Cantharidin Enhances Norepinephrine-Induced Vasoconstriction in an Endothelium-Dependent Fashion 1

ABSTRACT In this study we characterized the effects of the protein phosphatase (PP) type 1 and type 2A inhibitor cantharidin (Cant) and its structural analogs cantharidic acid and endothall on PP activity, force of contraction, and myosin light chain phosphorylation in rat aorta. All compounds inhibited PP activity in homogenates of rat aorta with a rank order of potency of Cant ϭ cantharidic acid Ͼ endothall. However, only Cant increased force of contraction and myosin light chain phosphorylation in intact isolated rat aortic rings. Based on these findings, we investigated the effects of Cant on ␣-adrenoceptormediated vasoconstriction. Cant (1 and 3 M) enhanced norepinephrine-induced contraction in endothelium-intact rat aorta. In contrast, Cant did not affect norepinephrine-induced contraction in endothelium-denuded rat aorta. We suggest that inhibition of PP1 and/or PP2A activities by Cant enhances vascular contractility in endothelium-intact rat aorta by increasing the phosphorylation state of endothelial regulatory proteins

Modulation of SR Ca2+ release by the triadin-to-calsequestrin ratio in ventricular myocytes

Calsequestrin (CSQ) is a Ca2+storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca2+signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TGCSQ) was associated with heart failure, attenuation of SR Ca2+release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TGCxT) could be beneficial for impaired intracellular Ca2+signaling and contractile function. Indeed, the depressed intracellular Ca2+concentration ([Ca]i) peak amplitude in TGCSQwas normalized by co-overexpression in TGCxTmyocytes. This effect was associated with changes in the expression of cardiac Ca2+regulatory proteins. For example, the protein level of the L-type Ca2+channel Cav1.2 was higher in TGCxTcompared with TGCSQ. Sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) expression was reduced in TGCxTcompared with TGCSQ, whereas JUN expression and [3H]ryanodine binding were lower in both TGCxTand TGCSQcompared with wild-type hearts. As a result of these expressional changes, the SR Ca2+load was higher in both TGCxTand TGCSQmyocytes. In contrast to the improved cellular Ca2+, transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TGCxTcompared with TGCSQ. Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca2+release compared with TGCSQmice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca2+signaling.</jats:p

Pertussis toxin sensitive and insensitive effects of adenosine and carbachol in murine atria overexpressing A(1)-adenosine receptors

1. It was investigated how A(1)-adenosine receptor overexpression alters the effects of carbachol on force of contraction and beating rate in isolated murine atria. Moreover, the influence of pertussis toxin on the inotropic and chronotropic effects of adenosine and carbachol in A(1)-adenosine receptor overexpressing atria was studied. 2. Adenosine and carbachol alone exerted negative inotropic and chronotropic effects in electrically driven left atrium or spontaneously beating right atrium of wild-type mice. 3. These effects were abolished or reversed by pre-treatment of animals with pertussis toxin which can interfere with signal transduction through G-proteins. 4. Adenosine and carbachol exerted positive inotropic but negative chronotropic effects in atrium overexpressing A(1)-adenosine receptors from transgenic mice. 5. The positive inotropic effects of adenosine and carbachol were qualitatively unaltered whereas the negative chronotropic effects were abolished or reversed in atrium overexpressing A(1)-adenosine receptors after pre-treatment by pertussis toxin. 6. Qualitatively similar effects for adenosine and carbachol were noted in the presence of isoprenaline, β-adrenoceptor agonist. 7. It is concluded that overexpression of A(1)-adenosine receptors also affects the signal transduction of other heptahelical, G-protein coupled receptors like the M-cholinoceptor in the heart. The chronotropic but not the inotropic effects of adenosine and carbachol in transgenic atrium were mediated via pertussis toxin sensitive G-proteins