Received 2002-04-11 Accepted 2002-06-06

*Corresponding author. Tel: +86-311-6062490; Fax: +86-311-6062490;

Acta Physiologica Sinica Dec. 2002, 54 (6), 460-466

Research Paper

Microinjection of adrenomedullin into rostral ventrolateral medulla increases blood pressure, heart rate and renal sympathetic nerve activity in rats

JI Shu-Mei, HE Rui-Rong*

Department of Physiology, Hebei Medical University, Shijiazhuang 050017

Abstract: The present study was undertaken to examine the effects of microinjection of adrenomedullin (AM) into rostral ventrolateral medulla (RVLM) on mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in 34 anesthetized Sprague-Dawley rats. The results obtained are as follows. (1) Following microinjection of AM (10 μmol/L, 200 nl) into the RVLM, MAP, HR and RSNA were significantly increased from 99.09±3.32 mmHg, 370.78±7.84 bpm and 100±0% to 113.57±3.64 mmHg (P<0.001), 383.28±7.38 bpm (P<0.001) and 123.72±2.74% (P<0.001), respectively. (2) Pretreatment with microinjection of calcitonin gene-related peptide receptor antagonist CGRP8-37 (100 μmol/L, 200 nl) did not change the effects of AM. (3) L-arginine (100 mg/kg, 0.2 ml, i.v.), an NO precursor, abolished the effects of AM. This study demonstrates that AM acting at the rostral ventrolateral medulla may produce significant cardiovascular responses, the effects are not mediated by CGRP receptor but may be abolished by NO.Key words: adrenomedullin; rostral ventrolateral medulla; mean arterial pressure; heart rate; renal sympathetic nerve; calcitonin gene-related peptide receptor antagonist; L-arginine

延髓腹外侧头端区注射肾上腺髓质素对大鼠血压、心率和肾交感神经活动的影响

季淑梅, 何瑞荣

河北医科大学基础医学研究所生理室, 石家庄 050017

摘要: 本研究在34只麻醉Sprague-Dawley大鼠观察了延髓腹外侧头端区内微量注射肾上腺髓质素(10 μmol/L, 200 nl)对平均动脉压(MAP)、心率(HR)和肾交感神经放电(RSNA)的影响。实验结果如下: (1) 延髓腹外侧头端区内微量注射肾上腺髓质素可引起MAP、HR、和RSNA明显增加, 分别由99.09±3.32 mmHg, 370.78±7.84 bpm 和100±0% 增至113.57±3.64 mmHg (P<0.001), 383.28 ±7.38 bpm (P<0.001) 和123.72±2.74% (P<0.001); (2) 降钙素基因相关肽受体阻断剂CGRP8-37 (100 μmol/L, 200 nl)不能阻断肾上腺髓质素的上述效应; (3) 静脉注射NO前体L-精氨酸(100 mg/kg, 0.2 ml)可消除肾上腺髓质素的上述效应。以上结果提示, 肾上腺髓质素作用于延髓腹外侧头端区可产生显著的心血管作用, 此作用不是由降钙素基因相关肽受体介导,但可被NO所阻断。

关键词: 肾上腺髓质素; 延髓腹外侧头端区; 平均动脉压; 心率; 肾交感神经; 降钙素基因相关肽受体拮抗剂; L-精氨酸

中图分类号: Q463

Adrenomedullin (AM) is a novel hypotensive peptide originally isolated from human pheochromocytoma. It consists of 52 amino acids in human and 50 amino acids in rats, belonging to calcitonin gene-related peptide (CGRP) family, and interacts with specific AM receptors and CGRP 1 receptors[1]. Abundant specific binding sites for AM, mRNA expression of receptors for AM and the production of AM were observed not only in peripheral tissues but also in the central nervous system (CNS)[1-6]. AM has been shown to be widely involved in the control of fluid and electrolyte homeostasis and cardiovascular function through its peripheral and central actions. Central administration of AM inhibited water drinking, appetite for salt and feeding[7, 8], and induced natriuresis and diuresis[9]. In contrast to its hypotensive effect in the periphery, intracerebroventricular (i.c.v.) administration of AM caused a dose-dependent increase in blood pressure[10-12]. The hypotensive effect of AM in periphery is mainly due to direct vasodilatation through action at specific AM receptors and CGRP receptors[13,14]. The mechanisms of AM-induced vasodilatation involved increases in intracellular cAMP, NO-cGMP pathway and activation of K+ channels depending on animal species and vascular bed studied[15-18]. The hypertensive effect elicited by i.c.v. administration of AM is induced by stimulating sympathetic nerve activity[12, 19]. However, the exact site and precise intracellular mechanism of action of AM in the brain that mediates these cardiovascular effects are not clear. Recently Smith et al.[20] reported that AM acted on the rat paraventricular nucleus (PVN) to decrease blood pressure. Allen et al.. showed that microinjection of AM into the area postrema (AP) increased blood pressure[21], and AM elicited direct excitatory effects on AP neurons in vitro[22].

It is generally accepted that rostral ventrolateral medulla (RVLM) plays an important role in the regulation of vascular tone and the maintenance of blood pressure. RVLM neurons have been shown to be involved in various sympathetic reflex and integration of the inputs from a variety of visceral, somatic and supramedullary structures. AM-like immunoreactivity and AM receptors have been demonstrated within RVLM neurons[1-5]. These data support the notion that RVLM is a likely site for the action of AM. However, the direct action of AM on RVLM has not yet been reported. The present study aimed to investigate the effect of microinjection of AM into RVLM on blood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA), and also to investigate the possible underlying mechanism.

1 MATERIALS AND METHODS

Sprague-Dawley rats (280-320 g) were anesthetized with urethane (1.0 g/kg, ip), with body temperature maintained at 36-37℃. The animals breathed spontaneously through an intratracheal tube. The right femoral artery and vein were cannulated for measurement of BP by a pressure transducer (MPU-0.5, Nihon Kohden) and for drugs infusion, respectively. HR was monitored by a heart rate counter (AT-601G, Nihon Kohden) triggered by differential signals of arterial pressure pulse.

1.1 Recording of RSNA

The left renal artery and vein were exposed via a retroperitoneal approach, and a branch of the renal nerve was isolated and clamped distally to eliminate the afferent activity. The nerve was placed on a bipolar platinum electrode for action potential recording, and immersed in liquid paraffin. The signals of RSNA were amplified by a biophysical amplifier (AB-621G, Nihon Kohden) and then fed to an integrator (EI-601G, Nihon Kohden), with an integrated time of 1 s. At the end of the experiments, the proximal end of the nerve was clamped to get the noise level of RSNA.

The raw neurogram and the integrated RSNA as well as the BP and HR were recorded on a polygraph system (RM-6000, Nihon Kohden) with a thermal-array recorder (WS-682G, Nihon kohden; band-pass width: 0-2.8 kHz).

1.2 Microinjection into the rostral ventrolateral medulla

The rats were fixed on a stereotaxic frame (Model 1C, Jiangwan) in a supine position. A glass micropipette (tip diameter 10-30 μm) was inserted into RVLM (stereotaxic coordinates: 1.5-2.0 mm lateral to midline, 2.6-3.3 mm posterior to interaural line, and 0.3-0.5 mm from the ventral surface of medulla oblongata) for microinjection. The injectio filled in the micropipette was delivered into the RVLM by a nanoliter injector (A203XVZ, World Precision, USA).

1.3 Histological examination

To identify the site of microinjection at postmortem, 2% pontamine sky blue was added to the injectio. At the completion of the experiments, the animals were deeply anesthetized, the brainstem was removed and stored in 10% formalin solution. After 7-10 d the frozen brain tissue was sectioned in the coronal plane (40 μm). Histological verification was carried out with reference to Paxinos and Watson's coordinates[23]. The stained area or the depth of the injecting track in RVLM was examined under the microscope. The results from the animals with injectio diffusing out of the RVLM were excluded from statistic data.

1.4 Experimental Protocols

The experimental animals were divided into three groups according to different protocols: (1) A volume of 200 nl of a vehicle (normal saline) was injected into RVLM as the control for volume. After 30 min, AM (10 μmol/L, 200 nl, Sigma) was microinjected into RVLM and the changes in BP, HR and RSNA were examined. (2) After microinjection of 200 nl saline was performed as vehicle control, CGRP8-37 (100 μmol/L, 200 nl, Sigma) was microinjected into RVLM. After 10 min, microinjection of AM into RVLM was carried out, and BP, HR and RSNA were examined. (3) BP, HR and RSNA were recorded following iv administration of saline and L-arginine (100 mg/kg, 0.2 ml, Sigma). After 15 min, microinjection of AM into RVLM was carried out, and BP, HR and RSNA were examined.

1.5 Statistics

All data were expressed as means±SE. Data were analyzed using paired Student's t test and two-way ANOVA followed by Student-Neuman-Keuls test. Statistical significance was accepted when P<0.05.

2 RESULTS

A total of 34 animals were used in the present study. Figure 1 presents the medullary coronal section summarizing the locations of RVLM sites for AM microinjection.

2.1 Effects of microinjection of AM into RVLM on BP, HR and RSNA (n=18)

Microinjection of vehicle into the RVLM caused insignificant changes in BP, HR and RSNA. AM (10 μmol/L, 200 nl) microinjected into the RVLM evoked significant increases in BP, HR and RSNA, the effects occurred within 120 s and lasted for about 60 min (Table 1, Fig. 2).2.2 Effects of CGRP8-37 on the actions of AM (n=8)

In order to rule out the possibility that AM elicited the responses via activation of CGRP receptors, AM was microinjected before and after administration of CGRP8-37 (100 μmol/L, 200 nl) into the same site in the same animal. Administration of the CGRP8-37 did not influence BP, HR and RSNA, nor did it influence the effects elicited by AM microinjection into the RVLM (Fig. 2 and Table 2).

Fig.1. Localization of microelectrode tips in RVLM. Numbers indicate caudal to interaural line in mm. Coronal sections of medulla are modified from Paxinos and Watson. IO, inferior olive; NA, nucleus ambiguus; py, pyramidal tract; RM, nucleus raphe magnus; RO, nucleus raphe obscurus; RP, nucleus raphe pallidus; closed circle, units responsive to adrenomedullin; triangle, units not in the nucleus paragigantocellularis lateralis (PGL).

Table１. Effects of microinjection of adrenomedullin (AM)(10μmol/L,200 nl) into

the RVLM on BP, HR and RSNA(n=18)

	n	MAP (mmHg)	HR (bpm)	RSNA (%)
Control	18	99.09±3.32	370.78±7.84	100±0
AM	18	113.57±3.64***	383.28±7.38***	123.72±2.74**

***P<0.001 vs control．

Fig.2. Effects of microinjection of AM into the RVLM on BP, HR and RSNA before and after administration of

CGRP8-37. A: Adrenomedullin before CGRP8-37.B: Adrenomedullin after CGRP8-37.

↓, injection of adrenomedullin; -↓, injection of CGRP8-37.

Table 2. Effects of microinjection of AM into the RVLM on BP, HR and RSNA before and after

administration of CGRP8-37 (n=8) or L-arginine (n=8)

	n	MAP (mmHg)	HR (bpm)	RSNA (%)
Control	8	96.38±4.57	376.13±6.59	100±0
AM	8	113.71±4.72**	386.00±8.31**	117.75±1.32**
CGRP8-37	8	95.74±4.51##	374.91±7.16##	98.78±0.84##
CGRP8-37+AM	8	112.49±4.70**++	385.13±8.13**++	117.00±1.39**++
Control	8	89.42±3.15	360.75±11.47	100±0
AM	8	101.53±3.74**	374.50±11.34**	116.50±1.95**
L-Arg	8	87.14±2.91##	358.27±8.94##	97.86±0.93##
L-Arg+AM	8	87.58±3.16##	357.63±11.23##	98.63±0.75##

*P<0.01 vs control; ## P<0.01 vs AM; ++P<0.01 vs CGRP8-37.

2.3 Effects of L-arginine on the actions of AM (n=8)

Following i.v. injection of L-arginine (100 mg/kg, 0.2 ml), the excitatory actions of AM on BP, HR and RSNA were abolished (Fig. 3 and Table 2), and L-arginine itself did not change BP, HR and RSNA.

Fig.3. Effects of microinjection of AM into RVLM on BP, HR and RSNA before and after intravenous administration of L-arginine. A： Adrenomedullin before L-arginine. B： Adrenomedullin after L-arginine. ↓, injection of adrenomedullin; -↓, injection of L-arginine.

3 DISCUSSION

The results of the present study showed that microinjection of AM into RVLM caused increases in BP, HR and RSNA. These effects are consistent with the cardiovascular effects elicited by i.c.v. administration of AM in anaesthetized and conscious rats[10-12]. Allen et al.[21] also demonstrated that microinjection of AM into the area postrema caused a dose-dependent increase in BP. In our experiment, microinjection of AM into the RVLM induced an increase in RSNA,suggesting that AM may directly stimulate the activity of neurons in RVLM, resulting in increases in BP, HR and RSNA.

AM is structurally homologous to CGRP and interacts with specific AM receptors and CGRP 1 receptors, AM displays a highly specific recognition at the specific AM receptors over CGRP receptors[1, 24, 25]. Some of the actions of AM have been shown to be blocked by the CGRP antagonist, CGRP8-37. The blocking effects of CGRP8-37 on the central action of AM were controversial. Takahashi et al.[10] and Saita et al.[11] reported that pretreatment with CGRP8-37 suppressed the central effect of AM; conversely, Samon et al.[12] did not have the same result. In our study, microinjection of AM into the RVLM still showed the effects on MAP, HR and RSNA after administration of the CGRP8-37, suggesting that AM exerts its cardiovascular effects in RVLM not through actions at CGRP receptors, but at specific AM receptors. Smith et al.[20] also demonstrated that AM exerted its cardiovascular effect in the PVN through direct actions at AM specific receptors instead of CGRP receptors.

In our experiment, the responses elicited by microinjection of AM were completely abolished by i.v. injection of L-arginine, implying that L-arginine:NO pathway may exert a suppressive action to the AM-activation mechanism in the RVLM. Our previous study demonstrated that[26] L-arginine:NO pathway exhibited a modulatory action on the activity of RVLM and that microinjection of N-nitro-L-arginine (L-NNA), a NO synthetase inhibitor, into the RVLM induced increases in MAP and RSNA, these effects could be reversed by prior intravenous injection of L-arginine.

In conclusion, our results show that AM directly stimulates the activity of the neurons in RVLM, resulting in increases in BP, HR and RSNA. These effects are not mediated by CGRP receptor and may be abolished by NO.

REFERENCES

[1] Owji AA, Smith DM, Coppock HA, Morgan DG, Bhogal R, Ghatei MA, Bloom SR. An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology, 1995,136:2127-2134.

[2] Oliver KR, Wainwright A, Heavens RP, Hill RG, Sirinathsinghji DJ. Distribution of novel CGRP1 receptor and adrenomedullin receptor mRNAs in the rat central nervous system. Brain Res Mol Brain Res, 1998,57:149-154.

[3] Shan J, Krukoff TL. Distribution of preproadrenomedullin mRNA in the rat central nervous system and its modulation by physiological stressors. J Comp Neurol, 2001,432:88-100.

[4] Serrano J, Uttenthal LO, Martinez A, Fernandez AP, Martinez de Velasco J, Alonso D, Bentura ML, Santacana M, Gallardo JR, Martinez-Murillo R, Cuttitta F, Rodrigo J. Distribution of adrenomedullin-like immunoreactivity in the rat central nervous system by light and electron microscopy. Brain Res, 2000,853:245-268.

[5] Satoh F, Takahashi K, Murakami O, Totsune K, Sone M, Ohneda M, Abe K, Miura Y, Hayashi Y, Sasano H. Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma. J Clin Endocrinol Metab, 1995,80:1750-1752.

[6] Takahashi K. Adrenomedullin from a pheochromocytoma to the eye: implications of the adrenomedullin research for endocrinology in the 21st century. Tohoku J Exp Med, 2001,193:79-114.

[7] Samson WK, Murphy TC. Adrenomedullin inhibits salt appetite. Endocrinology, 1997,138:613-616.

[8] Murphy TC, Samson WK. The novel vasoactive hormone, adrenomedullin, inhibits water drinking in the rat. Endocrinology, 1995,136:2459-2463.

[9] Israel A, Diaz E. Diuretic and natriuretic action of adrenomedullin administered intracerebroventricularly in conscious rats. Regul Pept, 2000,89(1-3):13-18.

[10] Takahashi H, Watanabe TX, Nishimura M, Nakanishi T, Sakamoto M, Yoshimura M, Komiyama Y, Masuda M, Murakami T. Centrally induced vasopressor and sympathetic responses to a novel endogenous peptide, adrenomedullin, in anesthetized rats. Am J Hypertens, 1994,7:478-482.

[11] Saita M, Shimokawa A, Kunitake T, Kato K, Hanamori T, Kitamura K, Eto T, Kannan H. Central actions of adrenomedullin on cardiovascular parameters and sympathetic outflow in conscious rats. Am J Physiol, 1998, 274(4 Pt 2):R979-R984.

[12] Samson WK, Murphy TC, Resch ZT. Central mechanisms for the hypertensive effects of preproadrenomedullin-derived peptides in conscious rats. Am J Physiol, 1998, 274(5 Pt 2):R1505-R1509.

[13] Van Rossum D, Hanisch UK, Quirion R. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neurosci Biobehav Rev, 1997,21:649-678.

[14] Mori Y, Takayasu M, Suzuki Y, Shibuya M, Yoshida J, Hidaka H. Effects of adrenomedullin on rat cerebral arterioles. Eur J Pharmacol, 1997,330(2-3):195-198.

[15] Hinson JP, Kapas S, Smith DM. Adrenomedullin, a multifunctional regulatory peptide.Endocr Rev, 2000,21:138-167.

[16] Hayakawa H, Hirata Y, Kakoki M, Suzuki Y, Nishimatsu H, Nagata D, Suzuki E,Kikuchi K, Nagano T, Kangawa K, Matsuo H, Sugimoto T, Omata M. Role of nitric oxide-cGMP pathway in adrenomedullin-induced vasodilation in the rat. Hypertension, 1999,33:689-693.

[17] Okamura T, Ayajiki K, Kangawa K, Toda N. Mechanism of adrenomedullin-induced relaxation in isolated canine retinal arteries. Invest Ophthalmol Vis Sci, 1997,38:56-61.

[18] Terata K, Miura H, Liu YP, Loberiza F, Gutterman DD. Human coronary arteriolar dilation to adrenomedullin: role of nitric oxide and K+ channels. Am J Physiol, 2000,279:H2620-2626.

[19] Wei YJ, Tian Q, Li QH, Li YL, Chang JK, Tang J, Ho SY. Hemodynamic effects of centrally administered adrenomedullin (13-52) in anesthetized rats. Biol Signals, 1995,4:338-344.

[20] Smith PM, Ferguson AV. Adrenomedullin acts in the rat paraventricular nucleus to decrease blood pressure. J Neuroendocrinol, 2001,13:467-471.

[21] Allen MA, Smith PM, Ferguson AV. Adrenomedullin microinjection into the area postrema increases blood pressure. Am J Physiol, 1997,272(6 Pt 2):R1698-R1703.

[22] Allen MA, Ferguson AV. In vitro recordings from area postrema neurons demonstrate responsiveness to adrenomedullin. Am J Physiol, 1996,270(4 Pt 2):R920-R925.

[23] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates.New York: Academic Press, 1982.

[24] Juaneda C, Dumont Y, Chabot JG, Quirion R. Autoradiographic distribution of adrenomedullin receptors in the rat brain. Eur J Pharmacol, 2001,421:R1-R2.

[25] Sone M, Takahashi K, Satoh F, Murakami O, Totsune K, Ohneda M, Sasano H, Ito H, Mouri T. Specific adrenomedullin binding sites in the human brain. Peptides, 1997,18:1125-1129.

[26] Yang LM (杨丽明), He RR(何瑞荣). Effects of microinjection of L-NNA and SNP into ventrolateral medulla on blood pressure, heart rate and renal sympathetic nerve activity in rats. Acta Physiol Sin (生理学报), 1996,48:368-376 (Chinese, English abstract).