| It is well established that the brain angiotensin system exerts regulatory influences in the control of blood pressure (BP) and thus plays a key role in the development and establishment of hypertension. The cardiovascular actions of angiotensin II (Ang II) within the brain involve increases in vasopressin release, dampening of baroreflexes and stimulation of sympathetic pathways via activation of angiotensin type 1 receptors (AT1R) in cardioregulatory hypothalamic and brain stem nuclei. The significance of the brain Ang II system in BP control and hypertension is further supported by studies with the spontaneously hypertensive rat (SHR). These rats exhibit increased expression and activity of AT1R in a number of hypothalamic and brain stem regions such as the paraventricular nucleus, subfornical organ, nucleus tractus solitarius. In addition, interruption of brain AT1R function by pharmacological or genetic means lowers BP in the SHR. Therefore, a large amount of efforts have been put forward to study the chronotropic action of Ang II in hypothalamic neurons. Recently, we found that reactive oxygen species (ROS) plays an important role in the chronotropic and pressor action of Ang II. Ang II via AT1 receptor stimulates NAD(P)H oxidase activity and increases the production of ROS, which triggers neuronal firing by modulation of potassium channel. Although there is a growing literature on the intracellular mechanisms that mediate the actions of Ang II via AT1-R in neurons, little is known about the mechanisms that diminish or "switch-off"the neuronal chronotropic action of Ang II. The aim of this study is to identify the intracellular negative regulator for the action of Ang II in neurons. First, our microarray study indicated that Ang II increased MIF expression. As a cytokine being found long time ago, MIF have been well studied in immune system, but not in neuronal system. Thus, we studied MIF expression in neurons using immunocytochemistry, Western Blot, ELISA, and Real time RT-PCR. Our results indicate that neurons high express MIF. Incubation of neurons with Ang II for 5 hours increased MIF mRNA and protein level. MIF exists inside of neurons and it is not secreted. These indicated that MIF exerts its biological function inside of neurons.What is the function of MIF in neuron? In order to study the action of MIF in neuron, MIF gene was cloned, expressed in E. Coli and MIF protein was purified by the technique reported. The activity and concentration of recombinant MIF (rMIF) were examined. Using rMIF we studied the neuronal function of MIF. Neuronal firing was recorded with current patch clamp technique before and after intracellular application of rMIF indicating that rMIF significantly inhibited neuronal firing rate. Thus, for the first we identified the inhibitory action of MIF on neuronal activity. Based on above experiments indicating that Ang II induces MIF expression and MIF inhibits neuronal firing, we hypothesize that MIF may exert a negative regulator for the action of Ang II in neurons. In order to study this question, we used two approaches. First, we examined the chronotropic action of Ang II in the neurons stimulated with Ang II for 5 hours. Endogenous MIF level was increased by the stimulation with Ang II (100nM, 5 hrs). In these neurons, Ang II lost the chronotropic action. However, intracellular addition of neutralizing MIF antibody can recover the chronotropic action of Ang II indicating that endogenous MIF inhibited the chronotropic action of Ang II. Second, direct intracellular application of MIF prevented the Ang II-induced neuronal activation. These results demonstrate that MIF is a negative regulator in the neuronal action of AngII. What is the underlying mechanism? It has been reported that MIF molecule has both tautomerase and oxido-reductase activity. In order to determine which enzyme property mediates the inhibitory action of MIF, we made another recombinant protein D-dopachrome tautomerase (DCT), which has Tautomerase property but no oxido-reductase property. Intracellular application of DCT had no effect on the chronotropic action of Ang II indicating that the mechanism of MIF inhibitory action is not from its Tautomerase activity. Next experiment is designed to examine whether the oxido-reductase activity inside MIF molecule mediates the inhibitory action of MIF. It has been found that amino acid residues C57 and C60 of the MIF molecule exert a thiol-protein oxidoreductase property that responsible for certain cellular actions of MIF. Intracellular addition of the peptide MIF(50-65) significantly inhibited the chronotropic action of Ang II. However, a mutant peptide in which the C57 and C60 residues have been replaced by serines and lacks the thiod-protein oxidoreductase property of MIF fails to affect the chronotropic action of Ang II after intracellular application. These data suggest that the inhibitory action of rMIF on neuronal firing may involve the C57 and C60 residues and the thiol-protein oxidoreductase property of the MIF molecule. In other experiment, we determined whether over expressing MIF in... |
PDF Full Text Request