New insights into myosin phosphorylation during cyclic nucleotide-mediated smooth muscle relaxation

Nitrovasodilators and agonists, via an increase in intracellular cyclic nucleotide levels, can induce smooth muscle relaxation without a concomitant decrease in phosphorylation of the regulatory light chains (RLC) of myosin. However, since cyclic nucleotide-induced relaxation is associated with a decrease in intracellular [Ca2+], and hence, a decreased activity of MLCK, we tested the hypothesis that the site responsible for the elevated RLC phosphorylation is not Ser19. Smooth muscle strips from gastric fundus were isometrically contracted with ET-1 which induced an increase in monophosphorylation from 9 +/- A 1 % under resting conditions (PSS) to 36 +/- A 1 % determined with 2D-PAGE. Electric field stimulation induced a rapid, largely NO-mediated relaxation with a half time of 8 s, which was associated with an initial decline in RLC phosphorylation to 18 % within 2 s and a rebound to 34 % after 30 s whereas relaxation was sustained. In contrast, phosphorylation of RLC at Ser19 probed with phosphospecific antibodies declined in parallel with force. LC/MS and western blot analysis with phosphospecific antibodies against monophosphorylated Thr18 indicate that Thr18 is significantly monophosphorylated during sustained relaxation. We therefore suggest that (i) monophosphorylation of Thr18 rather than Ser19 is responsible for the phosphorylation rebound during sustained EFS-induced relaxation of mouse gastric fundus, and (ii) that relaxation can be ascribed to dephosphorylation of Ser19, the site considered to be responsible for regulation of smooth muscle tone

Aging-related alterations in eNOS and nNOS responsiveness and smooth muscle reactivity of murine basilar arteries are modulated by apocynin and phosphorylation of myosin phosphatase targeting subunit-1

Aging causes major alterations of all components of the neurovascular unit and compromises brain blood supply. Here, we tested how aging affects vascular reactivity in basilar arteries from young (22months; o-BA) and old (>22months) heterozygous MYPT1-T-696A/+knock-in mice. In isometrically mounted o-BA, media thickness was increased by approximate to 10% while the passive length tension relations were not altered. Endothelial denudation or pan-NOS inhibition (100 mu mol/L L-NAME) increased the basal tone by 11% in y-BA and 23% in o-BA, while inhibition of nNOS (1 mu mol/L L-NPA) induced approximate to 10% increase in both ages. eNOS expression was approximate to 2-fold higher in o-BA. In o-BA, U46619-induced force was augmented (pEC(50) approximate to 6.9 vs. pEC(50) approximate to 6.5) while responsiveness to DEA-NONOate, electrical field stimulation or nicotine was decreased. Basal phosphorylation of MLC20-S19 and MYPT1-T-853 was higher in o-BA and was reversed by apocynin. Furthermore, permeabilized o-BA showed enhanced Ca2+-sensitivity. Old T-696A/+BA displayed a reduced phosphorylation of MYPT1-T696 and MLC20, a lower basal tone in response to L-NAME and a reduced eNOS expression. The results indicate that the vascular hypercontractility found in o-BA is mediated by inhibition of MLCP and is partially compensated by an upregulation of endothelial NO release

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Calc1(-/-) compared with Cold1(+/+) skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KO-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cold1(-/-) than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure

Quantitative Signal Intensity in Fluid-Attenuated Inversion Recovery and Treatment Effect in the WAKE-UP Trial

International audienceBackground and Purpose— Relative signal intensity of acute ischemic stroke lesions in fluid-attenuated inversion recovery (fluid-attenuated inversion recovery relative signal intensity [FLAIR-rSI]) magnetic resonance imaging is associated with time elapsed since stroke onset with higher intensities signifying longer time intervals. In the randomized controlled WAKE-UP trial (Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke Trial), intravenous alteplase was effective in patients with unknown onset stroke selected by visual assessment of diffusion weighted imaging fluid-attenuated inversion recovery mismatch, that is, in those with no marked fluid-attenuated inversion recovery hyperintensity in the region of the acute diffusion weighted imaging lesion. In this post hoc analysis, we investigated whether quantitatively measured FLAIR-rSI modifies treatment effect of intravenous alteplase. Methods— FLAIR-rSI of stroke lesions was measured relative to signal intensity in a mirrored region in the contralesional hemisphere. The relationship between FLAIR-rSI and treatment effect on functional outcome assessed by the modified Rankin Scale (mRS) after 90 days was analyzed by binary logistic regression using different end points, that is, favorable outcome defined as mRS score of 0 to 1, independent outcome defined as mRS score of 0 to 2, ordinal analysis of mRS scores (shift analysis). All models were adjusted for National Institutes of Health Stroke Scale at symptom onset and stroke lesion volume. Results— FLAIR-rSI was successfully quantified in stroke lesions in 433 patients (86% of 503 patients included in WAKE-UP). Mean FLAIR-rSI was 1.06 (SD, 0.09). Interaction of FLAIR-rSI and treatment effect was not significant for mRS score of 0 to 1 ( P =0.169) and shift analysis ( P =0.086) but reached significance for mRS score of 0 to 2 ( P =0.004). We observed a smooth continuing trend of decreasing treatment effects in relation to clinical end points with increasing FLAIR-rSI. Conclusions— In patients in whom no marked parenchymal fluid-attenuated inversion recovery hyperintensity was detected by visual judgement in the WAKE-UP trial, higher FLAIR-rSI of diffusion weighted imaging lesions was associated with decreased treatment effects of intravenous thrombolysis. This parallels the known association of treatment effect and elapsing time of stroke onset