Neurophysiological mechanisms of bradykinin-evoked mucosal chloride secretion in guinea pig small intestine.

AIM: To investigate the mechanism for bradykinin (BK) to stimulate intestinal secretomotor neurons and intestinal chloride secretion. METHODS: Muscle-stripped guinea pig ileal preparations were mounted in Ussing flux chambers for the recording of short-circuit current (Isc). Basal Isc and Isc stimulated by BK when preincubated with the BK receptors antagonist and other chemicals were recorded using the Ussing chamber system. Prostaglandin E2 (PGE2) production in the intestine was determined by enzyme immunologic assay (EIA). RESULTS: Application of BK or B2 receptor (B2R) agonist significantly increased the baseline Isc compared to the control. B2R antagonist, tetrodotoxin and scopolamine (blockade of muscarinic receptors) significantly suppressed the increase in Isc evoked by BK. The BK-evoked Isc was suppressed by cyclooxygenase (COX)-1 or COX-2 specific inhibitor as well as nonselective COX inhibitors. Preincubation of submucosa/mucosa preparations with BK for 10 min significantly increased PGE2 production and this was abolished by the COX-1 and COX-2 inhibitors. The BK-evoked Isc was suppressed by nonselective EP receptors and EP4 receptor antagonists, but selective EP1 receptor antagonist did not have a significant effect on the BK-evoked Isc. Inhibitors of PLC, PKC, calmodulin or CaMKII failed to suppress BK-induced PGE2 production. CONCLUSION: The results suggest that BK stimulates neurogenic chloride secretion in the guinea pig ileum by activating B2R, through COX increasing PGE2 production. The post-receptor transduction cascade includes activation of PLC, PKC, CaMK, IP3 and MAPK.

Catalpol regulates cholinergic nerve system function through effect on choline acetyl-transferase not M receptor affinity.

OBJECTIVE: To explore the effect of catalpol on choline acetyl-transferase and M receptor affinity in a PC12 cell model and a rat model induced by beta-amyloid 25-35 (Aß25-35). METHODS: In PC12 cells, catalpol (10µmol/l, 100µmol/) or saline was retained in the medium and Aß25-35 (final concentration 20µmol/l) was added. Choline acetyl-transferase (ChAT) expression was determined by immunocytochemistry, ChAT activity measured by radioenzymatic assay, and M receptor (muscarinic receptor) affinity determined by (3)H-QNB binding test. In Wistar rats, Aß25-35 was injected intracerebroventricularly to establish AD model. After injection of Aß25-35, the rats were injected catalpol at 5 and 10mg/kgd(-1) intraperitoneally for the next 7 days, and saline for the control rats. ChAT expression, ChAT activity and M receptor affinity were tested. Cells and rats all were divided into four groups: Group A (control), Group B (model), Group C (catalpol low dose), and Group D (catalpol high dose). RESULTS: Compared with control, both PC12 cell and rat AD models showed decreased expression and activity of ChAT (p<0.01), but M receptor affinity remained the same (p>0.05). Compared with model group, treatment of catalpol increased expression and activity of ChAT of PC12 cell and rat AD model induced by Aß25-35, p<0.05 or p<0.01 respectively. But there was no difference of M receptor affinity among the four groups (p>0.05). M receptor affinity remained the same as concentration of catalpol increased gradually in atropine competition experiments (p>0.05). CONCLUSIONS: Catalpol could regulate the cholinergic nerve system function from its effect on ChAT and may have beneficial effect for treatment of AD, but had no effect on M receptor affinity.

Preliminary detection of the anti­tumour activity of indoline­2,3­dione derivative DH­12a targeting aminopeptidase N.

Aminopeptidase N (APN) is important in tumour processes. The present study detected the anti­tumour activity of the novel APN inhibitor DH­12a, which is an indoline­2,3­dione derivative. In the present study, Bestatin, a clinical APN inhibitor was used as a positive control. The expression of APN in the ES-2 and 3AO cell lines were assessed using flow cytometry and the drug inhibition constants of DH­12a (Ki=13.15 µM) and Bestatin (Ki=16.57 µM) were assessed using a double reciprocal method of competitive inhibition. The in vitro effects of DH­12a on cell proliferation were assessed using a 3­(4,5­dimethyl­thiazol­2­yl)­2,5­diphenyl tetrazolium bromide assay on human cell lines of ES­2 (IC50=43.8 µM), A549 (inhibition rate=41.5% at 160 µM DH­12a), HL60 (inhibition rate=47.83% at 160 µM DH­12a) and 3AO (IC50=70.2 µM). The inhibition rates were consistently higher than those of Bestatin. The effects of DH­12a on cell migration (inhibition rates in ES­2 cells and 3AO cells were 56.4 and 76.5%, respectively at 15 µM) and invasion (inhibition rates in ES­2 cells and 3AO cells were 75.6 and 66.5%, respectively at 15 µM) were assessed using transwell plates. The in vivo effects of DH­12a on tumour proliferation and lung tumour metastasis were determined using an H22 xenograft mice model, where DH­12a was administered in combination with genotoxic 5­fluorouracil. The anti­tumour activities of DH­12a in vivo were also greater than those of Bestatin. In conclusion, the in vitro effects of DH­12a on tumour proliferation, migration and invasion were consistent with the in vivo effects. In addition, DH­12a exhibited greater anti­tumour properties compared with Bestatin.