Isolated Rat Vas Deferens Research Articles

Effects of noradrenaline, the calcium ionophore A23187, forskolin, sodium nitroprusside and glibenclamide on the degradation of extracellular adenosine 5'-triphosphate by the rat isolated vas deferens.

1. The effects of noradrenaline (NA), the calcium ionophore A23187, forskolin, sodium nitroprusside (SNP) and the K+-channel blocker glibenclamide on the degradation by ectonucleotidases of extracellular adenosine 5'-triphosphate (ATP) were studied in the rat vas deferens. 2. ATP (100 microM) was rapidly broken down by the rat vas deferens with a half-life of 5.83 +/- 0.40 min via adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP), with the final degradation product being inosine and with little adenosine being detected in the samples. 3. Preincubation for 1 h with NA (10 microM), A23187 (10 microM), or glibenclamide (100 microM) had no significant effect on the breakdown of ATP or the production of metabolites. However, both forskolin (10 microM) and SNP (1 microM) significantly increased the concentrations of AMP detected with time. In the case of SNP (1 microM) there was also a significant reduction in the rate of production of inosine, while in the case of forskolin (10 microM) there was a significant increase in the rate of removal of ATP. 4. These results suggest that preincubation with SNP may inhibit 5'-nucleotidase and so reduce the metabolism of AMP, while preincubation with forskolin may increase the activity of the ectonucleotidases responsible for production of AMP from ATP.

Role of Potassium Channels in the GABA Inhibitory Action on the Purinergic Response to Electrical Field Stimulation in Rat Isolated Vas Deferens

The involvement of potassium channels in the GABAB receptor-mediated modulatory effects of γ-aminobutyric acid (GABA) on the purinergic response of rat isolated vas deferens exposed to electrical field stimulation (EFS) was investigated. Non-selective Ca2+-dependent K+ (KCa) channel antagonists, tetraethylammonium chloride (TEA) and 4-aminopyridine (4-AP), and a selective large conductance Ca2+-activated K+ (BK) channel antagonist, iberiotoxin, reduced the inhibitory effects of GABA on the purigenic response to EFS. The GABA induced-inhibition of the EFS contractions was not affected by the small conductance Ca2+-activated K+ (SK) channel blocker, apamin, or by the ATP-sensitive K+ channel (KATP) channel blocker, glibenclamide. These observations seem to indicate a coupling between GABAB receptors and BK channels in rat vas deferens.

Roles of key functional groups in omega-conotoxin GVIA synthesis, structure and functional assay of selected peptide analogues.

The contributions of various functional groups to the pharmacophore of the N-type calcium-channel blocker, omega-conotoxin GVIA (GVIA), have been investigated using structural and in-vitro functional studies of analogues substituted at one or two positions with non-native residues. In most cases the structure of the analogue was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) were employed to monitor N-type calcium-channel activity. The data provide a more detailed picture of the roles in GVIA structure and activity of the crucial Lys2 and Tyr13, as well as all other positively charged residues, Tyr22, the hydroxyproline residues and the C-terminal amido moiety, many of which were identified as being important for activity in an alanine scan [Lew et al. (1997) J. Biol. Chem. 272, 12014-12023]. Substitutions of Lys2 with nonstandard amino acids and arginine quantified the roles of the length and charge of the Lys side chain. The orientation of the Tyr13 side chain and its hydroxyl moiety was shown to be important by substitution with d-Tyr and the d-form and l-form of the constrained analogue 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic(OH)]. The roles of the Hyp10 and Hyp21 hydroxyl groups, investigated by proline substitutions, appear to be more structural (as monitored by NMR) than functional, although small decreases in potency were observed in some assays. The reversibility of the channel blockade was also studied, and several analogues with faster wash-out characteristics than native GVIA were identified. Rapid reversibility (as in the case of omega-conotoxin MVIIA) may be beneficial for therapeutic applications. Disubstituted analogues revealed some interesting cooperative effects, which were not predicted from single-residue substitutions. A disubstituted chimera of GVIA and omega-conotoxin MVIIA was more potent than either native molecule. The more detailed description of the GVIA pharmacophore obtained here provides a better basis for the future design of truncated peptide and peptidomimetic analogues.

Effect of venlafaxine hydrochloride in different preparations of isolated guinea-pig and rat organ tissues.

A study was undertaken to know better the effects of venlafaxine hydrochloride on the responses of isolated rat vas deferens to noradrenaline and dopamine, those of isolated rat uterus to serotonin and histamine, and those of isolated guinea-pig ileum to acetylcholine and histamine. Venlafaxine hydrochloride increased the response of rat vas deferens to noradrenaline but not to dopamine. Venlafaxine did not alter the response of rat isolated uterus to serotonin. In rat uterus, venlafaxine did not modify the response to histamine but was able to increase it in guinea-pig ileum. An anticholinergic effect was observed with the lowest concentration tested. Although venlafaxine is a selective serotonine reuptake inhibitor in the central nervous system, serotonin uptake was not seen in the rat uterus. The anticholinergic effects observed in the present study might be consistent with some of the side-effects associated with venlafaxine.

DOPAMINE MODULATES PERIPHERAL PURINERGIC NEUROTRANSMISSION THROUGH MULTIPLE PRESYNAPTIC RECEPTORS: TISSUE-DEPENDENT EFFECTS

This study investigated the identity of presynaptic receptors involved in dopaminergic modulation of purinergic transmission in peripheral tissues including isolated rat vas deferens and urinary bladder. Isometric muscle twitches were established in the two tissues by low frequency electric field-stimulation (0.05 Hz, 1-ms duration, and supramaximal voltage). Exposure to prazosin, 50 nmol l−1(vas deferens), or atropine, 3 μmol l−1(urinary bladder), had no effect on the developed twitches. In contrast, desensitisation of P2X-purinoceptors by α,β-methylene ATP (α,β-mATP, 30 μmol l−1) abolished the twitches in both tissues, confirming their purinergic origin. Dopamine (1.8×10−7to 4.2×10−5mol l−1) reduced the twitch response in a concentration-related manner. Yohimbine (α2-adrenoceptor antagonist, 0.3 μmol l−1) significantly (P<0.05) attenuated the inhibitory effects of dopamine and caused an upward shift in the concentration–response curves in the vas deferens and the urinary bladder. On the other hand, a blockade of DA2-dopaminoceptors by domperidone (1 μmol l−1) produced significant (P<0.05) reductions in dopamine responses only in rat vas deferens, with no effect in the urinary bladder. These data suggest that dopamine exerts inhibitory influences on purinergically-mediated muscle twitches in rat vas deferens and urinary bladder. More importantly, the nature of presynaptic receptors (α2-adrenergic and/or DA2-dopaminergic) involved in mediating dopamine effects is dependent on the tissue under investigation.

Design, synthesis, and biological activity of prazosin-related antagonists. Role of the piperazine and furan units of prazosin on the selectivity for alpha1-adrenoreceptor subtypes.

Prazosin-related quinazolines 4-20 were synthesized, and their biological profiles at alpha1-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha1A), spleen (alpha1B), and aorta (alpha1D) and by binding assays in CHO cells expressing human cloned alpha1-adrenoreceptor subtypes. The replacement of piperazine and furan units of prazosin (1) by 1, 6-hexanediamine and phenyl moieties, respectively, affording 3-20, markedly affected both affinity and selectivity for alpha1-adrenoreceptor subtypes in functional experiments. Cystazosin (3), bearing a cystamine moiety, was a selective alpha1D-adrenoreceptor antagonist being 1 order of magnitude more potent at alpha1D-adrenoreceptors (pA2, 8.54 +/- 0.02) than at the alpha1A- (pA2, 7.53 +/- 0.01) and alpha1B-subtypes (pA2, 7.49 +/- 0. 01). The insertion of substituents on the furan ring of 3, as in compounds 4 and 5, did not improve the selectivity profile. The simultaneous replacement of both piperazine and furan rings of 1 gave 8 which resulted in a potent, selective alpha1B-adrenoreceptor antagonist (85- and 15-fold more potent than at alpha1A- and alpha1D-subtypes, respectively). The insertion of substituents on the benzene ring of 8 affected, according to the type and the position of the substituent, affinity and selectivity for alpha1-adrenoreceptors. Consequently, the insertion of appropriate substituents in the phenyl ring of 8 may represent the basis of designing new selective ligands for alpha1-adrenoreceptor subtypes. Interestingly, the finding that polyamines 11, 16, and 20, bearing a 1,6-hexanediamine moiety, retained high affinity for alpha1-adrenoreceptor subtypes suggests that the substituent did not give rise to negative interactions with the receptor. Finally, binding assays performed with selected quinazolines (2, 3, and 14) produced affinity results, which were not in agreement with the selectivity profiles obtained from functional experiments. This rather surprising and unexpected finding may be explained by considering neutral and negative antagonism.

Rilmenidine reveals differences in the pharmacological characteristics of prejunctional alpha2-adrenoceptors in the guinea-pig, rat and pig.

1. The alpha2A and alpha2D-adrenoceptor subtypes are thought to be species homologs most easily differentiated on the basis of the potency of antagonists. In the present study we have compared the effect of rilmenidine with two other selective alpha2-adrenoceptor agonists, UK-14304 (5-bromo-6- [2-imidazolin-2-ylamino]-quinoxaline) and clonidine, against electrically-evoked contractions in five isolated preparations from the rat, guinea-pig and pig, and, where possible, determined the receptor subtype involved. 2. UK-14034, clonidine and rilmenidine produced concentration-dependent inhibition of the electrically-evoked contractions of the rat isolated vas deferens and tail artery and the guinea-pig ileum. These inhibitory effects were reversed by the selective alpha2-adrenoceptor antagonist, RX-811058 (1 microM), except in the rat tail artery preparations where the remaining neurogenic response was inhibited; evidence for the involvement of 'innervated' alpha2-adrenoceptors. Both clonidine and UK-14304 produced concentration-dependent inhibition of responses in the porcine isolated tail artery and urinary bladder but clonidine was markedly less efficacious in these preparations. In contrast, rilmenidine failed to inhibit the neurogenic contractions in either preparation. 3. Although rilmenidine failed to elicit a detectable response in either the porcine isolated tail artery or urinary bladder, it (10 microM and 30 microM, respectively) competitively antagonised the inhibitory effects of UK-14304 with an estimated dissociation constant of (pK(B)) 5.82 and 5.93, respectively. 4. Prazosin (1 microM) failed to alter the effect of UK-14304 against neurogenic contractions in the porcine isolated urinary bladder, while rauwolscine (pK(B) 8.87) was 10 fold more potent than phentolamine (pK(B) 7.56). On the other hand, phentolamine (pK(B) 8.42) was only marginally more potent than rauwolscine (pK 8.05) against clonidine-induced inhibition of electrically-evoked contractions of the guinea-pig isolated ileum. This pharmacological evidence with antagonists supports the presence of alpha2D-adrenoceptors in the rat and guinea-pig and the alpha2A-adrenoceptors in the pig. 5. We have demonstrated that rilmenidine, unlike clonidine and UK-14304, is devoid of any agonist activity at prejunctional alpha2A-adrenoceptors in the pig, but is an efficacious agonist at alpha2D-adrenoceptors in the rat and guinea-pig.

Comparative Study of the Effects of Clozapine and Clothiapine in Different Preparations of Guinea Pig and Rat Isolated Organs

1. A study has been made to know the effects of clozapine and clothiapine on the responses of rat isolated vas deferens to norepinephrine, dopamine and potassium, those of the rat isolated uterus to serotonin and potassium, and that of guinea pig isolated ileum to histamine. 2. Clozapine was a noncompetitive antagonist to norepinephrine, dopamine, serotonin and histamine; it inhibited potassium-induced contraction in isolated rat uterus and vas deferens. 3. Clothiapine was a competitive antagonist to serotonin and a noncompetitive antagonist to norepinephrine, dopamine and histamine; it inhibited potassium-induced contractions in isolated rat uterus and vas deferens.

The characteristics of endothelin receptor subtypes on muscle contraction and neuro-transmission in rat vas deferens.

We observed endothelin (ET)-induced contractile responses on prostatic and epididymal segments, as well as the facilitation of an electrically stimulated tone on prostatic segments of isolated rat vas deferens. In both segments, the selective ET(B)-receptor agonists, IRL 1620 and sarafotoxin S6c, produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of contraction potencies (pD2 value) was ET-1 = ET-2 > ET-3 >> sarafotoxin S6c = IRL 1620. The maximum responses of ET-induced contractions in the prostatic segments were larger than those in the epididymal segments. The contractile response to ET-3 was antagonized by pretreatment for 30 min with BQ-123 (10 nM), a selective ET(A) receptor antagonist, and BQ-788 (1 microM), a selective ET(B) receptor antagonist. The contractile responses to ET-1 were antagonized by pretreatment with BQ-123 (10 microM), but not with BQ-788 (1 microM). The ET-3-induced facilitation on the twitch response to electrical stimulation in the prostatic segment of the vas deferens was antagonized by BQ-123 (0.1 microM) and BQ-788 (1 microM). The ET-1-induced facilitation was antagonized by pretreatment with BQ-123 (3 microM), but not with BQ-788 (10 microM). These results suggest that in rat vas deferens the ET(A) receptors are divided into BQ-123-sensitive ET(A1) and BQ-123-insensitive ET(A2) subtypes, and the production of a contractile response of smooth muscle as well as the facilitation of neurotransmission are accomplished through mediation by ET(A1)- and ET(A2)-subtypes.

Extraneuronal uptake inhibitor U-0521 decreases contractile responses in rat vas deferens

1. 1. The influence of catechol-O-methyltransferase inhibitor U-0521 on isotonic contraction of isolated rat vas deferens was examined to determine optimal concentration and nonspecific effects. 2. 2. Maximum responses to (-)-epinephrine were increased at 0.4 μM and 1 μM concentrations of U-0521. Epinephrine responses were progressively decreased in the presence of higher concentrations (10 μM, 30 μM and 100 μM) of U-0521. 3. 3. The response to the nonadrenergic agonist neurokinin A was similarly depressed in the presence of 100 μM U-0521. 4. 4. U-0521 not only inhibits COMT, at concentrations above 1 μM it nonspecifically depresses contraction of the rat vas deferens by both adrenergic and nonadrenergic agonists.

Involvement of ATP-sensitive K + channels in the inhibitory effect of calcitonin gene-related peptide on neurotransmission in rat vas deferens

This study investigated the inhibitory action of human calcitonin-gene-related peptide (CGRP) on neurotransmission in rat isolated vas deferens. The electrically stimulated contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers, were concentration-dependently inhibited by human CGRP (0.1–100 nM, IC 50=2.15±0.21 nM, n=17). Human CGRP at concentrations greater than 3 nM reduced the contractile responses to exogenous noradrenaline and ATP. The inhibitory effect of human CGRP on the electrically stimulated or agonist-induced contractions was antagonized by human CGRP-(8–37), the CGRP receptor antagonist. Glibenclamide (3–10 μM) decreased the effect of human CGRP at a concentration greater than 1 nM whilst glibenclamide did not affect the inhibitory effect of human CGRP on the agonist-induced contractions. These results indicate that human CGRP at low concentrations exerts its inhibitory action mainly by acting on CGRP receptors at the sympathetic nerve terminals supplying rat vas deferens and the activation of ATP-sensitive K + channels is at least in part involved in the action of human CGRP on neurotransmission.

Structure-Function Relationships of ω-Conotoxin GVIA: SYNTHESIS, STRUCTURE, CALCIUM CHANNEL BINDING, AND FUNCTIONAL ASSAY OF ALANINE-SUBSTITUTED ANALOGUES

The structure-function relationships of the N-type calcium channel blocker, omega-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2 and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.

Inhibition of contractions by tricyclic antidepressants and xylamine in rat vas deferens

The effects of noradrenaline uptake inhibitors on contractions evoked by electric field stimulation, noradrenaline, clonidine, 5-hydroxytryptamine, ATP, high K +, and BaCl 2 in the epididymal half of rat isolated vas deferens were examined. Protriptyline, amitriptyline and xylamine concentration-dependently inhibited monophasic contractions induced by low frequency electrical stimulation (0.3 Hz, 1 ms duration, 60 V). Protriptyline and xylamine inhibited in a noncompetitive manner the contractile response induced by noradrenaline (3×10 −8–3×10 −5 M) and the inhibitory effect of protriptyline was reversible, while xylamine produced long-lasting inhibition. All three noradrenaline uptake blockers inhibited the clonidine (3×10 −6 M) or 5-hydroxytryptamine (10 −5 M)-induced contraction. Protriptyline and amitriptyline at concentrations of 3×10 −6–3×10 −5 M reversibly inhibited the ATP (10 −4 M)-induced monophasic contraction. In contrast, xylamine ((1–3)×10 −5 M) had no effect. Protriptyline and amitriptyline but not xylamine concentration-dependently reduced the high K + (6×10 −2 M)-induced sustained contraction with respective IC 50 values of 1.81×10 −6 M and 8.6×10 −7 M. Protriptyline and amitriptyline at 10 −5 M reversibly inhibited BaCl 2 (3×10 −3 M)-induced phasic contractions and xylamine (10 −5 M) had no effect. These findings demonstrate that tricyclic antidepressants might exert direct inhibitory action on mechanical contraction pathway, whilst xylamine, a structurally different inhibitor of noradrenaline uptake, may act mainly at α-adrenoceptors and other amine receptors on the smooth muscle of the rat vas deferens as a long-lasting nonselective antagonist, and it at least in part blocks sympathetic transmission.

Effects of fluoxetine hydrochloride and fluvoxamine maleate on different preparations of isolated guinea pig and rat organ tissues

1. 1. Fluoxetine and fluvoxamine reinforced the response to norepinephrine of isolated rat vas deferens incubated in Krebs-Henseleit solution. 2. 2. This reinforcement disappeared when cocaine, 17β-estradiol, and propranolol were added to the incubation medium. 3. 3. Fluoxetine and fluvoxamine did not have antimuscarinic effect, but they did have antihistaminic effect, and at high concentrations they inhibited the contraction induced by potassium ion on the isolated rat uterus preparation (IC 50 3.99×10 −6 and 1.82×10 −5 M, respectively).

NS 1619 activates Ca 2+-activated K + currents in rat vas deferens

The effects of NS 1619, a newly developed activator of large-conductance Ca 2+-activated K + channels, were investigated on single smooth muscle fibers dissociated enzymatically from rat vas deferens and on contractions of the epididymal half of vas deferens. K + currents were recorded using whole-cell patch-clamp methods in near-physiological K + solutions (5.4 mM extracellular K +/145 mM intracellular K +). When cell membrane voltage was stepped to test potentials (−60 to +60 mV) from a holding potential of −10 mV, NS 1619 increased the outwardly rectifying K + current in a concentration-dependent manner. The increased portion of the K + current by NS 1619 was totally abolished by charybdotoxin (100 nM) but not by glibenclamide (3 μM). NS 1619 reduced electrically stimulated contractile responses of rat vas deferens in a concentration-dependent manner, and charybdotoxin but not glibenclamide partially inhibited the effect of NS 1619. NS 1619 (50 μM) inhibited the noradrenaline-induced contraction. Charybdotoxin (100 nM) partially reduced the NS 1619-induced inhibition while glibenclamide (3 μM) had no effect. NS 1619 (10–100 μM) reduced the high K +-induced contractions in a noncompetitive manner. The present results indicate that NS 1619 activates charybdotoxin-sensitive Ca 2+-activated K + channels and probably inhibits Ca 2+ influx. These two effects might account largely for the observed mechanical inhibition induced by NS 1619 in the epididymal half of isolated rat vas deferens. © 1997 Elsevier Science B.V.

Pharmacological Actions of Naringin on Alpha, Beta-adrenoceptors and Uptake of Noradrenaline in Rat Isolated Vas Deferens

In normal Krebs solution, naringin (1×10−7 Msc–2×10−6 M) enhanced noradrenaline-induced contractions in rat isolated vas deferens increasing this potentiation in a concentration dependent manner. This enhancing effect continued in the presence of yohimbine (10−6 M) or propranolol (10−6 M). Naringin did not modify significantly the dopamine dose-response curves. In a medium containing 10−8 M prazosin, naringin decreased noradrenaline dose-response curves underneath the control curve at all the doses tested. Naringin did not modify significantly the NA dose-response curve in a medium containing cocaine (10−5 M) or oestradiol (10−5 M). The potentiation produced by naringin on the NA-induced contractions in rat vas deferens may be due to alpha-1 receptor activation, together with an interference of naringin in catecholamine uptake process.

Presynaptic modulation by L‐glutamate and GABA of sympathetic co‐transmission in rat isolated vas deferens

1. The modulatory effects of L-glutamate and its structural analogues, and of gamma-aminobutyric acid (GABA), on sympathetic co-transmission were studied in the rat isolated vas deferens exposed to electrical field stimulation (EFS). 2. Application of exogenous L-glutamate caused a concentration-dependent (1 microM-3 mM) inhibition of the rapid twitch component of the biphasic EFS contraction. However, L-glutamate (1 microM-3 mM) had a minimal effect on the phasic contraction induced by exogenous adenosine 5'-triphosphate (ATP, 150 microM) and noradrenaline (50 microM). Unlike L-glutamate, D-glutamate had no effect on the EFS contraction. 3. The L-glutamate-induced inhibition of the EFS contractions was significantly attenuated by the glutamate decarboxylase (GAD) inhibitor 3-mercapto-propionic acid (150 microM) and was abolished in the presence of the GABA transaminase (GABA-T) inhibitor, 2-aminoethyl hydrogen sulphate (500 microM). 4. The L-glutamate-induced inhibition of the electrically evoked contraction was not affected by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(30 nM), reactive blue 2 (30 microM) or the GABAA receptor antagonist bicuculline (50 microM). However, the GABAB receptor antagonist 2-hydroxysaclofen (50 microM) significantly inhibited the L-glutamate effect. 5. Similar to L-glutamate, GABA also caused a concentration-dependent (0.1-100 microM) inhibition of the EFS contractions. This GABA-induced inhibition was not affected by either the GABAA receptor antagonist bicuculline (50 microM) or reactive blue 2 (30 microM). However, a significant attenuation of the GABA-mediated effect was recorded with the GABAB receptor antagonist 2-hydroxysaclofen (50 microM). Contractions of the vas deferens induced by exogenous ATP and noradrenaline were not affected by GABA (0.1-100 microM). 6. The L-glutamate analogues, N-methyl-D-aspartate (NMDA) (1 microM-1 mM) and quisqualate (Quis 0.1 microM-0.3 mM) had no effect, whilst kainate (Kain, 1 microM-1 mM) caused an inhibition of the EFS-induced contractions. Effects of Kain could be abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX, 10 microM). NMDA, Quis and Kain had no effect on the exogenous ATP- or noradrenaline-induced contractions. 7. It is concluded that the excitatory amino acid L-glutamate modulates the electrically evoked vas deferens contraction through conversion to the inhibitory amino acid GABA by a specific GABA transaminase. The GABA formed may then act on GABAB receptors and cause inhibition of the contraction through a presynaptic mechanism.

O-19-3 - Refractory depression a biochemical hypothesis

Ex vivo investigations were carried out to study the potentiating actions of the MAOIs moclobemide and tranylcypromine on peripheral a- and β-adrenergic effects of dopamine. Isolated rat vas deferens and atria were used. Preliminary experiments showed that both moclobemide and tranylcypromine did not affect the response to noradrenaline, while they enhanced the response to tyramine. By comparison of the responses to dopamine in control and reserpinized rats, it was shown that dopamine acts in a direct and indirect manner on both the tissues tested.Two concentrations (20, 40 mg/kg, i.p.) of moclobemide were tested and administered at different time periods (2, 5 or 24 h) before the animals were sacrificed. Two concentrations (2, 20 mg/kg, i.p.) of tranylcypromine were tested at different intervals of time (2, 24 h). The results obtained pointed out that: (1) Moclobemide 20 mg/kg potentiated the α-adrenergic response to dopamine only 2 h after administration while 40 mg/kg-exerted a potentiation which was evident not only 2 but also 5 h after administration. Both doses were ineffective 24 h after administration.(2) Moclobemide 20 and 40 mg/kg potentiated the β-adrenergic response to dopamine. The effect was present 2 and 5 h after administration but it was absent 24 h after.(3) Tranylcypromine20 mg/kg potentiated the α-adrenergic response to dopamine: this effect was present after 2 h from pretreatment and was still evident after 24 h. The lower concentration of tranylcypromine (2 mg/kg) enhanced the response to dopamine only after 24 h from pretreatment.(4) Tranylcypromine 20 mg/kg enhanced the β-adrenergic response to dopamine: this effect was present 2 h after pretreatment and lasted 24 h after pretreatment. The same results were obtained with tranylcypromine 2 mg/kg.Therefore there is strong evidence of differences between moclobemide and tranylcypromine in the time course of their enhancing effects of α- and β-adrenergic responses to dopamine.

S-51-1 - Modulation of cerebral Monoamine release by Mao inhibitors

The ability of L-3,4-dihydroxyphenylalanine (L-DOPA) to release noradrenaline (NA) from peripheral and CNS neurons was studied using isolated rat vas deferens and in vivo frontal cortex microdialysis. Application of L-DOPA (30 μM) to vas deferens increased basal NA efflux but not electrical field stimulation-evoked release of NA when the tissue was pretreated with an inhibitor of MAO-B (clorgyline 1 μM) or an inhibitor of MAO-A and MOA-B (AGN-1133 1 μM). No effect on NA efflux was seen when the tissue was treated with rasagiline (0.005 μM; selective inhibitor of MAO-B), but rasagiline, AGN-1133 and clorgyline increased basal efflux of dopamine (DA) following L-DOPA. In microdialysis experiments, systemic administration of L-DOPA/carbidopa combination (50/12 mg.kg−1) increased the efflux of 3,4-dihydroxyphenylglycol and 3-methoxy,4-hydroxyphenylglycol but reduced that of NA. Microdialysate levels of NA, however were increased following L-DOPA/carbidopa when desipramine (1 μM) was infused locally via the probe, or following systemic administration of yohimbine (2 mg.kg−1). The data are consistent with the hypothesis that administration of L-DOPA is followed by increased axoplasmatic levels of DA which release NA from storage sites into the free cytoplasmatic pool.

Internal calcium stores and norepinephrine overflow from isolated, field stimulated rat vas deferens

Ryanodine has been shown to selectively inhibit the initial phase of contraction of rat vas deferens smooth muscle stimulated by endogenous release of norepinephrine (NE) (1), and part of this effect could be pre-junctional. To assess this, its effect on NE overflow was measured in the same preparation. NE overflow from electrical field-stimulated isolated rat vas deferens was quantified by electrochemical detection using HPLC. In order to limit pre-junctional autoregulatory mechanisms, α 2 -adrenergic receptors were blocked and P 2x purinergic receptors were desensitized. In these experimental conditions, NE overflow was directly proportional to extracellular Ca 2+ concentration. Ryanodine only induced a modest decrease in NE overflow. Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum Ca 2+ -ATPase, slightly increased NE overflow but decreased smooth muscle contraction induced by electrical field stimulation. It is concluded that part of the effect of ryanodine on field stimulation-induced contraction may be due to an inhibition of NE release, although the major inhibitory effect of this alkaloid is post- junctional. For CPA, its inhibitory effect on field stimulation-induced contraction is entirely post-junctional. Its effect on NE overflow suggests that, in this preparation, internal Ca 2+ stores could function to accelerate termination of neurotransmitter release by sequestering cytosolic Ca 2+ .