6-Nitrodopamine potentiates contractions of rat isolated vas deferens induced by noradrenaline, adrenaline, dopamine and electric field stimulation

6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that is released from the rat isolated vas deferens, and has been characterized as a major modulator of the contractility of rat isolated epididymal vas deferens (RIEVD). Drugs such as tricyclic antidepressants, α1 and β1β﻿2 adrenoceptor blockers, act as selective antagonists of the 6-ND receptor in the RIEVD. In the rat isolated atria, 6-ND has a potent positive chronotropic action and causes remarkable potentiation of the positive chronotropic effects induced by dopamine, noradrenaline, and adrenaline. Here, whether 6-ND interacts with the classical catecholamines in the rat isolated vas deferens was investigated. Incubation with 6-ND (0.1 and 1 nM; 30min) caused no contractions in the RIEVD but provoked significant leftward shifts in the concentration-response curves to noradrenaline, adrenaline, and dopamine. Pre-incubation of the RIEVD with 6-ND (1 nM), potentiated the contractions induced by electric-field stimulation (EFS), whereas pre-incubation with 1 nM of dopamine, noradrenaline or adrenaline, did not affect EFS-induced contractions. In tetrodotoxin (1 μM) pre-treated (30 min) RIEVD, pre-incubation with 6-ND (0.1 nM) did not cause leftward shifts in the concentration-dependent contractions induced by noradrenaline, adrenaline, or dopamine. Pre-incubation of the RIEVD with the α2A-adrenoceptor antagonist idazoxan (30 min, 10 nM) did not affect dopamine, noradrenaline, adrenaline, and EFS-induced contractions. However, when idazoxan (10 nM) and 6-ND (0.1 nM) were simultaneously pre-incubated (30 min), a significant potentiation of the EFS-induced contractions of the RIEVD was observed. 6-nitrodopamine causes remarkable potentiation of dopamine, noradrenaline, and adrenaline contractions on the RIEVD, due to activation of adrenergic terminals, possibly via pre-synaptic adrenoceptors.


Introduction
The plexus formed by the sympathetic nerves represent the main excitatory component of the vas deferens (Quintas & Noël, 2009), and noradrenaline and ATP are the principal mediators co-released after nerve stimulation (Sneddon & Westfall, 1984). Electric-field stimulation (EFS) causes a biphasic muscular contraction, with an initial purinergic component (ATP), followed by an adrenergic (noradrenaline) component (French & Scott, 1983). The purinergic component is thought to play a major role in the prostatic portion of the vas deferens, whereas the adrenergic component would be more relevant in the epididymal part (Anton et al., 1977). In addition, other neurotransmitters such as dopamine (Morishita & Katsuragi, 1998), neuropeptide Y (Torres et al., 1992) and acetylcholine (Kaleczyc, 1998) may possibly modulate vas deferens motility.
6-nitrodopamine (6-ND) is a novel endogenous catecholamine that has a potent vasorelaxant action in human umbilical cord vessels (Britto-Júnior et al., 2021a), and aortic rings obtained from the tortoise Chelonoidis carbonarius , the snake Panterophis guttatus  and the marmoset Callithrix spp (Britto-Júnior et al., 2023a), where it acts as a truly selective dopamine D 2 -like receptor antagonist. However, in both human (Britto-Júnior et al., 2022b) and rat vas deferens (Britto-Júnior et al., 2021b), 6-ND causes contractions, which are blocked by tricyclic antidepressants (Britto-Júnior et al., 2021b). The contractions induced by 6-ND in the rat vas deferens are also blocked by α 1 - (Britto-Júnior et al., 2022c), β 1 -and β 1 /β 2 -adrenoceptor antagonists (Lima et al., 2022b). In the rat vas deferens, 6-ND is approximately 10 times less potent than noradrenaline and adrenaline, and equipotent to dopamine (Britto-Júnior et al., 2022b). In addition, in the rat isolated right atrium, 6-ND exerts a positive chronotropic effect being one 100 times more potent than noradrenaline and adrenaline, and 10.000 times more potent than dopamine (Britto-Júnior et al., 2022d). 6-ND also strongly potentiates the positive chronotropic effects induced by noradrenaline, adrenaline and dopamine (Britto-Júnior et al., 2023b). This potentiation was inhibited by incubating the atrium with the voltage-gated sodium channel blocker tetrodotoxin (Narahashi et al., 1967) or when the atrium was obtained from animals pre-treated with reserpine, indicating that 6-ND causes release of catecholamines from adrenergic terminals. Here, whether similar interactions between 6-ND and the classical catecholamines occur in the rat isolated epididymal vas deferens was investigated.

Animals
Adult male Wistar rats (280 to 320 g) were provided by the Central Animal House of the University of Campinas (CEMIB-UNICAMP; São Paulo, Brazil) and Animais de Laboratório Criação e Com. LTDA (ANILAB; Paulínia, São Paulo, Brazil). All experimental protocols were approved by the Ethics Committee for Animal Use of the UNICAMP (CEUA; Protocol No. 5942-1/2022) following the Brazilian Guidelines for the Production, Maintenance and Use of Animals for Teaching or Research from the National Council of Control in Animal Experimentation (CONCEA; Andersen, 2016) as well as by the ARRIVE guidelines (Percie du Sert et al., 2020). Animals were housed in cages (three per cage) located in ventilated cage shelters with a constant humidity of 55 ± 5 % and temperature of 24 ± 1°C under a 12-hour light-dark cycle. Animals received filtered water and standard rodent food ad libitum.

Isolation of epididymal vas deferens from rats
Euthanasia was performed by isoflurane overdose, in which the animals were exposed to a concentration greater than 5% until 1 min after which breathing stopped. Exsanguination was then performed. After euthanasia, both vas deferens were removed and immediately placed in Krebs-Henseleit's solution (KHS). The proximal portion of the vas deferens (close to the epididymis) was surgically dissected (length, 1.5 cm each; Britto-Júnior et al., 2021b). The rat isolated epididymal vas deferens (RIEVD) strip was mounted vertically in a glass chamber tissue bath containing 10-mL KHS with a fine sewing thread, one end attached through a loop to a metal hook and the other end tied to an isometric force transducer. The KHS was continuously gassed (95% O 2 : 5% CO 2 ) and kept at 37°C using a Heated Circulator (PolyScience, Illinois, USA). The tissues were allowed to equilibrate under a resting tension of 10 mN, and the isometric tension was registered using a PowerLab system (ADInstruments, Sydney, Australia). Following a 45-min stabilization period, the RIEVD strips were initially contracted with a single concentration of noradrenaline (NA, 10 μM) to verify the tissue viability.

Effect of the reserpine treatment in the interaction of 6-ND with catecholamines in the RIEVD preparations
Rats were treated with reserpine (5 mg/kg, i.p.) once daily, starting two days before the experiment (Murnaghan, 1968). After treatment, cumulative concentration-response curves to dopamine (10 nM -1mM), noradrenaline (10 nM -300 µM), and adrenaline (10 nM -300 µM), were performed in the RIEVD strips in the absence and the presence of 6-ND (100 nM, 30 min).

Interactions of idazoxan on the RIEVD contractions induced by catecholamines and electric-field stimulation (EFS)
Cumulative concentration-response curves to dopamine (10 nM -1mM), noradrenaline (10 nM -300 µM), and adrenaline (10 nM -300 µM), were performed in the RIEVD strips in the absence and in the presence of the α 2A -adrenoceptor antagonist idazoxan (10 nM, 30 min). The EFS-induced contractions of the RIEVD were also performed in the absence and in the presence of idazoxan (10 nM, 30 min).

Interactions of idazoxan and 6-ND on the RIEVD contractions induced by electric-field stimulation (EFS)
The EFS-induced contractions of the RIEVD were performed in control RIEVD and in the RIEVD previously co-incubated (30 min) with both idazoxan (10 nM) and 6-ND (0.1 nM).

Data analysis
Nonlinear regression analysis to determine the pEC 50 was carried out using GraphPad Prism (GraphPad Software, version 9.5, San Diego, California, USA) with the constraint that F = 0. All concentration-response data were evaluated for a fit to a logistics function in the form: E = E max / ([1 + (10c / 10x) n ] + F, where E represents the increase in response contractile induced by the agonist, E max is the effect agonist maximum, c is the logarithm of concentration of the agonist that produces 50% of E max , x is the logarithm of the concentration of the drug; the exponential term, n, is a curve fitting parameter that defines the slope of the concentration-response line, and F is the response observed in the absence of added drug. The values of pEC 50 data represent mean ± standard error of the mean (SEM) of n experiments. Values of E max were expressed in mN. Each animal provided two epidydimal vas deferens (right and left); one strip was used as the control response and the contralateral strip was incubated with an antagonist/inhibitor; n indicates both the number of paired strips (same animal) and the number of rats. Since the study has an exploratory character, the p values should be considered descriptive (Motulsky, 2014;Michel et al., 2020).
The incubation with 0.1 and 1 nM 6-ND did not contract the RIEVD. However, in tissues pre-incubated with 6-ND (0.1 and 1 nM), there were significant leftward shifts in the concentration-response curves to noradrenaline (  Tables 1, 2 and 3 show the potency (pEC 50 ) and the mean shifts of the concentration-response curves to noradrenaline, adrenaline and dopamine, respectively, in the presence of the various concentrations of 6-ND. No significant differences in the maximum response (E max ) were observed (Tables 1, 2 and 3). 6-ND at 10 and 100 nM did not contract the RIEVD, but also caused similar and significant leftward shifts of the noradrenaline concentrationresponse curves (Table 1 and Fig. S1A and Fig. S1D, respectively), adrenaline concentration-response curves (Table 2 and Fig. S1B and Fig. S1E, respectively) and dopamine concentration-response curves (Table 3 and Fig. S1C and Fig. S1F, respectively). The significant leftward shifts on the concentration-response curves of noradrenaline, adrenaline, and dopamine, caused by 6-ND were not proportional to the 6-ND concentrations used (0.1-100 nM), as evaluated by one-way ANOVA (p=0.74, p=0.53 and p=0.75 for noradrenaline, adrenaline, and dopamine, respectively).
The maximum responses (E max ) of the concentrationresponse curves to noradrenaline, adrenaline and dopamine, in the presence and in the absence of different concentrations of 6-ND (10 pM -100 nM), are shown in the supplementary tables (Tables 1, 2 and 3, respectively). In contrast to that observed with the pEC 50 values, pre-incubation with 6-ND did not cause significant differences in the E max (one-tail, unpaired t-test).

Effect of tetrodotoxin on the contractions of the RIEVD induced by noradrenaline, adrenaline, dopamine and 6-ND
Pre-incubation (30 min) of the RIEVD with the sodium channel blocker tetrodotoxin (TTX, 1 μM) caused significant rightward shifts in the concentration-dependent RIEVD contractions induced by 6-ND ( Table 4. Pre-incubation with TTX (30 min, 1 μM) did not cause significant differences in the E max values, as assessed by one-tail, unpaired t-test (Table 4).

Effect of tetrodotoxin on the potentiation by 6-ND of the RIEVD contractions induced by noradrenaline, adrenaline and dopamine
In TTX (30 min, 1 μM) pre-treated RIEVD strips, incubation with 6-ND (100 nM, 30 min) did not cause leftward shifts in the concentration-dependent contractions induced by noradrenaline (Fig. 4A), adrenaline (Fig. 4B), and dopamine ( Fig. 4 C). The potency (pEC 50 ), the mean shifts and E max induced by 6-ND in TTX pre-treated vas deferens are shown in Table S1.

Effects of the in vivo treatments with saline or reserpine on the potentiation by 6-ND of the contractions of the RIEVD induced by noradrenaline, adrenaline, and dopamine
In RIEVD obtained from animals pre-treated with saline, noradrenaline (Fig. 5A), adrenaline (Fig. 5C), and dopamine ( Fig. 5E), caused concentration-dependent contractions, which were significantly potentiated by pre-incubation with , and dopamine (DA) in the rat isolated epididymal vas deferens (RIEVD). Noradrenaline (10 nM -300 μM; Panels A, D and G), adrenaline (10 nM -300 μM; Panels B, E and H) and dopamine (10 nM -1 mM; Panels C, F and I) induced concentration-dependent RIEVD contractions. Pre-incubation of the RIEVD with 6-ND (30 min, 0.1 nM or 1 nM) caused significant leftward shift of the concentration-response curves to noradrenaline (Panels A and D, respectively), adrenaline (Panels B and E, respectively), and dopamine (Panels C and F, respectively). Pre-incubation of the RIEVD with 6-ND (30 min, 0.01 nM) had no effect on the concentrationdependent contractions induced by noradrenaline (Panel G), adrenaline (Panel H), and dopamine (Panel I). n represents the number of vas deferens employed in each experiment Table 1 The potency (pEC 50 ) and the maximum response (E max ) of noradrenaline in the rat isolated epididymal vas deferens in the absence (control) and presence of 6-nitrodopamine (6-ND; 0.01 to 100 nM) pEC50 is defined as the negative logarithm of the EC 50 . Emax is the maximal effect at the highest drug concentration. The pEC50, Emax and the mean shifts were expressed as mean ± SEM. n means the number of vas deferens strips. One-tail unpaired t-test was performed on control vs treated values 6-ND (100 nM), as indicated by leftward shifts of the pEC 50 (Table 5). In RIEVD obtained from animals pre-treated with reserpine, noradrenaline (Fig. 5B), adrenaline (Fig. 5D), and dopamine (Fig. 5F) caused concentration-dependent contractions, which were not significantly potentiated by pre-incubation with 6-ND (100 nM; Table 6). There were no significant differences in the Emax caused by noradrenaline, adrenaline, and dopamine, in the RIEVD obtained from saline-treated animals, compared to the RIEVD obtained from reserpine-treated animals (Table 6).

Effects of in vivo treatments with saline or reserpine on the potentiation by 6-ND of the contractions of the RIEVD induced by electric-field stimulation
In RIEVD obtained from animals pre-treated with either saline (Fig. 6A) or reserpine (Fig. 6B), EFS caused frequencydependent contractions of the RIEVD, although the amplitude of the RIEVD contractions were significantly smaller in reserpine-treated group compared with the saline-treated group (Table 7), as assessed by one-tail, unpaired t-test. In contrast to the saline-treated group, pre-incubation with 6-ND (1 nM, 30 min; Fig. 6B) did not cause significant changes in the amplitude of the EFS-induced contractions of the RIEVD obtained from reserpine-treated group (Fig. 6A, Table 7).

Effect of noradrenaline on the contractions of the RIEVD induced by 6-ND, adrenaline, and dopamine
6-nitrodopamine (10 nM -1 mM; Fig. S5A), adrenaline (10 nM -300 μM; Fig. S5B), and dopamine (10 nM -300 μM; Fig. S5C) induced concentration-dependent contractions of the RIEVD. Pre-incubation of the tissues with noradrenaline (30 min; 1 nM) did not affect the concentration-dependent Table 4 The effect of tetrodotoxin (TTX, 1 μM) on the potency (pEC 50 ) and the maximum response (E max ) of 6-nitrodopamine (6-ND), noradrenaline (NA), adrenaline (ADR) and dopamine (DA) in the rat isolated epididymal vas deferens pEC50 is defined as the negative logarithm of the EC 50 . Emax is the maximal effect at the highest drug concentration. The pEC50, Emax and the mean shifts were expressed as mean ± SEM. n means the number of vas deferens strips. One-tail unpaired t-test was performed on control vs treated values contractions of the RIEVD induced by 6-ND (Fig. S5A), adrenaline (Fig. S5B), and dopamine (Fig. S5C). The values for pEC 50 and E max for the concentration-response curves in the absence and presence of noradrenaline (1 nM) are described in Table S2.

Discussion
The results clearly demonstrated that 6-ND causes potentiation of the contractile action of the classical catecholamines noradrenaline, adrenaline, and dopamine in RIEVD strips at a remarkably low concentration (0.1 nM). This interaction is unique to 6-ND, since it was not observed with the classical catecholamines noradrenaline, adrenaline, and dopamine.
Synthesis of prostaglandins (PGs) has been identified in the rat vas deferens (Johnson & Ellis, 1977), and PGE 2 was the major prostaglandin synthetized by homogenates of this Fig. 4 Effect of the tetrodotoxin (TTX) on the potentiation by 6-nitrodopamine (6-ND) of the concentration-dependent contractions induced by noradrenaline (NA), adrenaline (ADR), and dopamine (DA) in the rat isolated epididymal vas deferens (RIEVD). In TTX (30 min, 1 μM) pre-treated preparations, incubation of the RIEVD with 6-ND (100 nM) had no effect on the concentration-dependent contractions induced by noradrenaline (panel A), adrenaline (panel B), and dopamine (panel C). n represents the number of vas deferens employed in each experiment  (Gerozissis & Dray, 1983). Although PGE 2 up to 60 μM did not alter the tonus of the rat or rabbit vas deferens, it did potentiate the contractions induced by noradrenaline (Swan & Poyser, 1983). In addition, pre-treatment of the rats with the cyclo-oxygenase inhibitor indomethacin (Vane,1971) shifted the noradrenaline concentrationdependent curves in the rat isolated vas deferens to the right (Radomirov & Venkova, 1986), indicating that endogenous PGs could modulate rat vas deferens smooth muscle contractility. However, the finding that treatment of the isolated tissue with indomethacin did not affect the potentiation induced by 6-ND indicates that prostaglandin release is not the mechanism responsible for the potentiation observed.
The smooth muscle of the rat vas deferens is mainly innervated by post-ganglionic adrenergic terminals, however it does receive some cholinergic nerves (Burnstock & Costa, 1975). Acetylcholine is released from rat vas deferens (Knoll et al., 1972) and acetylcholine itself contracts the rat vas Table 5 The potency (pEC 50 ) of noradrenaline (NA), adrenaline (ADR) and dopamine (DA), in the rat isolated epididymal vas deferens incubated or not with 6-nitrodopamine (6-ND, 100 nM), obtained from animals treated in vivo with saline pEC50 is defined as the negative logarithm of the EC 50 . Emax is the maximal effect at the highest drug concentration. The pEC50, Emax and the mean shifts were expressed as mean ± SEM. n means the number of vas deferens strips. One-tail unpaired t-test was performed on control vs treated values  deferens (Graham et al., 1968). Interestingly, acetylcholine and other cholinergic agonists such as carbachol and arecoline, potentiate the contractions induced by EFS of the rat vas deferens (Chung & Freer, 1983) via an atropine sensitive-muscarinic receptor, independent of an enhancement of adrenergic neurotransmission (Lee, 1985). The finding that the non-selective muscarinic receptor antagonist atropine did not affect the potentiation induced by 6-ND indicates that the activation of the cholinergic terminals is not responsible for the observed potentiation. Indeed, the role of the cholinergic system on rat vas deferens contractility is obscure, since atropine does not affect EFS-induced contractions of the rat isolated epididymal vas deferens (Britto-Júnior et al., 2021b). Adenosine triphosphate (ATP) contracts guinea-pig (Westfall et al., 1978) and rat (Göçmez et al., 2010) isolated vas deferens and sympathetic transmission is modulated by noradrenaline and ATP release, which are co-transmitters present in sympathetic nerves in the vas deferens of rat, rabbit and guinea-pig (Sneddon & Machalay, 1992). In addition, noradrenaline and ATP are synergistic in the mouse (Witt et al., 1991) and in the guinea-pig vas deferens (Kazic & Milosavljevic, 1980), and in the rat isolated prostatic vas deferens (Huidobro-Toro & Parada, 1988). Thus, one possibility could be that the potentiation induced by 6-ND could be due to activation of the purinergic pathway, such as enhancement of ATP release. Although this hypothesis deserves further investigation, it is unlikely that this mechanism would explain the potentiation induced by 6-ND, since a significant reduction in the potentiation induced by 6-ND was observed in animals that were pre-treated with reserpine. Sympathetic nerve-mediated release of ATP from the guinea-pig vas deferens was unaffected by reserpine (Kirkpatrick & Burnstock, 1987), and reserpine pre-treatment did not affect the contractions induced by electric field stimulation of prostatic segments of the rat isolated vas deferens (Pennefather, 1983). Table 7 Effect of in vivo treatment with reserpine on the potentiation by 6-nitrodopamine (6-ND; 1 nM) of the contractions induced by electric-field stimulation (2-16 Hz) in the rat isolated epididymal vas deferens. Control groups received saline instead of reserpine pEC50 is defined as the negative logarithm of the EC 50 . The pEC50 and the mean shifts were expressed as mean ± SEM. n means the number of vas deferens strips. One-tail unpaired t-test was performed on control vs 6-ND-treated strips. * indicates P-value <0.05 compared with respective control vs pre-treated with 6-ND 1 nM; # indicates P-value < 0.05 compared with respective control values (absence of 6-ND) in saline group  The finding that sodium channel blocker tetrodotoxin (Naharashi et al., 1967) abolished the potentiation induced by 6-ND of the contractions caused by noradrenaline, adrenaline, and dopamine clearly indicates that the 6-ND potentiation involves activation of nerve terminals. Since pre-treatment of the animals with reserpine attenuated the potentiation induced by 6-ND, one possible interpretation could be that 6-ND activates adrenergic terminals and promotes release of catecholamines, as observed in the rat isolated atria (Britto-Júnior et al., 2023b).
What is(are) the mechanism(s) involved in this activation of adrenergic terminals by 6-ND in the vas deferens? One possibility could be that 6-ND has a tyramine-like effect. Tyramine releases 3 H-noradrenaline from rat vas deferens in vitro (Barnett et al., 1969), indicating that the actions induced by tyramine in the rat vas deferens, like that observed in the rabbit heart (Lindmar & Muscholl, 1961) and cat aorta (Lockett & Eakins, 1960), are related to release of catecholamines. In addition, pretreatment of the animals with reserpine caused the complete disappearance of noradrenaline from adrenergic terminals of the rat vas deferens (Taxi & Droz, 1966) and abolished the contractions induced by tyramine (Patil et al., 1967). However, the finding that the potentiation induced by 6-ND of the contractions induced by catecholamine is sensitive to tetrodotoxin does not support this concept. Tetrodotoxin blocks axonal conduction in various nerve-smooth muscle preparations (Kuriyama et al., 1966;Tomita, 1966) and tetrodotoxin, although abolishes the contractions induced by nerve stimulation in the rat vas deferens (Bell, 1968;Lima et al., 2022a), did not alter the contractions induced by tyramine (Bell, 1968). In the synaptosomal fraction prepared from rabbit jejunum, tyramine released noradrenaline, which was insensitive to tetrodotoxin, and not affected by the removal of calcium ions from the bathing medium (Cheng et al., 1987). The release of noradrenaline by tyramine apparently involves a non-exocytotic mechanism, as described in the dog saphenous veins (Brandão et al., 1980). Basal release of noradrenaline has been identified from rat vas deferens (Britto-Júnior et al., 2021b) and human vas deferens (Britto-Júnior et al., 2022b), as measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Stimulated release of noradrenaline (followed electric field stimulation) from guinea-pig vas deferens was identified by liquid chromatography coupled to electrochemical detection (Gillespie & Macrae, 1983;Todorov et al., 1996). The release of noradrenaline from adrenergic terminals is modulated by presynaptic inhibitory α 2 -adrenoceptors (Langer, 1976). Three distinct genes are responsible for the synthesis of three subtypes of α 2 -adrenoceptors, named α 2A , α 2B and α 2C (Bylund et al., 1994), and they are widely distributed both in the CNS and in the periphery (McCune et al., 1993). The α 2A -adrenoceptor subtype has dominant role in the presynaptic inhibition of noradrenaline release (Altman et al., 1999), although in peripheral tissues all the three α 2 -adrenoceptor subtypes play a role in the feedback regulation of neurotransmitter release (Trendelenburg et al., 2003;Gyires et al., 2009). The α 2A -adrenoceptor antagonist idazoxan (10-1440 μg/kg, i.v.) potentiates electrically induced contractions of the rat vas deferens (Doxey et al., 1985). In vitro, idazoxan at 0.1 and 1 μM, also potentiates Fig. 8 The putative 6-ND receptors and actions in the rat isolated vas deferens the EFS-induced contractions of the rat isolated epididymal vas deferens (Britto-Júnior et al., 2022c). Thus, inhibition of presynaptic α 2 -adrenoceptors could be a possible mechanism for explaining the potentiating action of 6-ND. However, the finding that 6-ND at 0.1 nM, a concentration that does not potentiate EFS-induced vas deferens contractions, co-incubated with idazoxan at 10 nM, a concentration that does not potentiate EFS-induced vas deferens contractions, causes a significant potentiation of the EFS-induced contractions, reveals a remarkable synergistic effect. One possible explanation could be that 6-ND is acting on a different α 2subtype adrenoceptor than idazoxan, such as α 2B and/or α 2C adrenoceptors. Experiments with subtype selective α 2B and α 2C antagonists may clarify this issue.
Isoprenaline (12 nM) increases the stimulation-induced efflux of noradrenaline (Adler-Graschinsky & Langer, 1975), indicating that low concentration of b-adrenoceptor agonists facilitates noradrenaline release from nerve terminals (Majewski and Rand, 1981). Indeed, two pre-synaptic mechanisms modulate noradrenaline during nerve stimulation. The first one, mediated by b-adrenoceptors, operates at low frequencies of nerve stimulation, leading to an increase in transmitter release whereas the second one, mediated by α 2 -adrenoceptors, triggered when higher concentrations of the neurotransmitter are reached in the synaptic cleft, leads to inhibition of neurotransmitter release (Langer, 1976). Thus, 6-ND could be acting as an agonist of the β-presynaptic adrenoceptors. This is an interesting hypothesis, since the contractions induced by 6-ND on the rat isolated epididymal vas deferens are inhibited by β 1 -and β 1 /β 2 -adrenoceptors antagonists (Lima et al., 2022b). Whether these β-antagonists block the potentiation-induced by 6-ND remains to be investigated.
Our current understanding on the putative 6-ND receptors and actions in the rat isolated vas deferens is summarized in Figure 8. The direct contractile action of 6-ND in the vas deferens is due to its action on the 6-ND 1 receptor, which is located in the smooth muscle, and selectively blocked by tricyclic antidepressants (TCA; Britto-Júnior et al., 2021b;Britto-Júnior et al., 2022b), α 1 -adrenoceptor antagonists (Britto-Júnior 2022c) and β 1 and β 1 β 2 -adrenoceptor antagonists . 6-ND also acts on pre-synaptic receptors (6-ND 2 ), which increase release of classical catecholamines. Whether the 6-ND 2 receptor is associated to α 2 or β 1 pre-synaptic receptor, is yet to be determined.

Conclusion
6-nitrodopamine causes remarkable potentiation of dopamine, noradrenaline and adrenaline contractions on the RIEVD, due to activation of adrenergic terminals, most likely via pre-synaptic adrenoceptors.

Declarations
Ethical approval All experimental protocols were authorized by the Ethics Committee in Animal Use of UNICAMP (CEUA/UNICAMP, protocol numbers 5942-1/2022).

Consent to publish
The authors authorize the submission and publication of this article in Naunyn-Schmiedeberg's Archives of Pharmacology