Endogenous ACh tonically stimulates ANP secretion in rat atria.

Exogenous acetylcholine (ACh) is known to stimulate atrial natriuretic peptide (ANP) secretion concomitantly with a decrease in atrial pulse pressure. However, the role of intrinsic ACh in the regulation of ANP secretion remains unknown. Recently, it was shown that nonneuronal and neuronal ACh is present in the cardiac atria. From this finding we hypothesize that endogenously released ACh is involved in the regulation of ANP secretion in an autocrine or paracrine manner in the atria. Experiments were performed in isolated beating rat atria. ANP was measured using radioimmunoassay. To increase the availability of the ACh in the extracellular space of the atrium, its degradation was inhibited with an inhibitor of acetylcholinesterase. Acetylcholinesterase inhibition with physostigmine increased ANP secretion concomitantly with a decrease in atrial dynamics in a concentration-dependent manner. Inhibitors of M2 muscarinic ACh receptor (mAChR), methoctramine, and ACh-activated K(+) (KACh(+)) channels, tertiapin-Q, abolished the physostigmine-induced changes. The effects were not observed in the atria from rats treated with pertussis toxin. Furthermore, the physostigmine-induced effects were attenuated by an inhibitor of high-affinity choline transporter, hemicholinium-3, which is a rate-limiting step of ACh synthesis. Inhibitors of the mAChR signaling pathway and ACh synthesis also attenuated the basal levels of ANP secretion and accentuated atrial dynamics. These findings suggest that endogenously released ACh tonically stimulates ANP secretion from atrial cardiomyocytes via activation of M2 mAChR-Gi/o-KACh(+) channel signaling. It is also suggested that the ACh-ANP signaling is implicated in cardiac physiology and pathophysiology.

[Forskolin inhibits spontaneous contraction of gastric antral smooth muscle in rats].

The aim of the present study was to investigate the effects of cyclic adenosine monophosphate (cAMP) on rat gastric antral circular smooth muscle function. Forskolin, a direct activator of adenylyl cyclase (AC), was used to observe the influences of cAMP. Multi-channel physiological recorder was used to record spontaneous contraction activity of gastric antral circular muscle from Wistar rats. And ELISA method was used to detect the change of cAMP production in perfusate. The results showed that forskolin concentration-dependently suppressed the amplitude and frequency of the spontaneous contraction of the gastric antral muscle, and lowered the baseline of contraction movement significantly. Forskolin concentration-dependently increased the production of cAMP in the perfusate, which showed a significant negative correlation with the contraction amplitude of gastric antral ring muscle. The inhibitory effect of forskolin on spontaneous contraction activity of rat gastric antral circular muscle could be blocked by cAMP-dependent protein kinase (PKA) inhibitor H-89. These results suggest forskolin increases cAMP production and then activates PKA pathway, resulting in the inhibition of the spontaneous contraction activity of rat gastric antral circular smooth muscle.

Peroxiredoxin V Silencing Elevates Susceptibility to Doxorubicin-induced Cell Apoptosis via ROS-dependent Mitochondrial Dysfunction in AGS Gastric Cancer Cells.

BACKGROUND/AIM: Peroxiredoxin V (Prx V) plays crucial roles in cellular apoptosis and proliferation in various cancer cells by regulating the cellular reactive oxygen species (ROS) levels. MATERIALS AND METHODS: Here, we examined the possible regulatory effects of Prx V on doxorubicin (DOX)-induced cellular apoptosis and its mechanisms in the human gastric adenocarcinoma cell line (AGS cells). RESULTS: Our findings suggest that Prx V knockdown may significantly increase the DOX-induced apoptosis by aggravating intracellular ROS accumulation. We also found that DOX-induced mitochondrial ROS levels and membrane permeability were significantly higher in short hairpin Prx V cells than in mock cells, and these phenomena were dramatically reversed by ROS scavenger treatment. Prx V knockdown also significantly upregulated the cleaved caspase 9, 3, and B-cell lymphoma 2 (Bcl2)-associated agonist of cell death/Bcl2 protein expression levels, suggesting that Prx V knockdown activates mitochondria-dependent apoptotic signaling pathways. CONCLUSION: Taken together, this study suggests that Prx V may be a strong molecular target for gastric cancer (GC) chemotherapy, and further elucidates the role of Prx V in oxidative stress-induced cell apoptosis.

Natriuretic peptide-dependent cGMP signal pathway potentiated the relaxation of gastric smooth muscle in streptozotocin-induced diabetic rats.

A common gastrointestinal complication of diabetes is gastroparesis, and patients with gastroparesis may present with early satiety, nausea, vomiting, bloating, postprandial fullness, or upper abdominal pain. However, the pathogenesis is not clear yet. A recent study indicated that atrial natriuretic peptide (ANP) was secreted from the gastric mucosa and the ANP family plays an inhibitory role in the regulation of gastrointestinal motility, but the effect of the natriuretic peptide signal pathway on diabetic gastroparesis has not been reported. The study investigated the effect of C-type natriuretic peptide (CNP) particulate guanylyl cyclase (pGC) cyclic guanosine monophosphate (cGMP) signaling on gastroparesis in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats; 4 weeks after induction, the experiments were performed. The spontaneous contraction of gastric smooth muscle strips was recorded by using physiographs in control and diabetic rats. The pGC activity in response to CNP and cGMP production in gastric smooth muscle were measured by using radioimmunoassay (RIA) in normal and diabetic rats. CNP induced a longer lasting relaxation of gastric antral circular smooth muscle strips in STZ-induced diabetic rats. The inhibitory effect of CNP on spontaneous contraction revealed a dose-dependency, and the inhibitory percentages were 25.5 +/- 1.7%, 43.6 +/- 3.2%, 85.1 +/- 2.5% in diabetic rats and 20.5 +/- 1.5%, 31.1 +/- 1.7%, 58.9 +/- 3.7% in the control group at the concentrations of 0.01, 0.03, and 0.1 mumol/l, respectively. The cGMP production and pGC activity in response to CNP in gastric muscle tissues were significantly potentiated in STZ-induced diabetic rats. Natriuretic peptide receptor type B (NPR-B) gene was expressed in the gastric smooth muscles of normal and diabetic rats, and the expression was increased in diabetic rats. The results suggest that natriuretic peptide-dependent pGC-cGMP signal is upregulated and may contribute to diabetic gastroparesis in STZ-induced diabetic rats.

Pacinian corpuscles in the human fetal foot: A study using 3D reconstruction and immunohistochemistry.

PURPOSE: Our group had recently described human hand Pacinian corpuscles (PCs): the hand PCs are not simply arranged along the digital palmar nerves but often exhibited specific morphologies known uncommonly. However, there is still no or few information about human foot PCs. MATERIALS AND METHODS: We observed transverse sections of all five toes including the interdigital area obtained from 12 feet of eight fetuses at 28-33 weeks (crown-rump length 230-290mm). Serial sections were prepared for 3D reconstructions and measurement. RESULTS: Foot PCs were characterized by (1) a dense distribution in the interdigital area in contrast to a few PCs in the distal tip of the all five toes; (2) abundant dorsal PCs including those in the nail bed and: (3) a long chain of PCs in the flexor tendon sheath of all five toes. Therefore, a distal dominance was not evident in the foot in contrast to the hand and, a tendon sheath contained much greater numbers of PCs than the hand. A tree-like or bouquet-like arrangement of PCs along a short perforating artery to the palmar digital skin was seen in the foot as we had described in the hand. The tree of foot PCs was sometimes seen laying transversely along the digital skin surface, not toward the skin. CONCLUSION: It is still unknown that, in utero, how the PCs distribution became different between the hand and foot: it might be determined genetically in a region-specific manner.

Peroxiredoxin V Inhibits Emodin-induced Gastric Cancer Cell Apoptosis via the ROS/Bcl2 Pathway.

BACKGROUND/AIM: Peroxiredoxin (Prx) protein family is aberrantly expressed in various cancers including gastric cancer. Among the six family members, Prx V has been known as an antioxidant enzyme which scavenges intracellular reactive oxygen species (ROS) and modulates cellular apoptosis. This study aimed at investigating the role of Prx V in apoptosis of gastric cancer cells. MATERIALS AND METHODS: Stably constructed Prx V knockdown, over-expression and mock AGS cells (a human gastric adenocarcinoma cell line) were used to study the effect of Prx V on emodin-induced apoptosis by western blotting, cell viability, apoptosis and ROS detection assays. RESULTS: Overexpression of Prx V significantly decreased emodin-induced cellular apoptosis and ROS levels compared to Mock and Prx V knockdown AGS cells. Also, overexpression of Prx V down-regulated the expression of pro-apoptotic proteins, Bad and cleaved PARP, and increased the expression of anti-apoptotic protein, Bcl2. CONCLUSION: Prx V suppresses AGS cell apoptosis via scavenging intracellular ROS and modulating apoptosis-related markers.

K+ACh channel activation with carbachol increases atrial ANP release.

Although it has been known that atrial natriuretic peptide (ANP) release is regulated through muscarinic acetylcholine receptors (mAChR), the mechanism by which this neurotransmitter regulates atrial ANP release is largely unknown. This study tested the hypothesis that K(+)(ACh) channels mediate the action of mAChR on atrial myocyte ANP release. Experiments were performed in perfused beating rabbit atria. Carbachol (CCh), an agonist of cardiac mAChR, increased atrial myocyte ANP release concomitantly with a decrease in stroke volume and intra-atrial pulse pressure in a concentration-dependent manner. Isoproterenol, a beta-adrenoceptor agonist, decreased ANP release concomitantly with an increase in cAMP and mechanical dynamics. In the presence of isoproterenol, the CCh-induced increase in ANP release and decrease in cAMP efflux levels and mechanical dynamics were able to be repeated. The CCh-induced changes were blocked by selective M(2) mAChR antagonists. Tertiapin, a selective G-protein-gated K(+)(ACh) channel blocker, attenuated the CCh-induced increase in ANP release and decrease in mechanical dynamics in a concentration-dependent manner, but without a significant effect on the CCh-induced decrease in cAMP efflux levels. The CCh-induced changes in ANP release and atrial dynamics were inhibited in the atria from pertussis toxin-pretreated rabbits. These findings demonstrate that G-protein-gated K(+)(ACh) channels regulate atrial myocyte ANP release. The present study also shows that mAChR and adrenoceptors have opposing roles in the regulation of ANP release.

Identification of molecular markers for the oncogenic differentiation of hepatocellular carcinoma.

The aim of this study was to identify molecular markers associated with oncogenic differentiation in hepatocellular carcinoma (HCC). Using an unsupervised clustering method with a cDNA microarray, HCC (T) gene expression profiles and corresponding non-tumor tissues (NT) from 40 patients were analyzed. Of total 217 genes, 72 were expressed preferentially in HCC tissues. Among 186 differentially regulated genes, there were molecular chaperone and tumor suppressor gene clusters in the Edmondson grades I and II (GI/II) subclass compared with the liver cirrhosis (LC) subclass. The Edmondson grades III and IV (GIII/IV) subclass with a poor survival (P=0.0133) contained 122 differentially regulated genes with a cluster containing various metastasis- and invasion-related genes compared with the GI/II subclass. Immunohistochemical analysis revealed that ANXA2, one of the 72 genes preferentially expressed in HCC, was over-expressed in the sinusoidal endothelium and in malignant hepatocytes in HCC. The genes identified in the HCC subclasses will be useful molecular markers for the genesis and progression of HCC. In addition, ANXA2 might be a novel marker for tumor angiogenesis in HCC.

[Effect of microinjection of atrial natriuretic peptide into the paraventricular nucleus on baroreflex sensitivity in conscious rats].

The role of atrial natriuretic peptide (ANP) in the central regulation of the circulation is known to be a neurotransmitter or a neuromodulator, but its actions on baroreceptor reflex function are not fully resolved. The present study examined the role of ANP (6, 60 ng/0.2 microl) by direct microinjection into the hypothalamic paraventricular nucleus (PVN) in conscious rats. OPC-21268 (0.45 microg/3 microl), an antagonist of the V(1) receptor, was microinjected into the lateral ventricle to examine whether the effect of ANP on baroreflex sensitivity is mediated by vasopressin (VP). ANP significantly increased the baroreflex sensitivity, and OPC-21268 attenuated the increase of baroreflex sensitivity induced by ANP. Intravenous injections of ANP (60 ng/0.04 ml) did not affect baroreflex sensitivity. These results suggest that ANP in the PVN may produce a facilitative effect on baroreflex, and the effect may be via, at least in part, the central vasopressin.

[Effects of purinergic analogues on spontaneous contraction and electrical activities of gastric antral circular muscle in guinea-pig].

In order to investigate the role of non-adrenergic non-cholinergic nerves in regulating mechanical and electrical activity of gastric circular smooth muscle, the effects of ATP and its analogues on gastric motility and electrical activities were observed in guinea-pig. In organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured. Electrical activity of the muscle was recorded by using intracellular microelectrode. Electrical and mechanical activities were recorded by chart recorder. ATP and 2-MeSATP potentiated the mechanical activity but did not affect electrical activity in gastric circular smooth muscle. ATP and 2-MeSATP-induced contraction was effectively blocked by nonselective P2y-purinoceptor antagonist, reactive-blue-2 and suramin, but ATP-induced contraction was not blocked by alpha,beta-MeATP-induced desensitization of P2x-purinoceptors. However, alpha,beta-MeATP, P2x-purinoceptor agonist, attenuated slow waves with membrane hyperpolarization and inhibited contraction. The relaxation by beta,gamma-MeATP was blocked by alpha,beta-MeATP-induced desensitization of P2x-purinoceptors. ATP-induced contraction was blocked by external calcium-free, but not by nicardipine, a L-type calcium channel blocker. Indomethacin, a nonselective cyclooxygenase inhibitor, did not block ATP-induced contraction. The results suggest that: (1) ATP- and analogues-induced contraction is mediated by P2y-purinoceptor, whereas alpha,beta-MeATP-induced relaxation by P2x-purinoceptor in guinea-pig gastric antral circular smooth muscle. (2) ATP-induced contraction is dependent on extracellular calcium, but Ca2+ entry is not mediated by L-type calcium channel. (3) Prostaglandins are not involved in ATP- and analogue-induced contraction of gastric circular smooth muscle in guinea-pigs, and alpha,beta-MeATP-induced relaxation is related to membrane hyperpolarization.

Pro-oncogenic potential of NM23-H2 in hepatocellular carcinoma.

NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). There is abundant mRNA expression of NM23-H1, NM23-H2, or a read through transcript (NM23-LV) in the primary sites of hepatocellular carcinoma (HCC). Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study was to examine whether NM23-H2 is associated with hepatocarcinogenesis. The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Its subcellular localization was confined to mainly the cytoplasm and to a lesser extent in the nucleus. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes showed a transformed morphology, enhanced focus formation, and allowed anchorage-independent growth. Finally, NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice and showed c-Myc over-expression. In addition, NF-κB and cyclin D1 expression were also increased by NM23-H2. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Collectively, these results indicate that NM23-H2 may be pro-oncogenic in hepatocarcinogenesis.

C-type natriuretic-peptide-potentiated relaxation response of gastric smooth muscle in streptozotocin-induced diabetic rats.

AIM: To study the sensitivity of gastric smooth muscle to C-type natriuretic peptide (CNP) in streptozotocin (STZ)-induced diabetic rats. METHODS: The spontaneous contraction of a gastric smooth muscle strip was recorded by using physiological methods in rats. The expressions of CNP and natriuretic peptide receptor-B (NPR-B) in gastric tissue were examined by using immunohistochemistry techniques in the diabetic rat. RESULTS: At 4 wk after injection of STZ and vehicle, the frequency of spontaneous contraction of gastric smooth muscle was significantly reduced in diabetic rats, and the frequency was decreased from 3.10 +/- 0.14 cycle/min in controls to 2.23 +/- 0.13 cycle/min (n = 8, P < 0.01). However, the amplitude of spontaneous contraction was not significant different from the normal rat. CNP significantly inhibited spontaneous contraction of gastric smooth muscle in normal and diabetic rats, but the inhibitory effect was significantly potentiated in the diabetic rats. The amplitudes of spontaneous contraction were suppressed by 75.15% +/- 0.71% and 58.92% +/- 1.32% while the frequencies were decreased by 53.33% +/- 2.03% and 26.95% +/- 2.82% in diabetic and normal rats, respectively (n = 8, P < 0.01). The expression of CNP in gastric tissue was not changed in diabetic rats, however the expression of NPR-B was significantly increased in diabetic rats, and the staining indexes of NPR-B were 30.67 +/- 1.59 and 17.63 +/- 1.49 in diabetic and normal rat, respectively (n = 8, P < 0.01). CONCLUSION: The results suggest that CNP induced an inhibitory effect on spontaneous contraction of gastric smooth muscle, potentiated in diabetic rat via up-regulation of the natriuretic peptides-NPR-B-particulate guanylyl cyclase-cyclic GMP signal pathway.