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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 kinase (MLCK) to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase (MLCP) is involved in the rapid development of MLC phosphorylation and contraction during Ca2+ transient, we compared Ca2+ signal, MLC phosphorylation, and two MLCP-inhibiting phosphorylation of CPI-17 Thr38 and MYPT1 Thr853 during α1-agonist-induce contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from negligible to a peak value of 0.38±0.04 mol of Pi/mol within 7-second stimulation as rapidly as those of Ca2+ rise and MLC phosphorylation. This rapid phosphorylation of CPI-17 relied on both agonist-induced Ca2+-release from the sarcoplasmic reticulum and PKC activity, and followed by a slow Ca2+-independent and Rho-kinase/PKC-dependent phosphorylation. In contrast, MYPT1 phosphorylation had only a slow component that Rho-kinase-dependently 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 that of contraction. These results indicate that there are at least two (rapid in addition to slow) components of the Ca2+ sensitization through inhibition of MLCP. Our results support the hypothesis that the initial rapid Ca2+ rise induces a rapid inhibition of MLCP in coordination with the well-known Ca2+-induced MLCK activation to synergistically initiate a rapid MLC phosphorylation and contraction in artery.
A wide range of excitatory agonists including α1-adrenergic agonist activates both heterotrimeric Gq and G12/13 G proteins in smooth muscle following their bindings to G protein-coupled receptors (GPCRs).1 The former G protein further activates phospholipase Cβ to hydrolyze plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and concurrently generates two 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-depednet MLC kinase (MLCK).2,3 Although Ca2+ influx is subsequently increased through opening of surface membrane Ca2+ channels, [Ca2+]i is not kept constant but rather transient and/or oscillated during the agonist stimulation.4 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 to that of high K+ stimulation, suggesting an increase in the Ca2+ sensitivity of MLC phosphorylation via the inhibition of MLC phosphatase (MLCP).1,4 As the 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.5 The inhibition of MLCP thus appears to play a critical role in the maintenance of tonic contraction as important as does the Ca2+-dependent activation of MLCK.
MLCP is a holoenzyme composed of three subunits: a 38-kDa catalytic subunit (δ-isoform of type 1 protein phosphatase, PP1cδ), a large 110–130-kDa regulatory subunit (MYPT1), and a small 20-kDa subunit.6 MYPT1 is responsible for binding to and activation of PP1c and in 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 Both phosphorylations were shown to suppress the phosphatase activity in vitro.7 Several other kinases such as ILK, ZIPK, PAK, and 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 upon G protein activation, and not decreased by the Rho-kinase inhibitor Y-27632 that can significantly suppress agonist-induced Ca2+ sensitization of contraction and MLC phosphorylation.8,9 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.8,10
The second mechanism of MLCP inhibition is through phosphorylation of smooth muscle-specific MLCP inhibitor protein CPI-17.11 When this protein is phosphorylated at Thr38, the inhibitory effect on MLCP is increased by 1,000-fold. A major kinase for agonist-induced CPI-17 phosphorylation in smooth muscle tissues is protein kinase C (PKC),11,12 which is activated by the PLCβ-produced signaling messenger DAG. Therefore, a potential signaling pathway through CPI-17 phosphorylation toward inhibition of MLCP is Gq•PLCβ•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.8,9,12 Furthermore, over-expression of constitutively active RhoA in cultured arterial smooth muscle cells increases CPI-17 phosphorylation.13 Thus, multiple signaling pathways mediate the agonist-induced inhibition of MLCP via the phosphorylation of MYPT1 and CPI-17. However, the kinetics of two phosphorylations, CPI-17 at Thr38 and MYPT1 at Thr853 upon agonist stimulation has not been investigated in detail.
We hypothesized that PKC and Rho-kinase for CPI-17 or MYPT1 are activated in a specific time course upon agonist stimulation. We therefore examined the temporal relationship among Ca2+, MLC phosphorylation, CPI-17 (Thr38) phosphorylation, MYPT1 (Thr853) phosphorylation and contraction in response to α1-agonist phenylephrine 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 to evaluate the significance. 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 MLC phosphorylation. 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.
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 details.14 See the online data supplement for more details.
Results are expressed as the means ± S.E.M of n experiments. Statistical significance was evaluated using ANOVA analysis. A level of P < 0.05 was considered statistically significant.
Figure 1A illustrates an example of the simultaneous measurements of Fura-2 ratio signal and isometric contraction in response to 50 μM 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 moles of Pi/mole of MLC at 15 seconds; n=6), while the force at 7-second time point was developed to only 30% of the peak level at 5 minutes.
Figures 1C and D illustrate 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 sec 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 (Figures 1B & D). 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, i.e., MLCP concentration was 0.8 ± 0.1 μmo/L (n = 6) in rabbit femoral artery. Total expression level of CPI-17 in rabbit femoral artery is previously estimated 6 ± 1 μmol/L,17 thereby the cellular concentration of phosphorylated CPI-17 is increased to 2.3±0.2 μmole/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 moles of Pi/mole 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, while 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), while MYPT1 phosphorylation level was not decreased and was maintained up to 5 minutes (Figure 1D; 0.67±0.07 mol/mol; n=7).
A mixture of two Ca2+-channel inhibitors (2 μM ryanodine18 for the SR Ca2+-release channel plus 1 μM nicardipine19 for the voltage-dependent L-type Ca2+ channel) was applied to eliminate the intracellular Ca2+ 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 (B), and MLC phosphorylation at 7 and 15 second-time points (C). However, these Ca2+ blockers had no significant effect on MLC phosphorylation at 5 minutes after PE stimulation (C) and did not prevent the slow development of contraction (B). The basal levels of Ca2+ was slightly elevated possibly due to the leakage of Ca2+ through the ryanodine receptors18 or an increase in the Ca2+ influx by the depletion of Ca2+ stores.20 This may cause a slight increase in the resting MLC phosphorylation (0 sec in C) 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 (D), while 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 (E).
To further evaluate the effects of Ca channel blockers on the initial rapid rising and sustained phases of PE-induced contraction, arterial strips were subjected to individual channel blocker, nicardipine or ryanodine alone (Figure 3). Pretreatment with 1 μM nicardipine for 10 mintues primarily inhibited the sustained but not initial rapid phase of PE-induced contraction (A). Longer treatment with the Ca2+ entry blocker for 30–40 minutes caused a 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 (A). 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 to 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 D), but partially increased at 15 seconds (25±8% for ryanodine-treatment vs. 122±12% for control; n=3). At 5 minutes, CPI-17 became phosphorylated to 58±18% (n=3; E), which was not significantly different from the control at 5 minutes. The value was significantly higher than that of the channel blocker combination (D). The ryanodine treatment had no significant effect on the PE-induced MYPT1 phosphorylation at 7 seconds and 5 minutes (F and G).
The high K+-induced membrane depolarization of smooth muscle tissues is known to rapidly increase intracellular Ca2+ through direct opening the voltage-dependent Ca2+ channels, MLC phosphorylation and a contraction.15,16 We examined the effect of high (124 mM) K+ on the Ca2+ signal, 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 produced a rapid increase in contraction (see Figure 6), had neither significant effect on phosphorylation of CPI-17 nor MYPT1 at 7 seconds as compared to the respective resting value (D and F). Five-minutes stimulation with high K+, however, significantly elevated MYPT1 phosphorylation (G), while CPI-17 phosphorylation was still not significantly increased (E). 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 (D and E). 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 (G).
Two modes of PKC inhibitors, GF-109203X (binding to the catalytic domain of both conventional and novel PKCs)21 and calphostin C (binding to the regulatory domain of both conventional and novel PKCs)22 were used to examine the role of PKC in the PE-induced contraction. GF-109203X at 3 μM 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 (4C). The delay was much shorter than that by the Ca channel blocker combination (4C vs. 2B). Calphostin C (1 μM) had similar inhibitory effect on both initial rising and sustained phases of PE-induced contraction (4D) but without obvious delay in the Ca2+ signals (4B). GF-109203X significantly inhibited the PE-induced increase in MLC phosphorylation at all three time points, but had no effect at rest (hatched bar at 0 sec in 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 (F) similar to the effect of the Ca2+ blocker combination (D). Calphostin C at 1 μM also significantly inhibited PE-induced CPI-17 phosphorylation to 21±1% (n=3) at 7 seconds, but less effective than 3 μM GF-109203X. In contrast, the MYPT1 phosphorylation was not significantly decreased by either GF-109203X (G) or calphostin C (62±10% at 7 seconds and 118±6% at 5 minutes; n=3).
We examined the effect of Rho-kinase inhibitors (Y-2763223 and H-115224) on PE-induced contraction and phosphorylation. Y-27632 (10 μM) had a slight inhibitory effect on the initial rising phase of [Ca2+]i increase by 50 μM PE, but not in the sustained phase (Figure 5A) similar to the effect of GF-109203X. Both Y-27632 and H-1152 (3 μM) inhibited the sustained phase of PE-induced contraction while the inhibitors (even 30 μM Y-27632) had almost no effect on the initial rising phase of contraction (B and C). The effect of the three types of inhibitors (Ca2+ blockers, GF-109203X and Y-27632) on PE-induced contraction were additive in any combination of two and the pretreatment with all three types of inhibitors totally abolished the development of PE-induced contraction (not shown).
Y-27632 (10 μM) had no significant effect on phosphorylation of MLC and CPI-17 by 7-second time point after 50 μM PE stimulation, and thereafter phosphorylation of both proteins were significantly but partially inhibited (Figures 5D and E). H-1152 (3 μM) also significantly reduced CPI-17 phosphorylation to a level similar to Y-27632 (n=3; 30±5% vs. 42±7% in Y-27632) at 5 minutes. The MYPT1 phosphorylation at every time point including rest was almost abolished by Y-27632 (F). H-1152 had a similar inhibitory effect on MYPT1 phosphorylation at 5 minutes (n=3; 12±2% vs. 15±3% in Y-27632).
Recently, we have demonstrated that chicken smooth muscle tissues lack both CPI-17 expression and phorbol ester-induced contraction, unlike pigeon or other mammals.25 We examined the time course of PE-induced contraction of CPI-17-deficient chicken mesenteric artery compared to those of CPI-17-rich rabbit and pigeon. The rise in the high K+-induced contraction was not significantly different between CPI-17-rich rabbit (Figure 6A) and pigeon (C) and CPI-17-deficient chicken arteries (B) under the same conditions. However, the rise of PE-induced contraction in CPI-17-deficient chicken artery was much slower than high K+-induced contraction in chicken artery (B) and also much slower than PE-induced contraction in CPI-17-rich rabbit and pigeon arteries. These data suggest that the rapid phosphorylation of CPI-17 by PKC activation is necessary for the rapid response of PE-induced contraction.
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 two distinct Ca2+ sensitizing signal transduction pathways leading to inhibition of MLCP: rapid and slow mechanisms mainly driven by PKC and Rho-kinase, respectively (Figure 7). 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.
The initial rapid phosphorylation of CPI-17 was Ca2+-dependent. Treatment of arterial smooth muscle with ryanodine abolished the transient component of Ca2+ increase and the initial rapid but not slow increase in phosphorylation of CPI-17 in response to PE. 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. Together, these results suggest that the SR Ca2+ release is responsible for the initial rapid phase and the voltage-dependent Ca2+ influx plays a crucial role in the sustained phase of the CPI-17 phosphorylation and contraction (Figs. 2, ,33 and and7).7). 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 intracellular Ca2+ level during high K+-induced contraction was higher than that of PE. In fact, increase in the intracellular Ca2+ to 1 μM using the Ca2+/EGTA buffer in α-toxin-permeabilized strips did not significantly increase CPI-17 phosphorylation.12 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 exterminated by PKC inhibitor either GF-109203X or calphostin C but not by Rho-kinase inhibitor Y-27632. These results, in conjunction with the fact that both PKCα and PKCβ but not PKCγ are expressed as a major Ca2+-dependent PKC isoform in rabbit femoral artery (unpublished results), suggest that both IP3-induced SR Ca2+ release and DAG-induced activation of Ca2+-dependent PKC α– and/or β-isoforms are required for the CPI-17 phosphorylation. Removal of rapid CPI-17 phosphorylation by ryanodine treatment 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. 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. This Ca2+-dependent phosphorylation of CPI-17 together with the activation of MLCK initiates a rapid increase in MLC phosphorylation and contraction prior to the slow RhoA/Rho-kinase signaling pathway. In fact, the regulatory/contractile apparatus in the CPI-17-null chicken arterial smooth muscle lacks the rapid response to PE stimulation. Thus, the Ca2+-dependent phosphorylation of CPI-17 is necessary for the rapid contraction induced upon agonist stimulation.
In α-toxin-permeabilized smooth muscle, PDBu, GTPγS and agonists increase contraction and MLC phosphorylation at a given submaximal Ca2+.1,14,26 Since the Ca2+ release from the SR has a crucial role in the rapid phosphorylation of CPI-17 by PKC, the rapid Ca2+-dependent CPI-17 phosphorylation cannot be seen in permeabilized smooth muscle 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 Ca2+ loading of SR effect the rapid CPI-17 phosphorylation and thus the rapid Ca2+ sensitization of contraction in intact smooth muscle.
The Ca2+-dependent translocation of PKCα 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 about 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 to the translocation of PKCα. The issue, however, should be reevaluated in the fresh smooth muscle tissues or cells under conditions where CPI-17 is rapidly phosphorylated by PKC in response to agonists.
Phosphorylation of CPI-17 at the slow and sustained phase is rather insensitive to the Ca2+ blockers and PKC-inhibitors as compared with that of the initial phase, 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, suggesting that other mechanism(s) besides Ca2+-dependent PKC/CPI-17 phosphorylation are slowly developed at the late phase.
MYPT1 phosphorylation at Thr853 in response to either PE or high K+ is rather slowly augmented compared to 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. 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 to the observation that noradrenalin stimulation induces a slow increase in the amount of active GTP-bound form of RhoA in artery.28 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. This contraction was partially inhibited by either GF-109203X or Y-27632 and completely inhibited by a mixture of two inhibitors (not shown). These results suggest that this slow development of MLC phosphorylation indicates a slow inhibition of MLCP in intact artery via three possible Ca2+-independent mechanisms: nPKC/CPI-17, Rho-kinase/CPI-17 and Rho-kinase/MYPT1 (Figure 7).
In conclusion, α1-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: a down-regulation of phosphatase in coordination with the up-regulation of Ca2+-dependent kinase to synergistically increase a phosphorylation of the same target protein MLC and a large contraction.
We thank Katsuhide Mabuchi and Terry Tao (BBRI) for providing recombinant MYPT1 and Rho-kinase. This work was supported by National Institute of Health grants R01HL70881 and P01AR041637 to TK and R01HL083261 to ME.