Role of brain adrenoceptors in the corticortopin-releasing factor-induced central activation of sympatho-adrenomedullary outflow in rats

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Life Sciences 82 (2008) 487 – 494 www.elsevier.com/locate/lifescie

Role of brain adrenoceptors in the corticortopin-releasing factor-induced central activation of sympatho-adrenomedullary outflow in rats Mieko Yorimitsu, Shoshiro Okada ⁎, Naoko Yamaguchi-Shima, Takahiro Shimizu, Junichi Arai, Kunihiko Yokotani Department of Pharmacology, Graduate School of Medicine, Kochi University, Nankoku, Kochi 783-8505, Japan Received 28 August 2007; received in revised form 30 November 2007; accepted 4 December 2007

Abstract We investigated the role played by catecholamine-dependent pathways in modulating the ability of centrally administered corticotropin releasing factor (CRF) to activate sympatho-adrenomedullay outflow, using urethane-anesthetized rats. The CRF (1.5 nmol/animal, i.c.v.)-induced elevations of both plasma noradrenaline and adrenaline were attenuated by phentolamine (a non-selective α adrenoceptor antagonist) [125 and 250 µg (0.33 and 0.66 µmol)/animal], Heat (a selective α1 adrenoceptor antagonist) [10 and 30 µg (30 and 90 nmol)/animal, i.c.v.] and clonidine (a selective α2 adrenoceptor agonist) [100 µg (0.375 µmol)/animal, i.c.v.]. On the other hand, the CRF (1.5 nmol/animal, i.c.v.)-induced elevation of both catecholamines was not influenced by RS 79948 (a selective α2 adrenoceptor antagonist) [10 and 30 µg (7.2 and 72 nmol)/animal, i.c.v.]. Furthermore, the CRF (1.5 nmol/animal, i.c.v.)-induced elevation of noradrenaline was attenuated by sotalol (a non-selective β adrenoceptor antagonist) [125 and 250 µg (0.4 and 0.8 µmol)/animal, i.c.v.], while that of adrenaline was not influenced by sotalol. These results suggest that centrally administered CRF-induced elevation of plasma noradrenaline is mediated by an activation of α1 and β adrenoceptors in the brain, and that of plasma adrenaline is mediated by an activation of α1 adrenoceptors in the brain. Furthermore, central α2 adrenoceptors are involved in modulating the CRF-induced elevation of both plasma catecholamines. © 2007 Elsevier Inc. All rights reserved. Keywords: Adrenoceptor; Brain; Corticotropin-releasing factor; Plasma catecholamine

Introduction Corticotropin-releasing factor (CRF), a 41 amino acid peptide, has a long established functional role to stimulate the synthesis and secretion of adrenocorticotropic hormone, and thus initiates pituitary-adrenal responses to stress (Chadwick et al., 1993). The peptide also exhibits a broad distribution in brain acting as a neurotransmitter/neuromodulator (Swanson et al., 1983), and when administered centrally evokes indices of autonomic activation, resulting in stress-like increases in blood pressure and heart rate, plasma noradrenaline and adrenaline (Brown et al., 1982; Korte et al., 1993). In addition, intracere-

⁎ Corresponding author. Tel./fax: +81 88 880 2328. E-mail address: [email protected] (S. Okada). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.12.006

broventricularly administered CRF elicits Fos expression in cell groups that are involved in central autonomic control, including paraventricular hypothalamic nuclei and brainstem catecholaminergic cell groups (Bittencourt and Sawchenko, 2000). These observations suggest a role for CRF in regulation of several autonomic responses in the brain. However, the central mechanisms underlying these actions of CRF are still not fully defined. Noradrenaline has been implicated as a primary neurotransmitter of central autonomic regulation (McCall, 1988). Several studies indicated a close involvement of central noradrenergic neurons in the maintenance of circulatory control and the regulation of sympathetic outflow (Lightman et al., 1984; Woodruff et al., 1986). Furthermore, recent reports suggest a role for α2adrenergic receptor-mediated mechanisms in central autonomic responses (Schreihofer and Guyenet, 2000; Li et al., 2005).

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Anatomical evidence suggests that CRF-containing terminals synaptically contact cell bodies of noradrenergic neurons in the brainstem, including the locus coeruleus (Swanson et al., 1983; Van Bockstaele et al., 1996; Reyes et al., 2005). Electrophysiological evidence suggests that CRF can activate locus coeruleus noradrenergic neurons (Valentino et al., 1983; Page and Abercrombie, 1999). Furthermore, it has been demonstrated that centrally administered CRF stimulates the release of noradrenaline in the brain (Lavicky and Dunn, 1993). These observations suggest a functional interaction between CRF system and noradrenergic neurons in the brain (Koob, 1999). In the present study, we investigated a role for central adrenoceptors in modulating the elevation of plasma noradrenaline and adrenaline induced by intracerebroventricularly administered CRF using urethane-anesthetized rats. Materials and methods Experimental procedures Male Wistar rats weighing about 350 g were maintained in an air-conditioned room at 22–24 °C under a constant day-night rhythm for more than 2 weeks and given food (laboratory chow, CE-2; Clea Japan, Hamamatsu, Japan) and water ad libitum. Under urethane anesthesia (1.0 g/kg, i.p.), the femoral vein was cannulated for infusion of saline (1.2 ml/h) and the femoral artery was cannulated for collecting blood samples, as shown in our previous report (Okada et al., 2003a). After these procedures the animal was placed in a stereotaxic apparatus as shown previously (Yokotani et al., 2001). The skull was drilled for intracerebroventricular administration of test substances using a stainless-steel cannula (0.3 mm outer diameter). The stereotaxic coordinates of the tip of the cannula were as follows (in mm): AP-0.8, L 1.5, V 4.0 (AP, anterior from the bregma; L, lateral from the midline; V, below the surface of the brain), according to the rat brain atlas of Paxinos and Watson (1997). Three hours were allowed to elapse before the application of CRF or the application of blocking reagents such as phentolamine (non selective a adrenoceptor antagonist), Heat (selective α1 adrenoceptor antagonist), RS-79948 (selective α2 adrenoceptor antagonist), clonidine (selective α2 adrenoceptor agonist), and sotalol (non-selective β adrenoceptor antagonist). In the case of the application of blocking reagents, CRF was intracerebroventricularly administered 20 min after application of phentolamine, Heat, RS-79948, clonidine or sotalol. Correct placement of the cannula was confirmed at the end of experiments by verifying that a blue dye, injected through the cannula, had spread throughout the entire ventricular system. All experiments were conducted in compliance with the guiding principles for the care and use of laboratory animals approved by Kochi University.

mines in the plasma were extracted by the method of Anton and Sayre (1962) with a slight modification and were assayed electrochemically with high-performance liquid chromatography (HPLC) (Okada et al., 2003a). Briefly, after centrifugation, plasma (100 µl) was transferred to a sample tube containing 30 mg of activated alumina, 1 ng of 3,4-dihydroxybenzylamine as an internal standard and 3 ml of 0.5 M Tris Buffer (pH 8.6) containing 0.1 M disodium EDTA. The tube was shaken for 10 min and the alumina was washed three times with 4 ml of ice-cold double deionized water. Catecholamines adsorbed onto the alumina were eluted with 300 µl of 4% acetic acid containing 0.1 mM disodium EDTA. A pump (EP-300: Eicom, Kyoto, Japan), a sample injector (Model-231XL; Gilson, Villiers-le-Bel, France) and an electrochemical detector (ECD-300: Eicom) equipped with a graphite electrode were used with HPLC. Analytical conditions were as follows: detector +450 mV potential against an Ag/AgCl reference electrode; column, Eicompack CA-50DS, 2.1 × 150 mm (Eicom); mobile phase, 0.1 M NaH2PO4–Na2HPO4 buffer (pH 6.0) containing 50 mg/l EDTA dihydrate, 750 mg/l 1-octane sulfate sodium (Nacalai Tesque, Kyoto, Japan) and 15% methanol at a flow rate of 0.18 ml/min. The amount of catecholamines in each sample was calculated using the peak height ratio relative to that of 3,4-dihydroxybenzylamine, an internal standard. This assay could determine 0.5 pg of adrenaline and noradrenaline accurately. Treatment of data and statistics Results are expressed as the means ± S.E.M. of the net changes above the respective basal values. The data were analyzed by repeated-measure analysis of variance (ANOVA), followed by post-hoc analysis with the Bonferroni method (Figs. 1, 2, 3, 4, 5A and B). P values less than 0.05 were taken to indicate statistical significance. Reagents The following drugs were used: CRF (rat/human) (Peptide Institute, Osaka, Japan); phentolamine, sotalol (Sigma Aldrich Fine Chemicals, St. Louis, MO, U.S.A.), Heat; (2-{[b-(4hydroxyphenyl)ethyl]aminomethyl}-1tetralone, RS-79948; (8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6] naphthylridine (Tocris Cookson Inc. Northpoint, U.K.). All other reagents were the highest grade available (Nacalai Tesque, Kyoto, Japan). Results Effects of phentolamine, a non-selective α adrenoceptor antagonist, on the CRF-induced elevation of plasma levels of catecholamines

Measurement of plasma catecholamines Blood samples (250 μl) were collected through an arterial catheter and were preserved on ice during experiments. Plasma was prepared immediately after the final sampling. Catechola-

Previously, we reported that intracerebroventricularly (i.c.v.) administered CRF (0.5, 1.5 and 3.0 nmol/animal) dosedependently increased plasma noradrenaline and adrenaline (Yokotani et al., 2001; Okada et al., 2003a). In the present

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animal, i.c.v.)-induced elevation of plasma noradrenaline and adrenaline. Effect of heat, a selective α1 adrenoceptor antagonist, on the CRF-induced elevation of plasma catecholamines Pretreatment with heat [30 µg (90 nmol)/animal, i.c.v.] had no effect on the basal plasma levels of catecholamines (Fig. 2). Heat dose-dependently attenuated the CRF (1.5 nmol/animal, i.c.v.)induced elevation of plasma noradrenaline and adrenaline.

Fig. 1. Effect of phentolamine, a non-selective α adrenoceptor antagonist, on the CRF-induced elevation of plasma levels of catecholamines. Increase in Plasma Noradrenaline or Adrenaline; Increase of noradrenaline or adrenaline above the basal. Phentolamine [125 and 250 µg (0.33 and 0.66 µmol/animal, i.c.v.)] or vehicle-1 (5 µl saline/animal, i.c.v.) was administered 20 min before the administration of CRF (1.5 nmol/animal, i.c.v.) or vehicle-2 (10 µl saline/animal, i.c.v.). ●, vehicle-1 plus CRF (n = 6); ▲, phentolamine (125 µg/animal) plus CRF (n = 7); ■, phentolamine (250 µg/animal) plus CRF (n = 9); □, phentolamine (250 µg/animal) plus vehicle-2 (n = 4); ○, vehicle-1 plus vehicle-2 (n = 4). Arrow indicates intracerebroventricular administration of vehicles or reagents (phentolamine and CRF). Each point represents the mean ± S.E.M. ⁎Significantly different (p b 0.05) from vehicle-treated group with the Bonferroni method. The actual values for noradrenaline and adrenaline at 0 min were 362.0 ± 59.9 and 169.7 ± 43.0 pg/ml in the vehicle-1-pretreated group (n = 10) and 429.4 ± 47.2 and 197.3 ± 73.8 pg/ml in the phentolamine (125 µg/animal)-pretreated group (n = 7) and 402.1 ± 47.9 and 320.9 ± 67.7 pg/ml in the phentolamine (250 µg/animal)pretreated group (n = 13), respectively.

experiments, therefore, we used the dose of 1.5 nmol/animal of CRF. Administration by i.c.v. of vehicle-1 (5 µl of saline/animal), vehicle-2 (10 µl of saline/animal) or phentolamine [250 µg (0.66 µmol)/animal] followed by blood sampling five times over a 120 min-period had no effect on the basal plasma levels of either noradrenaline or adrenaline (Fig. 1). The phentolamine dose-dependently attenuated the CRF (1.5 nmol/

Fig. 2. Effect of heat, a selective α1 adrenoceptor antagonist, on the CRFinduced elevation of plasma catecholamines. Heat [3 and 30 µg (9 and 90 nmol/ animal, i.c.v.)] or vehicle-1 (5 µl saline/animal, i.c.v.) was administered 20 min before the administration of CRF (1.5 nmol/animal, i.c.v.) or vehicle-2 (10 µl saline/animal, i.c.v.). ●, vehicle-1 plus CRF (n = 6) (cited from Fig. 1) ; ▲, Heat (3 µg/animal) plus CRF (n = 5); ■, Heat (30 µg/animal) plus CRF (n = 7); □, Heat (30 µg/animal) plus vehicle-2 (n = 3); ○, vehicle-1 plus vehicle-2 (n = 4) (cited from Fig. 1). Arrow indicates intracerebroventricular administration of vehicles or reagents (Heat and CRF). ⁎Significantly different (p b 0.05) from vehicle-1-and CRF-treated group with the Bonferroni method. Other conditions were the same as those of Fig. 1. The actual values for noradrenaline and adrenaline at 0 min were 362.0 ± 59.9 and 169.7 ± 43.0 pg/ml in the vehicle-1pretreated group (n = 10) and 368.1 ± 42.4 and 67.4 ± 12.6 pg/ml in the Heat (3 µg/animal)-pretreated group (n = 5) and 262.8 ± 68.1 and 163.9 ± 19.6 pg/ml in the Heat (30 µg/animal)-pretreated group (n = 10), respectively.

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Effect of clonidine, a selective α2 adrenoceptor agonist, on the CRF-induced elevation of plasma catecholamines Pretreatment with clonidine [100 µg (375 nmol)/animal, i.c.v.] had no effect on the basal plasma levels of catecholamines (Fig. 4). Although the low dose [10 µg (37.5 nmol)/animal, i.c.v.] of clonidine had no effect on the CRF-induced elevation of both of catecholamines, the high dose [100 µg (375 nmol)/animal,

Fig. 3. Effect of RS 79948, a selective α2 adrenoceptor antagonist, on the CRFinduced elevation of plasma catecholamines. RS 79948 [3 and 30 µg (7.2 and 72 nmol/animal, i.c.v.)] or vehicle-1 (5 µl saline/animal, i.c.v.) was administered 20 min before the administration of CRF (1.5 nmol/animal, i.c.v.) or vehicle-2 (10 µl saline/animal, i.c.v.). ●, vehicle-1 plus CRF (n = 6) (cited from Fig. 1) ; ▲, RS 79948 (3 µg/animal) plus CRF (n = 9); ■, RS 79948 (30 µg/animal) plus CRF (n = 5); □, RS 79948 (30 µg/animal) plus vehicle-2 (n = 3); ○, vehicle-1 plus vehicle-2 (n = 4) (cited from Fig.1). Arrow indicates intracerebroventricular administration of vehicles or reagents (RS 79948 and CRF). ⁎Significantly different (p b 0.05) from vehicle-1-and CRF-treated group with the Bonferroni method. Other conditions were the same as those of Figs. 1 and 2. The actual values for noradrenaline and adrenaline at 0 min were 362.0 ± 59.9 and 169.7 ± 43.0 pg/ml in the vehicle-1-pretreated group (n = 10) and 521.6 ± 33.5 and 203.9 ± 42.7 pg/ml in the RS 79948 (3 µg/animal)-pretreated group (n= 9) and 420.7 ±55.3 and 151.8 ± 66 pg/ml in the RS 79948 (30 µg/animal)-pretreated group (n =8), respectively.

Effect of RS-79948, a selective α2 adrenoceptor antagonist, on the CRF-induced elevation of plasma catecholamines Pretreatment with RS-79948 [30 µg (72 nmol)/animal, i.c.v.] had no effect on the basal plasma levels of catecholamines (Fig. 3). RS-79948 [10 and 30 µg (24 and 72 nmol)/animal, i.c.v.] had no effect on the CRF-induced elevation of catecholamines.

Fig. 4. Effect of clonidine, a selective α2 adrenoceptor agonist, on the CRFinduced elevation of plasma catecholamines. Clonidine [10 and 100 µg (37.5 and 375 nmol/animal, i.c.v.)] or vehicle-1 (5 µl saline/animal, i.c.v.) was administered 20 min before the administration of CRF (1.5 nmol/animal, i.c.v.) or vehicle-2 (10 µl saline/animal, i.c.v.). ●, vehicle-1 plus CRF (n = 6) (cited from Fig. 1) ; ▲, clonidine (10 µg/animal) plus CRF (n = 5); ■, clonidine (100 µg/ animal) plus CRF (n = 6); □, clonidine (100 µg/animal) plus vehicle-2 (n = 4); ○, vehicle-1 plus vehicle-2 (n = 4) (cited from Fig. 1). Arrow indicates intracerebroventricular administration of vehicles or reagents (clonidine and CRF). ⁎Significantly different (p b 0.05) from vehicle-1-and CRF-treated group with the Bonferroni method. Other conditions were the same as those of Figs. 1, 2 and 3. The actual values for noradrenaline and adrenaline at 0 min were 362.0± 59.9 and 169.7 ± 43.0 pg/ml in the vehicle-1-pretreated group (n = 10) and 281.9 ± 30.4 and 63.4 ± 14.0 pg/ml in the clonidine (10 µg/animal)-pretreated group (n = 5) and 204.9 ± 50.8 and 120.7 ± 24.0 pg/ml in the clonidine (100 µg/ animal)-pretreated group (n = 10), respectively.

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Fig. 5. Effect of sotalol, a non-selective β adrenoceptor antagonist, on the CRF-induced elevation of plasma levels of catecholamines. Increase in Plasma Noradrenaline or Adrenaline; Increase of noradrenaline or adrenaline above the basal. Sotalol [125 and 250 µg (0.4 and 0.8 µmol/animal, i.c.v.)] or vehicle-1 (V-1) (5 µl saline/animal, i.c.v.) was administered 20 min before the administration of CRF (1.5 nmol/animal, i.c.v.) or vehicle-2 (V-2) (10 µl saline/animal, i.c.v.). (A) ●, vehicle-1 plus CRF (n = 6) (cited from Fig. 1); ▲, sotalol (125 µg/animal) plus CRF (n = 9); ■, sotalol (250 µg/animal) plus CRF (n = 8); □, sotalol (250 µg/animal) plus vehicle-2 (n = 4); ○, vehicle-1 plus vehicle-2 (n = 4) (cited from Fig.1). Arrow indicates intracerebroventricular administration of vehicles or reagents (sotalol and CRF). ⁎Significantly different (p b 0.05) from vehicle-1- and CRF-treated group with the Bonferroni method. Other conditions were the same as those of Figs. 1, 2, 3 and 4. (B) The area under the curve (AUC) of the CRF-induced elevation of plasma catecholamines above the basal in the presence or absence of sotalol is expressed as pg/2 h. Other conditions were the same as those of Figs. 1, 2, 3 and 4. The actual values for noradrenaline and adrenaline at 0 min in (A) were 362.0 ± 59.9 and 169.7 ± 43.0 pg/ml in the vehicle-1-pretreated group (n = 10) and 280.0 ± 37.2 and 205.2 ± 36.8 pg/ml in the sotalol (125 µg/animal)-pretreated group (n = 9) and 262.7 ± 28.8 and 143.6 ± 30.4 pg/ml in the sotalol (250 µg/animal)-pretreated group (n = 12), respectively.

i.c.v.] of clonidine significantly attenuated the CRF-induced elevation of both catecholamines. Pretreatment with intravenous clonidine [100 µg (375 nmol)/ animal] had no effect on the CRF-induced elevation of both catecholamines (results not shown). Effect of sotalol, a non-selective β adrenoceptor antagonist, on the CRF-induced elevation of plasma catecholamines

while the reagent had no effect on the elevation of adrenaline induced by this peptide (Fig. 5A). When using the area under the curve to observe the effect of sotalol, the reagent significantly and dose-dependently reduced the CRF-induced elevation of plasma noradrenaline levels (Fig. 5B). On the other hand, the sotalol did not significantly reduce the CRF-induced elevation of plasma adrenaline levels (Fig. 5B). Discussion

Pretreatment with sotalol [250 µg (0.8 µmol)/animal, i.c.v.] had no effect on the basal plasma levels of catecholamines (Fig. 5). The sotalol dose-dependently attenuated the elevation of noradrenaline induced by CRF (1.5 nmol/animal, i.c.v.),

Adrenoceptors can be divided into two broad categories, α and β, or more correctly classified into three major subcategories, α1, α2 and β (Docherty, 1998). We first investigated

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the effect of the non-selective α-adrenergic antagonist phentolamine on the CRF-induced elevations of both catecholamines. Central pretreatment with phentolamine effectively reduced the CRF-induced elevations of both plasma catecholamines. It has been demonstrated that intracerebroventricular pretreatment with phentolamine reduced the pressor responses induced by intracerebroventricular injection of catecholamines or acute cold water startle (McCall and Humphrey, 1981; Peres-Polon and Correa, 1987; Tan et al., 2003). In addition, several lines of evidence have indicated that centrally applied CRF induces an activation of noradrenergic neurons in the brain and an enhancement of noradrenaline release in some brain regions, including the anterior hypothalamus known to regulate autonomic responses (Emoto et al., 1993; Curtis et al., 1997). Taken together, it would be reasonable to assume that intracerebroventricularly administered CRF activates central noradrenergic neurons and the resulting noradrenaline that is released elevates both plasma catecholamines via an activation of the central α-adrenoceptors. Central α1-adrenoceptors have been proposed to be involved in central nervous system blood pressure control since central pretreatment with prazosin, a conventional selective α1 adrenergic antagonist, reduced the sympatho-excitation (McCall and Humphrey, 1981; Correa and Peres-Polon, 1995). Furthermore, a recent report suggests that activation of α1 adrenergic receptors increases the excitability of spinally projecting presympathetic neurons in the hypothalamic paraventricular nucleus (Chen et al., 2006). In the next experiment, we investigated the effect of selective α1 adrenergic antagonist heat (Lima et al., 2005) on the CRF-induced elevations of both catecholamines. Because of the water solubility for intracerebroventricular administration, we used heat instead of prazosin. Central pretreatment with heat clearly and effectively attenuated the CRF-induced elevations of both plasma catecholamines. Alpha1-adrenoceptors have been well established as proteins composed of seven transmembrane domains, with the third intracellular loop being crucial for coupling to the guanine nucleotide regulatory protein (G-protein) (Cotecchia et al., 1990; Blitzer et al., 1993). Activation of this G-protein promotes phospholipase C activation and the production of two distinct second messengers, diacylglycerol and inositol triphosphate (Summers and McMartin 1993). Previously, we reported that central pretreatment with inhibitors for phospholipase C and diacylglycerol lipase, related to signaling cascades downstream of the α1 adrenoceptors, effectively attenuated the CRFinduced elevations of both catecholamines (Okada et al., 2003a). Taking these observations into account, it would be reasonable to assume that central α1-adrenoceptor-mediated mechanisms play an important component of the intracerebroventricularly administered CRF-induced sympatho-adrenomedullary outflow in rats. Alpha2-adrenoceptors are distributed throughout the central nervous system and mediate a multitude of functions, such as sedation, analgesia, and sympatho-inhibition (Ruffolo et al., 1993). It is generally accepted that the blockade of the α2adrenoceptor function results in an enhanced noradrenaline release as well as in the stimulation of postsynaptic β-and α1receptors (Ruffolo et al., 1993). It has been shown that blockade

of α2-adrenoceptors in the locus coeruleus produces an increase in firing rate, resulting in enhancement of noradrenaline release from axon terminals (Simson et al., 1988). Furthermore, a recent report indicates that the sympatho-excitatory effect of α2adrenoceptor antagonists is due to the blockade of a tonic activation of α2A-adrenoceptors located into the rostral ventral pressor area (Vayssettes-Courchay et al., 2002). In the present study, however, pretreatment with RS-79948, a selective α2adrenoceptor antagonist (Uhlen et al., 1998), but non-selective among the α2-adrenoceptor subtypes, did not alter the effects of the CRF-induced elevation of both plasma catecholamines. Although we have no explanation for this result at present, one possibility is that the near maximal effect of high dose (1.5 nmol) of CRF could not be further increased by RS-79948 or another possibility is that RS-79948 may stimulate noradrenaline release presynaptically, but also block postsynaptic α2-adrenoceptors (Haller et al., 1998). Several studies indicated that stimulation of central α2adrenoceptors elicits a decrease in blood pressure in animals through a reduction of sympathetic nerve activity (Schmitt and Fenard, 1971; Timmermans et al., 1981; Gillis et al., 1985). In addition, it has been shown that the major effect of α2adrenoceptor stimulation is an inhibition of the firing activity of noradrenaergic neurons in the locus coeruleus (Svensson et al., 1975; Pineda et al., 1997). In the present study, pretreatment with a high dose of clonidine, a selective α2-adrenoceptor agonist, effectively attenuated the CRF-induced elevation of both plasma catecholamines. Taking these observations into account, it would be reasonable to assume that α2-adrenoceptors play a regulatory role in the CRF-induced sympathoadrenomedullary outflow. There is, however, contradictory evidence that microinjection of clonidine into the paraventricular hypothalamic nucleus increases the blood pressure in conscious rats (Ebihara et al., 1993). Furthermore, a recent report suggests that stimulation of α2-adrenoceptors primarily attenuates GABAergic inputs to spinally projecting neurons in the paraventricular hypothalamic nucleus, resulting in excitation of preautonomic neurons (Li et al., 2005). The difference in the results may be due to different experimental conditions. They used conscious rats or slice preparations, while we used anesthetized rats. However, whether this experimental difference can explain the difference between the present results and the results of Ebihara et al. (1993) and Li et al. (2005) remains unresolved. There is considerable evidence that β-adrenoceptors in the central nervous system play roles in autonomic regulations. It is well known that propranolol, a non-selective β-adrenoceptor antagonist, acts, in part, in the central nervous system as an antihypertensive agent (Scriabine et al., 1976). Furthermore, central β-adrenoceptors have been proposed to be involved in autonomic responses induced by acute stress (Koepke and DiBona, 1985; Tan et al., 2000; Rauls et al., 2005). In contrast to heat, pretreatment with sotalol, a non-selective β-adrenoceptor antagonist, effectively reduced the CRF-induced elevation of plasma noradrenaline, whereas the reagent was relatively ineffective in the CRF-induced elevation of plasma adrenaline. Previously, we reported that the CRF-induced elevation of plasma noradrenaline and adrenaline were separately regulated

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by brain prostanoid-mediated mechanisms (Yokotani et al., 2001; Yamaguchi-Shima et al., 2007). Since several studies indicate the involvement of β-adrenoceptor stimulation in the release of arachidonic acid and the production of prostanoids (Levine and Moskowitz, 1979; Weis and Malik, 1985; Schuller et al., 1999), the present results suggest the involvement of not only α1 adrenoceptors but also β adrenoceptors in the centrally administered CRF-induced elevation of plasma noradrenaline. Whether centrally administered β adrenergic agonist such as isoproterenol can also elevate plasma levels of noradrenaline, but not plasma adrenaline, in a brain prostanoid-dependent manner is an intriguing question that should be clarified in further studies. It is still obscure as to the brain sites that centrally administered CRF-induced brain catecholasmines released would act to cause sympatho-adrenomedullary outflow. Interestingly, it has been demonstrated that adrenal sympathetic preganglionic neurons can be physiologically segregated into two populations in responses to stimulation of rostral ventrolateral medulla (RVLM) (Morrison, 2001), a location of adrenal sympathetic premotor neurons (Strack et al., 1989). Furthermore, destruction of noradrenergic innervation of RVLM has been shown to reduce hypotensive responses by α2-adrenoceptor mechanisms (Chan et al., 2005). Collectively, one can assume that RVLM might be one of the brain sites responsible for the activation of adrenoceptors. In the present experiments, we used a rather high dose (1.5 nmol) of CRF (i.c.v.) according to our previous reports (Yokotani et al, 2001; Okada et al., 2003a,b; Yamaguchi-Shima et al., 2007). However, CRF administered centrally even in a dose of 0.75 pmol has been shown to increase arterial blood pressure and heart rate in conscious rats (Diamant et al., 1992). Therefore, the data obtained using the dose of CRF in the present study would need to be interpreted cautiously when discussing their physiological significance. Although the half-life of peptides such as CRF is short (Schulte et al., 1984; Nink et al., 1994), in the present experiment, the effect of CRF is shown to start immediately after its intracerebroventricular injection and lasts for over 2 h. Since we previously reported that brain inducible nitric oxide synthase is involved in the intracerebroventricularly administered CRF-induced sympatho-adrenomedullary outflow in rats (Okada et al., 2003b) and we have just found that brain nuclear factor — kappa B is also involved in these responses (submitted), these observations might be possible explanations for long lasting effects of intracerebroventricularly administered CRF. Conclusions We have demonstrated that centrally administered CRFinduced elevation of plasma noradrenaline is mediated by an activation of α1 and β adrenoceptors in the brain, and that of plasma adrenaline is mediated by an activation of α1 adrenoceptors in the brain. Furthermore, central α2 adrenoceptors are involved in modulating the CRF-induced elevation of both plasma catecholamines.

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