[Manual Acupuncture Stimulation Regulates Expression of Receptor Activity-modifying Protein 1 and 5-HT 1 D Receptor Proteins and Genes in Migraine Rats].

OBJECTIVE: To observe the effect of liver-soothing and mental-activity-regulating (LSMAR) needling on the expression of receptor activity-modifying protein 1 (RAMP 1, receptor of calcitonin gene-related peptide), 5-hydroxytryptamine 1 D receptor(5-HT 1 DR) in the spinal trigeminal nucleus (STN) and midbrain in migraine rats, so as to explore its underlying mechanism in relieving migraine. METHODS: A total of 40 male Wistar rats were randomly divided into control, model, LSMAR and conventional needling groups (n=10 rats in each). The migraine model was established by subcutaneous injection of nitroglycerin at the posterior neck. LSMAR was applied to "Baihui" (GV 20), bilateral "Fengchi" (GB 20), "Neiguan" (PC 6) and "Taichong" (LR 3) in the LSMAR group and conventional needling was applied to "Baihui" (GV 20) and bilateral "Fengchi" (GB 20) in the conventional acupuncture group for 30 min, once a day for 8 days before modeling. The expression levels of RAMP 1 and 5-HT 1 DR proteins and mRNAs in the STN and mesencephalon were detected by real-time fluorescent quantitative PCR and Western blot, separately. RESULTS: Compared with the control group, the expression levels of RAMP 1 protein and mRNA in STN and mesencephalon were significantly increased (P<0.05) and those of 5-HT 1 DR protein and mRNA considerably decreased (P<0.05) in the model group. After the acupuncture treatment, the increased levels of RAMP 1 protein and mRNA and the decreased levels of 5-HT 1 DR protein and mRNA in the STN and midbrain were obviously reversed in the LSMAR and conventional needling groups relevant to the model group (P<0.05). The effect of LSMAR needling was significantly superior to that of conventional needling in down-regulating the expression levels of RAMP 1 mRNA and protein in the STN and mesencephalon (P<0.05) and in up-regulating the expression levels of 5-HT 1 DR mRNA and protein in the two brain regions (P<0.05). CONCLUSION: Manual acupuncture stimulation of GV 20, GB 20, etc. can inhibit the expression of RAMP 1 protein and mRNA in the STN and midbrain, and up-regulate the expression of 5-HT 1 DR in the two brain regions of migraine rats, which may be related to its effect in relieving migraine.

The central anorexigenic mechanism of amylin in Japanese quail (Coturnix japonica) involves pro-opiomelanocortin, calcitonin receptor, and the arcuate nucleus of the hypothalamus.

Amylin is a 37-amino acid peptide hormone that exerts anorexigenic effects in humans and animals. We demonstrated that central injection of amylin into chicks affected feeding and related behaviors via the hypothalamus and brainstem, although the molecular mechanisms remained elusive. Thus, the objective of this study was to investigate the molecular mechanisms underlying anorexigenic effects of amylin in 7 day-old Japanese quail. Food but not water intake was reduced after intracerebroventricular amylin injection, and the behavior analysis indicated that this was associated with decreased food pecks and preening. Whole hypothalamus and hypothalamic nuclei including the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), dorsomedial nucleus (DMN) and lateral hypothalamic area (LH) were extracted from quail at 1h post-injection for total RNA isolation. Real time PCR was performed to quantify mRNA abundance of amylin receptors, appetite-associated neuropeptides and monoamine-synthesis-related enzymes. Central amylin injection increased the mRNA abundance of calcitonin receptor (CALCR), receptor activity modifying protein 1 (RAMP1), pro-opiomelanocortin (POMC), and aromatic l-amino acid decarboxylase (AADC) in the hypothalamus and individual hypothalamic nuclei. Relative quantities of CALCR and POMC mRNA were greater in the ARC of the amylin- than vehicle-treated group. Thus, amylin-mediated effects on food intake may involve POMC, monoamine synthesis, and amylin receptor 1 (a complex of CALCR and RAMP1) in the ARC. Together, these data provide novel insights on the hypothalamic-specific molecular mechanisms of amylin-induced food intake.

Effects of onion extract on endogenous vascular H2S and adrenomedulin in rat atherosclerosis.

OBJECTIVE: This study aimed to explore the effect of onion extract on endogenous hydrogen sulfide (H2S) and adrenomedulin (ADM) and on atherosclerotic progression in rats with atherosclerosis (AS). METHODS AND RESULTS: Male Sprague-Dawley rats were randomly divided into control, AS and AS+onion groups. Ultrastructure of aorta and atherosclerotic lesions both in aorta and in coronary artery were detected. Plasma and aortic H2S were detected by using a sulfide- sensitive electrode. Plasma and aortic ADM was determined with radioimmunoassay. Cystathionine-γ-lyase (CSE), calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein (RAMP1, RAMP2 and RAMP3) mRNA expressions were analysed. Glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO) and NO synthase (NOS) contents in plasma, SOD1, SOD2 and ICAM-1 expressions in aorta were detected. Rats in the AS group showed marked atherosclerotic lesions both in aorta and in coronary artery but decreased aortic H2S production. Decreased plasma and aortic ADM content, but increased levels of aortic CRLR, RAMP2 and RAMP3 mRNAs were observed. Plasma GSH-PX and SOD were reduced but MDA elevated. Plasma ICAM-1 and NO contents and iNOS activity were increased. Onion extract, however, lessened atherosclerotic lesions and increased endogenous aortic H2S production, but decreased plasma ADM content, aortic ADM content and aortic CRLR, RAMP2 and RAMP3 mRNAs. In addition, it increased plasma GSH-PX level and SOD activities but reduced MDA; it decreased inflammatory response but increased plasma eNOS activity and NO content. CONCLUSIONS: Onion extract exerted a marked antiatherogenic effect in association with the up-regulation of the endogenous CSE/H2S pathway but down-regulation of the ADM/CRLR family in atherosclerotic rats.

Functions of the cytoplasmic tails of the human receptor activity-modifying protein components of calcitonin gene-related peptide and adrenomedullin receptors.

Receptor activity-modifying proteins (RAMPs) enable calcitonin receptor-like receptor (CRLR) to function as a calcitonin gene-related peptide receptor (CRLR/RAMP1) or an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Here we investigated the functions of the cytoplasmic C-terminal tails (C-tails) of human RAMP1, -2, and -3 (hRAMP1, -2, and -3) by cotransfecting their C-terminal deletion or progressive truncation mutants into HEK-293 cells stably expressing hCRLR. Deletion of the C-tail from hRAMP1 had little effect on the surface expression, function, or intracellular trafficking of the mutant heterodimers. By contrast, deletion of the C-tail from hRAMP2 disrupted transport of hCRLR to the cell surface, resulting in significant reductions in (125)I-hAM binding and evoked cAMP accumulation. The transfection efficiency for the hRAMP2 mutant was comparable with that for wild-type hRAMP2; moreover, immunocytochemical analysis showed that the mutant hRAMP2 remained within the endoplasmic reticulum. FACS analysis revealed that deleting the C-tail from hRAMP3 markedly enhances AM-evoked internalization of the mutant heterodimers, although there was no change in agonist affinity. Truncating the C-tails by removing the six C-terminal amino acids of hRAMP2 and -3 or exchanging their C-tails with one another had no effect on surface expression, agonist affinity, or internalization of hCRLR, which suggests that the highly conserved Ser-Lys sequence within hRAMP C-tails is involved in cellular trafficking of the two AM receptors. Notably, deleting the respective C-tails from hRAMPs had no effect on lysosomal sorting of hCRLR. Thus, the respective C-tails of hRAMP2 and -3 differentially affect hCRLR surface delivery and internalization.