Mechanisms of cardiovascular actions of urocortins in the hypothalamic arcuate nucleus of the rat

Abstract

The presence of urocortins (UCNs) and corticotropin-releasing factor (CRF) receptors has been reported in the hypothalamic arcuate nucleus (ARCN). We have previously reported that UCNs are involved in central cardiovascular regulation. Based on this information, we hypothesized that the ARCN may be one of the sites where UCNs exert their central cardiovascular actions. Experiments were done in artificially ventilated, adult male Wistar rats anesthetized with urethane. Unilateral microinjections (30 nl) of UCN1 (0.12-2 mM) elicited decreases in mean arterial pressure (MAP) and heart rate (HR). Maximum cardiovascular responses were elicited by a 1 mM concentration of UCN1. Microinjections of UCN2 and UCN3 (1 mM each) into the ARCN elicited similar decreases in MAP and HR. UCN1 was used as a prototype for the other experiments described below. HR responses elicited by UCN1 were significantly attenuated by bilateral vagotomy. Prior microinjections of NBI-27914 (CRF-1 receptor antagonist) and astressin (CRF-1 receptor and CRF-2 receptor antagonist) (1 mM each) into the ARCN significantly attenuated the cardiovascular responses elicited by UCN1 microinjections at the same site. Microinjections of UCN1 into the ARCN decreased efferent renal sympathetic nerve activity. It was concluded that microinjections of UCN1, UCN2, and UCN3 into the ARCN elicited decreases in MAP and HR. Decreases in MAP, HR, and renal sympathetic nerve activity elicited by UCN1 microinjections into the ARCN were mediated via CRF receptors. Bradycardic responses to UCN1 were mediated via the activation of vagus nerves, and decreases in MAP may be mediated via decreases in sympathetic nerve activity.

Keywords: N-methyl-d-aspartic acid; blood pressure; heart rate; microinjection; sympathetic nerve activity.

Fig. 1.

Cardiovascular responses of different concentrations of urocortin 1 (UCN1). A: mean arterial pressure (MAP). *P < 0.01 compared with 0.12 and 0.25 mM. B: heart rate [HR; in beats/min (bpm)]. Microinjections (30 nl) of 0.12, 0.25, 0.5, 1.0, and 2.0 mM concentrations of UCN1 into the arcuate nucleus (ARCN) elicited decreases of 5 ± 1, 8 ± 1, 10 ± 1, 14 ± 2, and 15 ± 1 mmHg in MAP, respectively. The same concentrations of UCN1 at the same site elicited decreases of 6 ± 1, 9 ± 1, 14 ± 1, 19 ± 2, and 17 ± 1 beats/min in HR, respectively. *P < 0.001 compared with 0.12 and 0.25 mM.

Fig. 2.

Effect of microinjection of UCN1 (1 mM) into the ARCN on renal sympathetic nerve activity (RSNA). The top trace shows MAP (in mmHg), the middle top trace shows HR (in beats/min), the middle bottom trace shows integrated RSNA (∫RSNA; in μV/1s), and the bottom trace shows whole RSNA (in μV). A: phenylephrine (PE; 10 μg/kg iv) elicited a pressor response accompanied by reflex inhibition of HR and RSNA. B: 10 min later, microinjection of N-methyl-d-aspartic acid (NMDA; 10 mM) into the ARCN decreased MAP, integrated RSNA, and whole RSNA and increased HR. C: no responses were observed after microinjection of artificial cerebrospinal fluid (aCSF) into the ARCN. D: 20 min later, microinjection of UCN1 (1 mM) into the ARCN decreased MAP, HR, integrated RSNA, and whole RSNA. E: 60 min later, microinjection of astressin (1 mM) into the ARCN did not elicit any response. F: 2 min later, microinjection of UCN1 (1 mM) at the same site failed to elicit a response.

Fig. 3.

Group data showing changes in RSNA elicited by microinjection of UCN1 (1 mM) into the ARCN and its blockade by astressin. A: effect of PE (10 μg/kg iv). B: microinjection of NMDA (10 mM) into the ARCN. C: microinjection of aCSF into the ARCN. D: UCN1 (1 mM)-induced decreases in RSNA. E: effect of Astressin (1 mM) on RSNA. F: blockade of UCN1 (1 mM)-induced inhibition of RSNA by astressin (1 mM). G: astressin did not alter NMDA-induced decreases in RSNA. *P < 0.05 compared with D.

Fig. 4.

Effect of bilateral vagotomy (Vag-X) on NMDA- and UCN1-induced cardiovascular responses elicited from the ARCN. A: the decreases in MAP elicited by microinjections of NMDA (10 mM) into the ARCN before and after Vag-X were 11 ± 2 and 7 ± 1 mmHg, respectively. B: the decreases in MAP elicited by microinjections of UCN1 (1 mM) into the ARCN before and after Vag-X were 12 ± 2 and 10 ± 1 mmHg, respectively. C: the increases in HR elicited by microinjections of NMDA (10 mM) into the ARCN before and after Vag-X were 36 ± 7 and 11 ± 1 beats/min, respectively. *P < 0.01 compared with before Vag-X. D: the decreases in HR elicited by microinjections of UCN1 (1 mM) into the ARCN before and after Vag-X were 25 ± 2 and 5 ± 1 beats/min, respectively. **P < 0.001 compared with before Vag-X.

Fig. 5.

Effect of inhibition of the nucleus ambiguus (nAmb) on UCN1-induced bradycardic responses elicited from the ARCN. The top trace shows pulsatile arterial pressure (PAP; in mmHg), the middle trace shows MAP (in mmHg), and the bottom trace shows HR (in beats/min). A: microinjection of NMDA (10 mM) into the ARCN elicited decreases in PAP and MAP and increases in HR. After blood pressure (BP) and HR recovered to baseline levels, aCSF was microinjected into the ARCN, and no changes were observed (not shown). B: 20 min later, microinjection of UCN1 (1 mM) into the ARCN elicited decreases in PAP, MAP, and HR. C: 60 min later, the nAmb was identified with l-glutamate (l-Glu; 5 mM), and a decrease in HR was elicited with no changes in PAP and MAP. After the recovery of HR, aCSF was microinjected into the nAmb, and no changes were observed (not shown). Five minutes later, muscimol (1 mM) was microinjected into the nAmb, and an increase in HR was elicited (not shown): D: 5 min later, microinjection of UCN1 (1 mM) into the ARCN failed to elicit decreases in HR, whereas decreases in PAP and MAP persisted.

Fig. 6.

Histological identification of microinjection sites in the ARCN. A: coronal section at a level of 3.0 mm caudal to the bregma showing a microinjection site in the ARCN, which was marked by a microinjection of diluted (1:50) green retrobeads IX containing NMDA (10 mM, 30 nl, arrow). The center of the spot was 0.35 mm lateral to the midline and 9.5 mm deep from the dura. B–F: drawings of coronal sections 3.48 (B), 3.12 (C), 2.76 (D), 2.28 (E), and 1.92 mm (F) caudal to the bregma showing the ARCN microinjection sites as dark spots. Each spot represents a site in one animal; the microinjection sites were located in the ARCN, 0.1–0.6 mm lateral to the midline and 9.8–10.1 mm deep from the dura. 3V, third ventricle; f, fornix.

Fig. 7.

Schematic diagram showing the possible pathways mediating the cardiovascular responses to UCN1 in the ARCN (see details of pathways in the discussion). PVN, paraventricular nucleus; RVLM, rostral ventrolateral medullary pressor area; IML, spinal intermediolateral cell column; SNA, sympathetic nerve activity; NTS, nucleus tractus solitarius; CVLM, caudal ventrolateral medullary depressor area; SG, sympathetic ganglion; NPY, neuropeptide Y; MSH, α-melanocyte-stimulating hormone; NE, norepinephrine.