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There is increasing evidence that free radicals play an important role in neuronal damages induced by diabetes mellitus or cerebral ischemia insults. Antioxidants with free radical scavenging activities have been shown to be beneficial and neuroprotective for these pathological conditions. Here, we report free radical scavenging activity and neuroprotective potential of D138, one copper(II)/zinc(II) Schiff-base complex derived from N,N'-2(2-hydroxynaphthylmethylidene)-1,3-propanediamine. The data from three in vitro assays, 2,2-diphenyl-1-picrylhydrazyl assay, nitro blue tetrazolium assay and hydroxyl radical scavenging assay, indicated that D138 presented a potent free radical scavenging activity. The neuroprotective and antioxidative effects of D138 were further evaluated in vivo using bilateral common carotid artery occlusion (BCCAO) mouse model and streptozotocin (STZ) diabetic mouse model. Our results indicated that treatment of D138 significantly ameliorated the hippocampal neuronal damage and the oxidative stress levels in these animal models. Moreover, D138 also reversed the behavioral deficiencies induced by BCCAO or STZ, as assessed by Y-maze test and fear conditioning test. In conclusion, all these findings support that D138 exerts free radical scavenging and neuroprotective activities and has the potentials to be a potent therapeutic candidate for brain oxidative damage induced by cerebral ischemia or diabetes mellitus.

As a natural compound, Ornithogalum caudatum Ait is primarily used as an anti-inflammatory and antitumor agent in Chinese folk medicine. In 1992, OSW-1 was isolated from this compound, which is a new member of cholestane saponin family. In numerous recent studies, OSW-1 has been shown to have powerful cytotoxic anticancer effects against various malignant cells. However, the therapeutic efficacy of OSW-1 on colon cancer and the underlying mechanism are not understood. To explore the mechanism underlying OSW-1 in antitumor therapy, a therapeutic function analysis of OSW-1 on colon cancer was performed in vitro and in vivo. It was shown that with low toxicity on normal colonic cells, OSW-1 suppresses colon cancer cells in vitro and this inhibition was via the intrinsic apoptotic pathway, which increased cellular calcium, changed mitochondrial membrane potential, disrupted mitochondrial morphology, and led to the release of cytochrome c and the activation of caspase-3. Furthermore, in a nude mouse model, OSW-1 had a powerful effect on suppressing colon tumor proliferation without significant side effects through the apoptosis pathway. Taken together, these results demonstrate that OSW-1 is a potential drug for colon cancer treatment.

Background Selenoprotein S (SelS) is a transmembrane protein that is expressed in the liver, skeletal muscle, adipose tissue, pancreatic islets, kidney, and blood vessels. In addition to its transmembrane localization, SelS is also secreted from hepatoma HepG2 cells (but not L6 skeletal muscle cells, 3T3-L1 adipocytes, Min6 pancreatic β cells and human embryonic kidney 293 cells) and has been detected in the serum of some human subjects, with a detection rate of 31.1 %. These findings prove that serum SelS is secreted by hepatocytes. However, whether vascularly expressed SelS can be secreted has not been reported. Transmembrane SelS has been suggested to play different roles in the pathogenesis and progression of diabetes mellitus (DM) and atherosclerosis (AS), but the association of secreted SelS with DM and macroangiopathy remains unclear. Research design and methods Supernatants were collected from human umbilical vein endothelial cells (HUVECs), human aortic vascular smooth muscle cells (HA/VSMCs) and human hepatoma HepG2 cells that were untransfected or transfected with the indicated plasmid and concentrated for western blotting. Serum samples were collected from 158 human subjects with or without type 2 DM (T2DM) and/or AS. Serum SelS levels were measured using an enzyme-linked immunosorbent assay. Results Secreted SelS was only detected in the supernatants of hepatoma HepG2 cells. The SelS detection rate among the 158 human serum samples was 100 %, and the average SelS level was 64.81 ng/dl. The serum SelS level in the isolated DM subjects was lower than the level in the healthy control subjects (52.66 ± 20.53 vs 70.40 ± 21.38 ng/dl). The serum SelS levels in the DM complicated with SAS subjects (67.73 ± 21.41 ng/dl) and AS subjects (71.69 ± 27.00 ng/dl) were significantly increased compared with the serum SelS level in the isolated DM subjects. There was a positive interaction effect between T2DM and AS on the serum SelS level (P = 0.002). Spearman correlation analysis showed that the serum SelS level was negatively correlated with fasting plasma glucose. Conclusions Vascular endothelial and vascular smooth muscle cells could not secrete SelS. Serum SelS was primarily secreted by hepatocytes. SelS was universally detected in human serum samples, and the serum SelS level was associated with T2DM and its macrovascular complications. Thus, regulating liver and serum SelS levels might become a new strategy for the prevention and treatment of DM and its macrovascular complications. Electronic supplementary material The online version of this article (doi:10.1186/s12933-016-0388-3) contains supplementary material, which is available to authorized users.

The present study demonstrated the anti-tumor effects of the quinoline derivative [5-(3-chloro-oxo-4-phenyl-cyclobutyl)-quinoli-8-yl-oxy] acetic acid hydrazide (CQAH) against colorectal carcinoma. Substantial apoptotic effects of CQAH on HCT116 and LoVo human colon cancer cell lines were observed. Apoptosis was identified based on cell morphological characteristics, including cell shrinkage and chromatin condensation as well as Annexin V/propidium iodide double staining followed by flow cytometric analysis and detection of apoptosis-associated proteins by western blot analysis. CQAH induced caspase-3 and PARP cleavage, reduced the expression of the anti-apoptotic proteins myeloid cell leukemia-1 and B-cell lymphoma (Bcl) extra large protein and elevated the expression of the pro-apoptotic protein Bcl-2 homologous antagonist killer. In addition, pharmacological inhibition of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, significantly reduced CQAH-mediated cell death as well as cleavage of caspase-3 and PARP. Co-treatment of CQAH with the commercial chemotherapeutics 5-fluorouracil and camptothecin-11 significantly improved their efficacies. Comparison of the apoptotic effects of CQAH with those of two illustrated structure-activity associations for this compound type, indicating that substitution at position-4 of the azetidine phenyl ring is pivotal for inducing apoptosis. In conclusion, the results of the present study indicated CQAH and its analogues are potent candidate drugs for the treatment of colon carcinoma.

To observe the effects of Dachengqi Decoction (大承气汤, DCQD) on morphological changes in the network of enteric nerve-interstitial cells of Cajal (ICCs)-smooth muscle cells (SMC) of enteric deep muscular plexuses (DMP) in the rats with multiple organ dysfunction syndrome (MODS).One hundred Wistar rats of both sexes weighing 200 to 250 g were randomly divided into the control group, MODS group, and DCQD group. The morphologic changes of enteric nerve-ICC-SMC network in the DMP of intestine was observed using c-Kit and vesicular acetylcholine transporter/neuronal nitric oxide synthase immunohistochemical double-staining with whole-mount preparation technique, confocal laser scanning microscopy, and electron microscopy.Compared with the control group, the distribution and densities of cholinergic/nitrergic nerves and ICC in the DMP (ICC-DMP) of intestine in the MODS group were significantly decreased (P0.01), and the network of cholinergic nerve-ICC-SMC was disrupted; and the ultrastructural features of ICC-DMP, enteric nerve, and SMC were severely damaged. After treatment with DCQD, the damage in the network of enteric nerve-ICC-SMC was significantly recovered. Compared with the MODS group, the distribution and densities of cholinergic/nitrergic nerves and ICC-DMP in the DCQD group were significantly increased (P0.01); and the ultrastructural features of ICC-DMP, enteric nerve, smooth muscle cells were significantly recovered.DCQD can improve the gastrointestinal motility in MODS. The mechanism may be related to the effect of repairing the damages in the network of enteric nerve-ICC-SMC.

Dendroaspis natriuretic peptide (DNP), a newly-described natriuretic peptide, relaxes gastrointestinal smooth muscle. L-type calcium channel currents play an important role in regulating smooth muscle contraction. The effect of DNP on L-type calcium channel currents in gastrointestinal tract is still unclear. This study was designed to investigate the effect of DNP on barium current (I Ba ) through the L-type calcium channel in gastric antral myocytes of guinea pigs and cGMP-pathway mechanism. The whole-cell patch-clamp technique was used to record L-type calcium channel currents. The content of cGMP in guinea pig gastric antral smooth muscle and perfusion solution was measured using radioimmunoassay. DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in perfusion medium. DNP concentration-dependently inhibited I Ba in freshly isolated guinea pig gastric antral circular smooth muscle cells (SMCs) of guinea pigs. DNP-induced inhibition of I Ba was partially blocked by LY83583, an inhibitor of guanylate cyclase. KT5823, a cGMP-dependent protein kinase (PKG) inhibitor, almost completely blocked DNP-induced inhibition of I Ba . However, DNP-induced inhibition of I Ba was potentiated by zaprinast, an inhibitor of cGMP-sensitive phosphodiesterase. Taken together, DNP inhibits L-type calcium channel currents via pGC–cGMP–PKG-dependent signal pathway in gastric antral myocytes of guinea pigs.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid-β (Aβ) in the brain. Recent findings have revealed that nitric oxide (NO) modulates the processing of amyloid-β precursor protein (APP) and alters Aβ production; however, the previously presented data are contradictory and the underlying molecular mechanisms are still incomplete. Here, using human SH-SY5Y neuroblastoma cells stably transfected with wild-type APPwt695, we found that NO, derived from NO donor sodium nitroprusside (SNP), bi-directionally modulates APP processing in vitro. The data from ELISA and Western blot (WB) tests indicated that SNP at lower concentrations (0.01 and 0.1 μM) inhibits BACE1 expression, thus consequently suppresses APP β-cleavage and decreases Aβ production. In contrast, SNP at higher concentrations (10 and 20 μM) biases the APP processing toward the amyloidogenic pathway as evidenced by an increased BACE1 but a decreased ADAM10 expression, together with an elevated Aβ secretion. This bi-directional modulating activity of SNP on APP processing was completely blocked by specific NO scavenger c-PTIO, indicating NO-dependent mechanisms. Moreover, the anti-amyloidogenic activity of SNP is sGC/cGMP/PKG-dependent as evidenced by its reversal by sGC/PKG inhibitions, whereas the amyloidogenic activity of SNP is peroxynitrite-related and can be reversed by peroxynitrite scavenger uric acid. In summary, these present findings predict a double-edged role of NO in APP processing in vitro. Low (physiological) levels of NO inhibit the amyloidogenic processing of APP, whereas extra-high (pathological) concentrations of NO favor the amyloidogenic pathway of APP processing. This preliminary study may provide further evidence to clarify the molecular roles of NO and NO-related signaling in AD and supply potential molecular targets for AD treatment.

This study aimed to examine the role of alveolar type II epithelial cell (AEC II) apoptosis in severe pancreatitis-induced acute lung injury (ALI) and the intervening role of Qingyi decoction (QYT). An SAP model was established in male Sprague-Dawley rats. Immunohistochemical analysis was conducted to observe the pathological changes in the pancreas and lung tissue. AEC II apoptosis was detected by flow cytometry and the free Ca2+ concentration in AECs II was determined by laser scanning confocal microscopy. A radioimmunoassay was performed to determine serum TNF-α content. Quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis were performed to detect the mRNA and protein expression levels of Bax and caspase-8 in the lung tissue. Hematoxylin and eosin staining of lung tissue sections in the severe acute pancreatitis (SAP) group showed pathological changes from control tissue, consistent with acute lung injury (ALI). Flow cytometry showed that the level of AEC II apoptosis in the SAP group was significantly increased compared with that in the control group (P

Allergic asthma is a chronic inflammatory disease regulated by coordination of T-helper2 (Th2) type cytokines and inflammatory signal molecules. Silibinin is one of the main flavonoids produced by milk thistle, which is reported to inhibit the inflammatory response by suppressing the nuclear factor-kappa B (NF-κB) pathway. Because NF-κB activation plays a pivotal role in the pathogenesis of allergic inflammation, we have investigated the effect of silibinin on a mouse ovalbumin (OVA)-induced asthma model. Airway hyperresponsiveness, cytokines levels, and eosinophilic infiltration were analyzed in bronchoalveolar lavage fluid and lung tissue. Pretreatment of silibinin significantly inhibited airway inflammatory cell recruitment and peribronchiolar inflammation and reduced the production of various cytokines in bronchoalveolar fluid. In addition, silibinin prevented the development of airway hyperresponsiveness and attenuated the OVA challenge-induced NF-κB activation. These findings indicate that silibinin protects against OVA-induced airway inflammation, at least in part via downregulation of NF-κB activity. Our data support the utility of silibinin as a potential medicine for the treatment of asthma.

Dendroaspis natriuretic peptide (DNP), a newly-described natriuretic peptide, plays an inhibitory role in smooth muscle motility of the gastrointestinal tract. However, the effect of DNP on delayed rectifier potassium currents I(K(V)) is still unclear. In this study, we sought to investigate the effect of DNP on I(K(V)) and its mechanism in gastric antral circular smooth muscle cells using the whole-cell patch-clamp technique. DNP significantly inhibited I(K(V)) in a concentration-dependent manner. LY83583 (1 micromol/l), a guanylate cyclase inhibitor, significantly impaired DNP-induced inhibition of I(K(V)). Moreover, DNP-induced inhibition in I(K(V)) was potentiated by the cyclic guanosine monophosphate (cGMP) sensitive phosphoesterase inhibitor zaparinast (0.1 micromol/l). DNP-induced inhibition of I(K(V)) was completely blocked by KT5823, an inhibitor of cGMP-dependent protein kinase G(PKG), but not affected by KT-5720, a PKA-specific inhibitor. Taken together, our results suggest that DNP inhibits I(K(V)) via the cGMP/PKG-dependent signaling axis instead of the cAMP/PKA pathway.

Aim: To determine whether the natriuretic peptide receptor (NPR) is present in the stomach of guinea pigs and to investigate the effect of dendroaspis natriuretic peptide (DNP) on the gastric motility of guinea pigs and its mechanism. Methods: The distribution of the NPR was analyzed by autoradioimmunography. The spontaneous contraction of gastric antral circular muscles of guinea pigs was recorded by a 4-channel physiograph. The whole cell patch-clamp technique was introduced to record calcium-activated potassium currents in the gastric myocytes isolated by collagenase. Results: The NPR existed in the gastric fundus, gastric body, and gastric antrum of guinea pigs, and its density was largest in the gastric antrum. DNP inhibited spontaneous contraction and exhibited a dose-dependent manner. The DNP-induced inhibition was diminished by LY83583 (a guanylate cyclase inhibitor) and was potentiated by zaprinast (a cGMP-sensitive phosphoesterase inhibitor). The inhibitory effect of DNP on spontaneous contraction was also inhibited by tetraethylammonium (a non-selective potassium channel blocker); 10 nmol/L DNP increased the calcium-activated potassium currents in the gastric circular myocytes of guinea pigs. Conclusion: The NPR is most common in the gastric antrum of guinea pigs. DNP significantly inhibits gastric motility in the gastric antrum of guinea pigs. The inhibitory effect occurs via a cGMP-dependent pathway, and a calcium-activated potassium channel may be also involved in the relaxation induced by DNP in gastric antral circular smooth muscles.

In this study, we sought to investigate the effect of dendroaspis natriuretic peptide (DNP) on calcium-activated potassium current (I K(Ca)) and its mechanism in gastric antral circular smooth muscle cells (SMCs) using the whole-cell patch-clamp technique. DNP concentration-dependently increased macroscopic I K(Ca) and spontaneous transient outward currents (STOCs) in freshly isolated guinea pig gastric antral circular SMCs. The effects of DNP on I K(Ca) and/or STOCs were not blocked by applying calcium-free bath solution or the ryanodine receptor (RyR) antagonist ryanodine (10 microM), but they were inhibited by the inositol triphosphate receptor (IP3R) inhibitor heparin or the guanylate cyclase inhibitor LY83583. Moreover, a DNP-induced increase in STOCs was potentiated by the cyclic guanosine monophosphate (cGMP)-sensitive phosphoesterase inhibitor zaprinast. In conclusion, our results suggest that DNP increases I K(Ca) in gastric antral circular SMCs by increasing cGMP production and activating IP3Rs.

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.

To investigate the effect of natriuretic peptides on gastric motility and its possible mechanism.Spontaneous contraction of gastric antral circular muscle of rats was recorded by four channel physiograph. The concentration of cyclic guanosine monophosphate (cGMP) was measured by radioimmunoassay. The distribution of natriuretic peptide receptors (NPR) was analyzed by autoradiograph.NPR existed in different regions of rat stomach and its density was the largest in gastric antrum. Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) all inhibited the spontaneous contraction of gastric antral circular smooth muscle. Among them, the inhibitory effect of CNP on the spontaneous contraction was the most potent and exhibited a dose-dependent manner. CNP-induced inhibition was diminished by LY83583 (a kind of inhibitor of guanylate cyclase) and potentiated by zaprinist (a kind of inhibitor of cGMP sensitive phosphoesterase). CNP markedly enhanced the concentration of cGMP in antral smooth muscle. The inhibitory effect of CNP on spontaneous contraction was also inhibited by tetraethylammonium (a nonselective potassium channel blocker, TEA).The distribution density of NPR is the most in gastric antrum. CNP significantly inhibits gastric motility in rat gastric antrum. The inhibitory effect occurs via a cGMP dependent pathway and potassium channel is also involved in the relaxation induced by CNP in gastric antrial circular smooth muscle of rat.

To investigate the role of calcium mobilization in the calcium-activated potassium currents [IK(Ca)] increased by sodium nitroprusside (SNP), a nitric oxide (NO) donor, in gastric antral circular myocytes of the guinea pig.A perforated patch-clamp technique was used, and the myocytes were isolated by collagenase.SNP 100 mol/L significantly increased IK(Ca), and enhanced the spontaneous transient outward currents (STOC). SNP-induced increase of IK(Ca) was not blocked by extracellular calcium-free solution (containing egtazic acid 10 micromol/L and nicardipine 5 micromol/L, an L-type calcium channel blocker. And SNP 100 micromol/L suppressed the L-type calcium currents (ICa). SNP-induced increase of STOC was inhibited by heparin 3 g/L, a potent inhibitor of inositol triphosphate receptor (InsP3R). However, ryanodine 10 micromol/L, an inhibitor of calcium-induced calcium release (CICR), did not inhibit the effect of SNP-induced increase of STOC. Methylene blue (1 micromol/L), an inhibitor of soluble guanylate cyclase, also inhibited such an effect.The increase of IK(Ca) caused by SNP may be mediated by cGMP via IP3-sensitive calcium pools, however, extracellular Ca2+ may not be involved in the process.

AIM: To investigate the effect of natriuretic peptides on gastric motility in various animals, and the effect of C-type natriuretic peptide (CNP) on spontaneous contraction of gastric smooth muscle in rat, guinea-pig and human in vitro was compared. METHODS: Spontaneous contraction of gastric smooth muscle was recorded by four channel physiograph. RESULTS: In the guinea-pig and rat gastric antral circular smooth muscle, CNP markedly decreased the amplitude of spontaneous contraction but it didn’t affect the frequency, however, the contractile activity was completely inhibited by CNP in gastric antral longitudinal smooth muscle. In the human gastric antral circular and longitudinal smooth musle, CNP completely inhibited spontaneous contraction. In the circular smooth muscle of guinea-pig and rat gastric fundus, CNP obviously decreased the amplitude of spontaneous contraction but it didn’t affect the frequency, however, the contractile activity was completely inhibited by CNP in smooth muscle of fundus longitudinal. In the circular and longitudinal smooth muscle of guinea-pig gastric body, CNP at first induced a relaxation and then an increase in amplitude of spontaneous contraction (rebound contraction), but the frequency was not changed. After the circular smooth muscle of gastric body was pretreated with atropine, an M receptor blocker, the rebound contraction was abolished; In circular and longitudinal smooth muscle of rat gastric body, CNP induced a transient and slight relaxation and successively followed by the recovery in amplitude of spontaneous contraction but it also didn’t affect the frequency. After the smooth muscle was pretreated with atropine, the transient and slight relaxation was replaced by long term and complete inhibition; The percentage of CNP-induced inhibition was 76.77% ± 6.21% (fundus), 67.21% ± 5.32% (body) and 58.23% ± 6.21% (antral) in the gastric circular muscle, however, the inhibitory percentage was 100% ± 0.00% (fundus), 68.66% ± 3.55% (body) and 100% ± 0.00% (antrum) in the gastric longitudinal smooth muscle of guinea-pigs; In the rat, the percentage of CNP-induced inhibition was 95.87% ± 4.12% (fundus), 94.91% ± 5.08% (body) and 66.32% ± 7.32% (antrum) in the gastric circular smooth muscle, but in the longitudinal smooth muscle, CNP completely inhibited the spontaneous contraction. Using LY83583, a guanylate cyclase inhibitor, and zaparinast as a phosphoesterase inhibitor to inhibit the generation of cGMP, the effect of CNP on the spontaneous contraction was markedly weakened by LY83583, however, the inhibitory effect was enhanced by zaparinast. CONCLUSION: (1) CNP can obviously inhibit the spontaneous contraction of gastric antral circular and longitudinal smooth muscle in the rat, guinea-pig and human. The order of inhibitory potency is human > rat > guinea-pig. (2) In the same animals, the inhibitory effect of CNP on spontaneous contraction is the most powerful in fundus and the weakest in antrum, in the same position, the inhibitory effect on the circular smooth muscle is more powerful than that on longitudinal smooth muscle. (3) The inhibitory effect of CNP on spontaneous contraction in the gastric smooth muscle is mediated by a cGMP dependent pathway.

To investigate the effect of ectogenesis unsaturated fatty acid on carbachol-induced muscarinic current (ICCh) and its mechanism.Using the whole-cell patch-clamp technique, ICCh was recorded in single smooth muscle cell isolated from the antral circular smooth muscles of guinea-pig stomach.Arachidonic acid (AA) was added in external perfusing solution and AA inhibited ICCh to 46 %+/-8 %, 23 %+/-5 %, and 3.8 %+/-0.9 % at 1, 3, and 5 micromol/L. Another unsaturated fatty acid, linoleic acid (LA) also inhibited ICCh in a dose-dependant manner. LA inhibited ICCh to 69 %+/-10 %, 35 %+/-5 %, and 7.4 %+/-1.2 % at 1, 5, and 10 micromol/L, respectively. The same concentration (5 micromol/L) of AA, LA, and oleic acid (OA) suppressed ICCh to 3.8 %+/-0.9 %, 35 %+/-5 %, and 67 %+/-9 %, respectively. The inhibitory potency sequence of these unsaturated fatty acids was AALAOA. After 10-15 min of pretreatment with H-7 (a protein phosphorylation C inhibitor) 100 micromol/L or indomethacin (a cyclooxygenase inhibitor) 10 micromol/L, ICCh was inhibited by 5 micromol/L of AA to 5.5 %+/-0.7 % and 3.0 %+/-1.0 %, respectively.The unsaturated fatty acids directly inhibited ICCh, and the inhibitory potency was related to the number of double bonds in fatty acid chain.

To investigate the role of intracellular calcium in muscarinic currents increased by hypoosmotic membrane stretch in gastric antral circular myocytes of the guinea pig.The whole cell patch-clamp technique was used, and the myocytes were isolated by collagenase. Cells were swelled by hypoosmotic solution (200 Osmmol/kg).The hypoosmotic membrane stretch markedly increased carbachol-induced muscarinic currents (ICCh). The ICCh and the increase of ICCh were completely blocked by quinidine 3 micromol/L, a specific muscarinic current blocker. In external calcium-free solution hypoosmotic membrane stretch could not increase ICCh, but in the presence of nicadipine 5 micromol/L, a L-type calcium channel blocker or gadolinium chloride 100 nmol/L, a stretch-activated cation channel blocker, the ICCh was still increased by hypoosmotic membrane stretch. When both nicardipine and gadolinium chloride were added into external solution, ICCh were not increased by hypoosmotic membrane stretch any more. Ryanodine, a calcium-induced calcium release (CICR) agonist completely blocked hypoosmotic membrane stretch-induced increase of ICCh.Hypoosmotic membrane stretch increased ICCh and the increment was related to influx of external calcium and CICR.

To systematically investigate if cGMP/cGMP-dependent protein kinase G (PKG) signaling pathway may participate in dendroaspis natriuretic peptide (DNP)-induced relaxation of gastric circular smooth muscle.The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay; spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca(2+)-activated K(+) currents (I(K(Ca))) and spontaneous transient outward currents (STOCs) in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique.DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium. DNP induced relaxation in gastric antral circular smooth muscle, which was inhibited by KT5823, a cGMP-dependent PKG inhibitor. DNP increased I(K(Ca)). This effect was almost completely blocked by KT5823, and partially blocked by LY83583, an inhibitor of guanylate cyclase to change the production of cGMP. DNP also increased STOCs. The effect of DNP on STOCs was abolished in the presence of KT5823, but not affected by KT-5720, a PKA-specific inhibitor.DNP activates I(K(Ca)) and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/PKA pathway.

To investigate ion channel mechanism in CNP-induced relaxation of gastric circular smooth muscle in guinea pigs.Spontaneous contraction of gastric smooth muscle was recorded by a four -channel physiograph. The whole cell patch-clamp technique was used to record calcium-activated potassium currents and membrane potential in the gastric myocytes isolated by collagenase.C-type natriuretic peptide (CNP) markedly inhibited the spontaneous contraction in a dose-dependent manner in gastric circular smooth muscle in guinea pigs. Ly83583, an inhibitor of guanylate cyclase, weakened CNP-induced inhibition on spontaneous contraction but Zaparinast, an inhibitor of cGMP sensitive phosphoesterase, potentiated CNP-induced inhibition in gastric circular smooth muscles. The inhibitory effects of CNP on spontaneous contraction were blocked by tetrathylammonium (TEA), a nonselective potassium channel blocker. CNP hyperpolarized membrane potential from -60.0 mV+/-2.0 mV to -68.3 mV+/-3.0 mV in a single gastric myocyte. CNP increased calcium-activated potassium currents (I(K(ca))) in a dose-dependent manner in gastric circular myocytes. CNP also increased the spontaneously transient outward currents (STOCs). Ly83583 partly blocked CNP-induced increase of calcium-activated potassium currents, but Zaparinast potented the effect.CNP inhibits spontaneous contraction, and potassium channel may be involved in the process in gastric circular smooth muscle of guinea pigs. CNP-induced increase of I(K(ca)) is mediated by a cGMP dependent pathway.