Phos-Tag-Based Analysis of Myosin Regulatory Light Chain Phosphorylation in Human Uterine Myocytes

The 'phosphate-binding tag' (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.We have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin (Calp) induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here

Effect of sitagliptin on cardiovascular outcomes in type 2 diabetes

BACKGROUND: Data are lacking on the long-term effect on cardiovascular events of adding sitagliptin, a dipeptidyl peptidase 4 inhibitor, to usual care in patients with type 2 diabetes and cardiovascular disease. METHODS: In this randomized, double-blind study, we assigned 14,671 patients to add either sitagliptin or placebo to their existing therapy. Open-label use of antihyperglycemic therapy was encouraged as required, aimed at reaching individually appropriate glycemic targets in all patients. To determine whether sitagliptin was noninferior to placebo, we used a relative risk of 1.3 as the marginal upper boundary. The primary cardiovascular outcome was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. RESULTS: During a median follow-up of 3.0 years, there was a small difference in glycated hemoglobin levels (least-squares mean difference for sitagliptin vs. placebo, -0.29 percentage points; 95% confidence interval [CI], -0.32 to -0.27). Overall, the primary outcome occurred in 839 patients in the sitagliptin group (11.4%; 4.06 per 100 person-years) and 851 patients in the placebo group (11.6%; 4.17 per 100 person-years). Sitagliptin was noninferior to placebo for the primary composite cardiovascular outcome (hazard ratio, 0.98; 95% CI, 0.88 to 1.09; P<0.001). Rates of hospitalization for heart failure did not differ between the two groups (hazard ratio, 1.00; 95% CI, 0.83 to 1.20; P = 0.98). There were no significant between-group differences in rates of acute pancreatitis (P = 0.07) or pancreatic cancer (P = 0.32). CONCLUSIONS: Among patients with type 2 diabetes and established cardiovascular disease, adding sitagliptin to usual care did not appear to increase the risk of major adverse cardiovascular events, hospitalization for heart failure, or other adverse events

Effects of Once-Weekly Exenatide on Cardiovascular Outcomes in Type 2 Diabetes.

Abstract BACKGROUND: The cardiovascular effects of adding once-weekly treatment with exenatide to usual care in patients with type 2 diabetes are unknown. METHODS: We randomly assigned patients with type 2 diabetes, with or without previous cardiovascular disease, to receive subcutaneous injections of extended-release exenatide at a dose of 2 mg or matching placebo once weekly. The primary composite outcome was the first occurrence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. The coprimary hypotheses were that exenatide, administered once weekly, would be noninferior to placebo with respect to safety and superior to placebo with respect to efficacy. RESULTS: In all, 14,752 patients (of whom 10,782 [73.1%] had previous cardiovascular disease) were followed for a median of 3.2 years (interquartile range, 2.2 to 4.4). A primary composite outcome event occurred in 839 of 7356 patients (11.4%; 3.7 events per 100 person-years) in the exenatide group and in 905 of 7396 patients (12.2%; 4.0 events per 100 person-years) in the placebo group (hazard ratio, 0.91; 95% confidence interval [CI], 0.83 to 1.00), with the intention-to-treat analysis indicating that exenatide, administered once weekly, was noninferior to placebo with respect to safety (P<0.001 for noninferiority) but was not superior to placebo with respect to efficacy (P=0.06 for superiority). The rates of death from cardiovascular causes, fatal or nonfatal myocardial infarction, fatal or nonfatal stroke, hospitalization for heart failure, and hospitalization for acute coronary syndrome, and the incidence of acute pancreatitis, pancreatic cancer, medullary thyroid carcinoma, and serious adverse events did not differ significantly between the two groups. CONCLUSIONS: Among patients with type 2 diabetes with or without previous cardiovascular disease, the incidence of major adverse cardiovascular events did not differ significantly between patients who received exenatide and those who received placebo. (Funded by Amylin Pharmaceuticals; EXSCEL ClinicalTrials.gov number, NCT01144338 .)

Changes in Phosphorylated RLC Proportions in uterine myocytes.

<p>All data are shown as mean ± SEM.</p><p>*, **, and ***indicate significant differences (p<0.05) compared to 0pRLC, 1pRLC and 2pRLC in the untreated group, respectively.</p

Demonstration of Phospho-S19-RLC phospho-state distribution in uterine myocytes.

<p><b>A.</b> Detection of 0pRLC, 1pRLC and 2pRLC separated by Mn²⁺-phos-tag SDS-PAGE with an Ab toward the C-terminus of RLC (CtRLC) and two Abs (mouse MonoclonalAb [MMAb] and rabbit PolyclonalAb [RPAb]) directed toward phospho-S19-RLC. <b>B.</b> Representative WB demonstrating RLC phospho-states separated by Mn²⁺-phos-tag SDS-PAGE and probed using PRAb from panel A. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. <b>C.</b> Vehicle-corrected distribution data for 1pRLC¹⁹ and 2pRLC¹⁹ obtained by normalizing the data in panel A to the untreated group distribution. g-H (1 µM, n = 5). Calp (0.5 mU/mL, n = 10). OT (100 nM, n = 10). All data are shown as means ± SEMs. The corresponding numerical data are compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020903#pone-0020903-t002" target="_blank">Table 2</a>.</p

Quantification of RLC phospho-state distribution.

<p>Panels A-D correspond to WB data derived using an Ab directed toward the C-terminus of RLC. <b>A.</b> Representative WBs demonstrating RLC phospho-states separated by Mn²⁺-phos-tag SDS-PAGE. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. <b>B.</b> Quantification of bands identified in panel A (untreated; n = 12, g-H; n = 5, Calp; n = 8, OT; n = 7). The signals derived from 0pRLC^T, 1pRLC^T, and 2pRLC^T are expressed as the proportion of their sum total within each lane. <b>C.</b> Absolute magnitude of the difference in each phospho-state caused by the treatments in comparison to the corresponding vehicle (‘+’ indicates enhancement relative to vehicle, ‘−’ indicates diminution relative to vehicle). <b>D.</b> Vehicle-corrected distribution data obtained by combining the data in panel C to the untreated group distribution. g-H (glycyl-H-1152); ROK inhibitor (1 µM). Calp (calpeptin); rhoA activator (0.5 mU/mL). OT; oxytocin (100 nM). All data are shown as means ± SEMs. The corresponding numerical data are compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020903#pone-0020903-t001" target="_blank">Table 1</a>.</p

Changes in Relative Proportions of Phospho-S19-RLC Isotypes in uterine myocytes.

<p>All data are shown as mean ± SEM.</p><p>** and ***indicate significant differences (p<0.05) compared to 1pRLC and 2pRLC in the untreated group, respectively.</p

Validation of increased rhoA activity and phosphorylation of RLC by calpeptin.

<p><b>A.</b> Estimation of GTP-bound ‘active’ rhoA in uterine myocytes treated with Calp or vehicle (DMF) for 15 min (n = 2). RhoA content was quantified relative to α-actin loading control, and used to correct rhoA activity data. Error bars represent standard deviations. <b>B.</b> Quantification of pRLC and ppRLC in uterine myocytes treated with Calp and with increasing concentrations of a cell-permeable rhoA inhibitor (C3 transferase) by ICW (n = 4). * and ** indicate significant differences from vehicle or Calp alone (second histogram), respectively. Calp (calpeptin); rhoA activator (0.5 mU/mL). p<0.05 in all cases as determined by one-way ANOVA followed by Tukey test.</p

Demonstration of RLC phospho-states in human uterine myocyte lysates.

<p><b>A.</b> WBs produced by separation of proteins in lysates from unstimulated uterine myocytes by traditional SDS-PAGE. Single lanes were loaded with ∼25 µg of protein/lane. Abs directed against the C- terminus of total-RLC (CtRLC), phospho-S19-RLC (p19RLC), and diphospho-T18/S19-RLC (p18p19RLC) identify a single prominent band of 20 kDa. <b>B.</b> WBs produced after Mn²⁺-phos-tag SDS-PAGE. CtRLC, p19RLC, and p18p19RLC Abs identified three, two, and one specific band(s), respectively. The lower, middle, and upper bands in these blots correspond to non-, mono-, and di-phosphorylated RLC, denoted as 0pRLC, 1pRLC, and 2pRLC.</p