Activation of Myosin Light Chain Phosphatase in Intact Arterial Smooth Muscle During Nitric Oxide-induced Relaxation (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1997
Nitrovasodilators are hypothesized to induce smooth muscle relaxation by their metabolism to nitric oxide, which then w x activates soluble guanylyl cyclase, increases cGMP , and activates cGMP-dependent protein kinase. cGMP-dependent w 2q x Žw 2q x . 2q phosphorylation is then proposed to decrease intracellular Ca Ca and to reduce the Ca -sensitivity of contraction. i ity of MLC phosphorylation without increasing MLCK site A phosphorylation. Nitrovasodilators, per se, can induce site A MLCK phosphorylation, potentially by cGMP dependent activation of cAMP-dependent protein kinase.
The Journal of Physiology, 2009
Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca 2+ concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca 2+ , myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca 2+ and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca 2+ release delayed SNP-induced relaxation while inhibition of Ca 2+ channel, BK Ca channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca 2+ , contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca 2+ release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.
Biochimica Et Biophysica Acta-molecular Cell Research, 1989
The effect of reduction of ATP content on phosphorylation of the 20 kDa light chain of myosin (MLC) and force development in intact carotid arterial smooth muscle was investigated. With reduction of ATP to 23% of control by treatment with 2-deoxyglucose there was reduction in basal, in peak and 30 rain MLC phosphorylation during contraction (P < 0.001). The rate of force development was reduced, but maximal force was the same as control. By treatment with 50/,M iedoactetate, the resting ATP content was unchanged but fell to 22% after 30 rain contraction.Basal MLC phosphorylation was the same as control, but peak (P < 0.001) and 30 rain phosphorylation were lower (P < 0.005), even though the rate and wagnitude of force development were greater. The results indicate that neither rate nor magnitude of force development correlate with MLC phosphorylation. Basal and initial MLC phosphorylation may play a cooperative role in contractile function.
2009
The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC 50 ؍ 1.6 and 60 nM, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of -escin-permeabilized ileum SM at constant pCa 6.3 (EC 50 ؍ 2 M), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697-880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca 2؉ sensitization of smooth muscle force.
Stretch-induced phosphorylation of the 20,000-dalton light chain of myosin in arterial smooth muscle
Journal of Biological Chemistry, 1983
Stretching to 1.7 times the resting length of porcine carotid arteries reversibly prevents active tension development by K+ or norepinephrine stimulation. The 20,000-dalton light chain of myosin was maximally phosphorylated in the stretched noncontracting muscles, equal to that in the nonstretched contracting muscles challenged with K+ or norepinephrine. These results show that the contractile event is not a prerequisite for phosphorylation. Furthermore, stretching alone also induced maximal light chain phosphorylation even in the absence of K+ or norepinephrine. The stretch-induced light chain phosphorylation was not affected by exhaustive washing of the muscle with Ca2+-free physiological salt solution, treatment of the muscle with verapamil, or by a short exposure to ethylene glycol bis(8-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Prolonged EGTA treatment abolished the stretch-induced light chain phosphorylation. All evidence suggests that upon stretch, Ca2+ is released from intracellular sources and this Ca2+ activates the myosin light chain kinase producing phosphorylation of the light chain.
Specificity and kinetic effects of nitrophenol analogues that activate myosin subfragment 1
The Biochemical journal, 1997
2,4-Dinitrophenol (DNP) activates the myosin ATPase of mammalian skeletal muscle in the presence of Ca2+ or Mg2+, and inhibits it when the bivalent cations are replaced by K+ and EDTA. Activation of Mg2+ATPase is abolished by the presence of unregulated actin. 3-Nitrophenol (3-NP) is also an activator, whereas other analogues (2-nitrophenol, 2-NP, and 4-nitrophenol, 4-NP) are much less effective. Concentrations required for their half-maximal effects (K0.5) range from 2 to 15 mM for 3-NP and DNP in the presence of different cations, and the sequence for the analogues is 3-NP<=DNP<2-NP approximately 4-NP, which is apparently unrelated to either hydrophobicity or pK. DNP and 3-NP have almost identical effects on the ATPase activity of chymotryptic subfragment 1 as they do on myosin, which is an indication that their target is the globular head region rather than the tail, or the 18 kDa (regulatory) light chain. Analysis of the ATP concentration dependence for subfragment- 1 ATPa...
Cell Calcium, 2004
The effects of authentic nitric oxide (NO, 10 −6 M) and NO-donors such as sodium nitroprusside (SNP, 10 −5 M) and glyceryl trinitrate (GTN, 10 −4 M) on contractile force and free intracellular calcium level ([Ca 2+ ] i) were studied on precontracted with high potassium chloride (KCl, 70 mM) isolated rings of rat tail artery. The sensitivity of contractile myofilaments to Ca 2+ was measured using chemically permeabilized (␣-toxin, -escin, Triton X-100) vascular rings. [Ca 2+ ] i and contractile activity were measured simultaneously. The relationship of [Ca 2+ ] i and tension developed was studied in endothelium-denuded rings and controlled calcium response was evaluated in both endothelium-denuded and permeabilized vascular rings. Both authentic NO and NO-donors decreased [Ca 2+ ] i and high potassium-induced tension with a different time course. Inhibitor of soluble guanylyl cyclase (sGC) LY83583 (10 −5 M) did not affect SNP-induced relaxation whereas the other sGC inhibitor ODQ (10 −6 M) attenuated SNP-induced relaxation. Both inhibitors had no effect on NO-and SNP-induced reduction in [Ca 2+ ] i. On the contrary, GTN induced neither relaxation nor decrease in [Ca 2+ ] i on application of both LY83583 and ODQ. Tail artery rings permeabilized with ␣-toxin, -escin, but not with Triton X-100 were relaxed by authentic NO and NO-donors, but to a less extent than non-permeabilized rings. Dithioerythritol (DTE, 5 × 10 −3 M) that maintains sulfhydryl (SH) groups in reduced state preventing their nitrosylation attenuated NO-induced relaxation in both non-permeabilized and permeabilized tail artery rings. The cyclic heptapeptide mycrocystin-LR (MC-LR) (10 −5 M), an inhibitor of type 1 and 2A phosphatases, induced sustained increase in tension of -escin permeabilized rings in low Ca 2+ (10 −8 M) solution. The tension was not affected by authentic NO and SNP. We conclude that authentic NO and SNP relax rat tail artery smooth muscle (SM) in the presence of inhibitors of sGC via cyclic guanosine monophosphate (cGMP)-independent pathway, whereas relaxation induced by GTN is inhibited. The data demonstrate that cGMP-dependent pathway in vascular smooth muscle is ubiquitous, but not the only way of relaxation induced by NO. NO can modulate vascular tone directly by reducing sensitivity of contractile myofilaments to [Ca 2+ ] i and may involve activation of protein phosphatase(s).
Circulation Research, 2002
It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca 2+ . One mechanism that has been suggested to explain Ca 2+ sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is asso...
The Journal of Physiology, 1990
1. The [Ca2"] sensitivity of myosin light chain phosphorylation in vascular smooth muscle is dependent on the form of stimulation. Contractile agonist stimulation, when compared to high-KCl depolarization, is associated with an increase in [Ca2+] sensitivity of phosphorylation. I evaluated potential mechanisms for this stimulusspecific response by measuring aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and isometric stress in swine carotid media.