Inhibitory effects of rubratoxin A, a potent inhibitor of protein phosphatase 2, on the Ca2+-dependent contraction of skinned carotid artery from guinea pig

Abstract

Rubratoxin A, a potent inhibitor of PP2A, is known to suppress smooth muscle contraction. The inhibitory role of PP2A in smooth muscle contraction is still unclear. In order to clarify the regulatory mechanisms of PP2A on vascular smooth muscle contractility, we examined the effects of rubratoxin A on the Ca²⁺-induced contraction of β-escin skinned carotid artery preparations from guinea pigs. Rubratoxin A at 1 µM and 10 µM significantly inhibited skinned carotid artery contraction at any Ca²⁺ concentration. The data fitting to the Hill equation in [Ca²⁺]-contraction relationship indicated that rubratoxin A decreased F_max-Ca2+ and increased [Ca²⁺]₅₀, indices of Ca²⁺ sensitivity for the force and myosin-actin interaction, respectively. These results suggest that PP2A inhibition causes downregulation of the myosin light chain phosphorylation and direct interference with myosin-actin interaction.

Keywords: Ca2+ sensitivity; contractile proteins; protein phosphatase 2A; rubratoxin A; vascular smooth muscle.

Fig. 1.

Typical force traces of skinned carotid artery muscle preparations activated with 10 µM Ca²⁺ (a) or 1 µM Ca²⁺ (b), with 1 µM calmodulin. Rubratoxin A (10 µM) was applied for 2 min before exposure to Ca²⁺ subsequently for 15 min.

Fig. 2.

Effects of rubratoxin A on the Ca²⁺-induced contraction (a: 10 µM Ca²⁺, b: 3 µM, c: 2 µM, d: 1 µM). 1% DMSO without rubratoxin A (control: ), 10 nM (), 100 nM (), 1 µM (), or 10 µM () rubratoxin A was added to the artificial intracellular solutions. Values are the means ± S.E.M. of 4–7 experiments. Asterisk indicates significant difference of the active force compared with that of controls, where P values are less than 0.05.

Fig. 3.

Effects of rubratoxin A on the Ca²⁺concentration-relative tension relationship. 1% DMSO without rubratoxin A (control: ), 10 nM (), 100 nM (), 1 µM (), or 10 µM () rubratoxin A was added to the artificial intracellular solutions.

Fig. 4.

Effects of rubratoxin A (10 μM) on the latent time of the Ca²⁺-induced contraction (a: 10 µM Ca²⁺, b: 2 µM). Values are the means ± S.E.M. of 4–7 experiments. Asterisk indicates a significant difference of the latency compared with that of control, where P values are less than 0.05.