Force-inhibiting effect of Ser/Thr protein phosphatase 2A inhibitors on bovine ciliary muscle

Abstract

Ciliary muscle is a smooth muscle characterized by a rapid response to muscarinic receptor stimulation and sustained contraction. Although it is evident that these contractions are Ca2+-dependent, detailed molecular mechanisms are still unknown. In order to elucidate the role of Ser/Thr protein phosphatase 2A (PP2A) in ciliary muscle contraction, we examined the effects of okadaic acid and other PP2A inhibitors on contractions induced by carbachol (CCh) and ionomycin in bovine ciliary muscle strips (BCM). Okadaic acid inhibited ionomycin-induced contraction, while it did not cause significant changes in CCh-induced contraction. Fostriecin showed similar inhibitory effects on the contraction of BCM. On the other hand, rubratoxin A inhibited both ionomycin- and CCh-induced contractions. These results indicated that PP2A was involved at least in ionomycin-induced Ca2+-dependent contraction, and that BCM had a unique regulatory mechanism in CCh-induced contraction.

Protein phosphatases 1 and 2A and their naturally occurring inhibitors: current topics in smooth muscle physiology and chemical biology

“…It is therefore natural to assume that the relaxant effect of okadaic acid on intact smooth muscles is produced by specific inhibition of PP2A. This assumption was recently supported by Ishida et al [ 4 , 66 , 117 ], who examined the effects of other PP2A inhibitors on contractions of guinea pig taenia caeci and bovine ciliary muscle. For this purpose, they chose fostriecin [ 92 ] and rubratoxin A [ 93 ] because both of these protein phosphatase inhibitors also show exceedingly higher affinity to PP2A than to PP1 (Table 1 ).…”

“…Cell-permeable antagonists for serine/threonine phosphatases, such as okadaic acid and calyculin A (see “ Effects of PP2A inhibitors on smooth muscle contractio ” and “ Recent topics in sponge-derived protein phosphatase inhibitors ” sections), are powerful tools for studying their physiological and pathophysiological roles. In the smooth muscle physiology field, these inhibitor compounds have contributed to establishing the myosin phosphorylation theory of excitation-contraction coupling [ 62 , 63 ], to identifying and characterizing abnormal phosphorylation under pathological conditions [ 64 ] and to the discovery of new regulatory circuits in smooth muscle regulation [ 65 , 66 ] (see “ Effects of PP2A inhibitors on smooth muscle contraction ” section). Nonetheless, the limitation of these antagonists lies in their lack of ability to distinguish MLCP among over 300 cellular serine/threonine phosphatase holoenzymes, each of which regulates a specific subset of protein phosphorylation.…”

“…However it was soon shown that, when applied at 30–37 °C to preparations with intact cell membrane, relatively low concentrations (<3 μM) of okadaic acid strongly inhibited rather than enhanced contractions induced by agonist stimulation or high K + depolarization. Since the initial reports in 1989 [ 104 , 105 ], the relaxant effect of okadaic acid has been demonstrated (at 30–37 °C; for the temperature dependence of this effect, see below) in a variety of smooth muscles including rabbit aorta [ 104 ]; dog basilar artery [ 105 ]; pig coronary artery [ 105 ]; bovine trachea [ 106 ] and iris [ 107 ]; and guinea pig vas deferens [ 108 ], taenia caeci [ 66 , 109 ], ileum and pulmonary artery (A. Takai, unpublished observations). However, okadaic acid (0.1–3 μM) failed to inhibit the carbachol-induced contraction in bovine ciliary muscle [ 66 ].…”

“…At higher concentrations (>10 μM), okadaic acid was constantly shown to produce or enhance contractions in intact smooth muscle preparations [ 107 , 110 ] as well as in permeabilized preparations [ 62 , 65 , 78 , 111 , 112 ]. Okadaic acid (10 and 30 μM) showed a clear enhancing effect even on the CCh-induced contraction of bovine ciliary muscle [ 66 ], which was not inhibited by lower concentrations of the toxin (see above).…”

“…For this purpose, they chose fostriecin [ 92 ] and rubratoxin A [ 93 ] because both of these protein phosphatase inhibitors also show exceedingly higher affinity to PP2A than to PP1 (Table 1 ). In guinea pig taenia caeci and bovine ciliary muscle, Ishida et al [ 4 , 66 , 117 ] showed that fostriecin and rubratoxin A, as well as okadaic acid, dose-dependently relaxed the contractions induced by a Ca 2+ ionophore, ionomycin. The three toxins also strongly inhibited the carbachol-induced contraction in guinea pig taenia caeci [ 66 ].…”