Magdalena Lomnicka

1-Methylnicotinamide (MNA), a primary metabolite of nicotinamide, exerts anti-thrombotic activity mediated by a cyclooxygenase-2/prostacyclin pathway

1‐methylnicotinamide (MNA) has been considered to be an inactive metabolite of nicotinamide. Here we assessed the anti‐thrombotic activity of MNA in vivo.

Simultaneous determination of nicotinic acid and its four metabolites in rat plasma using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS)

A sensitive and specific liquid chromatography electrospray ionization-tandem mass spectrometry method for the simultaneous quantitation of nicotinic acid (NicA) and its metabolites nicotinamide (NA), 1-methylnicotinamide (MNA), 1-methyl-2-pyridone-5-carboxamide (M2PY) and 1-methyl-4-pyridone-5-carboxamide (M4PY) in rat plasma has been developed and validated. As an internal standard, 6-chloronicotinamide was used. The samples (100 microL) were subjected to deproteinization with acetonitrile (200 microL) and then, after centrifugation, 150 microL of the supernatant was transferred into conical vial and evaporated. Dry residue was reconstituted in 100 microL of the ACN/water (10:90, v/v) mixture. Chromatography was performed on a Waters Spherisorb 5 microm CNRP 4.6 x 150 mm analytical column with gradient elution using a mobile phase containing acetonitrile and water with 0.1% of formic acid. The full separation of all compounds was achieved within 15 min of analysis. Detection was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer set at unit resolution. The mass spectrometer was operated in the selected reactions monitoring mode (SRM), monitoring the transition of the protonated molecular ions m/z 153-110 for M2PY, 153-136 for M4PY, 124-80 for NicA, 123-80 for NA and 137-94 for MNA. The mass spectrometric conditions were optimized for each compound by continuously infusing the standard solution at the rate of 5 microL/min using a Harvard infusion pump. Electrospray ionization (ESI) was used for ion production. The instrument was coupled to an Agilent 1100 LC system. The precision and accuracy for both intra- and inter-day determination of all analytes ranged from 1.3% to 13.3% and from 94.43% to 110.88%. No significant matrix effect (ME) was observed. Stability of compounds was established in a battery of stability studies, i.e. bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for various applications. In particular using this method we detected increased concentration of MNA and its metabolites in rat plasma after treatment with exogenous MNA (100 mg/kg), as well as increased concentration of endogenous NA and MNA in rat plasma in the early phase of hypertriglyceridemia development in rats fed high-fructose diet.

Late-stage alterations in myofibrillar contractile function in a transgenic mouse model of dilated cardiomyopathy (Tgαq*44)

Mechanical and biochemical alterations were investigated in permeabilized cardiomyocytes along with the progression of dilated cardiomyopathy (DCM) in a transgenic mouse line overexpressing the activated Galphaq protein (Tgalphaq*44). The isometric force, its Ca(2+) sensitivity (pCa(50)) and the turnover rate of the actin-myosin cycle (k(tr)) were determined at sarcomere lengths (SLs) of 1.9 mum and 2.3 mum before (at 4 and 10 months of age) and after hemodynamic decompensation (at 14 and 18 months of age) in Tgalphaq*44 cardiomyocytes and in age-matched control cardiomyocytes. The SL-dependence of pCa(50) was not different in Tgalphaq*44 and control hearts. In contrast, a significant increase in pCa(50) was observed in the Tgalphaq*44 cardiomyocytes (DeltapCa(50): 0.10-0.15 vs. the controls) after 10 months of age that could be diminished by exposures to the catalytic subunit of protein kinase A (PKA). Accordingly, a decline in endogenous PKA activity and decreased troponin I phosphorylation were detected after 10 months in the Tgalphaq*44 hearts. Finally, the maximal Ca(2+)-activated force (F(o)) and k(tr) were lower and the passive force (F(passive)) was higher at 18 months in the Tgalphaq*44 cardiomyocytes compared to the control. These mechanical alterations were paralleled by a robust increase in beta-myosin heavy chain expression in the Tgalphaq*44 hearts. In conclusion, our data suggested that an initial decrease of PKA signaling and subsequent changes in myofilament protein expression may contribute to the development of dilated cardiomyopathy in Tgalphaq*44 hearts.

Functional alterations in NO, PGI2 and EDHF pathways in the aortic endothelium after myocardial infarction in rats

Previous work on endothelial dysfunction in post‐MI heart failure has shown conflicting results.

Anti-thrombotic effects of nebivolol and carvedilol: Involvement of β2 receptors and COX-2/PGI2 pathways

BACKGROUND Third generation β-adrenolytics, such as selective β1 adrenoceptor antagonist nebivolol and non-selective β1/β2 and α1 adrenoceptor antagonist carvedilol, display beneficial nitric oxide (NO)-dependent vasodilator activities that contribute to their therapeutic efficacy. In the present work, we analyzed whether nebivolol and carvedilol, as well as other β-adrenolytics with similar pharmacological profiles (selective β1 adrenoceptor antagonist - atenolol and non-selective α/β adrenoceptor antagonist - labetalol), possess the ability to induce PGI2-dependent anti-thrombotic activity in vivo in normotensive rats. METHODS Anti-thrombotic effects of nebivolol and carvedilol were studied in vivo in anaesthetized rats with extracorporeal circulation superfusing collagen strips. We also assessed vasodilation induced by these drugs in isolated perfused guinea pig hearts according to Langendorffs procedures. RESULTS Nebivolol (both d- and l-isomers) (0.1-1mgkg(-1)) and carvedilol (1-3mgkg(-1)), but not atenolol (1mgkg(-1)) or labetalol (3mgkg(-1)), induced a dose-dependent and sustained anti-thrombotic response in rat model of thrombosis with extracorporeal circulation. The cyclooxygenase (COX)-2 inhibitors, rofecoxib (1mgkg(-1)) and indomethacin (5mgkg(-1)) abrogated this response, while l-NAME (5mgkg(-1)) had no significant effect. In the presence of β1/β2 adrenoceptor antagonist nadolol (1mgkg(-1)), but not in the presence of selective β1 adrenoceptor antagonist atenolol (4mgkg(-1)), anti-thrombotic responses to nebivolol, as well as carvedilol, were lost. Neither nebivolol nor carvedilol affected platelet aggregation in vitro, however both nebivolol and carvedilol induced NO-dependent vasodilation in guinea pig coronary circulation that was not dependent on β2 adrenoceptors. CONCLUSIONS We demonstrated for the first time that nebivolol and carvedilol, independently of their adrenergic receptor blocking activities, induced anti-thrombotic effects in vivo that involved β2 adrenoceptors and the activation of the COX-2/PGI2 pathway.