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 remarkable 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 PGE2 was the major prostaglandin synthetized by homogenates of this tissue (Gerozissis & Dray, 1983). Although PGE2 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 concentration-dependent 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 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).
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 3H-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., 2022), did not alter the contractions induced by tyramine (Bell, 1968). In the synaptosomal fraction prepared rom 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).
The release of noradrenaline from adrenergic terminals is modulated by presynaptic inhibitory a2-adrenoceptors (Langer, 1977). Three distinct genes are responsible for the synthesis of three subtypes of α2-adrenoceptors, named α2A, α2B and α2C (Bylund et al., 1994), and they are widely distributed both in the CNS and in the periphery (McCune et al., 1993). The a2A-adrenoceptor subtype has dominant role in the presynaptic inhibition of noradrenaline release (Altman et al, 1999), although in peripheral tissues all the three a2-adrenoceptor subtypes play a role in the feedback regulation of neurotransmitter release (Trendelenburg et al., 2003; Gyires et al., 2009). The a2A-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 the EFS-induced contractions of the rat isolated epididymal vas deferens (Britto-Júnior et al., 2022c). Thus, inhibition of presynaptic a2-adrenoceptors could be a possible mechanism 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 a2-subtype adrenoceptor than idazoxan, such as a2B and/or a2C adrenoceptors. Experiments with subtype selective α2B and α2C 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 α2-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 b-presynaptic adrenoceptors. This is an interesting hypothesis, since the contractions induced by 6-ND on the rat isolated epididymal vas deferens are inhibited by b1- and b1/b2-adrenoceptors antagonists (Lima et al., 2022b). Whether these b-antagonists will block the potentiation-induced by 6-ND remains to be investigated.