosphorylation indicates a slow inhibition of MLCP in intact artery via 3 possible Ca2+-independent mechanisms: nPKC/CPI-17, Rho-kinase/CPI-17, and Rho-kinase/MYPT1 (Figure 6).
In conclusion, 1 agonist triggers the Ca2+ release from the SR to induce a rapid and Ca2+-dependent phosphorylation of the MLCP inhibitor protein CPI-17 in artery. This can lead to a dual regulation of MLC phosphorylation in a synchronous way (Figure 6): a downregulation of phosphatase in coordination with the upregulation of Ca2+-dependent kinase to synergistically increase a phosphorylation of the same target protein MLC and a large contraction with a small increase in Ca2+. The expression level of CPI-17, however, greatly varies with smooth muscle tissue types13; therefore, this rapid CPI-17 signaling appears to be vital in vascular smooth muscles, although insignificant in visceral phasic smooth muscles.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants R01HL70881 and P01AR041637 (to T.K.) and R01HL083261 (to M.E.).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received July 27, 2006; revision received November 15, 2006; accepted November 16, 2006.
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