Myometrial relaxation of mice via expression of two pore domain acid sensitive K⁺ (TASK-2) channels

Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K⁺ channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K⁺ current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K⁺ channels (TASK-2). NIOK in the presence of K⁺ channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery.

Mechanism of Relaxation Via TASK-2 Channels in Uterine Circular Muscle of Mouse

Plasma pH can be altered during pregnancy and at labor. Membrane excitability of smooth muscle including uterine muscle is suppressed by the activation of K+ channels. Because contractility of uterine muscle is regulated by extracellular pH and humoral factors, K+ conductance could be connected to factors regulating uterine contractility during pregnancy. Here, we showed that TASK-2 inhibitors such as quinidine, lidocaine, and extracellular acidosis produced contraction in uterine circular muscle of mouse. Furthermore, contractility was significantly increased in pregnant uterine circular muscle than that of non-pregnant muscle. These patterns were not changed even in the presence of tetraetylammonium (TEA) and 4-aminopyridine (4-AP). Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretchactivated channels in myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed increased immunohistochemical expression of TASK-2. Therefore, TASK-2, seems to play a key role during regulation of myometrial contractility in the pregnancy and provides new insight into preventing preterm delivery.

Regional Distribution of Interstitial Cells of Cajal, (ICC) in Human Stomach

We elucidated the distribution of interstitial cells of Cajal (ICC) in human stomach, using cryosection and c-Kit immunohistochemistry to identify c-Kit positive ICC. Before c-Kit staining, we routinely used hematoxylin and eosin (HE) staining to identify every structure of human stomach, from mucosa to longitudinal muscle. HE staining revealed that the fundus greater curvature (GC) had prominent oblique muscle layer, and c-Kit immunostaining c-Kit positive ICC cells were found to have typical morphology of dense fusiform cell body with multiple processes protruding from the central cell body. In particular, we could observe dense processes and ramifications of ICC in myenteric area and longitudinal muscle layer of corpus GC. Interestingly, c-Kit positive ICC-like cells which had morphology very similar to ICC were found in gastric mucosa. We could not find any significant difference in the distribution of ICC between fundus and corpus, except for submucosa where the density of ICC was much higher in gastric fundus than corpus. Furthermore, there was no significant difference in the density of ICC between each area of fundus and corpus, except for muscularis mucosa. Finally, we also found similar distribution of ICC in normal and cancerous tissue obtained from a patient who underwent pancreotomy and gastrectomy. In conclusion, ICC was found ubiquitously in human stomach and the density of ICC was significantly lower in the muscularis mucosa of both fundus/corpus and higher in the submucosa of gastric fundus than corpus.

Relaxation Patterns of Human Gastric Corporal Smooth Muscle by Cyclic Nucleotides Producing Agents

To elucidate the mechanism of cyclic nucleotides, such as adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5' -cyclic monophosphate (cGMP), in the regulation of human gastric motility, we examined the effects of forskolin (FSK), isoproterenol (ISO) and sodium nitroprusside (SNP) on the spontaneous, high K+ and acetylcholine (ACh)-induced contractions of corporal circular smooth muscle in human stomach. Gastric circular smooth muscle showed regular spontaneous contraction, and FSK, ISO and SNP inhibited its phasic contraction and basal tone in a concentration-dependent manner. High K+ (50 mM) produced sustained tonic contraction, and ACh (10 micrometer) produced initial transient contraction followed by later sustained tonic contraction with superimposed phasic contractions. FSK, ISO and SNP inhibited high K+-induced tonic contraction and also ACh-induced phasic and tonic contraction in a reversible manner. Nifedipine (1 micrometer), inhibitor of voltage-dependent L-type calcium current (VDCC(L)), almost abolished ACh-induced phasic contractions. These findings suggest that FSK, ISO and SNP, which are known cyclic nucleotide stimulators, inhibit smooth muscle contraction in human stomach partly via inhibition of VDCCL.

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

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 (VDCCL). In this study, we also studied the effect of spermidine on mechanical contractions and calcium channel current (IBa), 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 (IC50=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 IBa 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 Ca2+-induced Ca2+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 VDCCL and Ca2+releasing mechanism in guinea-pig stomach.