Effect And Mechanism Of Orexin A On High Voltage-activated Calcium Channel Currents In The Rat Hypothalamic NPY Neurons

Part IEffects of orexin A on HVAs of NPY neuronsBackground: Orexins, also called hypocretins, is a new neuropeptide that was found in lateral hypothalamus recently. Orexin system includes two separate peptides: orexin A and orexin B. The first study confirmed that intracerebroventricular injection of orexin A significantly increased food intake in experimental rats. Subsequent studies suggested that orexin A also involved in regulation of multiple physiological functions, such as sleep and wake, energy metabolism and hormone secretion. Many evidences have identified that orexin A elevated intracellular [Ca2+] in Chinese hamster ovary (CHO) cells expressed OXR. Orexin A also elevated intracellular [Ca2+] in neurons and regulated many different aspects of neuronal function. However, it is unclear whether electrophysiological feature of voltage-dependent calcium channels (VDCCs) was regulated by orexin A, especially in hypothalamic NPY neurons. The aim of this experiment is to examine effects of orexin A on high voltage activation channels (HVAs) in NPY neurons.Methods: First, the immunocytochemical method was adopted to identify NPY neurons in the rat hypothalamus. Then whole-cell patch clamp technique was used to examine the effects of orexin A on HVAs of NPY neurons, which we identified by morphologic features and immunocytochemical identification at the end of recording.Results: Immunocytochemical identification showed that NPY neurons are typically small and medium neurons with triangular or spindle-shaped perikaryons. Most of them have 1-3 slenderly and poorly ramified primary dendrites. Whole-cell patch clamp records showed that orexin A (0.1~100 nM) increased the IHVA in a concentration-dependent and time-dependent manner. The EC50 of the dose-response curve was 0.75±0.09 nM. After application 1 nM orexinA, IHVA was increased during 30~60 s and the amplitude of IHVA reached to peak in 2~3 minutes. We found that 1 nM orexin A shifted I-V curve of IHVA to the downward, but had no effect on the activation threshold potential of IHVA. We also found 1nM orexin A shifted the steady-state activation curve of IHVA to more negative potentials, while the steady-state inactivation curve was not altered.Conclusion: Orexin A increased the IHVA in a concentration-dependent and time-dependent manner. 1 nM orexin A shifted the steady-state activation curve of IHVA to more negative potentials, while the steady-state inactivation curve was not altered.Part IIMechanism of orexin A regulating IHVA of NPY neuronsBackground: The actions of orexins are mainly mediated by orexin-1 and orexin-2 receptors (OX1R and OX2 R) belonging to G-protein-coupled receptors superfamily. The aim of this experiment is to study the mechanism that orexin A regulated IHVA of NPY neurons.Methods: We used whole-cell patch clamp technique to examine the effects of SB334867 (antagonist of OX1R), U73122 (inhibitor of PLC), GF109203X (inhibitor of PKC), verapamil on IHVA induced by orexin A.Results: 10μM SB334867 significantly inhibited the effects of 1nM orexin A on IHVA. Simultaneously, 10μM U73122 and 10μM GF109203X also significantly inhibited the effects of 1 nM orexinA on IHVA. We also found that 1 nM orexin A slightly increased IHVA after neurons were pretreated with 10μM verapamil.Conclusion: Orexin A increased IHVA of NPY neurons by PLC/PKC-L-type calcium channel pathways.