Neuropeptides. 2015 Dec;54:59-66.
Role of cerebellar adrenomedullin in blood pressure regulation.
Leticia Figueira and Anita Israel*
School of Pharmacy, Laboratory of Neuropeptides, Universidad Central de Venezuela. Caracas – Venezuela
*Corresponding author: Apartado Postal 50176, Sabana Grande 1050A, Caracas, Venezuela. E-mail: email@example.com
Adrenomedullin (AM) and their receptor components, calcitonin-receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP1, RAMP2 and RAMP3) are widely expressed in the central nervous system, including cerebellum. We have shown that AM binding sites are altered in cerebellum during hypertension, suggesting a role for cerebellar adrenomedullinergic system in blood pressure regulation. To further evaluate the role of AM in cerebellum, we assessed the expression of AM, RAMP1, RAMP2, RAMP3 and CRLR in the cerebellar vermis of 8 and 16 weeks old spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. In addition, the effect of microinjection of AM into rat cerebellar vermis on arterial blood pressure (BP) was determined. Animals were sacrificed by decapitation and cerebellar vermis was dissected for quantification of AM, CRLR, RAMP1, RAMP2 and RAMP3 expression using western blot analysis. Another group of male 16 weeks old SHR and WKY rats were anesthetized and a cannula was implanted in the cerebellar vermis. Following recovery AM (0.02 to 200 pmol/5μL) or vehicle was injected into cerebellar vermis. BP was determined, before and after treatments, by non-invasive plethysmography. In addition, to establish the receptor subtype involved in AM action in vivo, animals received microinjections of AM22-52 (200 pmol/5µL), an AM1 receptor antagonist, or the CGRP1 receptor antagonist, CGRP8-37 (200 pmol/5µL) into the cerebellar vermis, administered simultaneously with AM or vehicle microinjection. Cannulation was verified post mortem with the in situ injection of a dye solution. Our findings demonstrated that the expression of CRLR, RAMP1 and RAMP3 was higher in cerebellum of SHR rats, while AM and RAMP2 expression was lower than those of WKY rats, both in 8 and 16 week old rats. In vivo microinjection of AM into the cerebellar vermis caused a profound, dose dependent, hypotensive effect in SHR but not in normotensive WKY rats. Co-injections of a putative AM receptor antagonist, AM22–52 abolished the decreases in mean arterial pressure (MAP) evoked by AM, showing that AM acts through its AM1 receptor in the vermis to reduce MAP. These findings demonstrate a dysregulation of cerebellar AM-system during hypertension, and suggest that cerebellar AM plays an important role in the regulation of BP. Likewise; they constitute a novel mechanism of BP control which has not been described so far.
Keywords: Adrenomedullin, CRLR, RAMPs, cerebellum, vermis, AM22–52, hypertension.
Adrenomedullin is a regulatory peptide derived from the adrenal medulla, which is capable to raise platelet cyclic adenosine monophosphate (cAMP) levels. This peptide is composed by 52- (human) or 50-amino acid (rat) and shows some homology with calcitonin gene-related peptide (CGRP) (Kitamura et al., 1993) and has therefore been included to the amylin, CGRP and calcitonin super-family (Ichiki et al., 1994). AM actions are mediated through two specific receptors, the AM1 and AM2 receptors, formed from the obligate co-expression of a class-B, G protein coupled receptor, the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) 2 or 3, respectively (McLatchie et al., 1998). The calcitonin gene-related peptide 1 (CGRP1) receptor is formed of a complex between CRLR and RAMP1 (McLatchie et al., 1998). AM1 receptors are highly selective for AM over CGRP and other peptides, while AM2 receptors binds both AM and AM2 (intermedin) with high affinities (Hong et al., 2012). AM also has appreciable affinity for the CGRP1 receptor (Gibbons et al., 2007). AM and its receptors are found in the central nervous system, particularly they are localized to the autonomic nuclei, including nucleus tractus solitarii (NTS), lateral parabrachial nucleus (LPBN) and rostral ventrolateral medulla (RVLM), in cerebral cortex, pituitary gland, thalamus, hypothalamus, brainstem, amygdala and cerebellum (Serrano et al., 2000; Juaneda et al., 2003).
Our interest was mainly pointed to the cerebellum, since there is functional evidence suggesting that this organ may play a role in the regulation of arterial blood pressure (BP). In effect, it has been shown that stimulation of several regions of the cerebellum produces changes in arterial BP and heart rate (Nisimaru, 2004; Rector et al., 2006). Electric stimulus of cerebellar cortex posterior to vermis IX lobe, in anaesthetized rabbits induces bradycardia, BP fall and transient inhibition of sympathetic renal activity (Bradley et al., 1987a; Rocha et al., 2008). On the contrary, electric stimulation of the rostral region of fastigial nucleus in anaesthetized rabbits produces a pressor response (Bradley et al., 1987b).
The rational was if cerebellum plays a role in BP regulation, there should be a possible relationship between cerebellar AM and the regulation of cardiovascular function. Our pioneer work supported this idea, because when we evaluated the distribution and levels of AM receptor binding sites in the brain of 16 weeks old normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive (SHR) rats, using 125I-hAM13-52 as radioligand, we found a higher levels of AM binding sites in cerebellum of SHR when compared with WKY rats, suggesting that AM binding sites density are altered in cerebellum during hypertension (Pastorello et al., 2007), and indicating a possible role for cerebellum AM in the BP regulation. This increase in cerebellar AM receptors during hypertension was interpreted as an “up-regulation” mechanism of cerebellar binding sites to compensate the increase in BP in SHR rats; or they could represent a primary alteration which would result in a secondary alteration in the autonomic regulatory mechanisms in cerebellum with the consequent increase in BP.
If AM binding sites are altered during hypertension, we hypothesized that the expression and function of adrenomedullinergic system components could be altered in pathological conditions and possibly during growth. Our results point to this possibility since they demonstrate that RAMP1, RAMP3 and CRLR expression in rat cerebellar vermis increases with age, without changes for AM and RAMP2 (Figueira and Israel, 2015). To establish the pattern of cerebellar AM, CRLR, RAMP1, RAMP2 and RAMP3 expression during hypertension, we assessed their expression in 16 weeks old WKY and SHR rats (Figueira and Israel, 2015). Our results support the notion of a dysregulation of AM cerebellar system during hypertension, since they demonstrate an “up-regulation” of cerebellar CGRP1 (CRLR+RAMP1) and AM2 (CRLR+RAMP3) receptors and a “down-regulation” of AM1 (CRLR+RAMP2) receptor and a decreased expression of AM peptide during hypertension (Figure 1). Effectively, in cerebellar vermis CRLR, RAMP1 and RAMP3 expression was increased significantly while RAMP2 was reduced in SHR rats compared with WKY. The reduction in AM expression in vermis of SHR rats could be responsible for the up-regulation of AM2 receptors and binding sites observed by autoradiography (Pastorello et al., 2007; Figueira and Israel, 2015).
Figure 1. Effect of hypertension on AM and its receptor components expression in the cerebellar vermis. Under normotension condition AM, CRLR, RAMP1, RAMP2 and RAMP3 are expressed; but during hypertension there is an increased expression of CRLR, RAMP1 and RAMP3 and a decreased expression of RAMP2 and AM. During hypertension both AM and AM1 receptor are “down regulated” while AM2 and CGRP1 receptors are “up regulated”.
It is known that CRLR/RAMP2 complex constitutes the pathway for AM biological activity, and it is believed that altered expression of RAMPs is associated with alterations of AM response (Gibbons et al., 2007). In effect, during hypertension cerebellar cell phenotype changes from a high response (high expression of RAMP2) to a low response to AM (high expression of RAMP3) (Gibbons et al., 2007; Figueira and Israel, 2013a, 2015), changes which may contribute to the development of hypertension. In effect, during hypertension there are dynamic changes in the expression of genes involved in AM signaling, particularly involving a switch from RAMP2 to RAMP3 expression, which may underlie the marked decrease in AM expression in cerebellum and/or represent an altered responsiveness (Gibbons et al., 2007; Figueira and Israel, 2013a, 2015). Furthermore, up-regulation of RAMP1/RAMP3 expression would promote the interaction of AM with CGRP1 and AM2 receptors, rather than AM1 receptors, thereby favoring the compensatory mechanism to increased BP. Alternatively, these changes could be the initial disturbance that would result in dysregulation of the mechanisms controlling BP, since these changes are present from the early stages of life (8 weeks) of hypertensive rats Figueira and Israel, 2015).
Immediately it came to our thoughts the question that if cerebellar adrenomedullinergic system exerts a role in BP regulation, it would be plausible to expect that AM administered into the cerebellum in vivo may cause cardiovascular responses. Our findings pointed to this possibility since they demonstrated, for the first time, that microinjection of AM into the cerebellar vermis of hypertensive rats causes a powerful and significant hypotensive response, which is specific and dose-dependent. This hypotensive effect is manifested only during hypertension since AM microinjection into the cerebellar vermis was not able to reduce BP in normotensive rats (Figure 2). The specificity of the hypotensive action of AM administered into the cerebellar vermis was based on the fact that microinjection of the peptide outside the vermis did not cause the hypotensive effects in SHR, and in situ administration of a pressor peptide such as angiotensin II into cerebellar vermis did not altered BP (Figueira and Israel, 2013b). The hypotensive action of AM when administered into the cerebellar vermis of SHR rats was mediated through AM1 receptor, as AM22-52, an AM receptor specific antagonist, blunted AM’s effect when both agents were microinjected together, while the CGRP1 receptor antagonist, CGRP8-37 had no effect. These results provide the first functional evidence in vivo of a role for AM in the cerebellar vermis in the control of BP.
Figure 2. Effect of AM microinjection into the cerebellar vermis of WKY and SHR rats.
The possible cause of the difference in the AM action among normotensive and hypertensive rats may be variable. The hypotensive effect induced by the intracerebellar administration of AM in SHR could be due to an increase in sensitivity and response in SHR compared with WKY rats. Alternatively, this differential response may be the manifestation of the cerebellar dysregulation of signaling pathways, or AM and AM1 receptor expression which are reduced during hypertension (Figueira and Israel, 2013a, 2015).
In conclusion, our study demonstrated a dysregulation of cerebellar vermis AM and its receptor components during hypertension. In addition, they show that under hypertensive conditions AM administered into cerebellar vermis exerts a profound, dose-dependent hypotensive effect, which is mediated through AM1 receptor. These data suggest the existence of a cerebellar adrenomedullinergic system of physiological relevance in BP regulation. Likewise, they constitute a novel mechanism of BP control which has not been described so far.
This study was supported by Grants from People’s Ministry of Science, Technology and Industry, Science Mission Project, Subproject 7, ECCV No. 2007001585, PEII-20122000760 and CDCH-UCV PG-06-06-8669-2013.
Bradley D, Ghelarducci B, Paton J, Spyer K. The cardiovascular response elicited from the posterior cerebellar cortex in the anaesthetized and decerebrated rabbit. J Physiology 1987a; 383:537 – 550.
Bradley D, Paton J, Spyer K. Cardiovascular responses evoked from the fastigial region of the cerebellum in anaesthetized and decerebrated rabbits. J Physiol 1987b; 392:475 – 491.
Figueira F, Israel A. Role of cerebellar adrenomedullin in blood pressure regulation. Neuropeptides 2015c; pii: S0143-4179(15)00076-1. doi: 10.1016/j.npep.2015.07.003.
Figueira L, Israel A. Desregulación del sistema adrenomedulinérgico cerebeloso en la hipertensión arterial. Revista Latinoamericana de Hipertensión 2013a; 8 (1): 9-15.
Figueira L, Israel A. Efecto hipotensor de la adrenomedulina cerebelosa. Revista Latinoamericana de Hipertensión 2013b; 8 (3): 62-67.
Gibbons C, Dackor R, Dunworth W, Fritz-Six K, Caron K. Receptor Activity – modifying proteins: RAMPing up adrenomedullin signaling. Mol Endocrinol 2007; 21 (4): 783-796.
Hong Y, Hay DL, Quirion R, Poyner DR. The pharmacology of adrenomedullin 2/intermedin. Br J Pharmacol 2012; 166(1):110-120.
Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett 1994; 338: 6 – 10.
Juaneda C, Dumont Y, Chabot J, Fournier A, Quirion R. Adrenomedullin receptor binding sites in rat brain and peripheral tissues. Eur J Pharmacol 2003; 474:165 – 174.
Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commum 1993; 192: 553 – 560.
McLatchie L, Fraser N, Main M, Wise A, Brown J, Thompson N, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998; 393 (6683): 333-339.
Nisimaru N. Cardiovascular modules in the cerebellum. Jpn J Physiol 2004; 54: 431 – 448.
Pastorello M, Díaz E, Csibi A, Garrido M, Chabot J, Quirion R, et al. Papel de la adrenomedulina cerebelosa en la hipertensión arterial. Arch Ven Farmacol Terap 2007; 26 (2): 98 – 104.
Rector D, Richard C, Harper R. Cerebellar fastigial nuclei activity during blood pressure challenges. J Appl Physiol 2006; 101: 549 – 555.
Rocha I, Goncalves V, Bettencourt M, Silva L. Effect of stimulation of sublobule IX-b of the cerebellar vermis on cardiac function. Physiol Res 2008; 57: 701 – 707.
Serrano J, Uttenthal O, Martínez A, Fernández P, Martínez J, Alonso D, et al. Distribution of adrenomedullin – like inmunoreactivity in the rat central nervous system by light and electron microscopy. Brain Res 2000; 853:245 – 268.
Dra. Anita Israel
Laboratory of Neuropeptides
School of Pharmacy
Universidad Central de Venezuela