Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle

Abstract

1 . In smooth muscle, both Ca²⁺ release from the sarcoplasmic reticulum (SR) and Ca²⁺ influx across the plasma membrane are responsible for the increase in the cytosolic Ca²⁺ level ([Ca²⁺]_i). To understand further the role of SR on smooth muscle contraction, the effects of an inhibitor of the SR Ca²⁺ pump, cyclopiazonic acid (CPA 10 μm), an inhibitor of the Ca²⁺-induced Ca²⁺ release, ryanodine, (10 μm), and an activator of the Ca²⁺-induced Ca²⁺ release, caffeine (20 mM), on [Ca²⁺]_i and contractile force were examined in the ferret portal vein loaded with a photoprotein, aequorin. 2 . CPA induced a small increase in the aequorin signal reaching a maximum at 7 min. Several minutes after the increase in the aequorin signal, muscle tension increased reaching a maximum at 21.5 min. In contrast, ryanodine changed neither the aequorin signal nor contraction. In the presence of ryanodine, caffeine induced a sustained increase in the aequorin signal and transient contraction. After washing ryanodine and caffeine, the aequorin signal and muscle tone returned to their respective control levels. After treatment with ryanodine and caffeine, the second addition of caffeine was almost ineffective whereas CPA still increased the aequorin signal and muscle tension. 3 . In the presence of external Ca²⁺, noradrenaline (NA, 10 μm) induced a transient increase followed by a sustained increase in the aequorin signal and sustained contraction. In contrast, KC1 (70 mM) induced sustained increases in the aequorin signal and sustained contraction. In Ca²⁺-free solution, NA induced a small transient increase in the aequorin signal and a small transient contraction. These changes were inhibited in the presence of CPA or on pretreatment of the muscle with ryanodine and caffeine. These results suggest that CPA or ryanodine and caffeine depleted Ca²⁺ in SR. High K⁺ was ineffective in the absence of external Ca²⁺. 4 . In the presence of external Ca²⁺ and CPA, NA and high K⁺ induced larger aequorin signals than in the absence of CPA, whereas the magnitude and shape of the contractions did not change. In contrast, pretreatment with ryanodine and caffeine did not have such an effect. In the muscle pretreated with ryanodine and caffeine, CPA changed the responses to high K⁺ and NA in a similar manner to that in the muscle without the pretreatment with ryanodine and caffeine. 5 . Dissociation of contraction from [Ca²⁺]_i as measured with aequorin suggests that NA and high K⁺ increase Ca²⁺ in two compartments: a compartment containing contractile elements (contractile compartment) and another compartment unrelated to contractile elements (non-contractile compartment). Because CPA augmented the stimulant-induced increase in aequorin signal without changing contraction, the non-contractile compartment may be located near the SR and the CPA-sensitive SR Ca²⁺ pump may regulate the Ca²⁺ level in this compartment. However, because CPA changed neither the magnitude nor shape of the contractions in the presence of external Ca²⁺, the SR Ca²⁺ pump may have little effect on regulation of Ca²⁺ level in the contractile compartment. Furthermore, the release of Ca²⁺ from SR seems to have little effect on the increase in the contractile Ca²⁺ because ryanodine and caffeine changed neither the aequorin signals nor contractions induced by NA and high K⁺ in the presence of external Ca²⁺ in the ferret portal vein.