Decrease in arterial pressure induced by adrenomedullin in the hypothalamic paraventricular nucleus is mediated by nitric oxide and GABA

Abstract

We tested the hypothesis that the decrease in arterial pressure induced by adrenomedullin (ADM) in the hypothalamic paraventricular nucleus (PVN) is mediated by nitric oxide (NO) and/or GABA. Unilateral microinjections of ADM into the PVN of anesthetized rats caused a significant decrease in mean arterial pressure (MAP). The ADM-induced decrease in MAP was significantly attenuated by pretreatment with N(psi)-nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), 7-nitroindazole sodium salt (7-NiNa, a selective neuronal NOS inhibitor), N5-(1-Iminoethyl)-L-ornithine (L-NIO, a selective endothelial NOS inhibitor) or bicuculline methiodide, but pretreatment with S-methylisothiourea (SMIT, a selective inducible NOS inhibitor) had no effect on this ADM-induced effect. In addition, coronal sections of rat brains were processed for combined NADPH-diaphorase (a marker of neuronal NOS-containing neurons) histochemistry and in situ hybridization for the receptor-activity-modifying protein 2 (a specific ADM receptor component). Double-labeled neurons were found in both parvocellular and magnocellular subdivisions of the PVN, confirming that NO-producing neurons in the PVN are capable of mediating ADM's effects. Thus, our data provide evidence that the ADM-induced decrease in MAP in the PVN is mediated by NO from neuronal and endothelial NOS, and by GABA.

Fig. 1

(A) Light-field microphotograph showing the site of termination of an injection that is considered to be within the PVN. (B, C and D) Schematic representations of serial sections from 7.60 to 6.88 mm anterior to interaural zero showing the sites of termination of injection tracts in 36 rats. Each square represents a site of termination of an injection that is considered to be within the PVN, while each circle represents a site of termination of an injection that is considered to be outside of the PVN. The scale bar in (A) = 0.1 mm. 3V, third ventricle; AHA, anterior hypothalamic area, anterior part; AHC, anterior hypothalamic area, central part; AHP, anterior hypothalamic area, posterior part; Arc, arcuate hypothalamic nucleus; BSTMPL, bed nucleus of the stria terminalis, medial division, posterolateral part; f, fornix; LA, lateroanterior hypothalamic nucleus; MPO, medial preoptic nucleus; Pe, periventricular hypothalamic nucleus; PVN, paraventricular nucleus; RCh, retrochiasmatic area; VMH, ventromedial hypothalamic nucleus. Drawing is modified from Paxinos and Watson [35].

Fig. 2

(A) Representative arterial pressure traces of rats that received ADM injections in the PVN after the pretreatment of saline or L-NAME at doses indicated. (B and C) Amplitude and AUC of decreases in mean arterial pressure (MAP) to microinjections of ADM into the PVN after the pretreatment of saline or L-NAME. Graphs represent mean ± S.E.M. N = 8 in each group. ** P < 0.01.

Fig. 3

(A) Representative arterial pressure traces of rats that received ADM injections in the PVN after the pretreatment of saline or 7-NiNa at doses indicated. (B and C) Amplitude and AUC of decreases in mean arterial pressure (MAP) to microinjections of ADM into the PVN after the pretreatment of saline or 7-NiNa. Graphs represent mean ± S.E.M. N = 8 in each group. **P < 0.01.

Fig. 4

(A) Representative arterial pressure traces of rats that received ADM injections in the PVN after the pretreatment of saline or L-NIO at doses indicated. (B and C) Amplitude and AUC of decreases in mean arterial pressure (MAP) to microinjections of ADM into the PVN after the pretreatment of saline or L-NIO. Graphs represent mean ± S.E.M. N = 8 in each group. **P < 0.01.

Fig. 5

(A) Representative arterial pressure traces of rats that received ADM injections in the PVN after the pretreatment of saline or SMIT at doses indicated. (B and C) Amplitude and AUC of decreases in mean arterial pressure (MAP) to microinjections of ADM into the PVN after the pretreatment of saline or SMIT. Graphs represent mean ± S.E.M. N = 8 in each group.

Fig. 6

(A) Representative arterial pressure traces of rats that received ADM injections in the PVN after the pretreatment of saline or BIC at doses indicated. (B and C) Amplitude and AUC of decreases in mean arterial pressure (MAP) to microinjections of ADM into the PVN after the pretreatment of saline or BIC. Graphs represent mean ± S.E.M. N = 8 in each group. *P < 0.05, **P < 0.01.

Fig. 7

(A) Dark-field photomicrograph of the PVN showing localization of RAMP-2 mRNA with in situ hybridization. (B) Dark-field photomicrograph showing the background signal in an area near the PVN. (C and D) Light-and dark-field photomicrographs of the parvocellular subdivision at higher magnification, indicated by the black box in (A) and showing single-labeled neurons (arrowhead) and a neuron double-labeled with RAMP-2 mRNA and NADPH-d (arrow). (E and F) Light- and dark-field photomicrographs of the magnocellular subdivision at higher magnification, indicated by the white box in (A) and showing a neuron double-labeled with RAMP-2 mRNA and NADPH-d (arrow). The scale bar in (A) = 100 μm; the scale bar in (B) = 10 μm and applies in (C, D, E and F). V, third ventricle.