Urocortin-Induced Decrease in Ca2+ Sensitivity of Contraction in Mouse Tail Arteries Is Attributable to cAMP-Dependent Dephosphory
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Lubomir T. Lubomirov, Katrin Reimann, Do
参见附件。
the Institute of Vegetative Physiology (L.T.L., K.R., D.M., V.H., R. Stehle, G.P.), University of Cologne, Germany
First Department of Internal Medicine (M.I.), Mie University School of Medicine, Japan
Muscle Biology Group (D.J.H.), University of Arizona, Tucson
Department of Physiology (H.G.), St. Kliment Ohridski University, Sofia, Bulgaria
Institute of Physiology (R. Schubert), University of Rostock, Germany.
Abstract
Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca2+ sensitivity through a still unclear mechanism. Here we show that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 μmol/L noradrenaline was associated with a 2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with -toxin were relaxed with urocortin by 39±3% at constant [Ca2+], which was associated with a decrease in myosin light chain (MLC20Ser19), MYPT1Thr696, and MYPT1Thr850 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 μmol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20Ser19 2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20Ser19 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca2+-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1.
Key Words: arteries calcium sensitivity urocortin PKA myosin phosphatase
Introduction
Urocortin, (now known as urocortin 1 [UCN1]) is a 40 amino acid polypeptide that belongs to the corticotropin-releasing factor (CRF) family.1 Urocortin-like immunoreactivity2 and expression of the peripheral subtype of CRF receptors, CRF-2R,3 has been detected in the circulatory system. Urocortin, which has a higher affinity for CRF-2R than CRF itself, relaxes blood vessels both in vitro4 and in vivo,5 causing a CRF-2R–mediated decrease in the mean arterial blood pressure.6 Interestingly, in CRF-2R–deficient mice, the resting blood pressure was elevated.6 These findings suggest that urocortin is an endogenous regulator of blood pressure and blood flow. In addition, urocortin, the plasma levels of which are increased in human heart failure,7 has beneficial effects in experimental heart failure.8
The mechanism by which urocortin relaxes blood vessels appears to be complex, eg, vasodilation has been reported to be both endothelium dependent and independent.3,4,9,10 The endothelium-independent vasodilation was suggested to be mediated by activation of the cAMP/protein kinase A (PKA) signaling cascade,2,11 which is in line with the observation that activation of CRF-2R increases cAMP levels in Ltk cells.12 In some vessels, activation of PKA was associated with activation of potassium channels and membrane potential hyperpolarization,4,9 which will then lead to a decrease in intracellular Ca2+ ([Ca2+]i). However, in rat tail arteries, relaxation occurred without a decrease in global [Ca2+]i,11 ie, by a phenomenon known as Ca2+ desensitization of myofilaments.13
The contractile state of smooth muscle is mainly governed by phosphorylation of the regulatory light chains of myosin (MLC20), which is determined by the opposing actions of the Ca2+ calmodulin–activated myosin light chain kinase (MLCK) and myosin phosphatase (SMPP-1M). Within this scheme, a decrease in Ca2+ sensitivity will ensue when either MLCK is inhibited or SMPP-1M is activated in a Ca2+-independent manner.13 PKA can decrease the activity of MLCK in vitro, but the physiological relevance is not clear (reviewed by Somlyo and Somlyo13). SMPP-1M was initially thought to be a permanently active unregulated enzyme.14 It is now well established that SMPP-1M is the target of several intracellular signaling cascades that modulate its activity. Thus, G protein–mediated inhibition of SMPP-1M is responsible for Ca2+ sensitization observed in the presence of contractile agonists.13,15 Furthermore, it was shown that cyclic nucleotides, whereby most studies focused on cGMP,13 can increase the activity of SMPP-1M in vascular smooth muscle16–19 through a not fully understood mechanism.
Among several mechanisms that can decrease the activity of SMPP-1M,13 the Rho/Rho-associated kinase pathway emerged as a central mechanism,14,13 which leads to phosphorylation of the regulatory subunit MYPT1 at Thr696 and Thr850 in vitro.20 To what extent vasoconstrictors increase phosphorylation of these sites is still a matter of debate (reviewed by Ito et al14). Clearly, if urocortin decreased phosphorylation of these sites, the activity of SMPP-1M should increase and relaxation should ensue. The aim of this study was to test this hypothesis and to further provide evidence that the increase in SMPP-1M activity is mediated by cAMP signaling.
Materials and Methods
Tension Measurements
Dissection, permeabilization with Staphylococcus aureus -toxin, and tension measurements of intact and permeabilized mouse tail arteries were performed as described.11,21
Determination of Protein Phosphorylation
At the desired time points, arteries mounted on wires and subjected to the same experimental protocol as for force measurements were shock frozen and processed as described before.22 Western blots were probed with antibodies against MYPT1 and MLC20 or phospho-specific antibodies against p-MYPT1Thr696 (eg, see Feng et al20), p-MYPT1Thr850, and p-MLC20Ser19. Immunoreactive bands were detected with enhanced chemiluminescence and evaluated by densitometry, as described by Wirth et al.23 The ratio of the optical densities between total MYPT1 or MLC20 and the respective phospho forms was taken as a measure of phosphorylation.
Statistics
Results are expressed as means±SEM. Statistical significance was evaluated against paired, time-matched control experiments with the Student t test, with P<0.05 being considered significant.
Further details regarding the experimental protocols, materials used, and data analysis methods can be found in the online data supplement available at http://circres.ahajournals.org.
Results
Urocortin-Induced Relaxation of Intact Mouse Tail Arteries Is Associated With Dephosphorylation of MYPT1Thr696
KCl (42 mmol/L) induced a submaximal contraction amounting to 66±2% of force elicited by 10 μmol/L noradrenaline. KCl-induced contraction was similar in arteries with and without endothelium (2.16±0.23 N/m versus 2.43±0.93 N/m, n=5 each, P=0.76). Adding urocortin cumulatively at the plateau of the KCl-induced contraction relaxed the arteries with pD2 (–log ED50) values of 9.02±0.02 and 8.86±0.01 (n=5 each, P=0.98) in preparations with and without endothelium, respectively (Figure 1a). As shown in Figure 1, stimulation with KCl (42 mmol/L) or noradrenaline (0.5 μmol/L) resulted in a 200% increase in MYPT1Thr696 phosphorylation above resting levels (P<0.01 each), which was decreased by addition of urocortin (100 nmol/L) to 127±18% in KCl-preconstricted (n=16, P<0.05) and 114±26% in noradrenaline-preconstricted (n=9, P<0.05) arteries. These values were not different from resting phosphorylation (KCl, P=0.16; noradrenaline, P=0.62).
These experiments suggest that urocortin relaxes arteries by decreasing SMPP-1M activity. To test this hypothesis, arteries were permeabilized with the pore-forming protein -toxin, in which Ca2+-independent effects of agonists on force and SMPP-1M activity can be more directly assessed.19,21,24 This is because the quasiintracellular [Ca2+] can be rigorously controlled with high concentrations of Ca buffers, although the pharmacomechanical coupling remains functionally intact.21
Urocortin Decreased Force and MLC20Ser19 Phosphorylation at Constant Submaximal [Ca2+] in -Toxin–Permeabilized Arteries in a Receptor- and PKA-Dependent Manner
Submaximal contractions amounting to 29.0±3.6% (n=6) of maximal force at pCa 4.3 were elicited by pCa 6.1. Addition of urocortin (100 nmol/L) after 15 minutes, ie, at the plateau of the contraction relaxed the preparations by 39.0±2.8% compared with the tension decline of 3.3±2.6% in control arteries (Figure 2a and 2b). When added cumulatively, the pD2 value of 8.54±0.2 (n=5) was comparable to that in the intact arteries (Figure I in the online data supplement). Maximal force was not affected by urocortin, indicating that urocortin decreases myofilament Ca2+ sensitivity. Submaximal force before addition of urocortin was not different between groups (P=0.42). Urocortin-induced relaxation was associated with a decrease in MLC20Ser19 phosphorylation by 60% (Figure 2c and 2d). We note that no immunoreactivity with the p-MLC20Ser19 antibody could be detected at pCa >8 (Figure 2c), which is in line with the notion that Ser19 is specifically phosphorylated in a Ca2+-dependent manner.13
We then tested (1) whether the effect on force and MLC20Ser19 phosphorylation was receptor mediated and (2) whether it could be attributed to activation of PKA. Preincubation for 15 minutes with (and in the continued presence of) the CRF receptor antagonist 9-41-CRF had no effect on Ca2+-activated force (n=5, P=0.75) but inhibited relaxation induced by urocortin (Figure 3a). Antisauvagine-30, a CRF-2R receptor antagonist,25 similarly blocked urocortin-induced relaxation and MLC20Ser19 dephosphorylation (Figure 3b through 3d).
Preincubation with the PKA inhibitor Rp-8-CPT-cAMPS completely inhibited relaxation (Figure 4a and 4b) and dephosphorylation of MLC20Ser19 (Figure 4c and 4d), whereas addition of the PKA activator Sp-5,6-DCl-cBIMPS at the plateau of the submaximal contraction (pCa 6.1) mimicked the effect of urocortin (Figure 4b). Relaxation was also inhibited by the peptide inhibitor of PKA (supplemental Figure III).
Urocortin and Sp-5,6-DCl-cBIMPS Dephosphorylate MYPT1 in Permeabilized Arteries
Consistent with a previous report,15 MYPT1 is phosphorylated on Thr696 and Thr850 in submaximally (pCa 6.1) activated arteries (Figure 5a and 5b). Addition of urocortin decreased phosphorylation of Thr696 by 28% and Thr850 by 52% (Figure 5a and 5b). The decrease in MYPT1 phosphorylation was completely prevented by pretreatment with Rp-8-CPT-cAMPS (Figure 5a and 5b). Sp-5,6-DCl-cBIMPS decreased Thr696 and Thr850 phosphorylation by, respectively, 52% and 68% (Figure 5a and 5b). Thus, the larger relaxing effect of Sp-5,6-DCl-cBIMPS (Figure 4b) is associated with a larger decrease in MYPT1 phosphorylation, particularly of Thr696.
In line with the in vitro observation that both sites are phosphorylated by Rho-associated kinase (ROK),20 we found that the relatively specific ROK inhibitor Y-27632 (10 μmol/L) decreased MYPT1Thr696/Thr850 phosphorylation to a similar extent as urocortin (Figure 5c and 5d) and nearly maximally relaxed the preparations. Subsequent addition of urocortin did not enhance relaxation (data not shown).
To exclude that dephosphorylation is the result of cAMP-dependent activation of a type 2A phosphatase (PP-2A),26 arteries were preincubated with 3 nmol/L okadaic acid, which specifically inhibits PP-2A. This prevented neither the urocortin-induced dephosphorylation of MYPT1 nor its relaxation (Figure 5e and 5f), whereas inhibition of PP-1 with 3 μmol/L okadaic acid completely blocked MYPT1 dephosphorylation (Figure 5e).
Urocortin Increases the Rates of Relaxation and Dephosphorylation Under Conditions of Inhibited MLCK
To test whether the urocortin-induced decrease in MYPT1 phosphorylation results in an increased activity of SMPP-1M, we measured the rates of relaxation and dephosphorylation of MLC20Ser19 in -toxin–permeabilized arteries, in which relaxation of maximally activated arteries (pCa 4.3) was induced by incubation in relaxing solution containing 10 mmol/L EGTA and ML-9 (200 μmol/L in force and 20 μmol/L in phosphorylation determinations). Under this condition, relaxation is expected to be mainly determined by the activity of SMPP-1M and cross-bridge detachment24,27 (for experimental details, see the online data supplement). In arteries pretreated with urocortin, which affected neither force nor MLC20Ser19 phosphorylation at pCa 4.3, the half-time of relaxation (t1/2) was decreased from 128±6 seconds to 90±5 seconds at 11°C (n=5, P<0.01) and from 29±5 seconds to 23±2 seconds at 22°C (n=3), which gives a Q10 value of 4.4. Pretreatment with 8-Br-cAMP (300 μmol/L) decreased t1/2 in a similar manner (n=4, P<0.05). As in rabbit femoral arteries,27 the tension decline in the mouse tail arteries was biphasic, with an initial quasilinear phase and a subsequent exponential decay (Figure 6a). The acceleration of relaxation seen with urocortin was caused by a decrease in the duration of the linear phase by 26% and an increase in the rate constants of the linear and exponential phase by 54% and 39% (determined at 11°C, n=5, all parameters P<0.05; Figure 6).
The increase in the rate of relaxation was associated with an increase in the rate of MLC20Ser19 dephosphorylation. The time course of apparent dephosphorylation (Figure 7c) could be well fitted with a monoexponential function with rate constants 0.047 sec–1 in control, and 0.10 sec–1 in urocortin-treated arteries (n=5 each, P<0.001). Pretreatment with 30 μmol/L Rp-8-CPT-cAMPS completely inhibited the effect of urocortin on MLC20Ser19 dephosphorylation determined 15 seconds after Ca2+ removal (Figure 7b and 7c). In contrast, Sp-5,6-DCl-cBIMPS mimicked its effect (Figure 7b and 7c).
Knowing that cAMP decreased MLCK activity in vitro, we examined whether urocortin decreased the rate of contraction in the presence of 10 μmol/L microcystin-LR (MC-LR), which completely inhibits SMPP-1M activity.19 As shown in Figure 8, urocortin did not affect the rate of contraction at pCa 6.95, which, under this condition, induced a maximal contraction; t1/2 was 132±18 seconds and 117±9 seconds for control and urocortin pretreated arteries, respectively (Figure 8; for experimental details, see the online data supplement).
These results indicate that urocortin via cAMP increases the activity of SMPP-1M. If this were attributable to a decrease in the inhibitory phosphorylation of MYPT1, as suggested by the steady-state experiments (cf, Figure 5), dephosphorylation of MYPT1 should precede dephosphorylation of MLC20. MYPT1 phosphorylation determined at pCa 4.3 was significantly lower in arteries pretreated with Sp-5,6-DCl-cBIMPS compared with control arteries (Figure 7d). Fifteen seconds after removal of Ca2+ with the ML-9 and EGTA-containing relaxing solution MYPT1 phosphorylation was not different from the values at pCa 4.3 in both groups. We note that the lower degree of MYPT1 phosphorylation had no effect on steady-state force or MLC20Ser19 phosphorylation, indicating that at maximal pCa 4.3 MLCK activity is much larger than SMPP-1M activity irrespective of the phosphorylation state of MYPT1. This is also supported by the previous observation that GTPS, which increases Ca2+ sensitivity by inhibiting SMPP-1M, has no effect on maximal force in tonic smooth muscles.24
Discussion
Consistent with previous in vivo5,6 and in vitro4,9,10 findings, urocortin potently relaxes mouse tail arteries in an endothelial-independent manner. We proposed previously11 that urocortin-induced relaxation was caused by a PKA-mediated decrease in the apparent Ca2+ sensitivity of contraction. The mechanism by which this occurs is, however, not fully understood. Here, we present evidence that urocortin decreases Ca2+ sensitivity by a cAMP-mediated increase in the activity of SMPP-1M, which, at least in part, is attributable to a reduction of the inhibitory phosphorylations of the regulatory subunit of SMPP-1M, MYPT1Thr696/850.
As previously reported (reviewed by Ito et al14), MYPT1Thr696 is phosphorylated under resting conditions. Phosphorylation was further increased during the maintained phase of both noradrenaline- and KCl-induced contractions, which is in line with the observation that the maintained phase of phenylephrine- and KCl-induced contraction of arteries requires the activation of Rho.28 Because stimulation with KCl was performed in the presence of blockers of - and -adrenoceptors, it is unlikely that the increase in MYPT1Thr696 phosphorylation was attributable to a depolarization-induced release of noradrenaline from sympathetic varicosities in the vascular wall. We note that contradictory results have been reported with regard to the agonist-induced phosphorylation of Thr696, ie, both an increase and no change in the level of Thr696 phosphorylation in response to agonists have been reported in different types of smooth muscle (for review, see article by Ito et al14 and references therein). The reason for this discrepancy is not clear at present. Urocortin reversed the activation-induced increase in phosphorylation to the basal levels but not below them. Thus, not only the NO/cGMP29 but also the agonist/cAMP pathway may block the agonist-induced increase in Thr696 phosphorylation.
It was previously reported that Rho may be activated by Ca2+ in rabbit aorta,30 and, hence, if urocortin decreased [Ca2+]i, inactivation of the Rho/ROK cascade could ensue. However, phosphorylation of MYPT1Thr696 and MYPT1Thr850 was also decreased in -toxin–permeabilized preparations in which the quasi-[Ca2+]i can be kept constant by high concentrations of EGTA. Dephosphorylation occurred irrespective of whether urocortin was preincubated (cf, Figure 7d) or added to preconstricted arteries (cf, Figure 5), whereby Thr850 appears to be dephosphorylated to a greater extent than Thr696. These results indicate that MYPT1 dephosphorylation cannot be ascribed to a urocortin- or cAMP-induced decrease in [Ca2+]i. Dephosphorylation of MPYT1 by urocortin was associated with a decrease in submaximal but not maximal Ca2+-induced contraction and MCL20Ser19 phosphorylation in the -toxin–permeabilized arteries. The effects were mediated by CRF receptor activation.
The degree of MYPT1Thr696/Thr850 phosphorylation is determined by the activity of ROK and other MYPT1 kinases,31 and a not-yet-identified MYPT1 phosphatase(s), as well as by interaction32 of the PKG/PKA phosphorylation site MYPT1Ser695 with MYPT1Thr696. It was shown that cAMP inactivates Rho in cultured smooth muscle cells33 and in nonmuscle cells,34,35 which in endothelial cells was associated with activation of myosin phosphatase.35 Unfortunately, it was not possible to obtain sufficient material from the collagen-rich tail arteries to determine the activity of Rho. However, inhibition of ROK by Y-27632 dephosphorylated MYPT1 to a similar extent as urocortin or the PKA activator, whereas urocortin did not further enhance the relaxing effect of Y-27632. These results are compatible with a mechanism that leads to inhibition of Rho/ROK signaling by urocortin. Our results also show that in the permeabilized mouse tail artery ROK is at least partially active in the absence of an agonist. The mechanism by which Rho/ROK is activated under this condition is not yet clear. It is possible that activation of purinergic receptors36 by the ATP-containing incubation solutions leads to activation of Rho. A Ca2+-dependent activation30 appears less likely because removal of Ca2+ by EGTA does not lead to dephosphorylation of MYPT1. It should also be noted that neither urocortin nor Y-27632 completely dephosphorylated MYPT1, suggesting that MYPT1 is phosphorylated by additional kinases.31
MYPT1 can be phosphorylated by PKA and PKG at Ser695, which has no effect on SMPP-1M activity but interferes with subsequent phosphorylation of MYPT1Thr696 and vice versa.32 This mechanism could also clearly account for the decrease in MYPT1 phosphorylation observed when urocortin was added before activation with pCa 4.3 and also possibly when urocortin was added to submaximally precontracted arteries, provided that there is a phosphorylation turnover of MYPT1Thr696. Our results indicate that a PKA-mediated activation of PP-2A26 does not appear to be functional in this preparation. They do suggest that a type 1 MYPT1 phosphatase is active. Thus, if Thr696 is dephosphorylated by this phosphatase, PKA could phosphorylate Ser695, which then would prevent rephosphorylation of Thr696. Future experiments must show whether urocortin acts through inactivation of the Rho/ROK pathway or through these interactive phosphorylation sites or both.
SMPP-1M is bound to myosin, and, hence, its activity may have a functional spatial restriction that is not present with proteins in solution, ie, in muscle lysates. Because of this, we determined the rate of MLC20Ser19 dephosphorylation in the permeabilized arteries under conditions of full MLCK inhibition as a surrogate of SMPP-1M activity.19,24 This rate was increased 2.2-fold in the presence of urocortin, which compares to the 1.4-fold increase in permeabilized rabbit femoral arteries treated with 8-bromo-cGMP19 and the 3- to 4-fold increase in the activity of SMPP-1M determined in lysates of sodium nitroprusside-stimulated carotid arteries.18 The 3-fold slower rate of relaxation, which was also accelerated by urocortin, suggests that tension decline like in other types of smooth muscle27 is rate limited by cross-bridge detachment.
Although it is assumed that phosphorylating reactions are rapidly inactivated by EGTA and ML-9,24 we cannot exclude the possibility that the rate of dephosphorylation may be confounded by kinase reactions. This possibility must be considered because urocortin via cAMP might decrease the activity of MLCK.13 However, if urocortin decreased the MLCK activity, the measured, ie, apparent rate constant of dephosphorylation of MLC20, would be lower in the presence of urocortin rather than higher as observed. This is because it is determined by the sum (k1+k2) of the rate constants of phosphorylating (k1) and dephosphorylating (k2) reactions.37 Furthermore, the Q10 value of the rate of relaxation of 4.4 is much higher than that of MLCK activity (Q10=1.727,37) and is closer to the Q10 of 5.1 for SMPP-1M activity.37 Finally, the rate of contraction under conditions of fully inhibited phosphatase was not decreased by urocortin, which would be the case if MLCK activity were decreased.19,23 We, therefore, propose that urocortin through activation of PKA increases the activity of SMPP-1M. Because MLC20 phosphorylation is proportional to k1/(k1+k2), a decrease in steady-state MLC20 phosphorylation and, hence, tension will ensue.
Our study is limited because the effects of urocortin on SMPP-1M activity were assessed with the maximum effective concentration (100 nmol/L) with regard to vasorelaxation and increased cAMP levels,12 which is 10- to 100-fold higher than reported pD2 values.4 Plasma levels in humans ranged from 14 pmol/L7 to 1 nmol/L.38 Following systemic application in sheep, the blood pressure–lowering effects were associated with plasma levels of 7.5 nmol/L.8 We cannot eliminate the possibility that vasorelaxation at lower concentrations of urocortin involves a different mechanism, but the dose-response relation for Ca2+-desensitization and MYPT1 dephosphorylation in permeabilized arteries (cf, supplemental Figures I and II) are similar to the relaxation of intact arteries in vitro (our study and Schilling et al4). Therefore, we propose that cAMP-dependent activation of SMPP-1M is an important factor contributing to urocortin-mediated vasorelaxation. Because of the limited specificity of the inhibitor and activator of PKA, this cannot be ascribed with certainty to activation of PKA. It is possible that urocortin/cAMP disinhibits SMPP-1M PKA independently, eg, by cross-activation of PKG,39 an unlikely possibility because cAMP-induced relaxation was not attenuated in PKG–/– mice,40 or perhaps by novel cAMP targets (cf, legend of supplemental Figure III and references therein). Thus, future investigations must unravel the precise mechanism that lead to urocortin/cAMP-induced dephosphorylation of MYPT1 and must also show whether MYPT1 dephosphorylation acts in conjunction with dephosphorylation of CPI-17, which, when phosphorylated, inhibits SMPP-1M.15 PKA-mediated phosphorylation of telokin, an endogenous putative activator of SMPP-1M,17 may be of minor importance because of its low expression in tonic smooth muscle.
Our study adds further evidence to the notion that SMPP-1M activity in addition to [Ca2+]i is critical for regulating vascular tone. Its activity is determined by the balance of activating signals, ie, NO/cGMP16,18,19 and agonist/cAMP (this study), and inhibitory signals originating from vasoconstrictors.13 Evidence is increasing that this balance is disturbed under certain pathological conditions.22,29,41 Prostacyclin, currently among the best drugs to treat primary pulmonary hypertension,42 elevates cAMP levels. Hence, its beneficial effect may, in part, be caused by disinhibition of SMPP-1M and normalization of this balance. Understanding the mechanisms that regulate SMPP-1M in vivo will likely result in new therapeutic options.
Acknowledgments
Funding was provided by the Deutsche Forschungsgemeinschaft (436 BUL 17/7/01 and SCHU 805/7-1 to R. Schubert; and PF226/10-1 to G.P.) and the Medical Faculty of University of Cologne (to G.P). The expert technical assistance of M. Kneese is gratefully acknowledged.
Footnotes
Original received July 28, 2004; resubmission received November 15, 2005; revised resubmission received March 8, 2006; accepted March 21, 2006.
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the Institute of Vegetative Physiology (L.T.L., K.R., D.M., V.H., R. Stehle, G.P.), University of Cologne, Germany
First Department of Internal Medicine (M.I.), Mie University School of Medicine, Japan
Muscle Biology Group (D.J.H.), University of Arizona, Tucson
Department of Physiology (H.G.), St. Kliment Ohridski University, Sofia, Bulgaria
Institute of Physiology (R. Schubert), University of Rostock, Germany.
Abstract
Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca2+ sensitivity through a still unclear mechanism. Here we show that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 μmol/L noradrenaline was associated with a 2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with -toxin were relaxed with urocortin by 39±3% at constant [Ca2+], which was associated with a decrease in myosin light chain (MLC20Ser19), MYPT1Thr696, and MYPT1Thr850 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 μmol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20Ser19 2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20Ser19 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca2+-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1.
Key Words: arteries calcium sensitivity urocortin PKA myosin phosphatase
Introduction
Urocortin, (now known as urocortin 1 [UCN1]) is a 40 amino acid polypeptide that belongs to the corticotropin-releasing factor (CRF) family.1 Urocortin-like immunoreactivity2 and expression of the peripheral subtype of CRF receptors, CRF-2R,3 has been detected in the circulatory system. Urocortin, which has a higher affinity for CRF-2R than CRF itself, relaxes blood vessels both in vitro4 and in vivo,5 causing a CRF-2R–mediated decrease in the mean arterial blood pressure.6 Interestingly, in CRF-2R–deficient mice, the resting blood pressure was elevated.6 These findings suggest that urocortin is an endogenous regulator of blood pressure and blood flow. In addition, urocortin, the plasma levels of which are increased in human heart failure,7 has beneficial effects in experimental heart failure.8
The mechanism by which urocortin relaxes blood vessels appears to be complex, eg, vasodilation has been reported to be both endothelium dependent and independent.3,4,9,10 The endothelium-independent vasodilation was suggested to be mediated by activation of the cAMP/protein kinase A (PKA) signaling cascade,2,11 which is in line with the observation that activation of CRF-2R increases cAMP levels in Ltk cells.12 In some vessels, activation of PKA was associated with activation of potassium channels and membrane potential hyperpolarization,4,9 which will then lead to a decrease in intracellular Ca2+ ([Ca2+]i). However, in rat tail arteries, relaxation occurred without a decrease in global [Ca2+]i,11 ie, by a phenomenon known as Ca2+ desensitization of myofilaments.13
The contractile state of smooth muscle is mainly governed by phosphorylation of the regulatory light chains of myosin (MLC20), which is determined by the opposing actions of the Ca2+ calmodulin–activated myosin light chain kinase (MLCK) and myosin phosphatase (SMPP-1M). Within this scheme, a decrease in Ca2+ sensitivity will ensue when either MLCK is inhibited or SMPP-1M is activated in a Ca2+-independent manner.13 PKA can decrease the activity of MLCK in vitro, but the physiological relevance is not clear (reviewed by Somlyo and Somlyo13). SMPP-1M was initially thought to be a permanently active unregulated enzyme.14 It is now well established that SMPP-1M is the target of several intracellular signaling cascades that modulate its activity. Thus, G protein–mediated inhibition of SMPP-1M is responsible for Ca2+ sensitization observed in the presence of contractile agonists.13,15 Furthermore, it was shown that cyclic nucleotides, whereby most studies focused on cGMP,13 can increase the activity of SMPP-1M in vascular smooth muscle16–19 through a not fully understood mechanism.
Among several mechanisms that can decrease the activity of SMPP-1M,13 the Rho/Rho-associated kinase pathway emerged as a central mechanism,14,13 which leads to phosphorylation of the regulatory subunit MYPT1 at Thr696 and Thr850 in vitro.20 To what extent vasoconstrictors increase phosphorylation of these sites is still a matter of debate (reviewed by Ito et al14). Clearly, if urocortin decreased phosphorylation of these sites, the activity of SMPP-1M should increase and relaxation should ensue. The aim of this study was to test this hypothesis and to further provide evidence that the increase in SMPP-1M activity is mediated by cAMP signaling.
Materials and Methods
Tension Measurements
Dissection, permeabilization with Staphylococcus aureus -toxin, and tension measurements of intact and permeabilized mouse tail arteries were performed as described.11,21
Determination of Protein Phosphorylation
At the desired time points, arteries mounted on wires and subjected to the same experimental protocol as for force measurements were shock frozen and processed as described before.22 Western blots were probed with antibodies against MYPT1 and MLC20 or phospho-specific antibodies against p-MYPT1Thr696 (eg, see Feng et al20), p-MYPT1Thr850, and p-MLC20Ser19. Immunoreactive bands were detected with enhanced chemiluminescence and evaluated by densitometry, as described by Wirth et al.23 The ratio of the optical densities between total MYPT1 or MLC20 and the respective phospho forms was taken as a measure of phosphorylation.
Statistics
Results are expressed as means±SEM. Statistical significance was evaluated against paired, time-matched control experiments with the Student t test, with P<0.05 being considered significant.
Further details regarding the experimental protocols, materials used, and data analysis methods can be found in the online data supplement available at http://circres.ahajournals.org.
Results
Urocortin-Induced Relaxation of Intact Mouse Tail Arteries Is Associated With Dephosphorylation of MYPT1Thr696
KCl (42 mmol/L) induced a submaximal contraction amounting to 66±2% of force elicited by 10 μmol/L noradrenaline. KCl-induced contraction was similar in arteries with and without endothelium (2.16±0.23 N/m versus 2.43±0.93 N/m, n=5 each, P=0.76). Adding urocortin cumulatively at the plateau of the KCl-induced contraction relaxed the arteries with pD2 (–log ED50) values of 9.02±0.02 and 8.86±0.01 (n=5 each, P=0.98) in preparations with and without endothelium, respectively (Figure 1a). As shown in Figure 1, stimulation with KCl (42 mmol/L) or noradrenaline (0.5 μmol/L) resulted in a 200% increase in MYPT1Thr696 phosphorylation above resting levels (P<0.01 each), which was decreased by addition of urocortin (100 nmol/L) to 127±18% in KCl-preconstricted (n=16, P<0.05) and 114±26% in noradrenaline-preconstricted (n=9, P<0.05) arteries. These values were not different from resting phosphorylation (KCl, P=0.16; noradrenaline, P=0.62).
These experiments suggest that urocortin relaxes arteries by decreasing SMPP-1M activity. To test this hypothesis, arteries were permeabilized with the pore-forming protein -toxin, in which Ca2+-independent effects of agonists on force and SMPP-1M activity can be more directly assessed.19,21,24 This is because the quasiintracellular [Ca2+] can be rigorously controlled with high concentrations of Ca buffers, although the pharmacomechanical coupling remains functionally intact.21
Urocortin Decreased Force and MLC20Ser19 Phosphorylation at Constant Submaximal [Ca2+] in -Toxin–Permeabilized Arteries in a Receptor- and PKA-Dependent Manner
Submaximal contractions amounting to 29.0±3.6% (n=6) of maximal force at pCa 4.3 were elicited by pCa 6.1. Addition of urocortin (100 nmol/L) after 15 minutes, ie, at the plateau of the contraction relaxed the preparations by 39.0±2.8% compared with the tension decline of 3.3±2.6% in control arteries (Figure 2a and 2b). When added cumulatively, the pD2 value of 8.54±0.2 (n=5) was comparable to that in the intact arteries (Figure I in the online data supplement). Maximal force was not affected by urocortin, indicating that urocortin decreases myofilament Ca2+ sensitivity. Submaximal force before addition of urocortin was not different between groups (P=0.42). Urocortin-induced relaxation was associated with a decrease in MLC20Ser19 phosphorylation by 60% (Figure 2c and 2d). We note that no immunoreactivity with the p-MLC20Ser19 antibody could be detected at pCa >8 (Figure 2c), which is in line with the notion that Ser19 is specifically phosphorylated in a Ca2+-dependent manner.13
We then tested (1) whether the effect on force and MLC20Ser19 phosphorylation was receptor mediated and (2) whether it could be attributed to activation of PKA. Preincubation for 15 minutes with (and in the continued presence of) the CRF receptor antagonist 9-41-CRF had no effect on Ca2+-activated force (n=5, P=0.75) but inhibited relaxation induced by urocortin (Figure 3a). Antisauvagine-30, a CRF-2R receptor antagonist,25 similarly blocked urocortin-induced relaxation and MLC20Ser19 dephosphorylation (Figure 3b through 3d).
Preincubation with the PKA inhibitor Rp-8-CPT-cAMPS completely inhibited relaxation (Figure 4a and 4b) and dephosphorylation of MLC20Ser19 (Figure 4c and 4d), whereas addition of the PKA activator Sp-5,6-DCl-cBIMPS at the plateau of the submaximal contraction (pCa 6.1) mimicked the effect of urocortin (Figure 4b). Relaxation was also inhibited by the peptide inhibitor of PKA (supplemental Figure III).
Urocortin and Sp-5,6-DCl-cBIMPS Dephosphorylate MYPT1 in Permeabilized Arteries
Consistent with a previous report,15 MYPT1 is phosphorylated on Thr696 and Thr850 in submaximally (pCa 6.1) activated arteries (Figure 5a and 5b). Addition of urocortin decreased phosphorylation of Thr696 by 28% and Thr850 by 52% (Figure 5a and 5b). The decrease in MYPT1 phosphorylation was completely prevented by pretreatment with Rp-8-CPT-cAMPS (Figure 5a and 5b). Sp-5,6-DCl-cBIMPS decreased Thr696 and Thr850 phosphorylation by, respectively, 52% and 68% (Figure 5a and 5b). Thus, the larger relaxing effect of Sp-5,6-DCl-cBIMPS (Figure 4b) is associated with a larger decrease in MYPT1 phosphorylation, particularly of Thr696.
In line with the in vitro observation that both sites are phosphorylated by Rho-associated kinase (ROK),20 we found that the relatively specific ROK inhibitor Y-27632 (10 μmol/L) decreased MYPT1Thr696/Thr850 phosphorylation to a similar extent as urocortin (Figure 5c and 5d) and nearly maximally relaxed the preparations. Subsequent addition of urocortin did not enhance relaxation (data not shown).
To exclude that dephosphorylation is the result of cAMP-dependent activation of a type 2A phosphatase (PP-2A),26 arteries were preincubated with 3 nmol/L okadaic acid, which specifically inhibits PP-2A. This prevented neither the urocortin-induced dephosphorylation of MYPT1 nor its relaxation (Figure 5e and 5f), whereas inhibition of PP-1 with 3 μmol/L okadaic acid completely blocked MYPT1 dephosphorylation (Figure 5e).
Urocortin Increases the Rates of Relaxation and Dephosphorylation Under Conditions of Inhibited MLCK
To test whether the urocortin-induced decrease in MYPT1 phosphorylation results in an increased activity of SMPP-1M, we measured the rates of relaxation and dephosphorylation of MLC20Ser19 in -toxin–permeabilized arteries, in which relaxation of maximally activated arteries (pCa 4.3) was induced by incubation in relaxing solution containing 10 mmol/L EGTA and ML-9 (200 μmol/L in force and 20 μmol/L in phosphorylation determinations). Under this condition, relaxation is expected to be mainly determined by the activity of SMPP-1M and cross-bridge detachment24,27 (for experimental details, see the online data supplement). In arteries pretreated with urocortin, which affected neither force nor MLC20Ser19 phosphorylation at pCa 4.3, the half-time of relaxation (t1/2) was decreased from 128±6 seconds to 90±5 seconds at 11°C (n=5, P<0.01) and from 29±5 seconds to 23±2 seconds at 22°C (n=3), which gives a Q10 value of 4.4. Pretreatment with 8-Br-cAMP (300 μmol/L) decreased t1/2 in a similar manner (n=4, P<0.05). As in rabbit femoral arteries,27 the tension decline in the mouse tail arteries was biphasic, with an initial quasilinear phase and a subsequent exponential decay (Figure 6a). The acceleration of relaxation seen with urocortin was caused by a decrease in the duration of the linear phase by 26% and an increase in the rate constants of the linear and exponential phase by 54% and 39% (determined at 11°C, n=5, all parameters P<0.05; Figure 6).
The increase in the rate of relaxation was associated with an increase in the rate of MLC20Ser19 dephosphorylation. The time course of apparent dephosphorylation (Figure 7c) could be well fitted with a monoexponential function with rate constants 0.047 sec–1 in control, and 0.10 sec–1 in urocortin-treated arteries (n=5 each, P<0.001). Pretreatment with 30 μmol/L Rp-8-CPT-cAMPS completely inhibited the effect of urocortin on MLC20Ser19 dephosphorylation determined 15 seconds after Ca2+ removal (Figure 7b and 7c). In contrast, Sp-5,6-DCl-cBIMPS mimicked its effect (Figure 7b and 7c).
Knowing that cAMP decreased MLCK activity in vitro, we examined whether urocortin decreased the rate of contraction in the presence of 10 μmol/L microcystin-LR (MC-LR), which completely inhibits SMPP-1M activity.19 As shown in Figure 8, urocortin did not affect the rate of contraction at pCa 6.95, which, under this condition, induced a maximal contraction; t1/2 was 132±18 seconds and 117±9 seconds for control and urocortin pretreated arteries, respectively (Figure 8; for experimental details, see the online data supplement).
These results indicate that urocortin via cAMP increases the activity of SMPP-1M. If this were attributable to a decrease in the inhibitory phosphorylation of MYPT1, as suggested by the steady-state experiments (cf, Figure 5), dephosphorylation of MYPT1 should precede dephosphorylation of MLC20. MYPT1 phosphorylation determined at pCa 4.3 was significantly lower in arteries pretreated with Sp-5,6-DCl-cBIMPS compared with control arteries (Figure 7d). Fifteen seconds after removal of Ca2+ with the ML-9 and EGTA-containing relaxing solution MYPT1 phosphorylation was not different from the values at pCa 4.3 in both groups. We note that the lower degree of MYPT1 phosphorylation had no effect on steady-state force or MLC20Ser19 phosphorylation, indicating that at maximal pCa 4.3 MLCK activity is much larger than SMPP-1M activity irrespective of the phosphorylation state of MYPT1. This is also supported by the previous observation that GTPS, which increases Ca2+ sensitivity by inhibiting SMPP-1M, has no effect on maximal force in tonic smooth muscles.24
Discussion
Consistent with previous in vivo5,6 and in vitro4,9,10 findings, urocortin potently relaxes mouse tail arteries in an endothelial-independent manner. We proposed previously11 that urocortin-induced relaxation was caused by a PKA-mediated decrease in the apparent Ca2+ sensitivity of contraction. The mechanism by which this occurs is, however, not fully understood. Here, we present evidence that urocortin decreases Ca2+ sensitivity by a cAMP-mediated increase in the activity of SMPP-1M, which, at least in part, is attributable to a reduction of the inhibitory phosphorylations of the regulatory subunit of SMPP-1M, MYPT1Thr696/850.
As previously reported (reviewed by Ito et al14), MYPT1Thr696 is phosphorylated under resting conditions. Phosphorylation was further increased during the maintained phase of both noradrenaline- and KCl-induced contractions, which is in line with the observation that the maintained phase of phenylephrine- and KCl-induced contraction of arteries requires the activation of Rho.28 Because stimulation with KCl was performed in the presence of blockers of - and -adrenoceptors, it is unlikely that the increase in MYPT1Thr696 phosphorylation was attributable to a depolarization-induced release of noradrenaline from sympathetic varicosities in the vascular wall. We note that contradictory results have been reported with regard to the agonist-induced phosphorylation of Thr696, ie, both an increase and no change in the level of Thr696 phosphorylation in response to agonists have been reported in different types of smooth muscle (for review, see article by Ito et al14 and references therein). The reason for this discrepancy is not clear at present. Urocortin reversed the activation-induced increase in phosphorylation to the basal levels but not below them. Thus, not only the NO/cGMP29 but also the agonist/cAMP pathway may block the agonist-induced increase in Thr696 phosphorylation.
It was previously reported that Rho may be activated by Ca2+ in rabbit aorta,30 and, hence, if urocortin decreased [Ca2+]i, inactivation of the Rho/ROK cascade could ensue. However, phosphorylation of MYPT1Thr696 and MYPT1Thr850 was also decreased in -toxin–permeabilized preparations in which the quasi-[Ca2+]i can be kept constant by high concentrations of EGTA. Dephosphorylation occurred irrespective of whether urocortin was preincubated (cf, Figure 7d) or added to preconstricted arteries (cf, Figure 5), whereby Thr850 appears to be dephosphorylated to a greater extent than Thr696. These results indicate that MYPT1 dephosphorylation cannot be ascribed to a urocortin- or cAMP-induced decrease in [Ca2+]i. Dephosphorylation of MPYT1 by urocortin was associated with a decrease in submaximal but not maximal Ca2+-induced contraction and MCL20Ser19 phosphorylation in the -toxin–permeabilized arteries. The effects were mediated by CRF receptor activation.
The degree of MYPT1Thr696/Thr850 phosphorylation is determined by the activity of ROK and other MYPT1 kinases,31 and a not-yet-identified MYPT1 phosphatase(s), as well as by interaction32 of the PKG/PKA phosphorylation site MYPT1Ser695 with MYPT1Thr696. It was shown that cAMP inactivates Rho in cultured smooth muscle cells33 and in nonmuscle cells,34,35 which in endothelial cells was associated with activation of myosin phosphatase.35 Unfortunately, it was not possible to obtain sufficient material from the collagen-rich tail arteries to determine the activity of Rho. However, inhibition of ROK by Y-27632 dephosphorylated MYPT1 to a similar extent as urocortin or the PKA activator, whereas urocortin did not further enhance the relaxing effect of Y-27632. These results are compatible with a mechanism that leads to inhibition of Rho/ROK signaling by urocortin. Our results also show that in the permeabilized mouse tail artery ROK is at least partially active in the absence of an agonist. The mechanism by which Rho/ROK is activated under this condition is not yet clear. It is possible that activation of purinergic receptors36 by the ATP-containing incubation solutions leads to activation of Rho. A Ca2+-dependent activation30 appears less likely because removal of Ca2+ by EGTA does not lead to dephosphorylation of MYPT1. It should also be noted that neither urocortin nor Y-27632 completely dephosphorylated MYPT1, suggesting that MYPT1 is phosphorylated by additional kinases.31
MYPT1 can be phosphorylated by PKA and PKG at Ser695, which has no effect on SMPP-1M activity but interferes with subsequent phosphorylation of MYPT1Thr696 and vice versa.32 This mechanism could also clearly account for the decrease in MYPT1 phosphorylation observed when urocortin was added before activation with pCa 4.3 and also possibly when urocortin was added to submaximally precontracted arteries, provided that there is a phosphorylation turnover of MYPT1Thr696. Our results indicate that a PKA-mediated activation of PP-2A26 does not appear to be functional in this preparation. They do suggest that a type 1 MYPT1 phosphatase is active. Thus, if Thr696 is dephosphorylated by this phosphatase, PKA could phosphorylate Ser695, which then would prevent rephosphorylation of Thr696. Future experiments must show whether urocortin acts through inactivation of the Rho/ROK pathway or through these interactive phosphorylation sites or both.
SMPP-1M is bound to myosin, and, hence, its activity may have a functional spatial restriction that is not present with proteins in solution, ie, in muscle lysates. Because of this, we determined the rate of MLC20Ser19 dephosphorylation in the permeabilized arteries under conditions of full MLCK inhibition as a surrogate of SMPP-1M activity.19,24 This rate was increased 2.2-fold in the presence of urocortin, which compares to the 1.4-fold increase in permeabilized rabbit femoral arteries treated with 8-bromo-cGMP19 and the 3- to 4-fold increase in the activity of SMPP-1M determined in lysates of sodium nitroprusside-stimulated carotid arteries.18 The 3-fold slower rate of relaxation, which was also accelerated by urocortin, suggests that tension decline like in other types of smooth muscle27 is rate limited by cross-bridge detachment.
Although it is assumed that phosphorylating reactions are rapidly inactivated by EGTA and ML-9,24 we cannot exclude the possibility that the rate of dephosphorylation may be confounded by kinase reactions. This possibility must be considered because urocortin via cAMP might decrease the activity of MLCK.13 However, if urocortin decreased the MLCK activity, the measured, ie, apparent rate constant of dephosphorylation of MLC20, would be lower in the presence of urocortin rather than higher as observed. This is because it is determined by the sum (k1+k2) of the rate constants of phosphorylating (k1) and dephosphorylating (k2) reactions.37 Furthermore, the Q10 value of the rate of relaxation of 4.4 is much higher than that of MLCK activity (Q10=1.727,37) and is closer to the Q10 of 5.1 for SMPP-1M activity.37 Finally, the rate of contraction under conditions of fully inhibited phosphatase was not decreased by urocortin, which would be the case if MLCK activity were decreased.19,23 We, therefore, propose that urocortin through activation of PKA increases the activity of SMPP-1M. Because MLC20 phosphorylation is proportional to k1/(k1+k2), a decrease in steady-state MLC20 phosphorylation and, hence, tension will ensue.
Our study is limited because the effects of urocortin on SMPP-1M activity were assessed with the maximum effective concentration (100 nmol/L) with regard to vasorelaxation and increased cAMP levels,12 which is 10- to 100-fold higher than reported pD2 values.4 Plasma levels in humans ranged from 14 pmol/L7 to 1 nmol/L.38 Following systemic application in sheep, the blood pressure–lowering effects were associated with plasma levels of 7.5 nmol/L.8 We cannot eliminate the possibility that vasorelaxation at lower concentrations of urocortin involves a different mechanism, but the dose-response relation for Ca2+-desensitization and MYPT1 dephosphorylation in permeabilized arteries (cf, supplemental Figures I and II) are similar to the relaxation of intact arteries in vitro (our study and Schilling et al4). Therefore, we propose that cAMP-dependent activation of SMPP-1M is an important factor contributing to urocortin-mediated vasorelaxation. Because of the limited specificity of the inhibitor and activator of PKA, this cannot be ascribed with certainty to activation of PKA. It is possible that urocortin/cAMP disinhibits SMPP-1M PKA independently, eg, by cross-activation of PKG,39 an unlikely possibility because cAMP-induced relaxation was not attenuated in PKG–/– mice,40 or perhaps by novel cAMP targets (cf, legend of supplemental Figure III and references therein). Thus, future investigations must unravel the precise mechanism that lead to urocortin/cAMP-induced dephosphorylation of MYPT1 and must also show whether MYPT1 dephosphorylation acts in conjunction with dephosphorylation of CPI-17, which, when phosphorylated, inhibits SMPP-1M.15 PKA-mediated phosphorylation of telokin, an endogenous putative activator of SMPP-1M,17 may be of minor importance because of its low expression in tonic smooth muscle.
Our study adds further evidence to the notion that SMPP-1M activity in addition to [Ca2+]i is critical for regulating vascular tone. Its activity is determined by the balance of activating signals, ie, NO/cGMP16,18,19 and agonist/cAMP (this study), and inhibitory signals originating from vasoconstrictors.13 Evidence is increasing that this balance is disturbed under certain pathological conditions.22,29,41 Prostacyclin, currently among the best drugs to treat primary pulmonary hypertension,42 elevates cAMP levels. Hence, its beneficial effect may, in part, be caused by disinhibition of SMPP-1M and normalization of this balance. Understanding the mechanisms that regulate SMPP-1M in vivo will likely result in new therapeutic options.
Acknowledgments
Funding was provided by the Deutsche Forschungsgemeinschaft (436 BUL 17/7/01 and SCHU 805/7-1 to R. Schubert; and PF226/10-1 to G.P.) and the Medical Faculty of University of Cologne (to G.P). The expert technical assistance of M. Kneese is gratefully acknowledged.
Footnotes
Original received July 28, 2004; resubmission received November 15, 2005; revised resubmission received March 8, 2006; accepted March 21, 2006.
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