The involvement of phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and MYPT1 isoform expression in NO/cGMP mediated differential vasoregulation of cerebral arteries compared to systemic arteries.

AIM: Constitutive release of NO blunts intrinsic and stimulated contractile activity in cerebral arteries (CA). Here, we explored whether phosphorylation and expression levels of the PKG-sensitive, leucine zipper positive (LZ+ ) splice variants of the regulatory subunit of myosin phosphatase (MYPT1) are involved and whether its expression is associated with higher cGMP sensitivity. METHODS: Vascular contractility was investigated by wire myography. Phosphorylation of MYPT1 was determined by Western blotting. RESULTS: Constitutive phosphorylation of MYPT1-T696 and T853 was lower and that of S695 and S668 was higher in cerebral arteries from the circulus arteriosus (CA-w) than in femoral arteries (FA), while total MYPT1 expression was not different. In CA-w but not in FA, L-NAME lowered phosphorylation of S695/S668 and increased phosphorylation of T696/T853 and of MLC20 -S19, plus basal tone. The increase in basal tone was attenuated in CA-w and basilar arteries (BA) from heterozygous MYPT1-T696A/+ mice. Compared to FA, expression of the LZ+ -isoform was ~2-fold higher in CA-w coincident with a higher sensitivity to DEA-NONOate, cinaciguat and Y27632 in BA and 8-Br-cGMP (1 µmol/L) in pre-constricted (pCa 6.1) α-toxin permeabilized CAs. In contrast, 6-Bnz-cAMP (10 µmol/L) relaxed BA and FA similarly by ~80%. CONCLUSION: Our results indicate that (i) regulation of the intrinsic contractile activity in CA involves phosphorylation of MYPT1 at T696 and S695/S668, (ii) the higher NO/cGMP/PKG sensitivity of CAs can be ascribed to the higher expression level of the LZ+ -MYPT1 isoform and (iii) relaxation by cAMP/PKA pathway is less dependent on the expression level of the LZ+ splice variants of MYPT1.

[Myosin phosphorylation as the main way of regulating smooth muscle contractions].

Rho-kinase is a key enzyme of the receptor-dependent signal cascades and is regarded today as the most prospective target for pharmacological therapy of smooth muscle contractility disorders.

CAP2, cyclase-associated protein 2, is a dual compartment protein.

Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.

[Urocortin decreases phosphorylation of MYPT1 and increases the myosin phosphatase activity via elevation of the intracellular level of cAMP].

Urocortin, a peptide hormone related to the corticotropin releasing factor, is suggested to be involved in blood pressure regulation by dilating the peripheral blood vessels. In rat tail arteries, urocortin-induced vasodilation is due to a decrease in myofilament Ca2+ sensitivity the mechanism of which is still unclear. In this study, the hypothesis was tested that the decrease in Ca2+ sensitivity in mouse tail arteries results from the activation of myosin light chain phosphatase. The relaxation of KCl-precontracted (42 mM) intact mouse tail arteries by urocortin (1 nM and 10 nM) was significantly inhibited by 1 microM antisauvagine30, a CRF-2 receptor antagonist (p < 0.05, n = 3). The addition of 1 microM KT 5720, an inhibitor of PKA, to intact rat tail arteries did not affect the KCl-induced force but significantly attenuated the urocortin-induced relaxation (n = 5). In alpha-toxin permeabilized mouse tail arteries, urocortin relaxed submaximally activated preparations at constant pCa 6.1 by 37.6 +/- 8.2% (n = 5) as compared to control vessels (n = 5, p < 0.001). The relaxation in permeabilized vessels was inhibited by pre-treatment with 30 microM Rp-8-CPT-cAMPS, an inactive analogue of cAMP. In permeabilized mouse tail arteries, treatment with 100 nM urocortin was associated with dephosphorylation of MLC20(Ser19) and MYPT1(Thr696/Thr850). The effect of urocortin on MYPTI dephosphorylation was completely abolished by 30 M Rp-8-CPT-cAMPS and mimicked by the cAMP analogue Sp-5,6-DCI-cBiMPS. Based on these findings, we propose that the urocortin-induced relaxation is due to a decrease in calcium sensitivity mediated by a cAMP-dependent increase in the activity of MLCP.

Mechanical properties of sarcomeres during cardiac myofibrillar relaxation: stretch-induced cross-bridge detachment contributes to early diastolic filling.

Sudden Ca2+ removal from isometrically contracting cardiac myofibrils induces a biphasic relaxation: first a slow, linear force decline during which sarcomeres remain isometric and then a rapid, exponential decay originating from sequential lengthening, i.e., successive mechanical relaxation, of individual sarcomeres (Stehle et al. 2002; Biophys J 83:2152-2162). Step-stretches were applied to the myofibrils, in order to study the mechanical properties of sarcomeres during this dynamic relaxation process. Stretch applied soon (approximately 10 ms) after Ca2+ removal accelerated the initiation of the rapid, exponential force decay and of the sequential sarcomere lengthening. After the stretch, a short, transient period (approximately 24 ms) remained, during which time force was enhanced and sarcomeres were homogenously elongated by the stretch. This period was similar to the duration of the switching-off of troponin C in myofibrils, as measured by stopped-flow. In contrast, when the stretch was applied during the rapid, exponential relaxation phase, force quickly decayed after stretch, back to the force level of isometric controls or even lower. Smaller stretches lengthened only those sarcomeres that were located at the wave front of the sequential sarcomere relaxation. The more the stretch-size was increased, the more of the contracting sarcomeres became lengthened by the stretch; those sarcomeres that were relaxed prior to stretch were barely elongated. These results indicate that the stretch accelerates myofibrillar relaxation by forcing the cross-bridges in contracting sarcomeres to detach. Subsequent rapid cross-bridge reattachment occurs during a short period after Ca2+ removal until troponin C is switched off. However, this switch off occurs approximately 5 times too fast to directly rate-limit the force relaxation under the isometric condition. After troponin C is switched off, stretching induces cross-bridge detachment without subsequent reattachment, and force rapidly decays below the isometric level. This may explain the rapid distention of the ventricular myocardium during early diastolic filling.

Regulation of the crossbridge cycle in vascular smooth muscle by cAMP signalling.

Urocortin, a novel vasodilatory peptide related to the corticotropin-releasing factor (CRF) increased cAMP levels to 220.8 +/- 27.6% of control in rat tail arteries. The effect was completely abolished by the adenylyl cyclase inhibitor, SQ22536 (100 microM). Urocortin also decreased phosphorylation of the regulatory light chains of myosin (MLC20) in rat tail arteries stimulated with high K+ from 27.5 +/- 0.9% (control) to 13 +/- 2% (n = 5). This suggests that urocortin relaxes blood vessels via cAMP-mediated dephosphorylation of MLC20. Previously we have shown that urocortin-induced vasodilation can be ascribed to a decrease in Ca2+ -sensitivity of tension and activation of smooth muscle myosin phosphatase (SMPP-1M). In this study, we provide evidence that urocortin-induced Ca2+ -desensitization does not affect agonist-induced Ca2+ -sensitization. Urocortin relaxed alpha-toxin permeabilized mouse tail arteries preconstricted with pCa 6.1, but did not prevent the Ca2+ -sensitization induced by 10 microM 5-HT, 100 microM norepinephrine (NE) or 1 microM GTPgammaS. In keeping, the maximally relaxing concentration of urocortin (100 nM) had no effect on the concentration dependence of the phenylephrine-induced Ca2+ -sensitization. By contrast, treatment with the cAMP analogue, cBIMPS (100 microM), or the Rho kinase inhibitor, H-1152 (3 microM) relaxed the mouse vessels to a greater extend and completely inhibited phenylephrine (PE) induced sensitization. The lack of effect of urocortin on agonist-induced sensitization could be due to a alpha-adrenergic receptor mediated inhibition of cAMP generation. Furthermore PE induced Ca2+ -sensitization was reported to occur independent of changes in MLC20 phosphorylation involving caldesmon. Our results are compatible with a model in which urocortin/cAMP signalling only affects the myosin linked regulation of vascular tone while cBIMPS may inactivate in addition the MLC20 phosphorylation independent pathway.

Endostatin, the proteolytic fragment of collagen XVIII, induces vasorelaxation.

Collagen XVIII is an important component of the extracellular matrix and is expressed in basement membranes. Its degradation results in the generation of endostatin claimed to possess antiangiogenic activity. To date, only limited knowledge exists with regard to the cellular signaling of this molecule. We show in single-cell measurements using the Ca2+ indicator fura-2 acetoxy methylester (fura-2 AM) and the nitric oxide (NO) indicator 4,5-diaminofluorescein diacetate that application of endostatin (ES) (5 pmol/L, 100 ng/mL) induced Ca2+ spikes and an increase of NO production in human and murine endothelial cells. The NO response was independent of an increase in cytosolic Ca2+ and blocked by the endothelial NO synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester and by incubation with pertussis toxin known to inhibit G(i/o) proteins. The physiological relevance of this novel signaling pathway of ES was assessed with isometric force measurements in large and small arteries of mouse. Physiological concentrations of ES were found to decrease vascular tone in an endothelium-dependent manner. This occurred via an Arg-Gly-Asp (RGD) peptide-independent pathway through activation of G(i/o) proteins, phosphatidylinositol 3-kinase, Akt, and eNOS. We conclude that the proteolytic matrix fragment ES is a prominent vasorelaxing agent. Because ES is constantly released into the blood, it is a novel regulator of blood pressure and, therefore, represents an interesting pharmacological target.

Dynamic behaviour of half-sarcomeres during and after stretch in activated rabbit psoas myofibrils: sarcomere asymmetry but no 'sarcomere popping'.

We examined length changes of individual half-sarcomeres during and after stretch in actively contracting, single rabbit psoas myofibrils containing 10-30 sarcomeres. The myofibrils were fluorescently immunostained so that both Z-lines and M-bands of sarcomeres could be monitored by video microscopy simultaneously with the force measurement. Half-sarcomere lengths were determined by processing of video images and tracking the fluorescent Z-line and M-band signals. Upon Ca2+ activation, during the rise in force, active half-sarcomeres predominantly shorten but to different extents so that an active myofibril consists of half-sarcomeres of different lengths and thus asymmetric sarcomeres, i.e. shifted A-bands, indicating different amounts of filament overlap in the two halves. When force reached a plateau, the myofibril was stretched by 15-20% resting length (L0) at a velocity of approximately 0.2 L0 s(-1). The myofibril force response to a ramp stretch is similar to that reported from muscle fibres. Despite the approximately 2.5-fold increase in force due to the stretch, the variability in half-sarcomere length remained almost constant during the stretch and A-band shifts did not progress further, independent of whether half-sarcomeres shortened or lengthened during the initial Ca2+ activation. Moreover, albeit half-sarcomeres lengthened to different extents during a stretch, rapid elongation of individual sarcomeres beyond filament overlap ('popping') was not observed. Thus, in contrast to predictions of the 'popping sarcomere' hypothesis, a stretch rather stabilizes the uniformity of half-sarcomere lengths and sarcomere symmetry. In general, the half-sarcomere length changes (dynamics) before and after stretch were slow and the dynamics after stretch were not readily predictable on the basis of the steady-state force-sarcomere length relation.

Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction-relaxation cycle in isolated cardiac myofibrils.

Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in sarcomeric proteins such as human cardiac troponin I (hcTnI). To elucidate the functional consequences of the mutation hcTnI(R145G) on crossbridge kinetics, force kinetics were analysed in murine cardiac myofibrils carrying either the mutant or the wild-type protein. The mutation was introduced into the myofibrils in two different ways: in the first approach, the endogenous Tn was replaced by incubation of the myofibrils with an excess of reconstituted recombinant hcTn containing either hcTnI(WT) or hcTnI(R145G). Alternatively, myofibrils were isolated either from non-transgenic or transgenic mice expressing the corresponding mcTnI(R146G) mutation. In myofibrils from both models, the mutation leads to a significant upward shift of the passive force-sarcomere length relation determined at pCa 7.5. Addition of 5 mm BDM (2,3-butandione-2-monoxime), an inhibitor of actomyosin ATPase partially reverses this shift, suggesting that the mutation impairs the normal function of cTnI to fully inhibit formation of force-generating crossbridges in the absence of Ca(2)(+). Maximum force development (F(max)) is significantly decreased by the mutation only in myofibrils exchanged with hcTnI(R145G) in vitro. Ca(2)(+) sensitivity of force development was reduced by the mutation in myofibrils from transgenic mice but not in exchanged myofibrils. In both models the rate constant of force development k(ACT) is reduced at maximal [Ca(2)(+)] but not at low [Ca(2)(+)] where it is rather increased. Force relaxation is significantly prolonged due to a reduction of the relaxation rate constant k(REL). We therefore assume that the impairment in the regulatory function of TnI by the mutation leads to modulations in crossbridge kinetics that significantly alter the dynamics of myofibrillar contraction and relaxation.

Inhibition of contraction and myosin light chain phosphorylation in guinea-pig smooth muscle by p21-activated kinase 1.

The p21-activated protein kinases (PAKs) have been implicated in cytoskeletal rearrangements and modulation of non-muscle contractility. Little, however, is known about the role of the PAK family members in smooth muscle contraction. Therefore, we investigated the effect of the predominant isoform in vascular smooth muscle cells, PAK1, on contraction and phosphorylation of the regulatory light chains of myosin (r-MLC) in Triton-skinned guinea-pig smooth muscle. We also investigated which of the three putative substrates at the contractile apparatus - MLCK, caldesmon or r-MLC - is phosphorylated by PAK1 in smooth muscle tissue. Incubation of Triton-skinned carotid artery and taenia coli from guinea-pig with an active mutant of PAK1 in relaxing solution for 30-60 min resulted in inhibition of submaximal force by about 50 %. The mechanism of inhibition of force was studied in the Triton-skinned taenia coli. In this preparation, inhibition of force was associated with a respective inhibition of r-MLC phosphorylation. In the presence of the myosin phosphatase inhibitor, microcystin-LR (10 microM), the rate of contraction and r-MLC phosphorylation elicited at pCa 6.79 were both decreased. Because under these conditions the rate of r-MLC phosphorylation is solely dependent on MLCK activity, this result suggests that the inhibitory effect of PAK1 on steady-state force and r-MLC phosphorylation is due to inhibition of MLCK. In line with this, we found that MLCK was significantly phosphorylated by PAK1 while there was very little 32P incorporation into caldesmon. PAK1 phosphorylated isolated r-MLC but not those in the skinned fibres or in purified smooth muscle myosin II. In conclusion, these results suggest that PAK1 attenuates contraction of skinned smooth muscle by phosphorylating and inhibiting MLCK.

Expression of members of the S100 Ca2+-binding protein family in guinea-pig smooth muscle.

The calcium-binding proteins of the S100 family show tissue-specific expression. In this study, the mRNA and protein expression of five S100 calcium-binding proteins was investigated in different guinea-pig smooth muscle preparations. Transcripts of cDNA of S100A1, S100A4, S100A6 and S100A10 were amplified from smooth muscle RNA of neonatal and adult urinary bladder, ileum, aorta and from freshly isolated and cultured urinary bladder smooth muscle cells. S100B was not detectable in smooth muscle RNA, but was seen in control RNA isolated from brain. The pattern of S100 mRNA expression did not change during postnatal development and cell culture of smooth muscle. The structural homology of guinea-pig S100 proteins reverse transcription-polymerase chain reaction (RT-PCR) sequences compared to other species was 85-93% (human), 83-88% (rat) and 81-87% (mouse). Protein expression of S100A4, S100A6 and S100A10 was investigated in aorta, ileum, bladder and cultured bladder smooth muscle cells by Western blot analysis using polyclonal antibodies against guinea-pig-specific S100 immunogenic peptide sequences raised in rabbits. The results show that the proteins S100A4, S100A6 and S100A10 are expressed in the smooth muscle of ileum, bladder and aorta. S100A4 and S100A6 proteins are also expressed in cultured smooth muscle cells. The results of this study suggest that the calcium-binding proteins S100A1, S100A4, S100A6 and S100A10, but not S100B, are expressed in guinea-pig smooth muscle, and could be potentially involved in the regulation of cytoplasmic Ca(2+)-concentration and/or in signal transduction in smooth muscle.

Force kinetics and individual sarcomere dynamics in cardiac myofibrils after rapid ca(2+) changes.

Kinetics of force development and relaxation after rapid application and removal of Ca(2+) were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca(2+) application, force developed with an exponential rate constant k(ACT) almost identical to k(TR), the rate constant of force redevelopment measured during steady-state Ca(2+) activation; this indicates that k(ACT) reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca(2+) removal were markedly biphasic. An initial slow linear decline (rate constant k(LIN)) lasting for a time t(LIN) was abruptly followed by an ~20 times faster exponential decay (rate constant k(REL)). k(LIN) is similar to k(TR) measured at low activating [Ca(2+)], indicating that k(LIN) reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also. Biophys. J. 83:2142-2151). Video microscopy revealed the following: invariably at t(LIN) a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P(i) on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P(i).

Thiophosphorylation-induced Ca(2+) sensitization of guinea-pig ileum contractility is not mediated by Rho-associated kinase.

1. Incubation of beta-escin-permeabilized guinea-pig longitudinal ileal smooth muscle with ATP gamma S under conditions that do not lead to thiophosphorylation of regulatory light chains of myosin (r-MLC) increased subsequent Ca(2+) sensitivity of force and r-MLC phosphorylation. In this study we tested whether this is due to activation of the Rho and/or Rho-associated kinase (ROK) as it is the case in agonist-induced Ca(2+) sensitization. 2. The increase in Ca(2+) sensitivity induced by pretreatment with ATP gamma S at pCa > 8 with the myosin light chain kinase (MLCK) inhibitor ML-9 in rigor solution was associated with (35)S incorporation into the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1, and several other high molecular mass proteins. No thiophosphorylation of r-MLC, MLCK, caldesmon, calponin and CPI-17 was detected. 3. While the relatively specific inhibitor of ROK, Y 27632, inhibited the carbachol-induced increase in Ca(2+) sensitivity with an IC(50) of 1.4 microM, the ATP gamma S-induced increase in Ca(2+) sensitivity and thiophosphorylation of MYPT1 was not inhibited. Inhibiton of Rho by exoenzyme C3 also had no effect. 4. Only staurosporine (2 microM), but not the PKC inhibitor peptide 19-31, nor genistein nor PD 98059, inhibited the ATP gamma S-induced Ca(2+) sensitization of force, r-MLC phosphorylation, and the (35)S incorporation into MYPT1. 5. The staurosporine-sensitive kinase(s) appeared to be tightly associated with the contractile apparatus because treatment of Triton-skinned preparations with ATP gamma S also induced a staurosporine-sensitive increase in Ca(2+) sensitivity of contraction. Since there was very little immunoreactivity with antibodies to p(21)-associated kinase (PAK) in Triton-skinned preparations, the staurosporine-sensitive kinase most probably is not PAK. 6. GTP gamma S had an additive effect on ATP gamma S-induced sensitization at saturating concentrations of ATP gamma S. The additional effect of GTP gamma S was inhibited by Y 27632. 7. We conclude that treatment with ATP gamma S under ATP-free conditions, unmasks a staurosporine-sensitive kinase which induces a large increase in Ca(2+) sensitivity that is most likely to be due to thiophosphorylation of MYPT1. The kinase is distinct from ROK. The physiological significance of this kinase, which is tightly associated with the contractile apparatus, is not known at present.

Invited review: regulation of myosin phosphorylation in smooth muscle.

Phosphorylation of the regulatory light chains of myosin II (rMLC) by the Ca(2+)/calmodulin-dependent myosin light-chain kinase (MLCK) and dephosphorylation by a type 1 phosphatase (MLCP), which is targeted to myosin by a regulatory subunit (MYPT1), are the predominant mechanisms of regulation of smooth muscle tone. The activities of both enzymes are modulated by several protein kinases. MLCK is inhibited by the Ca(2+)/calmodulin-dependent protein kinase II, whereas the activity of MLCP is increased by cGMP and perhaps also cAMP-dependent protein kinases. In either case, this results in a decrease in the Ca(2+) sensitivity of rMLC phosphorylation and force production. The activity of MLCP is inhibited by Rho-associated kinase, one of the effectors of the monomeric GTPase Rho, and protein kinase C, leading to an increase in Ca(2+) sensitivity. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades.

Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle: role of Rho proteins.

1. Clostridium difficile toxin B glucosylates the Ras-related low molecular mass GTPases of the Rho subfamily thereby inactivating them. In the present report, toxin B was applied as a tool to test whether Rho proteins participate in the carbachol-induced increase in the Ca2+ sensitivity of force and myosin light chain (MLC) phosphorylation in intact intestinal smooth muscle. 2. Small strips of the longitudinal muscle of guinea-pig small intestine were incubated in toxin B (40 ng ml-1) overnight. Carbachol-induced force and intracellular [Ca2+], and, in a separate series, force and MLC phosphorylation, were determined. 3. Carbachol induced a biphasic contraction: an initial rapid increase in force (peak 1) followed by a partial relaxation and a second delayed increase in force (peak 2). The peak of the Ca2+ signal measured with fura-2 preceded peak 1 of force and then declined to a lower suprabasal steady-state level. Peak 2 was not associated with a significant increase in [Ca2+]. Toxin B nearly completely inhibited peak 2 while peak 1 was not significantly inhibited. Toxin B had no effect on the Ca2+ transient. 4. In control strips, MLC phosphorylation at peak 2 was 27.7% which was significantly higher than the resting value (18.6%). The inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosphorylation. The resting MLC phosphorylation was not significantly different from that of the control strips. 5. The initial increase in MLC phosphorylation determined 3 s after exposure to carbachol was 54% in the control strips. Toxin B also inhibited this initial phosphorylation peak despite the fact that the Ca2+ transient and the initial increase in force were not inhibited by toxin B. This suggests that Rho proteins play an important role in setting the balance between MLC phosphorylation and dephosphorylation reactions even at high levels of intracellular Ca2+. 6. These findings are consistent with the hypothesis that the delayed rise in force elicited by carbachol is due to an increase in the Ca2+ sensitivity of MLC phosphorylation mediated by Rho proteins.