Investigação funcional e molecular dos adrenoceptores alfa-1 em modelo experimental de epididimite em ratos
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2019-04-23
Autores
Mueller, Andre
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Universidade Estadual Paulista (Unesp)
Resumo
Primordialmente, o epidídimo funciona para proteger e realizar a maturação de espermatozoides. A cauda do epidídimo (CE), região que armazena espermatozoides maduros até a ejaculação, recebe densa inervação de fibras pós-ganglionares simpáticas que liberam noradrenalina para contrair o músculo liso do epidídimo via ativação de adrenoceptores alfa-1A (alfa-1A-ARs). As contrações da musculatura lisa têm papel importante no transporte dos espermatozoides ao longo do ducto epididimário, bem como na contração da CE distal durante a fase de emissão seminal da ejaculação. Recentemente, foi demonstrado que a epididimite, a condição inflamatória do epidídimo, induz aceleração do tempo de trânsito espermático em ratos. Nesta tese, testamos a hipótese de que essa aceleração do trânsito espermático induzida pela epididimite é resultado de alterações na contratilidade do ducto epididimário. Dessa forma, o objetivo deste estudo foi investigar a contração da CE distal mediada por alfa-1-ARs em um modelo experimental de epididimite aguda induzida pelo lipopolissacarídeo (LPS) de E. coli em ratos. Após 6 e 24 h da injeção intraductal de LPS (25 µg) para indução da epididimite (in vivo), estudos de contração indicaram que a potência da noradrenalina aumentou ~6 vezes na CE distal inflamada. As contrações induzidas pela noradrenalina foram competitivamente antagonizadas pelo prazosin, um antagonista não-seletivo de alfa-1-ARs, com afinidades semelhantes nos grupos tratados com LPS e salina (pA2~8,90). No entanto, a potência do BMY 7378, um antagonista alfa-1D-seletivo, foi ~50 vezes maior na CE distal tratada com LPS (pA2 LPS = 8,70 vs pA2 Sal = 6,95), produzindo inclinação de Schild menor do que a unidade teórica. Além disso, a buspirona, um agonista seletivo para alfa-1D-ARs, foi ~3 vezes mais potente no grupo tratado com LPS (pEC50 LPS = 4,93 vs pEC50 Sal = 4,36) e as curvas concentração resposta (CCRs) para este agonista foram antagonizadas pelo BMY 7378, com uma potência estimada de ~8,70, consistente com a participação de alfa-1D-AR. Como suporte a esses achados, em segmentos da CE distal incubados com LPS (300 ng/mL) por 4 h in vitro, o BMY 7378 também antagonizou as contrações induzidas pela noradrenalina, produzindo pA2~8,50. Além disso, a oximetazolina (agonista parcial alfa-1A) foi ~6 vezes menos potente em segmentos expostos ao LPS em comparação controle (pEC50 LPS = 6,22 vs pEC50 Sal = 7,00). Interessantemente, em tecidos que foram pré-incubados com PDTC, um inibidor de NFKB, antes do desafio com LPS, o BMY 7378 em baixa concentração foi ineficaz em deslocar CCRs para noradrenalina. Ensaios de RT-qPCR indicaram que os níveis relativos de transcritos para adrenoceptores alfa-1a, -1b e -1d não foram alterados na CE distal com epididimite in vivo. Curvas de inibição da ligação específica de [3H]-Prazosin com ligantes seletivos foram realizadas. Apesar dos resultados comparáveis para prazosin e BMY 7378, sítios de baixa afinidade para RS 100329 (antagonista alfa-1A-seletivo) foram identificados, sugerindo um aumento na população de alfa-1D-ARs na CE distal inflamada. Em conclusão, a epididimite induzida por LPS alterou o subtipo funcional alfa-1-AR no músculo liso da CE distal, resultando em uma população heterogênea de receptores (alfa-1A e alfa-1D-ARs) envolvidos na resposta contrátil durante a inflamação aguda. A co-participação dos alfa-1D-ARs está correlacionada com a hipersensibilidade da noradrenalina na CE distal inflamada, e parece ser dependente do NFKB. Essa alteração pode cooperar para um aumento na contração do ducto, evitando, assim, a ascensão de bactérias para regiões proximais do epidídimo. Entretanto, isso pode contribuir para a aceleração do trânsito espermático no epidídimo, um efeito deletério para a maturação espermática.
The epididymis works primarly to protect and mature the spermatozoon. The cauda epididymis (CE), which stores mature spermatozoa until ejaculation, is densely innervated by sympathetic postganglionic fibers that release noradrenaline to contract epididymal smooth muscle via activation of alpha-1A adrenoceptors (1A-ARs). The smooth muscle contractions have an important role for the adequate transport of spermatozoa within the epididymal duct, as well as for distal CE contraction during the seminal emission phase of ejaculation. It was recently shown that epididymitis, the inflammatory condition of the epididymis, induces acceleration of the sperm transit time in the rat epididymis. We hypothesize that this epididymitis-induced sperm transit time acceleration is due to changes in the contractility of the epididymal duct. Thus, the aim of this study was to investigate the 1-ARs-mediated contraction of the distal CE in an experimental model of acute epididymitis in rats induced by lipopolysaccharide (LPS) from E. coli. Contraction studies indicated that the potency of noradrenaline increased ~6-fold in the inflamed distal CE at 6 h post-intravasal LPS injection. Noradrenaline-induced contractions were competitively antagonized by prazosin, a non-selective 1-ARs antagonist, with similar affinities in both LPS- and saline-treated groups (pA28.9). However, the potency of BMY 7378, a selective 1D-ARs antagonist, was ~50-fold higher in the distal CE treated with LPS (pA2 LPS = 8.70 vs pA2 Sal= 6.95), yielding a Schild slope less than unity. Furthermore buspirone, a selective 1D-ARs agonist, was ~3-fold more potent in the LPS-treated group (pEC50 LPS = 4.93 vs pEC50 Sal = 4.36) and the concentration-response curves (CRCs) for this agonist was antagonized by BMY 7378, with estimated potency of ~8.70, consistent with 1DAR participation. Supporting these findings, in segments of distal CE incubated with LPS (300 ng/ml) for 4 h in vitro, BMY 7378 potentially antagonized the noradrenaline-induced contractions, yielding a pA28.50. Moreover, oxymetazoline (1A partial agonist) was ~6-fold less potent in segments exposed to LPS in comparison to its control (pEC50 LPS = 6.22 vs pEC50 Sal = 7.00). Interestingly, in tissues that were pre-incubated with PDTC, an inhibitor of NFKB, prior to challenge with LPS, low concentration of BMY 7378 was ineffective in displace the CRCs for noradrenaline. RT-qPCR assays indicated that the relative levels of transcripts for 1a-, 1b- and 1d-ARs were not changed in the distal CE with in vivo epididymitis. Inhibition curves of the specific binding of [3H]-Prazosin to selective ligands were performed. Despite the comparable results for prazosin and BMY 7378, low affinity sites for RS 100329 (a selective 1A ligand) were identified, suggesting an increase of the 1D-ARs population in inflamed distal CE. In conclusion, LPS-induced epididymitis changed the functional 1-AR subtype in the smooth muscle of the distal CE, resulting in a heterogeneous population of receptors (1A- and 1D-ARs) involved in the contractile response during acute inflammation. The co-participation of 1D-ARs is correlated with noradrenaline super-sensitivity in inflamed distal CE, and seems to depend on the NFKB signaling pathway. This change may concur to increase the ductal constriction, thereby avoiding the ascent of bacteria to proximal regions of the epididymis. However, it may contribute to the acceleration of sperm transit in the epididymis, an event associated with poor sperm maturation outcomes.
The epididymis works primarly to protect and mature the spermatozoon. The cauda epididymis (CE), which stores mature spermatozoa until ejaculation, is densely innervated by sympathetic postganglionic fibers that release noradrenaline to contract epididymal smooth muscle via activation of alpha-1A adrenoceptors (1A-ARs). The smooth muscle contractions have an important role for the adequate transport of spermatozoa within the epididymal duct, as well as for distal CE contraction during the seminal emission phase of ejaculation. It was recently shown that epididymitis, the inflammatory condition of the epididymis, induces acceleration of the sperm transit time in the rat epididymis. We hypothesize that this epididymitis-induced sperm transit time acceleration is due to changes in the contractility of the epididymal duct. Thus, the aim of this study was to investigate the 1-ARs-mediated contraction of the distal CE in an experimental model of acute epididymitis in rats induced by lipopolysaccharide (LPS) from E. coli. Contraction studies indicated that the potency of noradrenaline increased ~6-fold in the inflamed distal CE at 6 h post-intravasal LPS injection. Noradrenaline-induced contractions were competitively antagonized by prazosin, a non-selective 1-ARs antagonist, with similar affinities in both LPS- and saline-treated groups (pA28.9). However, the potency of BMY 7378, a selective 1D-ARs antagonist, was ~50-fold higher in the distal CE treated with LPS (pA2 LPS = 8.70 vs pA2 Sal= 6.95), yielding a Schild slope less than unity. Furthermore buspirone, a selective 1D-ARs agonist, was ~3-fold more potent in the LPS-treated group (pEC50 LPS = 4.93 vs pEC50 Sal = 4.36) and the concentration-response curves (CRCs) for this agonist was antagonized by BMY 7378, with estimated potency of ~8.70, consistent with 1DAR participation. Supporting these findings, in segments of distal CE incubated with LPS (300 ng/ml) for 4 h in vitro, BMY 7378 potentially antagonized the noradrenaline-induced contractions, yielding a pA28.50. Moreover, oxymetazoline (1A partial agonist) was ~6-fold less potent in segments exposed to LPS in comparison to its control (pEC50 LPS = 6.22 vs pEC50 Sal = 7.00). Interestingly, in tissues that were pre-incubated with PDTC, an inhibitor of NFKB, prior to challenge with LPS, low concentration of BMY 7378 was ineffective in displace the CRCs for noradrenaline. RT-qPCR assays indicated that the relative levels of transcripts for 1a-, 1b- and 1d-ARs were not changed in the distal CE with in vivo epididymitis. Inhibition curves of the specific binding of [3H]-Prazosin to selective ligands were performed. Despite the comparable results for prazosin and BMY 7378, low affinity sites for RS 100329 (a selective 1A ligand) were identified, suggesting an increase of the 1D-ARs population in inflamed distal CE. In conclusion, LPS-induced epididymitis changed the functional 1-AR subtype in the smooth muscle of the distal CE, resulting in a heterogeneous population of receptors (1A- and 1D-ARs) involved in the contractile response during acute inflammation. The co-participation of 1D-ARs is correlated with noradrenaline super-sensitivity in inflamed distal CE, and seems to depend on the NFKB signaling pathway. This change may concur to increase the ductal constriction, thereby avoiding the ascent of bacteria to proximal regions of the epididymis. However, it may contribute to the acceleration of sperm transit in the epididymis, an event associated with poor sperm maturation outcomes.
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Palavras-chave
adrenoceptores alfa-1, epididimite, contração, músculo liso, alpha-1 adrenoceptors, epididymitis, contraction, smooth muscle