| Objectives: To investigate the physiological significance of brain-derived adrenomedullin (ADM) and its receptors in cardiovascular regulation and their roles in stress-induced hypertension by observing the dynamic changes of ADM and its specific receptor components in medulla oblongata during the stress and the changes of systolic blood pressure (SBP), heart rate (HR) and spike frequency of singe neurons after microinjection and iontophoretic application of rat ADM into the rostral ventrolateral medulla (rVLM).Methods: We established the stress-induced hypertension model by foot electric shock associated with noise for 15 consecutive days. To detect the dynamic changes of preproadrenomedullin (ppADM) gene encoding ADM and its specific receptor components, calcitonin receptor-like receptor (CRLR) and the receptor activity modifying protein 2 and 3 (RAMP2 and RAMP3) in the medulla oblongata within consecutive stress for 15 days, we used reverse transcription-polymerase chain reaction (RT-PCR), Radioimmunoassay(RIA), in situ hybidization (ISH) and immunohistochemistry methods. Then we observed the cardiovascular effect and its potential mechanism of ADM by microinjection and iontophoretic application of rat adrenomedullin (rADM), ADM receptor antagonists or nitric oxide synthase antagonists and electric stimulating the dorsal periaqueductal gray (dPAG) of midbrain as well as extracellular single-unit recordings methods.Results: (1) Consecutively stressing for 15 days induced a significant increase in SBP at the 5th, 10th and 15th day and unique changes in ppADM and its receptor components mRNAs as well as ADM levels in medulla oblongata studied by RT-PCR and RIA methods. As compared with the control group, the level of ppADM mRNA, normalized against a glyceraldehydes-3-phosphate dehydrogenase (GAPDH) control, was down-regulated in the medulla oblongata in the stress group, especially at the 5th and 15th day (P<0. 01). The relative amount of CRLR, RAMP2 mRNAwas higher in the medulla oblongata. Chronic stress for 15 consecutive days produced a decrease in the level of RAMP2 and CRLR mRNA expression at the 5th day and an increase at the 15th day in stress group as compared with the control (P <0. 05), but no significant stress-related changes in RAMP3 mRNA. In addition, the levels of ADM in medulla oblongata are relatively high, about 1.45 ± 0.12 pmol. g-1 fresh tissues. During the stress, the levels of ADM in the stress group had fluctuated to some degree in every time point we observed. However, only at the 15th day after stress, we found a significant decrease in ADM levels in medulla oblongata of the stress group compared with the control group(P <0. 05) ; (2) With ISH and IHC method, we detected that ADM-like immunoreactivity and ppADM mRNA are widely distributed throughout the medulla oblongata. Highly labeled neurons were found in the rVLM, ventrolateral reticular nucleus, hypoglossal nucleus. Moderately labeled neurons were seen in the facial, arabiguus, lateral reticular, paragigantocellular reticular, and inferior olivary nuclei. Weak signal was present over neurons the nucleus of the solitary tract (NTS). Foot shock and noise stress significantly reduced ppADM mRNA and ADM-like immunoreactivity at the 5th day compared with the control group (P <0. 01) ; (3) 100 nl rat ADM (0.01 μmol/L, 0.1 μ mol/L or 1 μ mol/L) was unilaterally microinjected into the rVLM through micropipettes placed inside guide cannulas and induced a dose-dependent increase of SBP and HR in normotensive rats for about 40 rain or lh compared with the rats microinjected with 0.9%NS {P < 0.05 or P < 0.01, respectively). However, when applying these three doses to stress-induced hypertensive rats, only 1umol/L 100 nl rADM could induced a significant increase of SBP and HR for about 30 min compared with control group (P <0.05) and these effects were partly blocked by pretreatment of human ADM(22-52), a selective ADM receptor antagonist, and 7-nitroindazole (7-Ni), a highly selective antagonist for neuronal nitric oxide synthase (nNOS). In addition, if we increased the dosage to 10 u mol/L, then higher increases of SBP and HR in stress induced hypertensive rats for a long lasting time, about lh, were observed (P <0.05); (4) After electric stimulated dorsal periaqueductal gray (dPAG) of midbrain, the SBP and HR of normotensive rats rapidly zoomed up and then down to normal level. After...
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