The induction of apoptosis in SGC-7901 cells through the ROS-mediated mitochondrial dysfunction pathway by a Ir(III) complex.

A new ligand BTCP and its iridium(III) complex [Ir(ppy)2(BTCP)]PF6 (Ir-1) were synthesized and characterized by elemental analysis, ESI-MS, IR, 1H NMR and 13C NMR. The cytotoxic activity in vitro of the ligand and its complex against SGC-7901, HeLa, HOS, PC-12, BEL-7402, MG-63, SiHa, A549, HepG2 and normal cell LO2 were evaluated by MTT method [MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]. The apoptosis was assayed with AO/EB and Hoechst 33258 staining methods. The reactive oxygen species (ROS), mitochondrial membrane potential, autophagy and cell invasion were studied under fluorescent microscope. The expression of caspases and Bcl-2 family proteins were investigated by western blot. The IC50 values of complex toward SGC-7901, BEL-7402 and MG-63 cells are 3.9 ± 0.5, 5.4 ± 1.2 and 4.2 ± 0.6 µM. The complex can increase the levels of ROS, and induce a decrease in the mitochondrial membrane potential. Ir-1 inhibits the cell growth at G0/G1 phase in SGC-7901 cells, and the complex can induce both autophagy and apoptosis and inhibit the cell invasion. And the complex induces apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

Muscarinic activity modulated by C-type natriuretic peptide in gastric smooth muscles of guinea-pig stomach.

Natriuretic peptides (NPs) are a cyclic guanosine monophosphate (cGMP) generation system like nitric oxide (NO) and play an inhibitory regulation in gastrointestinal motility but the effect of NPs on muscarinic activity is still unclear. This study was designed to investigate effect of C-type natriuretic peptide (CNP) on muscarinic control of gastric motility and its ion channel mechanism. The spontaneous contraction of gastric smooth muscle strip was recorded by using physiograph in guinea-pig. Membrane currents and potential were recorded by using whole-cell patch-clamp technique. CNP significantly inhibited muscarinic M receptor agonist carbachol (Cch)-induced contractions of gastric smooth muscle strips and dramatically hyperpolarized Cch-induced depolarization of membrane potential in gastric single smooth muscle cell. Muscarinic currents induced by both Cch and GTPgammaS, a G-protein agonist were significantly suppressed by CNP. 8-Br-cGMP mimicked the effect of CNP on Cch-induced muscarinic currents, and the peak holding current was decreased from -200.66+/-54.35 pA of control to -67.35+/-24.82 pA. LY83583, a guanylate cyclase nonspecific inhibitor, significantly weakened the inhibitory effect of CNP on muscarinic current while zaprinast, a cGMP sensitive phosphoesterase inhibitor, potentiated the inhibitory effect of CNP on muscarinic current. cGMP production was dramatically enhanced by CNP and this effect was suppressed by LY83583 in gastric smooth muscle. These results suggest that CNP modulates muscarinic activity via CNP-NPR-particulate guanylate cyclase (pGC)-cGMP pathway in guinea-pig.

Effects of polyamines on contractility of guinea-pig gastric smooth muscle.

This study was designed to investigate the effects of polyamines on mechanical contraction and voltage-dependent calcium current (VDCC) of guinea-pig gastric smooth muscle. Mechanical contraction and calcium channel current I(Ba) were recorded by isometric tension recording and whole-cell patch clamp technique. Spermine, spermidine and putrescine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner. Spermine (2 mM) reduced high K+ (50 mM)-induced contraction to 16+/-6.4% of the control (n=9), and significantly inhibited I(Ba) in a reversible manner (p<0.05; IC50=0.8 mM). Pre- and post-treatment of tissue with spermine (2-5 mM, n=10) also inhibited acetylcholine (10 microM)-induced phasic contraction to 5+/-6.4% of the control. Inhibitory effect of spermine on I(Ba) was observed at a wide range of test potentials of current/voltage (I/V) relationship (p<0.05), and steady-state activation of I(Ba) was shifted to the right by spermine (p<0.05). Spermidine and putrescine (1 mM each) also inhibited I(Ba) to 51+/-5.7% and 81+/-5.3% of the control, respectively. And putrescine (1 mM) inhibited I(Ba) at whole tested potentials (p<0.05) without significant change of kinetics (p<0.05). Finally, 5 mM putrescine also inhibited high K+-induced contraction to 53+/-7.1% of the control (n=4). These findings suggest that polyamines inhibit contractions of guinea-pig gastric smooth muscle via inhibition of VDCC.

Relationship between atrial natriuretic peptide-immunoreactive cells and microvessels in rat gastric mucosa.

AIM: To investigate the ultrastructural localization of atrial natriuretic peptide (ANP)-synthesizing cells and the relationship between ANP-synthesizing cells and microvessels in rat gastric mucosa. METHODS: Immunohistochemistry techniques and postembedding immunoelectron microscopy techniques were used to validate the findings regarding the expression of ANP-synthesizing cells and the ultrastructural localization of ANP-synthesizing cells in the gastric mucosa. Histochemistry techniques and the tannic acid-ferric chloride method (TA-Fe staining method) were used to reveal microvessel density and the distribution of ANP-synthesizing cells in different regions of the stomach. RESULTS: Cells expressing ANP were localized and ANP-synthesizing cells were identified as enterochromaffin (EC) cells in the gastric mucosa. ANP-synthesizing cells existed in different regions of the stomach. The percentage ANP-synthesizing cells in the mucosa was greatest in the fundus (46.7%+/-5.3%), intermediate in the antrum (40.1%+/-4.5%), and least in the body (21.6%+/-3.6%). There was a positive relationship between the percentage of ANP-synthesizing cells and the density of microvessels in the antral mucosa, but not in the fundus or body mucosa. CONCLUSION: ANP is synthesized by EC cells in rat gastric mucosa, and ANP-synthesizing cells are most dense in the gastric fundus. ANP may act not only as a regional autocrine and/or paracrine regulator, but also as an endocrine regulatory peptide in the gastrointestinal tract.

Role of arachidonic acid in hyposmotic membrane stretch-induced increase in calcium-activated potassium currents in gastric myocytes.

AIM: To study effects of arachidonic acid (AA) and its metabolites on the hyposmotic membrane stretch-induced increase in calcium-activated potassium currents (I(KCa)) in gastric myocytes. METHODS: Membrane currents were recorded by using a conventional whole cell patch-clamp technique in gastric myocytes isolated with collagenase. RESULTS: Hyposmotic membrane stretch and AA increased both I(K(Ca))) and spontaneous transient outward currents significantly. Exogenous AA could potentiate the hyposmotic membrane stretch-induced increase in I(K(Ca)). The hyposmotic membrane stretch-induced increase in I(K(Ca)) was significantly suppressed by dimethyleicosadienoic acid (100 micromol/L in pipette solution), an inhibitor of phospholipase A2. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly suppressed AA and hyposmotic membrane stretch-induced increases in I(K(Ca)). External calcium-free or gadolinium chloride, a blocker of stretch-activated channels, blocked the AA-induced increase in I(K(Ca)) significantly, but it was not blocked by nicardipine, an L-type calcium channel blocker. Ryanodine, a calcium-induced calcium release agonist, completely blocked the AA-induced increase in I(K(Ca)); however, heparin, a potent inhibitor of inositol triphosphate receptor, did not block the AA-induced increase in I(K(Ca)). CONCLUSION: Hyposmotic membrane stretch may activate phospholipase A2, which hydrolyzes membrane phospholipids to ultimately produce AA; AA as a second messenger mediates Ca(2+) influx, which triggers Ca(2+)-induced Ca(2+) release and elicits activation of I(K(Ca)) in gastric antral circular myocytes of the guinea pig.