Microinjection of orexin into the rat nucleus tractus solitarius causes increases in blood pressure

Brain Research 950 (2002) 261–267 www.elsevier.com / locate / bres

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Microinjection of orexin into the rat nucleus tractus solitarius causes increases in blood pressure Pauline M. Smith, Barbara C. Connolly, Alastair V. Ferguson* Department of Physiology, Queen’ s University, Kingston, Ont. K7 L3 N6, Canada Accepted 4 June 2002

Abstract Orexin A (OX-A) and orexin B (OX-B), also known as hypocretin-1 and hypocretin-2, have been suggested to play a role cardiovascular control. The nucleus tractus solitarius (NTS), located in the dorsal medulla plays an essential role in neural control of the cardiovascular system. Orexin-immunoreactive axons have been demonstrated within this nucleus suggesting that NTS may be a site through which OX acts to influence cardiovascular control. We report here that microinjection of OX-A into the NTS of urethane anesthetized rats causes increases in blood pressure (10 29 M, mean AUC5607.1665.65 mmHg s, n55) and heart rate (10 29 M, mean AUC516.1563.3 beats, n55) which returns to baseline within 90 s. We show that these effects are dose related and site specific. Microinjection of OX-B into NTS elicited similar increases in BP (mean AUC5680.86128.5 mmHg s, n54) to that of OX-A suggesting specific actions at the OX 2 R receptor. These observations support the conclusion that orexins act as chemical messengers in the NTS likely influencing the excitability of cardiovascular neurons in this region and thus regulating global cardiovascular function.  2002 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Hypocretin; Cardiovascular regulation; Autonomic control

1. Introduction Orexin A (OX-A) and orexin B (OX-B), also known as hypocretin-1 and hypocretin-2, belong to a family of recently discovered hypothalamic neuropeptides derived from a single precursor, prepro-orexin, primarily localized in the lateral hypothalamus (LH) [29,22]. Proteolytic processing of the 130-amino-acid prepro-orexin produces OX-A, a 33-amino-acid peptide with two disulphide bonds in the N-terminal region and OX-B, a 28-amino-acid peptide devoid of any disulphide bonds. The name orexin was coined as a result of the initial observation that administration of the peptide into the lateral ventricle of rats resulted in an increase in food consumption. Since this initial discovery, these peptides have been shown to play a role in controlling the sleep– *Corresponding author. Tel.: 11-613-533-2803; fax: 11-613-5336880. E-mail address: [email protected] (A.V. Ferguson).

wake cycle [12,25], hormone secretion [21], and locomotor activity [9]. Two different orexin receptors have been described, the orexin-1 receptor (OX 1 R) and orexin-2 receptor (OX 2 R), both of which belong to the G-protein coupled cell-surface receptor family and are found only in the brain [22]. OX 2 R is considered to be nonselective in that it binds OX-A and OX-B with equal affinities. The OX 1 R, on the other hand, shows a selective affinity (30–100 times) for OX-A over OX-B and is therefore considered to be selective for OX-A [22]. Orexin producing neurons have been specifically localized within the dorsal and lateral hypothalamic and the perifornical area (LH / PFA) [29,22]. In contrast, the distribution of orexin receptors and orexin immunoreactive neuronal fibres are widely distributed throughout the brain [29,18], suggesting this peptide may play complex roles in the coordination of integrated physiological systems. Intracerebroventricular (i.c.v.) and intrathecal [2] administration of orexin has been shown to have clear effects on the

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03048-2

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cardiovascular system causing increases in blood pressure (BP) and heart rate (HR) [2,23,24,15] through modulation of sympathetic outflow [2,15]. In addition to these cardiovascular roles, OX-A and OX-B have been found to stimulate luteinizing hormone secretion in ovarectomized rats [19], while OX-A decreased circulating growth hormone and prolactin and increased circulating corticosterone [13] and insulin [16]. OX-A has also been shown to increase water consumption [11] and stimulate gastric acid secretion [27]. Evidence suggesting that the nucleus tractus solitarius (NTS) may mediate some of the actions of orexin stems from studies demonstrating the presence of orexin-immunoreactive axons [18,4] and both OX 1 R and OX 2 R mRNA [14] within this nucleus. In addition, i.c.v. administration of orexin induced c-fos expression in the NTS, indicating neuronal activation in this region [4]. The NTS is a medullary structure that has been implicated in a number of autonomic functions including cardiovascular regulation. Electrical stimulation in this region has been shown to cause increases in BP and HR [6]. Direct application of a number of vasoactive peptides influence a variety of cardiovascular variables [1,8]. Electrophysiological single unit recordings have also demonstrated the ability of many different peptides to influence the activity of NTS neurons in vivo and in vitro [17,10,20]. Taken together the above observations suggest roles for orexins as potential chemical messengers, released in the NTS from LH / PFA neurons, controlling central autonomic pathways involved in cardiovascular control. In this study we have therefore examined the effects of exogenous application of this peptide on BP and HR in anaesthetized rats.

2. Materials and methods

2.1. Animals and surgery Urethane anesthetized (1.4 g / kg) male Sprague–Dawley rats (150–300 g) were fitted with indwelling femoral arterial catheters for the measurement of BP and HR. Body temperature was maintained at 37 8C throughout the duration of the experiment using a feedback controlled heating blanket. Animals were placed in a stereotaxic frame with their head in the nose down position. The medulla was exposed at the level of the obex and a microinjection cannula (stainless steel, tip diameter 150 mm) was advanced into the region of NTS (midline–1.0 mm lateral, 0.3–2.0 mm ventral). Following a minimum 5 min stable

baseline recording period, a bolus (0.5 ml) microinjection of OX-A (10 211 –10 28 M, Phoenix Pharmaceuticals), dissolved in sterile saline, was administered and the effects on BP and HR assessed. A second, identical, bolus microinjection (OX-A, 10 29 M) was administered to the same site in several animals to determine the repeatability of the responses. In order to determine the receptor subtype (OX 1 R or OX 2 R) responsible for mediating the cardiovascular effects elicited in response to OX-A, a second set of experiments was performed whereby a bolus microinjection of OX-B (10 29 M, Phoenix Pharmaceuticals) was delivered into the region of NTS. At the end of the experiment animals were overdosed with anesthetic and perfused with 0.9% saline, followed by 10% formalin, through the left ventricle of the heart. The brain was removed and placed in formalin for at least 24 h. Using a vibratome, 50-mm coronal sections were cut through the region of NTS, mounted, and cresyl violet stained. The anatomical location of the microinjection site was verified at the light microscope level by an observer unaware of the experimental protocol or the data obtained.

2.2. Data analysis Animals were placed into one of two anatomical groups (NTS or non-NTS) according to the histological location of the microinjection site. NTS sites were those in which the tip of the injection pipette was located within the anatomical boundaries of commissural or medial NTS, while non-NTS sites included animals where this tip was located at least 0.2 mm from the border of this nucleus. Normalized BP and HR data (mean baseline BP (91.963.6 mmHg, n532) and HR (227.868.3 bpm, n532) was calculated for 60 s prior to injection and subtracted from all data points prior to and post injection) were obtained for each animal 60 s prior to the time of microinjection (control period) until 90 s postmicroinjection. The area under the curve (AUC, area between baseline and each BP or HR response) was calculated for each animal, for each dose, treatment, and anatomical location, and the mean AUC was then calculated for each of these factors as previously described [1,26]. A one-way ANOVA (followed by Newman–Keuls posthoc analysis) was used to determine whether any cardiovascular changes elicited by OX-A or OX-B microinjection were significant depending upon the OX isoform (OX-A or OX-B), concentration of OX administered, or anatomical location of the microinjection site.

Fig. 1. (A) Photomicrograph of the superimposed location of orexin microinjection in NTS (d, OX-A; h, OX-B), and non-NTS (s, OX-A sites). Scale bar5500 mm. Normalized BP (B) and HR (C) traces from a single animal demonstrate that a bolus (0.5 ml) microinjection of OX-A (10 29 M) into NTS causes a rapid, reversible increase in BP and HR (solid lines). A second, identical microinjection of OX-A into the same anatomical location in the same animal elicited a similar hypertensive and tachycardic effect (dashed line). Time of microinjection is indicated by the arrow.

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3. Results A total of 49 animals were used in the present study of which 20 were shown to have histologically confirmed NTS sites and 12 were determined to be non-NTS sites (see Fig. 1A). The remaining animals were excluded from analysis as the microinjection sites could not be reliably classified as either NTS or non-NTS sites. OX-A microinjection (0.5 ml, 10 29 M) into the NTS resulted in rapid increases in BP (mean AUC5 607.1665.64 mmHg s, n55) and HR (mean AUC5 16.1563.3 beats, n55) which returned to baseline within 90 s of OX-A administration as seen in Fig. 1B and C. These effects were normally found to be repeatable, as a subsequent identical microinjection into the same region of

NTS in the same animal caused similar hypertensive (see Fig. 1B) and tachycardic (see Fig. 1C) responses. These increases in BP elicited by OX-A microinjection into NTS were found to be dose related (10 28 M: mean AUC5754.06151.3 mmHg s, n53, P,0.01 compared to 10 211 M; 10 29 M, mean AUC5607.1665.64 mmHg s, n55, P,0.01 compared to 10 211 M; 10 210 M, mean AUC5275.26183.7 mmHg s, n55, P,0.05 compared to 10 211 M and 10 211 M, mean AUC5265.6640.7 mmHg s, n54, one-way ANOVA P,0.001) with an EC 50 of 2.05310 210 M (see Fig. 2). Similarly, the increases in HR were also dose related (10 28 M, mean AUC513.1262.48 beats, n53, P,0.05 compared to 10 211 M, P,0.01 compared to 10 210 M; 10 29 M, mean AUC516.1563.3 beats, n55, P,0.01 compared to 10 211 M and 10 210 M;

Fig. 2. Normalized mean BP traces demonstrate that cardiovascular effects elicited in response to microinjection of OX-A into the NTS are dose related (♦, 10 28 M, mean AUC5754.06151.3 mmHg s, n53; d, 10 29 M, mean AUC5607.1665.65 mmHg s, n55; m, 10 210 M, mean AUC5275.26183.7 mmHg s, n55; ., 10 211 M, mean AUC5265.6640.7 mmHg s, n54). Time of microinjection is indicated by the arrow. Inset: data were fitted to a sigmoid dose response function (EC 50 52.05310 210 M) and the corresponding curve was overlaid. (one-way ANOVA P,0.001, Newman–Keuls posthoc analysis, ***, P,0.001; *, P,0.05 vs. 10 211 M).

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10 210 M, mean AUC526.4763.9 beats, n53 and 10 211 M, mean AUC520.9262.83 beats, n54, one-way ANOVA P,0.0001) with an EC 50 55.7310 210 M. Importantly these hypertensive effects were also found

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to be site-specific as in strict contrast to the effects of OX-A (10 29 M) into NTS sites (see above), the peptide was found to be without effect on BP (mean AUC52 19.1654.9 mmHg s, n512, P.0.05, Fig. 3A) or HR

Fig. 3. (A) Normalized mean BP traces in response to a microinjection of OX-A (10 29 M) into NTS (d) or non-NTS (s) sites. Microinjection of OX-A into NTS causes a rapid reversible increases in BP whereas an identical microinjection into the AP is without significant effect. The time of microinjection is indicated by the arrow. Inset bar graph shows mean BP AUC for NTS (mean AUC5607.1665.65 mmHg s, n55, solid bar, ***, P,0.001) and non-NTS (mean AUC5219.1654.9 mmHg s, n512, open bar) elicited by OX-A microinjection into that anatomical location. (B) Normalized mean BP traces demonstrate that microinjection of 10 29 M OX-B (h) into the NTS elicits similar increases in BP as administration of 10 29 M OX-A (d) into the NTS, suggesting that OX 2 R is responsible for mediating the cardiovascular effects of orexin in the NTS. Time of microinjection is indicated by the arrow. Inset bar graph summarizes the mean AUC for BP following OX-A (mean AUC5607.1665.65 mmHg s, n55) or OX-B (mean AUC5680.8662.50 mmHg s, n55) microinjection into the NTS.

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(mean AUC524.862.4 beats n512, P.0.05) when microinjected into non-NTS sites. In order to determine the receptor subtype responsible for the increases in BP seen in response to OX-A administration into NTS, the effects of a bolus microinjection of OX-B (0.5 ml, 10 29 ) into this region was examined. As illustrated in Fig. 3B, microinjection of OX-B into NTS elicited similar increases in BP (mean AUC5680.86128.5 mmHg s, n55) to that of OX-A (mean AUC5 607.1665.65 mmHg s, n55: P.0.05).

4. Discussion Previous studies demonstrating that intracisternal injections of OX-A and OX-B dose dependently increased BP and HR in urethane anesthetized male Sprague–Dawley rats [3] suggest a medullary site of action. Our results reported here clearly demonstrate that microinjection of OX-A and OX-B into the rat NTS results in large, rapid, repeatable increases in BP and HR, while previous studies have shown longer lasting increases in BP and HR of similar magnitude following OX-A microinjection into the rostral ventrolateral medulla increased BP and HR [3]. The different profile of these responses could be explained by, different underlying mechanisms of action, different densities and distributions of OX receptors in these two regions, or differential diffusion to a common alternative site of action. The fact that hypertensive effects following NTS injections were shown to be site-specific (similar microinjections into anatomically adjacent regions dorsal, ventral and lateral to effective NTS sites were without similar cardiovascular effects) argues strongly against the latter possibility. In addition, the fact that the lowest concentration of OX-A delivered was without effect on BP or HR demonstrates that effects were not the result of mechanical disruption of the region. It is interesting to note that many of the non-NTS sites were located within the area postrema, a circumventricular structure containing high densities of orexin-immunoreactive fibres [11]. Despite these characteristics, and the well documented involvement of the area postrema in cardiovascular control [7,28], microinjection of orexins into this region did not have an effect on BP or HR. However, the fact that intravenous administration of orexins does not influence BP [3], while central administration of these peptides do, perhaps precludes the involvement of this or other CVOs as a target for circulating orexin actions in central control of cardiovascular regulation. In addition, the close proximity of immunoreactive OX-A and OX-B fibres to the lumen of the third and lateral ventricles suggests that this peptide may enter ventricular circulation and influence the activity of neurons distant from the hypothalamus, and that systemic orexin has little involvement in central control of BP. Although the receptor subtype (OX 1 R or OX 2 R) respon-

sible for this hypertensive effect was not conclusively elucidated in the present study, the fact that similar increases in BP were elicited by both OX-A and OX-B microinjection into the NTS, suggests that the OX 2 R is responsible for this effect. The fact that neither OX-A nor OX-B are effective in stimulating food intake when microinjected directly into the NTS [5], suggests that the hypertensive effect elicited by these peptides is the result of activation of a specific group of cardiovascular neurons in this region likely receiving orexigenic input from the LH / PFA. Our data demonstrate that exogenous administration of OX into the NTS results in significant increases in BP and HR. These observations clearly establish a novel role for the orexins in controlling the cardiovascular system as a direct result of actions in the NTS. While such observations alone do not prove a neurotransmitter role for this peptide in cardiovascular function, they do satisfy one of the important primary criterion leading to such a conclusion. In order to confirm such a role in neuronal communication, future studies will need to address the consequences of potential cardiovascular effects of endogenous synaptic release of orexins into NTS following activation of LH / PFA neurons.

Acknowledgements This work was funded by a grant from the Heart and Stroke Foundation of Ontario.

References [1] M.A. Allen, P.M. Smith, A.V. Ferguson, Adrenomedullin microinjection into the area postrema increases blood pressure, Am. J. Physiol. 272 (1997) R1698–R1703. [2] V.R. Antunes, G.C. Brailoiu, E.H. Kwok, P. Scruggs, N.J. Dun, Orexins / hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro, Am. J. Physiol. Regul. Integr. Comp. Physiol. 281 (2001) R1801–R1807. [3] C.T. Chen, L.L. Hwang, J.K. Chang, N.J. Dun, Pressor effects of orexins injected intracisternally and to rostral ventrolateral medulla of anesthetized rats, Am. J. Physiol. Regul. Integr. Comp. Physiol. 278 (2000) R692–R697. [4] Y. Date, Y. Ueta, H. Yamashita, H. Yamaguchi, S. Matsukura, K. Kangawa, T. Sakurai, M. Yanagisawa, M. Nakazato, Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems, Proc. Natl. Acad. Sci. USA 96 (1999) 748–753. [5] M.G. Dube, S.P. Kalra, P.S. Kalra, Food intake elicited by central administration of orexins / hypocretins: identification of hypothalamic sites of action, Brain Res. 842 (1999) 473–477. [6] A.V. Ferguson, P. Marcus, Area postrema stimulation induced cardiovascular changes in the rat, Am. J. Physiol. 255 (1988) R855–R860. [7] A.V. Ferguson, K.M. Wall, Central actions of angiotensin in cardiovascular control: multiple roles for a single peptide, Can. J. Physiol. Pharmacol. 70 (1992) 779–785.

P.M. Smith et al. / Brain Research 950 (2002) 261–267 [8] R.F. Furchgott, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53 (1983) 557–573. [9] J.J. Hagan, R.A. Leslie, S. Patel, M.L. Evans, T.A. Wattam, S. Holmes, C.D. Benham, S.G. Taylor, C. Routledge, P. Hemmati, R.P. Munton, T.E. Ashmeade, A.S. Shah, J.P. Hatcher, P.D. Hatcher, D.N. Jones, M.I. Smith, D.C. Piper, A.J. Hunter, R.A. Porter, N. Upton, Orexin A activates locus coeruleus cell firing and increases arousal in the rat, Proc. Natl. Acad. Sci. USA 96 (1999) 10911–10916. [10] A.A. Hegarty, L.F. Hayward, R.B. Felder, Influence of circulating angiotensin II and vasopressin on neurons of the nucleus of the solitary tract, Am. J. Physiol. 270 (1996) R675–R681. [11] K. Kunii, A. Yamanaka, T. Nambu, I. Matsuzaki, K. Goto, T. Sakurai, Orexins / hypocretins regulate drinking behaviour, Brain Res. 842 (1999) 256–261. [12] L. Lin, J. Faraco, R. Li, H. Kadotani, W. Rogers, X. Lin, X. Qiu, P.J. de Jong, S. Nishino, E. Mignot, The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene, Cell 98 (1999) 365–376. [13] L.K. Malendowicz, A. Hochol, A. Ziolkowska, M. Nowak, L. Gottardo, G.G. Nussdorfer, Prolonged orexin administration stimulates steroid-hormone secretion, acting directly on the rat adrenal gland, Int. J. Mol. Med. 7 (2001) 401–404. [14] J.N. Marcus, C.J. Aschkenasi, C.E. Lee, R.M. Chemelli, C.B. Saper, M. Yanagisawa, J.K. Elmquist, Differential expression of orexin receptors 1 and 2 in the rat brain, J. Comp. Neurol. 435 (2001) 6–25. [15] K. Matsumura, T. Tsuchihashi, I. Abe, Central orexin-A augments sympathoadrenal outflow in conscious rabbits, Hypertension 37 (2001) 1382–1387. [16] K.W. Nowak, P. Mackowiak, M.M. Switonska, M. Fabis, L.K. Malendowicz, Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies, Life Sci. 66 (2000) 449–454. [17] S. Papas, P. Smith, A.V. Ferguson, Electrophysiological evidence that systemic angiotensin influences rat area postrema neurons, Am. J. Physiol. 258 (1990) R70–R76. [18] C. Peyron, D.K. Tighe, A.N. van den Pol, L. de Lecea, H.C. Heller, J.G. Sutcliffe, T.S. Kilduff, Neurons containing hypocretin (orexin) project to multiple neuronal systems, J. Neurosci. 18 (1998) 9996– 10015. [19] S. Pu, M.R. Jain, P.S. Kalra, S.P. Kalra, Orexins, a novel family of hypothalamic neuropeptides, modulate pituitary luteinizing hormone

[20]

[21]

[22]

[23]

[24]

[25] [26]

[27]

[28]

[29]

267

secretion in an ovarian steroid-dependent manner, Regul. Pept. 78 (1998) 133–136. L. Qu, A.J. Mcqueeney, K.L. Barnes, Presynaptic or postsynaptic location of receptors for angiotensin II and substance P in the medial solitary tract nucleus, J. Neurophysiol. 75 (1996) 2220–2228. S.H. Russell, M.S. Kim, C.J. Small, C.R. Abbott, D.G. Morgan, S. Taheri, K.G. Murphy, J.F. Todd, M.A. Ghatei, S.R. Bloom, Central administration of orexin A suppresses basal and domperidone stimulated plasma prolactin, J. Neuroendocrinol. 12 (2000) 1213– 1218. T. Sakurai, A. Amemiya, M. Ishii, I. Matsuzaki, R.M. Chemelli, H. Tanaka, S.C. Williams, J.A. Richardson, G.P. Kozlowski, S. Wilson, J.R. Arch, R.E. Buckingham, A.C. Haynes, S.A. Carr, R.S. Annan, D.E. McNulty, W.S. Liu, J.A. Terrett, N.A. Elshbagy, D.J. Bergsma, M. Yanagisawa, Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior [see comments], Cell 92 (1998) 573–585. W.K. Samson, B. Gosnell, J.K. Chang, Z.T. Resch, T.C. Murphy, Cardiovascular regulatory actions of the hypocretins in brain, Brain Res. 831 (1999) 248–253. T. Shirasaka, M. Nakazato, S. Matsukura, M. Takasaki, H. Kannan, Sympathetic and cardiovascular actions of orexins in conscious rats, Am. J. Physiol. 277 (1999) R1780–R1785. J.M. Siegel, Narcolepsy: a key role for hypocretins (orexins) [comment], Cell 98 (1999) 409–412. P.M. Smith, A.V. Ferguson, Adrenomedullin acts in the rat paraventricular nucleus to decrease blood pressure, J. Neuroendocrinol. 13 (2001) 467–471. N. Takahashi, T. Okumura, H. Yamada, Y. Kohgo, Stimulation of gastric acid secretion by centrally administered orexin-A in conscious rats, Biochem. Biophys. Res. Commun. 254 (1999) 623–627. K. Takeda, T. Nakata, T. Takesako, H. Itoh, M. Hirata, S. Kawasaki, J. Hayashi, M. Oguro, S. Sasaki, M. Nakagawa, Sympathetic inhibition and attenuation of spontaneous hypertension by PVN lesions in rats, Brain Res. 543 (1991) 296–300. L. de Lecea, T.S. Kilduff, C. Peyron, X. Gao, P.E. Foye, P.E. Danielson, C. Fukuhara, E.L. Battenberg, V.T. Gautvik, F.S. Bartlett, W.N. Frankel, A.N. van den Pol, F.E. Bloom, K.M. Gautvik, J.G. Sutcliffe, The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity, Proc. Natl. Acad. Sci. USA 95 (1998) 322–327.