Toshio Kitazawa

Cyclic GMP Causes Ca2+ Desensitization in Vascular Smooth Muscle by Activating the Myosin Light Chain Phosphatase

Using permeabilized, arterial smooth muscle strips where membrane-associated pathways remain intact but intracellular Ca2+ stores are depleted, we investigated mechanism(s) for the Ca2+ desensitization of contractile force by cGMP. The nonhydrolyzable analog 8-bromo-cGMP, when applied to these strips with submaximal Ca2+ levels clamped, dramatically and reversibly reduced the steady state levels of phosphorylation at 20-kDa myosin light chain and contractile force, with a nanomolar concentration required to obtain 50% reduction. Supramaximal concentrations of 8-bromo-cGMP (10 μM), however, did not change the steady state relationship between phosphorylation and force. When light chain phosphatase activity was blocked at pCa 6.7, 10 μM 8-bromo-cGMP did not affect the rates of rise of light chain phosphorylation and contractile force. When light chain kinase activity was blocked, 10 μM 8-bromo-cGMP significantly accelerated light chain dephosphorylation and force relaxation from the maximal contraction steady state. The light chain phosphorylation time course of a pCa 6.0-induced contraction in the presence of 8-bromo-cGMP exhibited kinetics that are predictable from a mathematical model in which only light chain phosphatase activity is increased. The results of this study strongly suggest that cGMP indirectly activates light chain phosphatase, the first proposed mechanism for cGMP-induced Ca2+ desensitization in vasodilatation.

Expression of CPI‐17 and myosin phosphatase correlates with Ca2+ sensitivity of protein kinase C‐induced contraction in rabbit smooth muscle

1 Various smooth muscles have unique contractile characteristics, such as the degree of Ca2+ sensitivity induced by physiological and pharmacological agents. Here we evaluated six different rabbit smooth muscle tissues for protein kinase C (PKC)‐induced Ca2+ sensitization. We also examined the expression levels of myosin light chain phosphatase (MLCP), the MLCP inhibitor phosphoprotein CPI‐17, and the thin filament regulator h‐calponin. 2 Immunohistochemical and Western blot analyses indicated that CPI‐17 was found primarily in smooth muscle, although expression varied among different tissues. Vascular muscles contained more CPI‐17 than visceral muscles, with further distinction existing between tonic and phasic subtypes. For example, the tonic femoral artery possessed approximately 8 times the cellular CPI‐17 concentration of the phasic vas deferens. 3 In contrast to CPI‐17 expression patterns, phasic muscles contained more MLCP myosin‐targeting subunit than tonic tissues. Calponin expression was not statistically different. 4 Addition of phorbol ester to α‐toxin‐permeabilized smooth muscle caused an increase in contraction and phosphorylation of both CPI‐17 and myosin light chain (MLC) at submaximal [Ca2+]i. These responses were several‐fold greater in femoral artery as compared to vas deferens. 5 We conclude that the expression ratio of CPI‐17 to MLCP correlates with the Ca2+ sensitivities of contraction induced by a PKC activator. PKC stimulation of arterial smooth muscle with a high CPI‐17 and low MLCP expression generated greater force and MLC phosphorylation than stimulation of visceral muscle with a relatively low CPI‐17 and high MLCP content. This implicates CPI‐17 inhibition of MLCP as an important component in modulating vascular muscle tone.

Phosphorylation of the myosin phosphatase targeting subunit and CPI‐17 during Ca2+ Sensitization in Rabbit Smooth Muscle

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca2+ sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr695 of the MLCP regulatory subunit (MYPT1) and at Thr38 of the MLCP inhibitor protein CPI‐17 results in inhibition of MLCP activity. We have previously demonstrated that CPI‐17 Thr38 phosphorylation plays an important role in G‐protein‐mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site‐ and phospho‐specific antibodies, phosphorylation of MYPT1 Thr695 and CPI‐17 Thr38 was examined along with MYPT1 Thr850, which is a non‐inhibitory Rho‐kinase site. We found that both CPI‐17 Thr38 and MYPT1 Thr850 were phosphorylated in response to agonists or GTPγS concurrently with contraction and myosin phosphorylation in α‐toxin‐permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr695 did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho‐kinase inhibitor Y‐27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr850 and CPI‐17 Thr38, respectively, in intact VD while MYPT1 Thr695 phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr695 is independent of stimulation of G‐proteins, Rho‐kinase or PKC. In the phasic PV, phosphorylation of CPI‐17 Thr38 may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI‐17 concentration, probably utilizes other Ca2+ sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI‐17.

Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in Triton X-100-demembranated rabbit arterial smooth muscle

1 Triton X‐100‐demembranated smooth muscle loses Ca2+‐sensitizing responsiveness to protein kinase C (PKC) activators while intact and α‐toxin‐permeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca2+ sensitization by PKC in the demembranated preparations. 2 Western blot analyses showed that the content of the PKC α‐isoform (PKCα) was markedly reduced and that the smooth muscle‐specific protein phosphatase‐1 inhibitor protein CPI‐17 was not detectable, while the amount of calponin and actin still remained similar to those of intact strips. 3 Unphosphorylated recombinant CPI‐17 alone induced a small but significant contraction at constant Ca2+. Isoform‐selective PKC inhibitors inhibited unphosphorylated but not pre‐thiophosphorylated CPI‐17‐induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI‐17. 4 Exogenously replenishing PKCα alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca2+. 5 PKC in the presence of CPI‐17, but not the [T38A]‐CPI mutant, markedly induced potentiation of both contraction and MLC phosphorylation. CPI‐17 itself was phosphorylated. 6 In in vitro experiments, CPI‐17 was a much better substrate for PKCα than calponin, caldesmon, MLC and myosin. 7 Our results indicate that PKC requires CPI‐17 phosphorylation at Thr‐38 but not calponin for reconstitution of the contractile Ca2+ sensitization in the demembranated arterial smooth muscle.

SULF1 and SULF2 regulate heparan sulfate-mediated GDNF signaling for esophageal innervation

Heparan sulfate (HS) plays an essential role in extracellular signaling during development. Biochemical studies have established that HS binding to ligands and receptors is regulated by the fine 6-O-sulfated structure of HS; however, mechanisms that control sulfated HS structure and associated signaling functions in vivo are not known. Extracellular HS 6-O-endosulfatases, SULF1 and SULF2, are candidate enzymatic regulators of HS 6-O-sulfated structure and modulate HS-dependent signaling. To investigate Sulf regulation of developmental signaling, we have disrupted Sulf genes in mouse and identified redundant functions of Sulfs in GDNF-dependent neural innervation and enteric glial formation in the esophagus, resulting in esophageal contractile malfunction in Sulf1-/-;Sulf2-/- mice. SULF1 is expressed in GDNF-expressing esophageal muscle and SULF2 in innervating neurons, establishing their direct functions in esophageal innervation. Biochemical and cell signaling studies show that Sulfs are the major regulators of HS 6-O-desulfation, acting to reduce GDNF binding to HS and to enhance GDNF signaling and neurite sprouting in the embryonic esophagus. The functional specificity of Sulfs in GDNF signaling during esophageal innervation was established by showing that the neurite sprouting is selectively dependent on GDNF, but not on neurotrophins or other signaling ligands. These findings provide the first in vivo evidence that Sulfs are essential developmental regulators of cellular HS 6-O-sulfation for matrix transmission and reception of GDNF signal from muscle to innervating neurons.

Ca2+-Dependent Rapid Ca2+ Sensitization of Contraction in Arterial Smooth Muscle

Ca2+ ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca2+ with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca2+ transient, we compared Ca2+ signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during &agr;1 agonist–induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38±0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca2+ rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca2+ release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca2+-dependent protein kinase C. This was followed by a slow Ca2+-independent and Rho-kinase/protein kinase C–dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29±0.09 at rest to the peak of 0.68±0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca2+ sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca2+ rise induces a rapid inhibition of MLC phosphatase coincident with the Ca2+-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.

BAG3 and Hsc70 Interact With Actin Capping Protein CapZ to Maintain Myofibrillar Integrity Under Mechanical Stress

Rationale: A homozygous disruption or genetic mutation of the bag3 gene, a member of the Bcl-2–associated athanogene (BAG) family proteins, causes cardiomyopathy and myofibrillar myopathy that is characterized by myofibril and Z-disc disruption. However, the detailed disease mechanism is not yet fully understood. Objective: bag3−/− mice exhibit differences in the extent of muscle degeneration between muscle groups with muscles experiencing the most usage degenerating at an accelerated rate. Usage-dependent muscle degeneration suggests a role for BAG3 in supporting cytoskeletal connections between the Z-disc and myofibrils under mechanical stress. The mechanism by which myofibrillar structure is maintained under mechanical stress remains unclear. The purpose of the study is to clarify the detailed molecular mechanism of BAG3-mediated muscle maintenance under mechanical stress. Methods and Results: To address the question of whether bag3 gene knockdown induces myofibrillar disorganization caused by mechanical stress, in vitro mechanical stretch experiments using rat neonatal cardiomyocytes and a short hairpin RNA–mediated gene knockdown system of the bag3 gene were performed. As expected, mechanical stretch rapidly disrupts myofibril structures in bag3 knockdown cardiomyocytes. BAG3 regulates the structural stability of F-actin through the actin capping protein, CapZ&bgr;1, by promoting association between Hsc70 and CapZ&bgr;1. BAG3 facilitates the distribution of CapZ&bgr;1 to the proper location, and dysfunction of BAG3 induces CapZ ubiquitin–proteasome–mediated degradation. Inhibition of CapZ&bgr;1 function by overexpressing CapZ&bgr;2 increased myofibril vulnerability and fragmentation under mechanical stress. On the other hand, overexpression of CapZ&bgr;1 inhibits myofibrillar disruption in bag3 knockdown cells under mechanical stress. As a result, heart muscle isolated from bag3−/− mice exhibited myofibrillar degeneration and lost contractile activity after caffeine contraction. Conclusions: These results suggest novel roles for BAG3 and Hsc70 in stabilizing myofibril structure and inhibiting myofibrillar degeneration in response to mechanical stress. These proteins are possible targets for further research to identify therapies for myofibrillar myopathy or other degenerative diseases.

Desensitization and muscarinic re-sensitization of force and myosin light chain phosphorylation to cytoplasmic Ca2+ in smooth muscle

In alpha-toxin-permeabilized guinea-pig ileum smooth muscle, a step increase in Ca2+ caused a rapid rise in force and myosin light chain (LC20) phosphorylation, followed by their spontaneous decline to a low steady level even though Ca2+ remained constant. Carbachol resensitized the muscles to Ca2+, causing an increase in both the steady state force and LC20 phosphorylation at constant Ca2+. In beta-escin permeabilized preparations, calmodulin and okadaic acid converted the phasic responses to Ca2+ to more tonic ones. We conclude that Ca2(+)-sensitivity of force is modulated through changes in LC20 kinase/phosphatase activity ratio by Ca2+ itself (desensitization) and by agonists (sensitization).

Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells

Phosphorylation of myosin light chain (MLC) and contraction of differentiated smooth muscle cells in vascular walls are regulated by Ca2+-dependent activation of MLC kinase, and by Rho-kinase- or protein-kinases-C-dependent inhibition of MLC phosphatase (MLCP). We examined regulatory pathways for MLC kinase and MLCP in cultured vascular smooth muscle cells (VSMCs), and for isometric force generation of VSMCs reconstituted in collagen fibers. Protein levels of RhoA, Rho-kinase and MYPT1 (a regulatory subunit of MLCP) were upregulated in cultured VSMCs, whereas a MLCP inhibitor protein, CPI-17, was downregulated. Endothelin-1 evoked a steady rise in levels of Ca2+, MLC phosphorylation and the contractile force of VSMCs, whereas angiotensin-II induced transient signals. Also, Thr853 phosphorylation of MYPT1 occurred in response to stimuli, but neither agonist induced phosphorylation of MYPT1 at Thr696. Unlike fresh aortic tissues, removal of Ca2+ or addition of voltage-dependent Ca2+-channel blocker did not inhibit contractions of reconstituted VSMC fibers induced by agonists or even high concentrations of extracellular K+ ions. Inhibitors of Ins(1,4,5)P3-receptor and Rho-kinase antagonized agonist-induced or high-K+-induced contraction in both reconstituted fibers and fresh tissues. These results indicate that both Ins(1,4,5)P3-induced Ca2+ release and Rho-kinase-induced MYPT1 phosphorylation at Thr853 play pivotal roles in MLC phosphorylation of cultured VSMCs where either Ca2+-influx or CPI-17-MLCP signaling is downregulated.

CPI-17-deficient smooth muscle of chicken.

Ca2+ sensitivity of arterial contractility is governed by regulating myosin phosphatase activity in response to agonist stimuli. CPI‐17, a myosin phosphatase inhibitor phosphoprotein, is phosphorylated concomitantly with agonist‐induced contractile Ca2+ sensitization in mammalian artery. CPI‐17 has not been detected in chicken artery, but is readily detectable in pigeon artery. To evaluate a role of CPI‐17, we compared contractility of the arteries of ‘CPI‐17‐deficient’ chicken with those of CPI‐17‐rich rabbit and pigeon, and studied the effect of CPI‐17‐reconstitution in chicken artery. Other major regulatory/contractile proteins for Ca2+ sensitization are expressed in both chicken and rabbit arteries. Agonists, such as an α1‐agonist and endothelin‐1, produced significant contraction in arteries of all species under physiological Ca2+‐containing conditions. Depletion of Ca2+ abolished these contractions in chicken but partially inhibited them in rabbit and pigeon arteries. Unlike CPI‐17‐rich tissues, chicken arteries exerted little Ca2+ sensitization in response to α1‐agonist or endothelin‐1. GTPγS produced a slight Ca2+ sensitizing effect in chicken artery, but this was significantly smaller compared with CPI‐17‐rich tissues. A PKC activator (PDBu) did not generate but rather reduced a contraction in both intact and α‐toxin‐permeabilized chicken artery in contrast to a large contraction in CPI‐17‐rich arteries. Myosin light chain phosphorylation was reduced by PDBu in chicken but elevated in rabbit artery. Addition of recombinant CPI‐17 into β‐escin‐permeabilized chicken artery restored PDBu‐induced and enhanced GTPγS‐induced Ca2+ sensitization. Thus, CPI‐17 is essential for G protein/PKC‐mediated Ca2+ sensitization in smooth muscle.