Vasomotion in human arteries and their regulations based on ion channel regulations: 10 years study.

Vasomotion is the oscillation of vascular tone which gives rise to flow motion of blood into an organ. As is well known, spontaneous contractile organs such as heart, GI, and genitourinary tract produce rhythmic contraction. It imposes or removes pressure on their vessels alternatively for exchange of many substances. It was first described over 150 years ago, however the physiological mechanism and pathophysiological implications are not well understood. This study aimed to elucidate underlying mechanisms and physiological function of vasomotion in human arteries. Conventional contractile force measurement, immunohistochemistry, and Western blot analysis were employed to study human left gastric artery (HLGA) and uterine arteries (HUA). RESULTS: Circular muscle of HLGA and/or HUA produced sustained tonic contraction by high K+ (50 mM) which was blocked by 2 µM nifedipine. Stepwise stretch and high K+ produced nerve-independent spontaneous contraction (vasomotion) (around 45% of tested tissues). Vasomotion was also produced by application of BayK 8644, 5-HT, prostagrandins, oxytocin. It was blocked by nifedipine (2 µM) and blockers of intracellular Ca2+ stores. Inhibitors of Ca2+ -activated Cl- channels (DIDS and/or niflumic acid) and ATP-sensitive K+ (KATP ) channels inhibited vasomotion reversibly. Metabolic inhibition by sodium cyanide (NaCN) and several neuropeptides also regulated vasomotion in KATP channel-sensitive and -insensitive manner. Finally, we identified TMEM16A Ca2+ -activated Cl- channels and subunits of KATP channels (Kir 6.1/6.2 and sulfonylurea receptor 2B [SUR2B]), and c-Kit positivity by Western blot analysis. We conclude that vasomotion is sensitive to TMEM16A Ca2+ -activated Cl- channels and metabolic changes in human gastric and uterine arteries. Vasomotion might play an important role in the regulation of microcirculation dynamics even in pacemaker-related autonomic contractile organs in humans.

Physiological function and molecular composition of ATP-sensitive K+ channels in human gastric smooth muscle.

Gastric motility is controlled by slow waves. In general, the activation of the ATP-sensitive K+ (KATP) channels in the smooth muscle opposes the membrane excitability and produces relaxation. Since metabolic inhibition and/or diabetes mellitus are accompanied by dysfunctions of gastric smooth muscle, we examined the possible roles of KATP channels in human gastric motility. We used human gastric corpus and antrum smooth muscle preparations and recorded the mechanical activities with a conventional contractile measuring system. We also identified the subunits of the KATP channels using Western blot. Pinacidil (10 µM), a KATP channel opener, suppressed contractions to 30% (basal tone to -0.2 g) of the control. The inhibitory effect of pinacidil on contraction was reversed to 59% of the control by glibenclamide (20 µM), a KATP channel blocker. The relaxation by pinacidil was not affected by a pretreatment with L-arginine methyl ester, tetraethylammonium, or 4-aminopyridine. Pinacidil also inhibited the acetylcholine (ACh)-induced tonic and phasic contractions in a glibenclamide-sensitive manner (42% and 6% of the control, respectively). Other KATP channel openers such as diazoxide, cromakalim and nicorandil also inhibited the spontaneous and ACh-induced contractions. Calcitonin gene-related peptide (CGRP), a gastric neuropeptide, induced muscle relaxation by the activation of KATP channels in human gastric smooth muscle. Finally, we have found with Western blot studies, that human gastric smooth muscle expressed KATP channels which were composed of Kir 6.2 and SUR2B subunits.

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.

Interstitial cells of Cajal (ICC)-like-c-Kit positive cells are involved in gastritis and carcinogenesis in human stomach.

This study was executed to prove the existence of c-Kit-positive interstitial cells of Cajal (ICC)-like cells [c-Kit (+) ICC-like cells] and their possible role associated with gastric inflammation and/or carcinogenesis in human gastric mucosa. c-Kit (+) ICC-like cells were observed throughout all the layers of the gastric fundus along the greater curvature. Dense fusiform cell bodies with many processes were found in each layer. We also studied the c-Kit-positive immunoreactivity distribution pattern in the mucosa. c-Kit (+) cells were found mainly around the epithelial repair zone of the normal gastric fundus/corpus and of the fundus/corpus with non-metaplastic chronic gastritis. Notably, they were found attached to the epithelia of the repair zone in non-metaplastic chronic gastritis. In chronic gastritis with intestinal metaplasia, they were found scattered everywhere in the stroma of the gastric mucosa and did not attach to the metaplastic epithelium. We found c-Kit (+) ICC-like cells in human mucosa. They were present mainly in the stroma around the repair zone of the glands in chronic gastritis as well as in normal mucosa, whereas they seemed to redistribute over the whole mucosa in gastritis with intestinal metaplasia. These cells around the repair zone were found to be tightly attached to epithelial cells, but not to metaplastic epithelial cells. Thus, c-Kit (+) ICC-like cells appear to have a role in the epithelial recovery process and may be associated with carcinogenesis of human gastric mucosa.

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 microM) 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 microM), 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 VDCC(L).

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 (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.