| Neuropeptides exist widely and could regulate physiological function of many organs. In recent years, the regulation of neuropeptides on cardiovascular system has becoming a hot spot of research, one of which is the regulation of OFQ (nociceptin, Orphanin FQ). OFQ is a heptadecapeptide whose primary structure is indeed similar to that of dynorphin A, but binds to classical opioid receptors with low affinity. The OFQ receptor is referred as opioid receptor-like 1(ORL1) receptor, which shares high structural homogeneity with the opioid receptors and also belong to G protein-coupled membrane receptors family. Both Intracerebroventricular and and intravenous administration of OFQ decreased the blood pressure and heart rate obviously. It is showed OFQ not only played a role in cardiovascular regulation via central or peripheral neverous mechanism, but also possibly had direct inhibition of cardiovascular system. The recent studies have showed OFQ increased markedly in myocardial tissue and blood plasma during acute myocardial ischemia and heart failure, which suggest OFQ may regulate the heart function directly and the mechanism need to be explored. Therefore we established the current study to investigate the effects of OFQ on heart function in vivo and explore the possible mechanism.Objective: The purpose of this study was to investigate the effects of OFQ by intravenous administration on heart function in rats and explore the possible mechanism from cardiac ion channels.Methods: The study was carried out in a series of experiments:1. In vivo research: Healthy adult male SD (weighing 250-280g) rats were used for this experiment. The rats were randomly divided into four groups: the control group (normal saline i.v.), 1nM group (1nmol/kg, i.v.), 10 nM group(10 nmol/kg, i.v.) and 50 nM group(50 nmol/kg, i.v.). For the measurement of left ventricular pressures, a polyethylene catheter (Epidural catheter) was inserted into the left carotid artery and gently pushed into the left ventricle of rat anesthetized with urethane. After Hemodynamics indexes came to a steady state, the LVSP, LVDP, +dP/dtmax, -dP/dtmax and HR were recorded.2. Immunofluorescence assay: OFQ and ORL1 receptor in the myocardial tissue were observed through immunofluorescence histochemistry, and ORL1 receptor was also located in the isolated ventricular myocytes from SD rats with confolcal laser scanning microscopy.3. Patch clamp experiment: Ventricular myocytes were isolated from SD rats by Langendorff perfusion of the heart with an enzyme-containing solution as previous described. L-Ca current (ICa-L), inward rectifyimg potassium current (Ik1) and transient outward potassium current (Ito) of ventricular myocytes were induced through the particular procedure and recorded with the whole-cell voltage-clamp technique, respectively. The effects of OFQ on ICa-L, Ik1 and Ito were investigated respectively. All experiments were performed at room temperature and solutions were gased with 100% oxygen.4. Effect of OFQ on PKA activity assay: Peptides were extracted from the isolated ventricular myocytes treated with and without OFQ, and using the Assay kit for Non-Radioactive Detection of cAMP Dependent Protein Kinase, PKA activity was measured via gel electrophoresis.Results:1. LVSP, LVDP, +dP/dtmax, -dP/dtmax and HR were significantly decreased in a dose-dependent manner after intravenous administration with OFQ. There was significant difference among groups (P<0.05). The OFQ induced inhibitions of LVSP, LVDP, +dP/dtmax, -dP/dtmax and HR were completely blocked by pre treatment with UFP-101, an potent, specific antagonist.2. Immunofluorescence Histochemistry showed OFQ and ORL1 receptor existed in the myocardial tissue, furthermore the ORL1 receptor was confirmed in isolated myocytes with confolcal laser scanning microscopy.3. OFQ obviously inhibited ICa-L in rat ventricular myocytes but not in a dose-depended manner, and the inhibition elicited by OFQ can be reversed by pre treatment with UFP-101. There were no significant effect on Ik1 and Ito in rat ventricular myocytes treated with OFQ.4. There was not obvious variation on PKA activity in rat ventricular myocytes treated with OFQ. The increase of PKA activity induced by Forskolin, a PKA activator, was not reversed by treatment with OFQ.Conclusions: OFQ inhibited the Left ventricular systolic and diastolic function, which may result from inhibition of ICa-L induced by OFQ in rat ventricular myocytes. Furthermore research showed OFQ had no significant effect on PKA activity of ventricular myocytes, which suggest the effect of OFQ on the heart function may not be mediated by cAMP-PKA passway. There was no significant effect on Ik1 and Ito in rat ventricular myocytes by treatment with OFQ.
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