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

The effect of the protein phosphatases inhibitor cantharidin on β-adrenoceptor-mediated vasorelaxation

1. Cantharidin, an inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A), increased basal tone of bovine isolated coronary artery rings (CARs) with and without endothelium in a time- and concentration-dependent manner with pEC(50) values of about 5.1 and 5.2, respectively, for both preparations. 2. β-Adrenoceptor stimulation with isoprenaline (Iso; 0.03–100 μM) or inhibition of phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX; 10–1000 μM), respectively, relaxed CARs precontracted with KCl (75 mM). CARs with and without endothelium showed no difference in the relaxing response to Iso and IBMX, respectively. 3. Cantharidin (3 μM) attenuated vasorelaxation induced by Iso (0.03–100 μM) in CARs with and without endothelium in a time-dependent manner, whereas vasorelaxation induced by IBMX (10–1000 μM) was not attenuated by 3 μM cantharidin. 4. Cantharidin (3 μM) did not affect cyclic AMP content in bovine cultured vascular cells, i.e. coronary artery smooth muscle cells (BCs), aortic endothelial cells (BAECs) and aortic smooth muscle cells (BASMCs), either under basal conditions, after β-adrenoceptor stimulation (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. 5. Cantharidin inhibited protein phosphatase activity in homogenates from bovine coronary artery rings with a pIC(50) of about 6.0. In homogenates of bovine cultured vascular cells pIC(50) values of cantharidin amounted to about 6.5 for BCs, 6.7 for BAECs and 6.7 for BASMCs, respectively. 6. It was concluded that cantharidin differently affects vasorelaxation due to stimulation of β-adrenoceptors (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. The attenuation of β-adrenoceptor-mediated vasorelaxation by phosphatase inhibition is not due to diminished adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) generation but could be evidence for different subcellular compartments of cyclic AMP