Α2a-Adrenoceptors, but Not Nitric Oxide, Mediate the Peripheral Cardiac Sympatho-Inhibition of Moxonidine

European Journal of Pharmacology 782 (2016) 35–43

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

α2A-adrenoceptors, but not nitric oxide, mediate the peripheral cardiac sympatho-inhibition of moxonidine

Luis E. Cobos-Puc a,n, Hilda Aguayo-Morales b, Yesenia Silva-Belmares a, Maria A. González-Zavala a, David Centurión b a Facultad de Ciencias Químicas, Universidad Autónoma de Coahuila, Boulevard Venustiano Carranza esquina con Ing. José Cárdenas Valdés, Colonia Re- pública, Saltillo, Coahuila C.P. 25280, México b Departamento de Farmacobiología, Cinvestav-Coapa, Czda. de los Tenorios 235,Col. Granjas‑Coapa, Deleg. Tlalpan, C.P. 14330, México D.F., México article info abstract

Article history: Moxonidine centrally inhibits the sympathetic activity through the I1-imidazoline receptor and nitric

Received 20 January 2016 oxide. In addition, inhibits the peripheral cardiac sympathetic outﬂow by α2-adrenoceptors/I1-imida- Received in revised form zoline receptors, although the role of α2-adrenoceptor subtypes or nitric oxide in the cardiac sympatho- 18 April 2016 inhibition induced by moxonidine are unknown. Therefore, the cardiac sympatho-inhibition induced by Accepted 20 April 2016 moxonidine (10 μg/kg min) was evaluated before and after of the treatment with the following an- Available online 22 April 2016 tagonists/inhibitor: (1) BRL 44408, (300 μg/kg, α ), imiloxan, (3000 μg/kg, α ), and JP-1302, (300 μg/ 2A 2B ω Keywords: kg, α2C), in animals pretreated with AGN 192403 (3000 μg/kg, I1 antagonist); (2) N -nitro-L-arginine α -adrenoceptor 2A methyl ester (L-NAME; 34, 100, and 340 μg/kg min); and (3) the combinations of the highest dose of L- Heart rate NAME plus AGN 192403 or BRL 44408. Additionally, the expression of the neuronal (nNOS) and inducible I -Imidazoline receptor 1 (iNOS) nitric oxide synthase in the stellate ganglion was determined after treatment with moxonidine (i. Moxonidine μ Nitric oxide p. 0.56 mg/kg daily, during one week). The cardiac sympatho-inhibition of 10 g/kg min moxonidine was: (1) unaffected by imiloxan and JP-1302, under pretreatment with AGN 192403, or L-NAME (34, 100 Chemical compounds studied in this article: and 340 μg/kg min) given alone; (2) partially antagonized by the combination of 340 μg/kg min L-NAME Moxonidine hydrochloride plus BRL 44408; and (3) abolished by BRL 44408 under treatment with AGN 192403. Furthermore, (PubChem CID: 11231255) moxonidine did not modify the nNOS or iNOS protein expression in the stellate ganglion, the main source BRL 44408 maleate salt of postganglionic sympathetic neurons innervating the heart. In conclusion, our results suggest that the (PubChem CID: 71311805) peripheral cardiac sympatho-inhibition induced by moxonidine is mediated by α -adrenoceptor sub- imiloxan hydrochloride 2A (PudChem CID: 172975) type but not by nitric oxide. JP-1302 dihydrochloride & 2016 Elsevier B.V. All rights reserved. (PubChem CID: 540335) AGN 192403 hydrochloride (PubChem CID: 11957452) L-NAME hydrochloride (PubChem CID: 135193)

1. Introduction et al., 2009a; Raasch et al., 2003). Although, the mechanisms of action involved in the bradycardic effect induced by moxonidine Moxonidine, an antihypertensive drug, reduces blood pressure are not fully understood. and heart rate by inhibition of sympathetic activity. The blood At peripheral level, we have demonstrated that moxonidine fl pressure reduction of moxonidine is due to activation of I1-imi- inhibits the cardioaccelerator sympathetic out ow in the pithed dazoline receptors (Edwards et al., 2012; Ernsberger, 2000) and rat (Cobos-Puc et al., 2009a). The sympathetic outflow to the heart synthesis of nitric oxide in the rostral ventrolateral medulla is maintained by noradrenaline release from sympathetic neurons (RVLM) (Peng et al., 2009). Regarding heart rate decreases, some arising right and left stellate ganglion, reaching the sinus and at- rioventricular nodes, and the left ventricle, respectively (Tri- findings suggest that the sites of action of moxonidine could be poskiadis et al., 2009). The α -adrenoceptors regulate the nora- central (Chan et al., 2005; Moreira et al., 2007; Peng et al., 2009; 2 drenaline release by a negative feedback mechanism. Three α - Szabo et al., 2001; Totola et al., 2013) and peripheral (Cobos-Puc 2 adrenoceptor subtypes have been identified: α2A, α2B, and α2C (Alexander et al., 2015). Nevertheless, solely the α2A- and 2C- n fl Corresponding author. adrenoceptors inhibit the cardiac sympathetic out ow (Cobos-Puc E-mail address: [email protected] (L.E. Cobos-Puc). et al., 2007; Hein et al., 1999; Smith et al., 1995). http://dx.doi.org/10.1016/j.ejphar.2016.04.043 0014-2999/& 2016 Elsevier B.V. All rights reserved. 36 L.E. Cobos-Puc et al. / European Journal of Pharmacology 782 (2016) 35–43

Besides the α2A- and 2C-adrenoceptors, also nitric oxide inhibits 2.3. Experimental protocols the cardiac sympathetic neurotransmission (Rastaldo et al., 2007; Sears et al., 2004). Nitric oxide is synthesized by the enzyme nitric 2.3.1. Electrical stimulation of the cardioaccelerator sympathetic oxide synthase (NOS). Three types of NOS have been described: outﬂow neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) The pithing rod was replaced by an enamelled electrode except (Knowles and Moncada, 1994). The three nitric oxide synthase for 1 cm length 7 cm from the tip, so that the uncovered segment isoforms are expressed in neurons (Alderton et al., 2001). Not- was situated at C7-T1 of the spinal cord to allow selective stimu- withstanding, experiments performed in the isolated guinea-pig lation of the cardioaccelerator sympathetic outﬂow (Gillespie double atrial/right stellate ganglion preparation have shown that et al., 1970). Prior to electrical stimulation, the animals received only nNOS inhibition increases the chronotropic and inotropic gallamine (25 mg/kg, i.v.) to avoid electrically-induced muscular responses induced by sympathetic stimulation (Choate et al., twitching. Since the sympatho-inhibitory responses are particu- 2008; Choate and Paterson, 1999; Mohan et al., 2000). larly more pronounced at lower frequencies of stimulation, all the

Previously, we have shown that the α2-adrenoceptors and I1- animals were systematically pretreated with desipramine (50 μg/ imidazoline receptors are involved in the peripheral cardiac kg, i.v.; a noradrenaline-reuptake inhibitor) before each stimulus– sympatho-inhibition of moxonidine (Cobos-Puc et al., 2009a). On response curve, as previously described (Cobos-Puc et al., 2009a). this basis, the present study has further investigated the role of After a stable haemodynamic condition for at least 30 min, base-

α2A, α2B, and α2C-adrenoceptor subtypes or nitric oxide in this line values of diastolic and systolic blood pressure and heart rate response. For this purpose, we used the α2-adrenoceptor antago- were determined. Then, the preganglionic cardiac sympathetic nists BRL 44408 (α2A), imiloxan (α2B), JP-1302 (α2C), under outflow was stimulated to elicit tachycardic responses by applying blockade of the I -imidazoline receptor with AGN 192403, and trains of 10s trains of monophasic, rectangular pulses (2 ms, 1 ω non-selective nitric oxide synthase inhibitor, N -nitro-L-arginine 50 V), at increasing frequencies of stimulation (0.03, 0.1, 0.3, methyl ester (L-NAME). 1.0 and 3.0 Hz). When the heart rate had returned to baseline levels, the next frequency was applied; this procedure was systematically performed until the stimulus–response curve had been 2. Materials and methods completed (about 30 min). At this point, the rats (n¼84) were subdivided into three 2.1. Animals subgroups (n¼24, 24 and 36, respectively). The first subgroup (n¼24) was divided into four sets (n¼6 each), which received an i. 116 male Wistar normotensive rats (250–300 g) were used in v. continuous infusion of, respectively: (1) saline (0.02 ml/min the present research. The animals were purchased from Harlan twice); (2) moxonidine (10 μg/kg min); (3) L-NAME (non-selective Laboratories (Harlan, Mexico) and maintained at a 12/12h light– NOS inhibitor; 34 and 100 μg/kg min); and (4) L-NAME (340 μg/ dark cycle (with light beginning at 7 a.m.) and housed in a special kg min). 20 min after starting the infusion of each compound, a room at constant temperature (2272 °C) and humidity (50%), stimulus–response curve was performed during the infusion of the with food and water freely available in their home cages. All ani- correspondent treatment. mal procedures and the protocols of the present investigation The second subgroup (n¼24) was systematically pretreated were approved by our Institutional Ethics Committee and followed with AGN 192403 (3000 μg/kg) to block I1-imidazoline receptors. the regulations established by the Mexican Official Norm for the Then, the animals were subdivided into four sets (n¼6 each) that Use and Welfare of Laboratory Animals (NOM-062-ZOO-1999), in received an i.v. bolus injection of, respectively: (1) physiological accordance with the Guide for the Care and Use of Laboratory saline (1 ml/kg twice); (2) BRL 44408 (α2A antagonist, 300 μg/kg); Animals in the USA. (3) imiloxan (α2B antagonist, 3000 μg/kg); and (4) JP-1302 (α2C antagonist, 300 μg/kg). 10 min after the administration of the 2.2. Pithed rat preparation abovementioned antagonists a stimulus–response curve was performed during the i.v. infusion of moxonidine (10 μg/kg min). 84 rats were anesthetized with i.p. injection of a mixture of The animals of the third subgroup (n¼36) were subdivided ketamine (100 mg/kg) and xylazine (10 mg/kg), and a stainless- into six sets (n¼6 each) that received the follow treatment, re- steel rod was introduced through an orbit to destroy the brain and spectively: (1) saline (0.02 ml/min); (2) L-NAME (34 μg/kg min); the spinal medulla. The rats were artificially ventilated with a (3) L-NAME (100 μg/kg min); (4) L-NAME (340 μg/kg min); (5) L- rodent ventilator (Intecs Instrumentación, Mexico) immediately NAME (340 μg/kg min) plus AGN 192403 (3000 μg/kg); and (6) L- after the pithing. Then, the rats were subjected to bilateral sym- NAME (340 μg/kg min) plus BRL 44408 (300 μg/kg). Then, a sti- pathectomy and vagotomy, and the left carotid artery was ligated. mulus–response curve was performed during the i.v. infusion of Catheters were inserted into the left jugular vein for intravenous 10 μg/kg min moxonidine. (i.v.) infusion of L-NAME, and in the left and right femoral veins, for the continuous infusion of moxonidine and i.v. administration of 2.3.2. Western blotting the corresponding pharmacological treatments. Finally, a catheter The nNOS or iNOS protein expression was determined by was inserted into the right carotid artery for monitoring arterial western blot. The dose used in this set of experiments is blood pressure throughout the experiment. Arterial blood pressure equivalent to the infusion of 10 μg/kg min moxonidine, was recorded through P23 Gould transducer. Heart rate was throughout an hour, period in which the experiments in pithed measured with a tachograph (7P4, Grass Instrument Co., Quincy, rats were performed. Briefly, 32 Wistar rats were divided into Ma, USA) from the blood pressure signal. Both blood pressure and two subgroups (n¼16 each) that were treated daily with mox- heart rate were recorded simultaneously by a model 7 Grass onidine (i.p. 0.6 mg/kg) or vehicle (1 ml/kg) during one week. polygraph. The tachycardic responses induced by sympathetic Then, the rats were euthanized in CO2 chamber, and under mi- stimulation were elicited using a sequential schedule, in 0.5 log croscope dissection, both right and left stellate ganglion (SG) unit increments, and completed in about 30 min without any were removed and cleaned of connective tissue in ice-cold Hanks’ change in resting heart rate. During all experiments, body tem- balanced salt solution, and lysed in buffer containing 50 mM Tris– perature was maintained at 37–38 °C by a heating lamp (Cobos- HCl pH 7.4, 1 mM EDTA, 0.5% Triton X-100, and a cocktail of Puc et al., 2009a). protease inhibitors. Protein from ganglia of four rats was pooled L.E. Cobos-Puc et al. / European Journal of Pharmacology 782 (2016) 35–43 37 into separate tubes (n¼4) and concentrations were measured 3. Results using Bradford method. 50 μg of protein were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a ni- 3.1. Systemic haemodynamic variables trocellulose membrane. The membrane was blocked with 5% non-fat dry milk in 25 mmol/L Tris-buffered saline, pH 7.2, plus The baseline values of diastolic and systolic blood pressure and heart rate in 7 7 7 0.1% Tween 20 (TBS-T) for 1 h at room temperature, followed by pithed rats were 47 5 and 79 8 mm Hg, and 275 12 beats/min, respectively. These variables were not significantly modified by the i.v. administration of gal- incubation with primary antibody (anti-nNOS or anti-iNOS, 1; lamine, desipramine, and saline (given as continuous infusion or bolus injection) 1000) for overnight at 4 °C. Recovering to room temperature on as previously reported (Cobos-Puc et al., 2009a, 2007). In contrast, moxonidine the second day, the membrane was washed in TBS-T, and in- produced significant increases in the blood pressure. Indeed, in the group treated cubated with the secondary antibody, horseradish peroxidase with moxonidine the baseline values of diastolic and systolic blood pressure were 4378and7674 mm Hg, respectively, which was significantly increased to (HRP)-conjugated (1:5000) for 1 h at room temperature. Chemi- 73711* and 10476* mm Hg after 10 μg/kg min of moxonidine. Similarly, L- luminescent signals were collected on autoradiography film. The NAME produced dose-dependent increases in the diastolic and systolic blood band intensity was determined using ImageQuan v8.1. Protein pressure (Table 1). On the other hand, the i.v. infusions of moxonidine or L-NAME fi expression of nNOS and iNOS for each sample was normalized to failed to signi cantly modify the baseline value of heart rate (see Table 1). It β should be remarked that the antagonists AGN 192403 (Cobos-Puc et al., 2009a), -actin levels. BRL 44408 (Cobos-Puc et al., 2009b), imiloxan, and JP-1302 (Villamil-Hernández et al., 2013) did not modify the baseline values of heart rate and blood pressure. 2.4. Compounds

3.2. Effect of moxonidine or L-NAME on the tachycardic responses produced by The compounds or antibodies used in the present study (ob- sympathetic stimulation tained from the sources indicated) were: anti-iNOS; anti-nNOS; anti-β-actin; anti-mouse IgG (Abcam, MA, USA); film (PierceTM Fig. 1 shows the tachycardic responses produced by sympathetic stimulation ECL, thermofisher Inc. MA, USA); desipramine hydrochloride; before (control) and during the i.v. continuous infusions of saline (0.02 ml/min ω twice), moxonidine (10 μg/kg min), and L-NAME (34, 100 and 340 μg/kg min). In gallamine triethiodide; N -nitro-L-arginine methyl ester hydro- animals infused with saline (Fig. 1(A)), or L-NAME (Fig. 1(C) and (D)), the tachy- chloride (L-NAME hydrochloride); 4-chloro-6-methoxy-2-methyl- cardic responses to sympathetic stimulation remained unchanged when a sub- 5-(2-imidazolin-2-yl)aminopyrimidine hydrochloride (mox- sequent stimulus–response curve was performed. In contrast, the i.v. continuous μ fi onidine hydrochloride); dimethyl sulfoxide (DMSO); (7)2-(2- infusion of moxonidine (10 g/kg min) signi cantly inhibited the tachycardic responses to sympathetic stimulation (Fig. 1(B)) at all frequencies of stimulation. chlorophenyl)2-(methylamino)cyclohexanone hydrochloride (ketamine); 2-(2,6-dimethylphenylamino)5,6-dihydro-4H-thia- α zine hydrochloride, 5,6-Dihydro-2-(2,6-xylidino)4H1,3-thia- 3.3. Effect of the 2-adrenoceptor antagonists on the cardiac sympatho-inhibition produced by moxonidine zine hydrochloride (xylazine); protease inhibitor cocktail; Tris hydrochloride; ethylenediaminetetraacetic acid (EDTA); 4-(1,1,3,3 Fig. 2 illustrates the effect of an i.v. bolus injection of saline (1 ml/kg) or the tetramethyl butyl)phenyl-polyethylene glycol solution (triton antagonists BRL 44408 (300 μg/kg), imiloxan (3000 μg/kg) and JP-1302 (300 μg/ μ X-100); Tris buffered saline; sodium chloride; polyethylene glycol kg) under blockade of I1-imidazoline receptor with AGN 192403 (3000 g/kg) on sorbitan monolaurate (Tween 20) (Sigma Chemical Co., St. Louis, the tachycardic responses induced by sympathetic stimulation during the i.v. continuous infusion of moxonidine (10 μg/kg min). The i.v. bolus injection of saline MO, USA); N-[4-(4-Methyl-1-piperazinyl)phenyl]9-acridinamine (Fig. 2(A)), imiloxan (Fig. 2(C)) and JP-1302 (Fig. 2(D)), failed to modify the cardiac dihydrochloride (JP-1302 dihydrochloride); 2-[(4,5dihydro-1H- sympatho-inhibition induced during the continuous infusion of moxonidine. In imidazol-2-yl)methyl]2,3-dihydro-1-methyl-1H-isoindole mal- marked contrast, BRL 44408 abolished the cardiac sympatho-inhibition to mox- eate (BRL 44408 maleate); (2-(1-ethyl-2-indazoyl)methyl-1,4- onidine (Fig. 2(B)). benzodioxan hydrochloride (imiloxan hydrochloride); (7)2- endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane hydrochloride 3.4. Effect of L-NAME on the cardiac sympatho-inhibition produced by moxonidine (AGN 192403 hydrochloride) (Tocris Cookson, Ellisville, MO, USA); Propylene glycol (JT Baker, Mexico). All pharmacological com- Fig. 3 shows the effect of an i.v. continuous infusion of saline (0.02 ml/min) or L- μ pounds were dissolved in physiological saline. When needed, NAME (34, 100 and 340 g/kg min) and the highest dose of L-NAME plus AGN 192403 or BRL 44408 on the tachycardic responses induced by sympathetic sti- some drops of DMSO (10% v/v) or PPG (20% v/v) were used to mulation during the i.v. continuous infusion of moxonidine (10 μg/kg min). The i.v. dissolve BRL 44408 and moxonidine or JP-1302, respectively. The infusion of saline (Fig. 3(A)) or L-NAME (Fig. 3(B), (C), and (D)) failed to modify the resulting solutions were finally diluted with physiological saline. cardiac sympatho-inhibition induced by moxonidine. In addition, the combination μ These vehicles had no effect on the baseline values of diastolic and of the highest dose of L-NAME (340 g/kg min) plus AGN 192403 (Fig. 3(E)) did not block the sympatho-inhibition induced by moxonidine. In contrast, the combina- systolic blood pressure or heart rate. Fresh solutions were pre- tion of L-NAME plus BRL 44408 partially antagonized this effect at 0.3–3Hz(Fig. 3 pared for each experiment. The doses mentioned in the text refer (F)). to the salt of substances, except in the case of moxonidine, where it refers to the free base. 3.5. Effect of moxonidine on the nNOS and iNOS protein expression in the stellate ganglion 2.5. Data presentation and statistical evaluation Fig. 4 shows the results of western blot analysis of the nNOS and iNOS protein All data are presented as mean7standard error of the mean. expression in the stellate ganglion from rats treated with moxonidine or its vehicle. The protein level of β-actin (43 kDa) from stellate ganglion was not different be- The maximum changes in heart rate, systolic and diastolic blood tween the rats treated with vehicle or moxonidine. Furthermore, 155 and 130 kDa pressure produced by electrical stimulation were determined be- protein bands compatible with nNOS and iNOS respectively, were identified in the fore and after the administration of different treatments. The dif- total protein sample from both groups (Fig. 4(A) and (B)). Additionally, the level of fi ference between the changes in heart rate within one subgroup of expression for nNOS or iNOS in the stellate ganglion was not modi ed by moxonidine (Fig. 4(C) and (D), respectively). Finally, the nNOS protein expression was animals was evaluated by using Student-Newman-Keuls's test, 2-fold higher than iNOS protein expression in vehicle-treated group (Fig. 4(E), once a two-way repeated measures analysis of variance (rando- P¼0.003). The specificity of the antibodies was evaluated in appropriated positive mized block design) had revealed that the samples represent dif- and negative controls (data not shown). ferent populations. Data for western blot were analyzed using Student's t-test for comparison of two groups. Statistical sig- nificance was accepted at Po0.05. 38 L.E. Cobos-Puc et al. / European Journal of Pharmacology 782 (2016) 35–43

Table 1 induced tachycardic responses, but this possibility seems highly Effect of the intravenous infusion of saline (0.02 ml/min), moxonidine (10 μg/kg min) unlikely since this effect is maintained regardless of treatment μ and L-NAME (34, 100 and 340 g/kg min) on diastolic (DBP) and systolic (SBP) blood with desipramine (Schwarz et al., 1995). A reasonable explanation pressure, and heart rate (HR). a Po0.05 vs baseline values. for these differential effects in vivo or in vitro is that noradrenaline DBP (mm Hg) SBP (mm Hg) HR (beats/min) released from adrenal medulla, by systemic action of L-NAME

Before After Before After Before After (Elayan et al., 2002), through the α2-adrenoceptors, in postganglionic cardiac sympathetic nerves, compensates the increases Saline (0.02 ml/min) 52755377797783782717827074 μ 7 7 a 7 7 a 7 7 on noradrenaline release induced by inhibition of nitric oxide Moxonidine (10 g/ 43 873 11 76 4104 6 265 6 268 9 α kg min) synthase isoforms. Undoubtedly, future experiments using 2 an- L-NAME (34 μg/kg 45746078a 77799177 2827727975 tagonists and/or adrenalectomy, which fall beyond the scope of min) the present investigation, need to be performed to evaluate this μ 7 7 a 7 7 a 7 7 L-NAME (100 g/kg 42 673 10 74 898 5 283 9 289 6 hypothesis. min) L-NAME (340 μg/kg 50798575a 8175113710a 27577 28978 min) 4.4. Role of α2-adrenoceptor subtypes on the cardiac sympatho-inhibition induced by moxonidine

4. Discussion The cardiac sympatho-inhibition of moxonidine, under I1-imidazoline receptor blockade, remained unchanged after treatment

4.1. General with imiloxan and JP-1302, α2B and α2C antagonists, respectively. In contrast, the treatment with BRL 44408, a selective α2A an- Moxonidine inhibits the noradrenaline release from peripheral tagonist, abolished the cardiac sympatho-inhibition induced by sympathetic neurons through the α2-adrenoceptors and I1-imi- moxonidine. The α2A-adrenoceptors by coupling to Gi/o proteins dazoline receptors (Cobos-Puc et al., 2009a; Schafer et al., 2002). inhibit noradrenaline release and other neurotransmitters. There

Besides, the present work demonstrates that the peripheral car- are four types of α2A agonists structurally different: catechola- diac sympatho-inhibition induced by moxonidine was abolished mines, imidazolines (moxonidine), dichlorophenylguanidines by BRL 44408 (α2A antagonist) under blockade of the I1-imidazo- (guanfacine) and oxazoloazepines (B-HT 933). For their structural line receptor with AGN 192403 (I1 antagonist) and unaffected by L- differences, these drugs can trigger the same signaling pathway NAME given alone or in combination with AGN 192403. As well, L- with different efficacy. For instance, moxonidine is equi-efficacious NAME in combination with BRL 44408 partially antagonized this to noradrenaline and B-HT 933 and 9-fold more efficacious than effect. Additionally, moxonidine was unable to modify the nNOS or guanfacine to inhibit forskolin-induced cyclic AMP elevation iNOS protein expression in the main autonomic ganglion by which through the α2A-adrenoceptors in HEL 92.1.7 erythroleukemia cells postganglionic sympathetic efferent neurons innervates the heart. (Kukkonen et al., 2001). Moreover, we have previously reported Hence, the autonomic cardiac sympatho-inhibition induced by that BRL 44408 antagonized the cardiac sympatho-inhibition in- moxonidine is mediated mainly by the α2A-adrenoceptor subtype duced by B-HT 933 (Cobos-Puc et al., 2007). Taken together all but not by nitric oxide. above findings suggest that the α2A-adrenoceptor is the main subtype involved on the cardiac sympatho-inhibition induced by 4.2. Systemic haemodynamic changes moxonidine. Interestingly, the cardiac sympatho-inhibition induced by Imidazoline receptors have been identified in rat blood vessels moxonidine was unaffected by 3000 μg/kg of AGN 192403 given (Regunathan et al., 1995; Sarac et al., 2015) but their stimulation alone as previously reported (Cobos-Puc et al., 2009a). This result produces vasorelaxation rather than vasoconstriction (Chen et al., suggests that expression of the α2A-adrenoceptors on cardiac 2014; Sarac et al., 2015); thus, their role in the vasopressor effect sympathetic neurons is higher than I1-imidazoline receptors. induced by moxonidine seems unlikely. Thereby, the increases in Certainly, we did not analyze the expression of I1-imidazoline re- the blood pressure induced by moxonidine can be attributed to ceptors or α2A-adrenoceptors in sympathetic neurons. However, stimulation of the vascular α1/2-adrenoceptors (Ernsberger, 2000; functional studies have shown that moxonidine stimulates pre- Kennedy et al., 2006), although this issue needs to be investigated. ferentially the α2-adrenoceptors in postganglionic sympathetic Similarly, L-NAME produced increases in the blood pressure. neurons innervating the heart (Schafer et al., 2002). Furthermore,

The vasopressor mechanisms of L-NAME are associated with an the I1-imidazoline receptors density is 4-fold higher than α2- enhancement of the vascular tone by eNOS activity inhibition adrenoceptors in RVLM (Piletz et al., 2003). In contrast, the α2- (Richard et al., 1995; Wakeﬁeld et al., 2003), and the releasing of adrenoceptors density is 3-fold higher than I1-imidazoline re- adrenaline from sympatho-adrenal system (Elayan et al., 2002; ceptors in the hypothalamus, ventral and dorsal pons, and the Huang et al., 2003). hippocampus (Piletz et al., 2003; Tolentino-Silva et al., 2000).

Therefore, the lower density of I1-imidazoline receptors in sym- 4.3. Effects of i.v. infusions of moxonidine and L-NAME on the sympathetic neurons could explain the fact that moxonidine even pathetically-in duced tachycardic responses displaying high selectivity to imidazoline I1 receptors produces inhibitory effects via α2-adrenoceptors. I.v. continuous infusion of moxonidine inhibited the tachycardic responses induced by sympathetic stimulation as previously dis- 4.5. No evidence for the role of nitric oxide in the cardiac sympatho- cussed (Cobos-Puc et al., 2009a). On the other hand, the increases inhibition induced by moxonidine on heart rate induced by sympathetic stimulation remained unchanged after i.v. infusion of L-NAME. In contrast, the adminis- We analyzed the role of nitric oxide in the cardiac sympatho- tration of NOS inhibitors in isolated guinea-pig double atrial/right inhibition induced by moxonidine using L-NAME, a non-selective stellate ganglion preparation increased the tachycardic response nitric oxide synthase inhibitor, with or without blockade of the I1- induced by sympathetic stimulation (Choate and Paterson, 1999). imidazoline receptor (AGN 192403) or α2A-adrenoceptor (BRL Therefore, it could be argued that treatment with desipramine 44408). Thus, considering that the half-life of L-NAME is 20 min in may have masked the effect of L-NAME on the sympathetically- the rat (Vitecek et al., 2012), we used 34, 100, and 340 μg/kg min L.E. Cobos-Puc et al. / European Journal of Pharmacology 782 (2016) 35–43 39

Fig. 1. Tachycardic responses produced by stimulation of the cardiac sympathetic outﬂow before (control) and during i.v. continuous infusions of: saline (0.02 ml/min twice; (A), moxonidine (10 μg/kg min; (B), and L-NAME (34, 100, and 340 μg/kg min; (C and D). *Po0.05 vs. the corresponding control response. 40 L.E. Cobos-Puc et al. / European Journal of Pharmacology 782 (2016) 35–43

Fig. 2. Effects of i.v. bolus injections of: saline (1 ml/kg; (A), BRL 44408 (300 μg/kg; (B), imiloxan (3000 μg/kg; (C), and JP-1302 (300 μg/kg; (D) on the sympathetically- induced tachycardic responses in the presence of an i.v. continuous infusion of moxonidine (10 μg/kg min). In all cases, the rats were systematically pretreated with AGN 192403 (3000 μg/kg). *Po0.05 vs. the corresponding control response.

of L-NAME to achieve a steady state concentration about to 1, 3, main source of postganglionic sympathetic efferent neurons in- and 10 μM, respectively. Concentrations high enough of L-NAME to nervating the heart. This ratio of expression remained unaltered inhibit differentially the three NOS isoforms (IC50 ¼0.29, 0.35, and after of the treatment with moxonidine, suggesting that under our 3.1 μM for human nNOS, eNOS, and iNOS) (Alderton et al., 2001). experimental conditions, moxonidine did not modulate the pro- Nonetheless, the cardiac sympatho-inhibition induced by mox- tein expression of nNOS or iNOS in the stellate ganglion. Not- onidine was unaffected by L-NAME. It should be pointed out that L- withstanding, L-NAME, at all doses used, increased the basal values NAME is 28, 32, and 3-fold more potent than the soluble Naþ salt of diastolic and systolic blood pressure indicating that eNOS was of 7-nitroindazole (7-NiNA; IC50 ¼8.3, 11.8 and 9.7 μM for human effectively inhibited and hence nNOS and iNOS. The eNOS protein nNOS, eNOS, and iNOS) to inhibit the nNOS, eNOS, and iNOS iso- expression was not determined, but the fact that the cardiac forms, respectively (Alderton et al., 2001). Moreover, L-NAME sympatho-inhibition of moxonidine was unaffected after the failed to modify the sympatho-inhibition of moxonidine under treatment with L-NAME suggests that nitric oxide, synthesized by blockade with AGN 192403 whilst partially antagonized this effect the three nitric oxide synthase in sympathetic neurons, is not in combination with BRL 44408. This last ﬁnding was related to- involved. tally with blockade of α2A-adrenoceptor. In addition, moxonidine up-regulates the iNOS protein ex- Additionally, our western blot analysis revealed that nNOS is pression in the rostral ventrolateral medulla but not in the nucleus expressed 2-fold more than iNOS in the rat stellate ganglion, the tractus solitarius (Peng et al., 2009). As well, imidazoline L.E. Cobos-Puc et al. / European Journal of Pharmacology 782 (2016) 35–43 41

Fig. 3. Effects of the i.v. treatments of: saline (0.02 ml/min; (A) L-NAME (34, 100, and 340 μg/kg min; (B–D), L-NAME plus AGN 192403 (340 μg/kg min and 3000 μg/kg, respectively; (E) and L-NAME plus BRL 44408 (340 μg/kg min and 300 μg/kg, respectively; (F) on the sympathetically-induced tachycardic responses in the presence of an i.v. continuous infusion of moxonidine (10 μg/kg min). *Po0.05 vs. the corresponding control response.

Fig. 4. Representative blotting of nNOS, iNOS and β-actin (A and B) and densitometry analysis of the nNOS and iNOS protein expression in the stellate ganglion from rats treated with moxonidine (Mox; 0.56 mg/kg, i.p.,) or vehicle (Veh; 1 ml/kg, i.p.) during one week (C and D). The basal protein expression of nNOS and iNOS was determined in vehicle-treated rats (E). β-actin normalization was performed.*Po0.05 vs nNOS. 42 L.E. Cobos-Puc et al. / European Journal of Pharmacology 782 (2016) 35–43 compounds: (1) increase nNOS in the locus coeruleus (Bender and Cobos-Puc, L.E., Villalón, C.M., Sánchez-López, A., Ramírez-Rosas, M.B., Lozano- Abdel-Rahman, 2010); (2) reduce nNOS and increase iNOS in Cuenca, J., Pertz, H.H., Gornemann, T., Centurión, D., 2009b. Pharmacological characterization of ergotamine-induced inhibition of the cardioaccelerator glioma cells (Venturini et al., 2000); and (3) increase eNOS in the sympathetic outflow in pithed rats. Naunyn Schmiede. Arch. 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