Activation of Dopamine D3 Receptor Subtypes Inhibits the

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Activation of dopamine D receptor subtypes inhibits the neurogenic systemic vasodilation induced by stimulation of the perivascular CGRPergic discharge Guadalupe Manrique-Maldonado, Alain Hassan Altamirano-Espinoza, Eduardo Rivera-Mancilla, Oswaldo Hernandez-Abreu, and Carlos M. Villalon ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00277 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 26, 2019

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ACS Chemical Neuroscience

ACS Chemical Neuroscience (MS ID: cn-2019-00277q)

Revised: 23rd July, 2019

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Activation of dopamine D3 receptor subtypes inhibits the neurogenic systemic

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vasodilation induced by stimulation of the perivascular CGRPergic discharge

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Guadalupe Manrique-Maldonado, Alain H. Altamirano-Espinoza, Eduardo Rivera-Mancilla,

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Oswaldo Hernández-Abreu and Carlos M. Villalón*

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Department of Pharmacobiology, Cinvestav-Coapa, Czda. Tenorios 235, Col. Granjas Coapa,

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Deleg. Tlalpan, 14330 Mexico City, Mexico

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GMM ([email protected]), AHAE ([email protected]), ERM ([email protected]),

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OHA ([email protected]), CMV ([email protected])

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*Correspondence:

Prof. Dr. Carlos M. Villalón at the above address

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Telephone:

(Int.)-(52)-(55)-5483-2854

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Fax:

(Int.)-(52)-(55)-5483-2863

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URL: http://farmacobiologia.cinvestav.mx/PersonalAcadémico/DrCarlosMVillalónHerrera.aspx 1 ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Manrique-Maldonado et al.: Neurogenic vasodilation is inhibited by D3 receptors

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Abstract

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The sensory nervous system controls cardiovascular homeostasis via capsaicin-sensitive neurons

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that release calcitonin gene-related peptide (CGRP), which subsequently activates CGRP

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receptors. How this perivascular CGRPergic discharge is modulated, nevertheless, remains

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unclear. In pithed rats, systemic vasodilation induced by CGRPergic discharge stimulation results

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in diastolic blood pressure (diastolic BP) decrements that are inhibited by the dopamine D2-like

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receptor agonist quinpirole. Since this inhibition is mediated by raclopride- or haloperidol-

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sensitive D2-like receptors (comprising the D2, D3, and D4 subtypes), the present study has

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pharmacologically investigated the specific contribution of these subtypes to the modulation of the

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systemic CGRPergic vasodilation, using highly specific antagonists. To that end, 55 male Wistar rats

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were pithed for thoracic (T9-T12) spinal stimulation of the perivascular CGRPergic discharge. The

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resulting frequency-dependent decrements in diastolic BP were inhibited by quinpirole, and this

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sensory-inhibition was: (a) unchanged after i.v. injections of the antagonists L-741,626 (D2) or

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L-745,870 (D4); and (b) completely blocked by SB-277011-A (D3). Accordingly, we suggest the

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main role of the D3 receptor subtypes in the inhibition by quinpirole of the neurogenic CGRPergic

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systemic vasodilation. These findings contribute to a better understanding of the dopaminergic

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modulation of the rat perivascular CGRPergic discharge producing systemic vasodilation.

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Key words: CGRP; D2-like receptors; Pithed rat; SB-277011-A; Sensory CGRPergic discharge

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ACS Chemical Neuroscience Manrique-Maldonado et al.: Neurogenic vasodilation is inhibited by D3 receptors

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Introduction

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Understanding the neural control of cardiovascular homeostasis is key for the development of new

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therapeutic strategies aimed at treating numerous illnesses such as hypertension, hearth failure and

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other cardiovascular diseases. Within this context, although calcitonin gene-related peptide

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(CGRP) seems not to play an important physiological role on blood pressure modulation, its potent

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vasodilator effect could function as a protective mechanism during the development and

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establishment of cardiovascular diseases1. Thus, the comprehension of how this perivascular

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CGRPergic discharge is modulated may provide valuable information for the design of more

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efficient therapeutic strategies to treat a wide range of cardiovascular illnesses. Accordingly, the

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modulation of systemic vascular tone by the perivascular CGRPergic discharge has been widely

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studied in pithed rats2,3. In this animal model, the decrements in diastolic BP generated by selective

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thoracic (T9-T12) spinal stimulation of the perivascular sensory CGRPergic discharge involve:

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(i) the release of CGRP from primary sensory nerves2; and (ii) activation of vascular CGRP

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receptors that are dose-dependently blocked by the non-peptide CGRP receptor antagonist

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olcegepant (which also potentiated the noradrenergic increments in diastolic BP)3.

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These findings, besides suggesting that CGRP may be involved in the control of systemic

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vascular tone, also emphasize the current importance of the pithed rat as a reliable model for

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studying the neurogenic systemic vasodilation and its modulation. Moreover, since pithed rats lack

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intrinsic sympathetic nerve activity or centrally-mediated baroreflex compensatory mechanisms3,4,

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this experimental model has been suitable for showing that activation of prejunctional

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α2A/2C-adrenergic5, serotonin 5-HT1B/1F6, histamine H37 and dopamine D2-like8 receptors can

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modulate the neurogenic systemic vasodilation by inhibiting the decrements in diastolic BP

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produced by stimulation of the perivascular CGRPergic discharge.




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Certainly, the prejunctional inhibition by dopamine D2-like receptors of the systemic

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perivascular CGRPergic discharge8 is of particular interest as, admittedly, the interaction between

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the dopaminergic and CGRPergic neurotransmission systems has not been comprehensively

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investigated. Thus far, several studies have shown that dopaminergic neurotransmission plays a

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main role in cardiovascular function as well as in the development and maintenance of

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cardiovascular diseases9. Notably, its contribution to the modulation of vascular tone and blood

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pressure depends mainly on the dopamine receptor (sub) types involved and their location. For

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example, activation of D1-like receptors located in blood vessels results in smooth muscle

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relaxation10, while stimulation of D2-like receptors on perivascular sympathetic nerves inhibits

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noradrenaline release with a subsequent decrease in blood pressure due to a fall in peripheral

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vascular resistance10. Likewise, some studies suggest that activation of D3 receptor subtypes

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induced an endothelium-independent vasorelaxation, while costimulation of the D1 and D3

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receptor subtypes resulted in an additive vasorelaxation11. Moreover, dopamine can also modulate

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(by presynaptic inhibition via the D4 receptor subtype) primary C- and Aδ-fiber nociceptive

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(CGRPergic) sensory pathways12. Interestingly, mRNAs for dopamine D1-like (including the D1

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and D5 subtypes) and D2-like (including the D2, D3 and D4 subtypes) receptors have been detected

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in dorsal root ganglia (DRG)13, where the cell bodies of perivascular sensory neurons are located.

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In view that dopamine D2-like receptors are activated by the agonist quinpirole8, can inhibit

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perivascular neuronal activity 14, 15 and consist of the D2, D3 and D4 subtypes16,17, the present study

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has pharmacologically investigated the specific contribution of these subtypes to the inhibition by

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quinpirole of the perivascular CGRPergic discharge. This approach included the use of selective

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antagonists at the D2 (L-741,626), D3 (SB-277011-A) and D4 (L-745,870) receptor subtypes

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(Table 1), which were administered in i.v. doses that produce a complete blockade of their

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respective subtypes in pithed rats18,19. 4 ACS Paragon Plus Environment

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Results and Discussion

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1.1.

General

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Although Manrique-Maldonado et al.8 had already reported that prejunctional dopamine D2-like

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receptors mediate quinpirole-induced inhibition of the systemic perivascular CGRPergic

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discharge, they also acknowledged that the specific contribution of the D2, D3, and D4 subtypes to

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this response would require additional studies. Hence, in the present pharmacological study we

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designed an experimental approach to discern the specific role of the D2, D3, and D4 subtypes in

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the above CGRPergic-inhibition by quinpirole using the specific antagonists L-741,626 (D2

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preferring), SB-277011-A (D3) and L-745,870 (D4) (Table 1), in doses suitable to completely block

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their respective subtypes in pithed rats18,19. Our results suggest, for the first time, that the dopamine

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D2-like receptors inhibiting the neurogenic perivascular CGRPergic discharge resemble the D3

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(but not the D2 or D4) receptor subtype.

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1.2.

Changes in cardiovascular haemodynamics

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To elicit neurogenic CGRPergic vasodepressor responses in pithed rats, it is absolutely essential to

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pre-treat these animals with hexamethonium and the vasoconstrictor methoxamine. We have

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previously described and discussed in detail this particular experimental condition3,5-8 as it is

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fundamental for eliciting and studying the rat systemic perivascular CGRPergic discharge.

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Resting diastolic BP and heart rate (HR) values in the 55 rats were 76±2 mm Hg and

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234±8 beats/min, respectively. These values remained unchanged (P>0.05) after administration of

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gallamine or hexamethonium, as previously reported8. Twenty min after starting the infusion of

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methoxamine, diastolic BP increased in all cases (147±3 mm Hg; P0.05) from that induced by higher

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doses; and (ii) failed to inhibit the decrements in diastolic BP produced by exogenous CGRP.

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These findings established that the above inhibition by quinpirole is prejunctional8.

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1.3.

The CGRPergic-inhibition by quinpirole is mediated by the D3 receptor subtype

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The decrements in diastolic BP by electrical stimulation (0.56-5.6 Hz): (i) lasted 5-10 min and

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were accompanied, if any, by insignificant HR effects (not shown); (ii) remained unaltered during

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the infusion of saline (Figure 1A); and (iii) were significantly inhibited (at 1.8, 3.1 and 5.6 Hz)

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during the infusion of quinpirole (Figure 1B). These results are similar to those reported by

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Manrique-Maldonado et al.8, who also showed that higher doses of quinpirole produced no further

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inhibition of these responses. Moreover, Figure 2 shows the neurogenic decrements in diastolic BP

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after i.v. treatment with 5% ascorbic acid (vehicle), L-741,626 (D2), SB-277011-A (D3) or 6 ACS Paragon Plus Environment

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L-745,870 (D4), during a saline infusion. Clearly, the neurogenic decrements in diastolic BP

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remained without significant changes after vehicle (Figure 2A), L-741,626 (Figure 2B),

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SB-277011-A (Figure 2C) or L-745,870 (Figure 2D) during the saline infusion. Thus, the doses of

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the above compounds were devoid of any effects by themselves on these neurogenic responses.

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Furthermore, Figure 3 illustrates the neurogenic decrements in diastolic BP after 5% ascorbic

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acid, L-741,626, SB-277011-A or L-745,870 given i.v. during a continuous infusion of quinpirole.

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The inhibition induced by quinpirole was: (a) unchanged after vehicle (Figure 3A), L-741,626

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(Figure 3B) or L-745,870 (Figure 3D); and (b) clearly abolished after SB-277011-A (Figure 3C).

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On this basis, by using subtype-selective antagonists, our study suggests that the D2-like receptors

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inhibiting the systemic CGRPergic discharge resemble the pharmacological properties of the

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dopamine D3 receptor subtype. Admittedly, quinpirole is a D2-like receptor agonist with little

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selectivity to discriminate among the D2, D3 and D4 receptor subtypes (Table 1) and, in general,

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dopamine receptor ligands display a high degree of cross-interactions with many other targets

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including G-protein coupled receptors, transporters, enzymes and ion channels20. Hence, from a

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pharmacological perspective, it may be argued that our study would have been more appropriate

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using a specific D3 receptor agonist. However, the D3 receptor agonists commercially available

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thus far (including PD128907) are also capable of interacting with the D2 receptor subtype21.

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Certainly, this is not surprising if we consider that the D2 and D3 receptor subtypes are highly

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homologous, sharing 78% of sequence homology in their transmembrane domains22. Thus, a more

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reliable pharmacological strategy designed for our study consisted in the use of specific antagonists

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for the D2, D3 and D4 receptor subtypes (Table 1). Accordingly, the fact that SB-277011-A

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abolished quinpirole-induced inhibition (displaying 2.5 log units higher affinity for the D3 subtype;

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Table 1), while the other antagonists were devoid of any effect on this inhibition (Figure 3),

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allowed us to unequivocally suggest the main role of the D3 receptor subtype. 7 ACS Paragon Plus Environment



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As previously shown, the doses used of these antagonists reversed other neurogenic

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responses that were also quinpirole-sensitive. For example, Ruiz-Salinas et al.19: (i) demonstrated

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that the same doses of SB-277011-A (D3) and L-745,870 (D4) abolished and weakly blocked,

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respectively, the quinpirole-induced inhibition of the vasopressor sympathetic discharge; and

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(ii) excluded the involvement of the D2 receptor subtype. On the other hand, Altamirano-Espinoza

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et al.18 reported that the same dose of L-741,626 (D2) abolished the quinpirole-induced inhibition

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of the cardiac sympathetic discharge, while they excluded the role of the D3 and D4 subtypes. These

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findings are important because it could be argued that the doses of these antagonists were not

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enough to reach the target tissues (i.e. CGRPergic fibers and/or DRG). Nevertheless, using the

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same experimental conditions, the inhibition by quinpirole of the vasopressor sympathetic

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discharge19, cardioaccelerator sympathetic discharge18 and CGRPergic discharge (present study)

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involves different D2-like receptor subtypes. Although we cannot unambiguously explain such

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pharmacological differences, the spinal origins of these neurogenic discharges (i.e. T7-T9 for

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vasopressor, C7-T1 for cardioaccelerator and T9-T12 for CGRPergic discharge) may play a role.

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Undoubtedly, further research will be required to ascertain: (i) the possible physiological relevance

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of these differences; and (ii) the net effect of the dopaminergic transmission on the systemic

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vascular tone under in vivo conditions, particularly when considering that activation of

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prejunctional D3 receptors results in inhibition of both the sympathetic vasopressor discharge19

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and the vasodepressor sensory CGRPergic discharge (present results).

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Notably, other authors have reported that the dopamine D3 receptor subtype modulates

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nociception transmission12,23,24. These findings could be relevant within the context of our study,

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since: (i) nociceptive stimuli are transmitted by sensory C and Aδ fibres25,26; and (ii) capsaicin-

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sensitive sensory C fibres are involved in the neurogenic CGRPergic decrements in diastolic BP2.

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Likewise, by using conscious transgenic (D3 receptor mutant; D3-/-) and wild-type (D3+/+) mice, 8 ACS Paragon Plus Environment

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Zhu et al.23 suggested that activation of the D3 receptor subtype can modulate the nociceptive

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transmission at different levels, as these receptors are widely distributed in the central and

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peripheral nervous systems13,14. Moreover, the fact that D3 receptors are localized at specific areas

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of the spinal cord involved in sensorimotor processing suggests that these receptors may play a

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role in sensory function27.

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1.4.

Possible location of the D3 subtype inhibiting the perivascular CGRPergic discharge

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Since quinpirole inhibited the neurogenic decrements in diastolic BP without affecting those by

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intravenously administered α-CGRP8, it is reasonable to suggest that the D3 receptor subtype is

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inhibiting the CGRPergic discharge, as previously reported for other Gi/o protein-coupled

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receptors5-7. Indeed, subchronic pre-treatment with capsaicin, which depletes CGRP content and

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destroys sensory C fibres, blocked the decrements in diastolic BP induced by stimulation of the

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perivascular CGRPergic discharge2. This finding implied the involvement of capsaicin-sensitive

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sensory C fibres. Nevertheless, when analysing the neurogenic decrements in diastolic BP in pithed

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rats, it is most likely that quinpirole is activating the D3 receptors located on perivascular

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CGRPergic nerves and/or DRG14,15. Evidently, other studies not contemplated in the present

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investigation may elucidate the precise location of the D3 receptors inhibiting the CGRPergic

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discharge; in this respect, the potential problem of such studies is the presence mRNAs for all

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dopamine receptor subtypes (i.e. D1, D2, D3, D4 and D5) in rat DRG neurons13.

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Whatever the case, in physiological terms, we could speculate that the most likely source of

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dopamine to activate prejunctional D3 receptors on perivascular sensory CGRPergic neurons

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would be the dopamine contained (as the precursor of noradrenaline biosynthesis) in perivascular

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sympathetic nerve terminals. In this respect, some classical studies reported by Manelli et al.28 and

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Bell29 have shown that dopamine released from sympathetic nerves: (i) may interact with 9 ACS Paragon Plus Environment



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prejunctional D2-like receptors inhibiting noradrenaline release; and (ii) is considerably augmented

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under stress-induced increase in sympathetic activity.

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Finally, our study provides no evidence for the signalling mechanisms involved in

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quinpirole-induced inhibition by activation of D3 receptors. Notwithstanding, D2-like (i.e. D2, D3

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and D4) receptors are coupled to heterotrimeric Gi/o proteins that downregulate the intracellular

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adenylyl cyclase activity, inactivating Ca2+ channels and/or activating inwardly rectifying K+

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channels14,16. As previously pointed out, these signal transduction pathways are linked to inhibition

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of both neuronal activity and neurotransmitter release15,30, as specifically suggested in other studies

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for inhibition of CGRP release5-7. On the whole, our results suggest that quinpirole inhibits the

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perivascular CGRPergic discharge by activation of the D3 receptor subtype, while the role of the

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D2 or D4 subtypes seems unlikely. These findings contribute to a better understanding of the

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dopaminergic modulation of the rat perivascular CGRPergic innervation producing systemic

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vasodilation (i.e. neurogenic decrements in diastolic BP).

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2.

Materials and methods

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2.1.

Animals and ethical approval of the experimental protocols

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Experiments were performed in a total of 55 male normotensive Wistar rats (300-350 g; 16–20

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weeks of age), following the procedures previously reported by our group3,5-8. The animals:

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(i) were housed in a special room at a constant temperature (22+2 °C) and humidity (50%); (ii) had

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water and food freely available; and (iii) were maintained at a 12/12-h light-dark cycle (light

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beginning at 7 a.m.). The experimental procedures were approved by our Institutional Ethics

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Committee (CICUAL-Cinvestav, permission protocol number 507-12), in accordance with the

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regulations established by: (i) the Mexican Official Norm (NOM-062-ZOO-1999)31; (ii) the guide


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for the Care and Use of Laboratory Animals in U.S.A.32; and (iii) the ARRIVE guidelines for

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reporting experiments involving animals33.

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2.2.

General methods

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After being deeply anesthetized with sodium pentobarbital (60 µ/kg, i.p.), and provided with

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artificial respiration by endotracheal intubation, all rats were pithed as reported by Villalón et al5.

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This procedure consists of inserting a thin stainless-steel bar through the orbit, until reaching the

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vertebral foramen to exclude the influence of the central nervous system. Immediately thereafter,

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the animals were ventilated with room air through a 7025 Ugo Basile pump (56 strokes/min; stroke

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volume: 20 ml/kg)34. After bilateral cervical vagal denervation, catheters were placed in: (i) the

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left and right femoral veins for the continuous infusions of methoxamine and quinpirole,

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respectively; (ii) the left jugular vein for the continuous infusion of hexamethonium; and (iii) the

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right jugular vein, for the bolus injections of gallamine or the dopamine receptor antagonists.

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Subsequently, the left carotid artery was connected to a Grass pressure transducer (P23 XL), for

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the recording of BP. Both, HR (measured with a 7P4F tachograph) and BP were recorded

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simultaneously by a model 7D Grass polygraph (Grass Instrument Co., Quincy, MA, U.S.A.).

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The stimulus-response curves (S-R curves), which consisted of frequency dependent

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decrements in diastolic BP, were completed in about 50 min. Each electrical stimulation was given

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every 5-10 min3,8. Each pithed animal was monitored with a rectal thermometer to maintain their

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body temperature at 37°C.

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2.3.

Experimental protocol: stimulation of the rat perivascular CGRPergic discharge

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After the hemodynamic conditions were stable for at least 30 min, baseline values of diastolic BP

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(a specific peripheral indicator of the systemic vascular tone) and HR were determined, as

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previously reported3,5-8. Then, the pithing rod was replaced by an electrode coated except for 11 ACS Paragon Plus Environment



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1.5 cm length 9 cm from the tip, placing the uncoated section at T9-T12 of the spinal cord to

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stimulate selectively the rat perivascular CGRPergic discharge3,8. Before applying the electrical

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stimuli, the animals received (i.v.): (i) 25 mg/kg of gallamine to avoid the electrically-induced

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muscular spasms; (ii) a continuous infusion of 2 µ/kg.min of hexamethonium (10 min later) to

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block the increments in diastolic BP resulting from stimulation of the preganglionic sympathetic

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discharge; and (iii) a continuous infusion of 20 µg/kg.min of the vasoconstrictor methoxamine

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(10 min later) to increase diastolic BP at around 135 mm Hg, as previously described3,8,35. At this

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point, the 55 rats were divided into three main groups (n=15; 20 and 20; see Figure 4).

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The first group (n=15) was subdivided into three subgroups (n=5 each) that received:

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(a) only the electrical stimuli (control group); (b) an i.v. infusion of saline (control with vehicle;

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0.02 ml/min); and (c) an i.v. infusion of 0.1 µg/kg.min quinpirole, which produces a maximal

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inhibition of the decrements in diastolic BP produced by stimulation of the rat perivascular

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CGRPergic discharge8. Twenty min later, this CGRPergic discharge was electrically stimulated to

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elicit decrements in diastolic BP by applying 10s trains of monophasic rectangular pulses (2 ms,

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50 V) at 0.56, 1, 1.8, 3.1 and 5.6 Hz as reported in previous studies3,8. When diastolic BP returned

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to baseline levels, the next frequency was applied until the S-R curve was completed.

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The second group (n=20) was subdivided into four subgroups (n=5 each) that received an

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i.v. bolus injection of, respectively: (a) 5% ascorbic acid (control; 1 ml/kg); (b) L-741,626

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(300 µg/kg); (c) SB-277011-A (300 µg/kg); and (d) L-745,870 (100 µg/kg). After 10 min, an S-R

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curve was elicited as described above during an infusion of saline (0.02 ml/min) to analyse the

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antagonists’ effects by themselves on the neurogenic decrements in diastolic BP.

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The third group (n=20) was subdivided into four subgroups (n=5 each) that received an i.v.

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bolus injection of, respectively: (a) 5% ascorbic acid (vehicle; 1 ml/kg); (b) L-741,626 (300


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µg/kg); (c) SB-277011-A (300 µg/kg); and (d) L-745,870 (100 µg/kg). After 10 min, an S-R curve

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was elicited as described above during an infusion of quinpirole (0.1 µg/kg.min).

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In view that the neurogenic decrements in diastolic BP are highly tachyphylactic, only one

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S-R curve was performed per animal3,8. Once the experiments ended, all animals were

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disconnected from the artificial respirator and nulled cardiovascular variables were verified.

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Hexamethonium, methoxamine, saline, and quinpirole were infused at a rate of 0.02 ml/min

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by a WPI model sp100i pump (World Precision Instruments Inc., Sarasota, FL, U.S.A.). Moreover,

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during the methoxamine infusion (20 µg/kg.min) a sustained increment in diastolic BP was

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produced in all cases. Regarding the electrical stimuli that were applied, the interval between the

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different frequencies of stimulation depended on the duration of the decrements induced

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(5-10 min), as we waited until diastolic BP returned to baseline values.

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2.4.

Compounds

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The compounds used in the present study (all obtained from Sigma Chemical Co., St. Louis, MO,

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U.S.A.) were: gallamine triethiodide, hexamethonium chloride, methoxamine hydrochloride,

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(±)-quinpirole dihydrochloride, (±)-3-[4-(4-chlorophenyl)-4-hydroxypiperidinyl]methylindole

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(L-741,626),

17

hydrochloride

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yl)ethyl]cyclohexyl]-4-quinolininecarboxamide} hydrochloride (SB-277011-A). All compounds

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were dissolved in physiological saline, except L-745,870, SB-277011-A, and L-741,626 which

20

were dissolved in 5% ascorbic acid in saline. Each solution was prepared the same day and right

21

before each experiment. The doses of the above compounds refer to the free base of reagents, except

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in the case of gallamine and hexamethonium, where they refer to the corresponding salts.

3-[[4-(4-chlorophenyl) (L-745,870)

and

piperazin-1-yl]methyl]-1H-pyrrolo[2,3-b]pyridine

{trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-

23




1

2.5.

Data presentation and statistical evaluation

2

All data are presented as means ± S.E.M. Changes in diastolic BP produced by electrical

3

stimulation in saline- and quinpirole-treated animals were expressed as percent change from

4

baseline at the steady-state effect of methoxamine, as previously reported3,8. The difference

5

between the variations in diastolic BP induced by electrical stimulation within the different

6

subgroups of animals was compared with a two-way repeated measures analysis of variance

7

calculated by the software Sigma Plot 12.0. Such analysis was followed, if applicable, by the

8

Student-Newman-Keul’s post hoc test. Statistical significance was accepted at P

Activation of Dopamine D3 Receptor Subtypes Inhibits the

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