Relaxant Effect of Spermidine on Acethylcholine and High K-induced Gastric Contractions of Guinea-Pig

Abstract

In our previous study, we found that spermine and putrescine inhibited spontaneous and acetylcholine (ACh)-induced contractions of guinea-pig stomach via inhibition of L-type voltage-dependent calcium current (VDCC(L)). In this study, we also studied the effect of spermidine on mechanical contractions and calcium channel current (I(Ba)), and then compared its effects to those by spermine and putrescine. Spermidine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner (IC(50)=1.1+/-0.11 mM). Relationship between inhibition of contraction and calcium current by spermidine was studied using 50 mM high K(+)-induced contraction: Spermidine (5 mM) significantly reduced high K(+) (50 mM)-induced contraction to 37+/-4.7% of the control (p<0.05), and inhibitory effect of spermidine on I(Ba) was also observed at a wide range of test potential in current/voltage (I/V) relationship. Pre- and post-application of spermidine (5 mM) also significantly inhibited carbachol (CCh) and ACh-induced initial and phasic contractions. Finally, caffeine (10 mM)-induced contraction which is activated by Ca(2+)-induced Ca(2+) release (CICR),' was also inhibited by pretreatment of spermidine (5 mM). These findings suggest that spermidine inhibits spontaneous and CCh-induced contraction via inhibition of VDCC(L) and Ca(2+) releasing mechanism in guinea-pig stomach.

Keywords: Ca2+ release; Calcium current; Relaxation; Spermidine; Stomach.

Fig. 1

Effect of spermidine on isometric and high K⁺ (50 mM)-induced contraction in guinea-pig gastric smooth muscle. Effects of spermidine on isometric contraction were studied by using vertical chamber system. (A) Spermidine causes relaxation in a concentration-dependent manner. Relative contractions at various concentrations of spermine were plotted and fitted by the non-linear regression equation (IC₅₀=1.1 mM). (B) High K⁺ (50 mM)-induced contraction was compared by pretreatment of spermidine (5 mM) before re-application of High K⁺ solution. Such a high K⁺ (50 mM)-induced contraction was significantly inhibited by the pretreatment with 5 mM spermidine (p<0.05).

Fig. 2

Inhibitory effect of spermidine on muscaranic receptor agonist-stimulated contraction via regulation of VDCC_L. Acethylcholine (ACh, 10 µM) and carbachol (CCh, 50 µM) were applied to guinea-pig gastric smooth muscle. ACh- and CCh-induced transient initial and sustained phasic contractions were studied in the presence or absence of spermidine. (A and C) When 5 mM spermidine was applied to ACh- and CCh-induced sustained contractions, those contractions were inhibited to 12% and 43% of the control, respectively. (B) When 5 mM spermidine was pretreated before the second application of ACh, ACh-induced transient initial and sustained phasic contractions were inhibited to 76% and 17% of the control, respectively. (D) When 5 mM spermidine was applied before the second application of CCh, CCh-induced transient initial and sustained phasic contractions were also inhibited to 58% and 42% of the control, respectively.

Fig. 3

Effect of spermidine on I_Ba and caffeine-induced contraction in guinea-pig gastric myocytes. (A) I_Ba was recorded under the condition in which extracellular Ca²⁺ was replaced by 10 mM Ba²⁺. I/V relationship of I_Ba by spermidine is shown by averaged responses in the presence and absence of spermidine (closed circle, Control; open circle, 1 mM spermidine). Inhibition of I_Ba was observed at whole potential ranges tested. (B) Caffeine (10 mM)-induced contraction was compared with pretreatment of spermidine (5 mM) before re-application of caffeine. Caffeine-induced contraction was significantly inhibited by pretreatment with 5 mM spermidine (p<0.05).