β1- and β1/β2-adrenergic receptor antagonists block 6-nitrodopamine-induced contractions of the rat isolated epididymal vas deferens

6-Nitrodopamine (6-ND) is an endogenous modulator of the contractility in the rat isolated epididymal vas deferens (RIEVD) and considered to be the main peripheral mediator of the emission process. Use of selective and unselective β-adrenergic receptor antagonists has been associated with ejaculatory failure. Here, the effects of selective β1- and β1/β2-adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline, and electric-field stimulation (EFS) were investigated. The selective β1-adrenergic receptor antagonists atenolol (0.1 and 1 µ﻿M), betaxolol (1 µ﻿M), and metoprolol (1 µ﻿M) and the unselective β1/β2-adrenergic receptor antagonists propranolol (1 and 10 µ﻿M) and pindolol (10 µ﻿M) caused significant rightward shifts of the concentration–response curve to 6-ND (pA2 6.41, 6.91, 6.75, 6.47, and 5.74; for atenolol, betaxolol, metoprolol, propranolol, and pindolol), but had no effect on dopamine-, noradrenaline-, and adrenaline-induced contractions. The effects of selective β1- and β1/β2-adrenergic receptor antagonists at a higher concentration (atenolol 1 µ﻿M, betaxolol 1 µ﻿M, metoprolol 1 µ﻿M, propranolol 10 µ﻿M, and pindolol 10 µ﻿M) also reduced the EFS-induced RIEVD contractions in control, but not in RIEVD obtained from L-NAME-treated animals. The selective β1-adrenoceptor agonist RO-363, the selective β2-adrenoceptor agonist salbutamol, and the selective β3-adrenoceptor agonist mirabegron, up to 300 µ﻿M, had no effect on the RIEVD tone. The results demonstrate that β1- and β1-/β2-adrenoceptor receptor antagonists act as 6-ND receptor antagonists in RIEVD, further confirming the main role of 6-ND in the RIEVD contractility.

In the rat epididymal vas deferens, 6-ND has been characterized as a major endogenous modulator of the contractility of this tissue (Britto-Jr et al. 2021b;Britto-Jr et al. 2022). Tricyclic antidepressants such as clomipramine (Millan et al. 2001), desipramine (Cusack et al. 1994), and amitriptyline (Sánchez and Hyttel 1999) and α 1 -adrenergic receptor antagonists such as doxazosin (Elliott et al. 1982;Wilt and MacDonald 2006), tamsulosin (Lepor et al. 1988;Dunn et al. 2002), terazosin (Frishman et al. 1988;O'Leary 2001), and alfuzosin (Ramsay et al. 1988) act as 6-ND receptor antagonist in the rat vas deferens (Britto-Jr et al. 2021b;2022). One known adverse reaction of these two classes of drugs is the impairment of the ejaculatory process (Beaumont 1977;Cavallini 1995;Hsieh et al. 1999;Debruyne 2000). Indeed, both classes of drugs are used for the treatment of premature ejaculation (Hellstrom and Sikka 2006;Basar et al. 2005), indicating a major role for 6-ND in the ejaculatory process. In pre-clinical studies, male rats treated for 16 weeks with the non-selective β-adrenoceptor antagonist propranolol (1.25 mg/ day) exhibited an impairment in the ejaculation and copulatory pattern (Srilatha et al. 1999). Subcutaneous administration of the non-selective β-blocker pindolol (4 mg/kg, 30 min) to male rats was also associated with inhibition of the sexual behavior, as evidenced by an increase in mounts, intromissions, and time to ejaculate (Ahlenius and Larsson 1991). In patients with arterial hypertension, coronary artery disease, or heart failure, meta-analytic data have shown that β-blockers are associated with a small, but significant, increase in risk of reported sexual dysfunction, which was not related to the lipid-soluble β-blockers (Ko et al. 2002). The use of the β 1 -, β 2 -, and α 1 -adrenergic receptor antagonist labetalol was associated with ejaculatory failure soon after the initiation of therapy that resolved with drug discontinuation (O'Meara and White 1988). In a double-blind, placebo-controlled trial comprising eighty-six paroxetine-refractory patients, pindolol, at the dose of 7.5 mg/day, increased significantly the mean intravaginal ejaculatory latency time after 6 weeks of treatment (Safarinejad 2008). Thus, both the experimental and clinical observations open the interesting possibility that β-blockers could act as 6-ND receptor antagonists in the vas deferens, as observed with tricyclic antidepressants and α 1 -adrenergic receptor antagonists.

Animals
Adult male Wistar rats (280-320 g) were obtained from the animal care of University of Campinas (UNICAMP; Campinas, São Paulo, Brazil) and Animais de Laboratorio Criação e Com. LTDA (ANILAB; Paulinia, São Paulo, Brazil). All experimental protocols were authorized by the Ethics Committee in Animal Use of UNICAMP (CEUA/UNICAMP, protocol numbers 5952-1/2022 and 5831-1/2021) and followed the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines (Percie du Sert et al. 2020). Animals were housed in cages (three per cage) located in ventilated cage shelters with constant humidity of 55% ± 5% and temperature of 24 ± 1 °C under a 12-h light-dark cycle. Animals received filtered water and standard food ad libitum.

Chronic L-NAME treatment
Animals were treated with L-NAME dissolved in the drinking filtered water at a concentration of approximately 20 mg/rat/day for a minimum of 4 weeks (Ribeiro et al. 1992). Control animals received filtered water alone. Vas deferens obtained from these chronically treated animals present lower release of 6-ND, as quantified by LC-MS/ MS (Britto-Júnior et al. 2021a, 2021b.

Rat isolated epididymal vas deferens (RIEVD) isolation and preparation
Euthanasia was performed by isoflurane overdose, in which animals were exposed to a concentration greater than 5% until 1 min after the breathing stops. Exsanguination was performed to confirm the euthanasia. The vas deferens was 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) for the functional studies (Burnstock and Verkhratsky 2010). The RIEVD strips were suspended vertically between metal hooks in 10-mL custom designed glass chambers containing KHS, continuously gassed with a mixture of 95%O 2 :5%CO 2 at 37 °C using a heated circulator (PolyScience, IL, USA). 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.

Data analysis
Nonlinear regression analysis to determine the pEC 50 was carried out using GraphPad Prism (GraphPad Software, version 9.0, San Diego, CA, USA) with the constraint that F = 0. All concentration-response data were evaluated for a fit to a logistics function in the form: 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 standard deviation (SD) of n experiments. Values of E max were expressed in mN. Each animal provided two epididymal 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. Student's two-tail unpaired t-test was employed and the differences between groups. In addition, standard ANOVA, followed by the Newman-Keuls post-test, were used when more than two groups were involved. A p value of less than 0.05 was considered statistically significant. Since the study has an exploratory character, the p values should be considered descriptive (Motulsky 2014;Michel et al. 2020). For 6-ND, the pA 2 values of the antagonists were calculated from the intercept on the concentration axis and by application of the equation: pA 2 = log (antagonist concentration) − log (CR-1) − log (antagonist concentration) (Arunlakshana and Schild 1959).

Effect of atenolol on RIEVD contractions induced by catecholamines and EFS
Atenolol (0.1 and 1 µM) produced concentration-dependent rightward shifts on the concentration-response curves to 6-ND ( Fig. 1A; p = 0.0284 and p = 0.0068, respectively) with a pA 2 value of 6.51 ± 0.54 (n = 4). Atenolol (1 µM) had no effect on the RIEVD contractions induced by dopamine Atenolol (0.1 µM) had no effect on the EFS-induced contractions of RIEVD (Fig. 1F), but at a higher concentration (1 µM), atenolol caused significant reductions on the EFSinduced contractions of the RIEVD in all frequencies tested (Fig. 1F), which was not observed in RIEVD obtained from animals chronically treated with L-NAME (Fig. 1G).

Effect of metoprolol on RIEVD contractions induced by catecholamines and EFS
Metoprolol at 0.1 µM had no effect on 6-ND-induced RIEVD contractions, but at 1 µM it caused a significant rightward shift on the concentration-response curve to 6-ND ( Fig. 3A; p = 0.0159) with a pA 2 value of 6.75 ± 0.08 (n = 4). Metoprolol Fig. 1 Effect of atenolol in the rat isolated epididymal vas deferens (RIEVD). Atenolol (0.1 and 1 µM) caused significant concentrationdependent rightward shifts of the concentration-response curves to 6-ND (A). Atenolol (1 µM) had no effect on the RIEVD contractions induced by dopamine (DA; B), noradrenaline (NA; C), and adrenaline (ADR; D) concentration-response curves. Atenolol (0.1 µM; E) had no effect on the EFS-induced contractions but atenolol (1 µM) reduced the contractions induced by EFS (F). Atenolol (1 µM) had no effect on the EFS-induced contractions of the RIEVD obtained from animals chronically treated with L-NAME (G). Data are expressed as mean ± SD. *p < 0.05 compared with respective control values. ANOVA followed by the Newman-Keuls post-test was applied in A whereas the unpaired t-test was applied in B-G. n means the number of vas deferens strips 1 3 (1 µM) had no effect on the RIEVD contractions induced by dopamine ( Fig. 3B; p = 0.4540), noradrenaline ( Fig. 3C; p = 0.1887), and adrenaline ( Fig. 3D; p = 0.3795).
Metoprolol at 0.1 µM had no significant effect on EFSinduced RIEVD contractions (Fig. 3E); however, at 1 µM metoprolol caused significant reductions of the EFS-induced contractions in all frequencies tested (Fig. 3F). In RIEVD obtained from animals chronically treated with L-NAME, metoprolol (1 µM) had no effect on the EFS-induced contractions (Fig. 3G).
Propranolol at 1 µM had no significant effect on EFSinduced RIEVD contractions (Fig. 4E); however, at 10 µM propranolol caused significant reductions of the EFS-induced contractions at the frequencies of 4 to 16 Hz (Fig. 4F), which was not observed in RIEVD obtained from animals chronically treated with L-NAME (Fig. 4G).   Fig. 3 Effect of metoprolol in the rat isolated epididymal vas deferens (RIEVD). Metoprolol (1 µM) caused significant concentration-dependent rightward shifts of the concentrationresponse curves to 6-ND (A). Metoprolol (1 µM) had no effect on the RIEVD contractions induced by dopamine (DA; B), noradrenaline (NA; C), and adrenaline (ADR; D) concentration-response curves. Metoprolol (0.1 µM) had no effect on the EFS-induced contractions (E), but at higher concentration (1 µM) significantly reduced the contractions in all frequencies tested (F). In RIEVD obtained from animals chronically treated with L-NAME, metoprolol (1 µM) failed to affect the EFSinduced contractions (G). Data are expressed as mean ± SD. *p < 0.05 compared with respective control values. ANOVA followed by the Newman-Keuls post-test was applied in A whereas the unpaired t-test was applied in B-G. n means the number of vas deferens strips 1 3
Lower concentrations of pindolol (1 and 3 µM) had no significant effect on the contractions induced by 6-ND (Fig. 5A). Pindolol (10 µM) had no effect on the RIEVD contractions induced by dopamine ( Fig. 5B; p = 0.2102), noradrenaline ( Fig. 5C; p = 0.3951), and adrenaline ( Fig. 5D; p = 0.2394). Fig. 4 Effect of propranolol in the rat isolated epididymal vas deferens (RIEVD). Propranolol (1 and 10 µM) caused significant concentrationdependent rightward shifts of the concentration-response curves to 6-ND (A). Propranolol (10 µM) had no effect on the RIEVD contractions induced by dopamine (DA; B), noradrenaline (NA; C), and adrenaline (ADR; D) concentrationresponse curves. Propranolol (1 µM) had no effect on the EFS-induced contractions (E), but at higher concentration (10 µM) significantly reduced the contractions in all frequencies tested (F). In RIEVD obtained from animals chronically treated with L-NAME, propranolol (10 µM) failed to affect the EFS-induced contractions (G). Data are expressed as mean ± SD. *p < 0.05 compared with respective control values. ANOVA followed by the Newman-Keuls post-test was applied in A whereas the unpaired t-test was applied in B-G. n means the number of vas deferens strips Pindolol at 1 µM had no significant effect on EFSinduced RIEVD contractions (Fig. 5E); however, at 10 µM, pindolol caused significant reductions of the EFS-induced contractions at the frequencies of 4 to 16 Hz (Fig. 5F), which was not observed in RIEVD obtained from animals chronically treated with L-NAME (Fig. 5G).

Effect of RO-363, salbutamol, and mirabegron on RIEVD tone
The selective β 1 -adrenoceptor agonist RO-363 (Fig. 6A), the selective β 2 -adrenoceptor agonist salbutamol (Fig. 6B), and the selective β 3 -adrenoceptor agonist mirabegron (Fig. 6C), up to 300 µM, had no effect on the RIEVD tone. Fig. 5 Effect of pindolol in the rat isolated epididymal vas deferens (RIEVD). Pindolol (10 µM) caused significant concentration-dependent rightward shifts of the concentrationresponse curves to 6-ND (A). Pindolol (10 µM) had no effect on the RIEVD contractions induced by dopamine (DA; B), noradrenaline (NA; C), and adrenaline (ADR; D) concentration-response curves. Pindolol (1 µM) had no effect on the EFS-induced contractions (E), but at higher concentration (10 µM) significantly reduced the contractions in all frequencies tested (F). In RIEVD obtained from animals chronically treated with L-NAME, pindolol (10 µM) failed to affect the EFS-induced contractions (G). Data are expressed as mean ± SD. *p < 0.05 compared with respective control values. ANOVA followed by the Newman-Keuls post-test was applied in A whereas the unpaired t-test was applied in B-G. n means the number of vas deferens strips

Discussion
The results clearly indicate that both selective and nonselective β-blockers can antagonize the contractions of the rat epididymal vas deferens induced by 6-ND, as observed with α 1 -adrenergic receptor antagonists and tricyclic depressants. These findings also reinforce the role of 6-ND as the major modulator of rat epididymal vas deferens contractility, since the contractions induced by electric-field stimulation were inhibited by the β-blockers only at the concentrations that caused right-shifts of the 6-ND concentration-response curves. The inhibition of RIEVD contractions by the β-blockers was not observed in the vas deferens obtained from animals chronically treated with L-NAME, further supporting the concept that the inhibition of EFS-induced by β-receptor antagonists is due to blockade of 6-ND action. β 1 -Adrenergic receptors are not considered relevant for contractile activity in the rat vas deferens, since this tissue contains a homogenous population of β 2 -adrenoceptors that inhibit field-stimulated contractions (Vohra 1979). Radioligand binding using [ 125 I]-pindolol in the rat vas deferens labeled a single class of high affinity binding sites with properties consistent with a population of β 2 -adrenoceptors (May et al. 1985). In the rat vas deferens, β 2 -adrenergic antagonists such as carazolol is more potent to displace [ 3 H]-dihydroalprenolol binding compared to β 1 -adrenergic antagonists such as atenolol and practolol (Chang and Lotti 1983). Indeed, the finding that the selective β 1 -adrenergic receptor agonist RO-363 (Iakovidis et al. 1980) had no contractile activity per se confirms the relative unimportance of modulatory role for this subclass of receptors in the vas deferens. The lack of contractile activity of RO-363 clearly demonstrates that the contractions induced by 6-ND are not due to activation of β 1 -adrenergic receptors. Similar results were obtained with the selective β 2 -adrenergic Fig. 6 Effect of the selective β 1 -adrenergic receptor agonist RO-363 (A), selective β 2adrenergic agonist salbutamol (B), and of the selective β 3adrenergic agonist mirabegron (C) in the rat isolated epididymal vas deferens tone. n means the number of vas deferens strips Fig. 7 Electric-field stimulation (EFS) caused frequency-dependent contractions of the isolated rat epididymal vas deferens (RIEVD), which were abolished by pre-treatment with tetrodotoxin (TTX, 1 µM). Data are expressed as mean ± SD. *p < 0.05 compared with respective control values in the unpaired t-test. n means the number of vas deferens strips agonist salbutamol and the selective β 3 -adrenergic agonist mirabegron, indicating that the contractile activity induced by 6-ND is independent of β-adrenergic receptor activation. 6-ND has been considered the endogenous mediator of EFSinduced contractions in the rat vas deferens (Britto-Júnior et al. 2021b), and these results further support the concept that 6-ND is acting on a specific 6-ND receptor.
In the rat vas deferens, the adrenergic axons are clearly identified within smooth muscle cells, and some are completely ensheathed in the smooth muscle cells (Furness and Iwayama 1971). Thus, the high concentrations of β-blockers required to inhibit EFS-induced contractions could reflect restricted access to the β-adrenergic receptors located in deeper layers of smooth muscle cells. However, this anatomical hypothesis is unlikely since EFS-induced contractions are inhibited by much lower concentrations of α-adrenergic antagonists (Britto-Júnior et al. 2022).
Although ejaculatory disorders have been reported with the use of selective and unselective β-blockers, the incidence is rather low (reported cases) when compared to α 1adrenergic receptor antagonists (4-11%; Höfner et al. 1999). This major difference in incidence (Djavan et al. 2004) could be easily attributed to the observed major potency (over 100 times) of α 1 -blockers (the pA 2 values are 9. 66, 9.15, 8.86, 7.70, 7.20, and 8.82 for tamsulosin, doxazosin, alfuzosin, silodosin, terazosin, and prazosin;Britto-Júnior et al 2022) in blocking 6-ND contractile activity compared to the β-blockers (6.41, 6.91, 6.75, 6.47, and 5.74; for atenolol, betaxolol, metoprolol, propranolol, and pindolol, respectively; this manuscript), further supporting a major role of 6-ND in the ejaculatory process. Although β 1 -, β 2 -, and β 3adrenergic receptor agonists per se did not induce contractions in the rat vas deferens, these findings do not exclude a potential modulatory role on vas deferens contractility. The finding that the sodium channel antagonist tetrodotoxin (Narahashi et al. 1967;Lee and Ruben 2008) abolished EFSinduced contractions in the RIEVD (Belevych et al. 1999) does not necessarily indicate that 6-ND is coming from the nerve terminals. Although 6-nitronoradrenaline has been extracted from rat brain (Shintani et al. 1996), it is also possible that some neurotransmitter induces 6-ND release from other cells.
The results here reported extend the findings that tricyclic antidepressants (Britto-Júnior et al. 2021b) and α 1adrenergic antagonists (Britto-Júnior et al. 2022) inhibited EFS-induced contractions only at the concentrations that inhibited 6-ND-induced contractions and that these drugs had no effect in the EFS-induced contractions of RIEVD obtained from L-NAME chronically treated animals. 6-ND does not act on adrenergic receptors, as demonstrated in human umbilical cord vessels (Britto-Júnior et al. 2021a). All these pieces of evidence support the concept that 6-ND is acting on a specific receptor. Purification and sequencing of the 6-ND receptor, the identification of the metabolic pathways involved in the 6-ND synthesis and the mechanisms responsible for 6-ND storage and release should further clarify its physiological role in the ejaculatory process.

Conclusion
The inhibitory effect of β 1 -and β 1 /β 2 -adrenergic receptor antagonists on the RIEVD contractions induced by both the EFS and 6-ND is due to blockade of the 6-ND receptor.