Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg^− 1, ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca²⁺ transients. Sibutramine 5 and 20 mg kg^− 1 reduced ejaculation latency whereas 50 mg kg^− 1 increased ejaculation latency. Sibutramine 3 to 30 μM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 μM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl₂. In HEK293 cells, sibutramine 10 to 100 μM inhibited intracellular Ca²⁺ transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca²⁺ may be involved in the delayed ejaculation observed with high doses of sibutramine.     

Effects of long‐term treatment with fluoxetine and venlafaxine on rat isolated vas deferens

1 Antidepressant therapy is considered as one of the factors leading to male infertility. 2 In this study, the effects of long‐term treatment with fluoxetine or venlafaxine were investigated on electrical field stimulation (EFS, 1–64 Hz), noradrenaline (10^?8 to 10^?4 m ), serotonin (10^?8 to 10^?4 m ), adenosine 5′‐triphosphate [ATP (10^?8 to 10^?4 m )] and 80 mm KCl‐induced contractile responses in the epididymal and prostatic portions of rat isolated vas deferens strips. 3 Serotonin‐induced contractile responses were significantly increased in the epididymal portion of the vas deferens obtained from the fluoxetine‐treatment group, whereas in the prostatic portion there was no change. However, venlafaxine treatment had no effect on serotonin responses in the either portion of the vas deferens. Both fluoxetine and venlafaxine treatment significantly inhibited ATP‐evoked contractions of the prostatic and epididymal portions of the rat vas deferens, but had no effect on EFS, noradrenaline‐ and KCl‐evoked contractions of the vas deferentia in both portions. 4 In conclusion, these results suggest that chronic treatment with fluoxetine and venlafaxine affects vas deferens motility. Purinoceptors may, at least in part, responsible for the impaired motility in chronic treatment of venlafaxine and fluoxetine.     

Attenuation of contractility in rat epididymal vas deferens by Rho kinase inhibitors

The involvement of Ca(2+) sensitization mediated through Rho kinase in the contractility of rat epididymal vas deferens was investigated using Rho kinase inhibitors, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinilcyclohexanecarboxamide dihydrochloride (Y-27632) and 1-(5-isoquinolinesulphonyl)homopiperazine (HA 1077), in comparison with myosin light chain kinase (MLCK) inhibitors, wortmannin and 1-(5-chloronaphthalenesulphonyl)homopiperazine (ML-9) and agents that affect protein kinase C (PKC) and non-receptor tyrosine kinase intracellular signalling. 2 In Ca(2+)-free/ethyleneglycol-bis-(beta-aminoethylether)N,N,N('),N(')-tetraacetic acid (EGTA) (1 mM) medium, noradrenaline evoked sustained contractions. Y-27632 and HA 1077 caused a concentration-dependent inhibition and complete relaxation (IC(50), 1.08 and 1.75 microM respectively). The Ca(2+)-free contraction was reduced by wortmannin (10 microM) or ML-9 (10 microM) but not by inhibitors of diacylglycerol metabolism, 3-[2-[4[bis(4-Fluoropheny)methylene]-1-piperidinyl]-2,3-dihydro-2-thioxi-4(H)-quinazolinone (R59949) (10 microm) or 1,6-bis(cyclohexyloximinocarbonylamino)hexane (RHC-80267) (10 microM) or by the phospholipase A(2) (PLA(2)) inhibitor, quinacrine (up to 100 microM) or tyrosine kinase inhibitor, genistein (30 microM). 3 In the presence of Ca(2+) (2.5 mM), noradrenaline (100 microM) evoked rhythmic activity and biphasic tonic contractions. Y-27632 (1-10 microM) or HA 1077 (1-10 microM) reduced the amplitude of rhythmic activity and tonic contractions. ML-9 (10 microM) attenuated the occurrence of rhythmic activity and modestly reduced the tonic contractions. ML-9 (10 microM) combined with Y-27632 (10 microM) significantly reduced the tonic contractions. ML-9 (30 microM) alone (or combined with Y-27632 10 microM) suppressed the rhythmic activity and substantially reduced (or abolished) the tonic contractions. 4 Contractions evoked by high [K(+)](o) (120 mM) or alpha,beta-methylene ATP (10 microM) were reduced significantly by Y-27632 (1-3 microM) indicating that the Rho kinase signalling pathway is activated by direct tissue depolarization or by stimulation of ligand-gated P(2X) purinoceptors. 5 Collectively, these results indicate that Ca(2+)-sensitization mediated by Rho kinase is involved in agonist- or depolarization-induced contraction of rat epididymal vas deferens. It is the major contractile mechanism underlying noradrenaline-induced Ca(2+)-free responses. It contributes to Ca(2+)-dependent rhythmic contractility and optimizes the development of full contractile tension triggered through calmodulin/MLCK activation by stimulated influx of Ca(2+).     

Modulatory effect of protein kinase C activator on contractility of rat vas deferens

The modulatory effect of the protein kinase C activator was examined on contraction of rat isolated vas deferens induced by constrictive agonists, noradrenaline (NA), ATP, BaCl2 and high K+. Phorbol 12,13-diacetate (PDA, 1 micromol/l) induced a transient extracellular Ca(2+)-dependent contraction while the inactive analogue, 4alpha-phorbol (1 micromol/l) had no effect. PDA significantly enhanced the peak amplitude of the contractile response to NA (0.1-10 micromol/l), ATP (100 micromol/l), Ba2+ (3 mmol/l) or high K+ (30 mmol/l). Staurosporine at 30 nmol/l reduced the enhancing effect of PDA on the agonist-induced contraction. NA (10 micromol/l) produced a phasic contraction followed by a sustained contraction, while ATP induced monophasic contraction. Pretreatment with nifedipine (10 nmol/l) had no effect on the phasic contraction induced by NA, but it significantly reduced ATP- or high K(+)-induced contraction. Staurosporine (30 nmol/l) alone attenuated the peak contractile response induced by NA or ATP but not by Ba2+. NA produced a transient contraction in Ca(2+)-free Krebs solution, and PDA (1 micromol/l) markedly enhanced this effect. These novel data indicate that activation of a protein kinase C-dependent mechanism not only affects contraction mediated by Ca2+ influx through voltage-sensitive Ca2+ channels, but also promotes intracellular Ca2+ release or intracellular Ca(2+)-mediated contractile mechanism in rat vas deferens.     

Influence of castration on the response of the rat vas deferens to fluoxetine.

Antidepressant drugs such as desipramine and fluoxetine increase norepinephrine (NE) contractile response in rat vas deferens by inhibiting neuronal amine uptake. Fluoxetine, unlike other antidepressants, also inhibits calcium fluxes, which results in an inhibition of maximal NE effect. Since the contractile response of the reproductive tract is under the influence of testosterone, the effect of fluoxetine could be modified according to the endocrine status of the animal. In the present study we evaluated the influence of castration and testosterone replacement (1 mg per 100 g body wt.) on the peripheral action of fluoxetine. Castration was followed by a decrease in vas deferens weight and the appearance of spontaneous activity. Testosterone replacement reversed these effects. Concentration-response curves to NE and calcium were obtained in the absence and the presence of fluoxetine in vasa deferentia from normal, castrated and testosterone-treated castrated rats. After castration the effect of fluoxetine on vas deferens contractility was markedly altered. The spontaneous activity that appears after castration was prevented by fluoxetine and the stimulatory effect on NE-induced contractions was not observed. In contrast, the inhibitory action of fluoxetine on maximal NE effect was increased. Testosterone replacement restored vas deferens response to NE in the presence of fluoxetine. Fluoxetine did not modify the binding parameters of [(3)H]prazosin in vasa deferentia from normal or castrated animals. Cocaine shifted the NE concentration-response curve to the left in all groups, suggesting that the changes in fluoxetine effect following castration were not the result of an alteration of the neuronal uptake mechanism. The nitric oxide synthase inhibitor l-NMMA did not modify vas deferens response to NE in castrated animals either in the absence or presence of fluoxetine. An increased sensitivity to the inhibitory effect of fluoxetine was observed in the calcium concentration-response curves in vasa deferentia from castrated rats, an effect that was reversed by testosterone replacement. The results suggest that the alteration in the responsiveness of vasa deferentia from castrated rats to calcium could be responsible for increased sensitivity to the inhibitory effect of fluoxetine. It is concluded that vas deferens contractile response is testosterone dependent and that this behaviour modifies the effect of drugs such as fluoxetine that have dual effect on contractility.     

Effect of full agonists following calcium deprivation in rat vas deferens

The contractile effects of maximum doses of adrenaline, noradrenaline, methacholine, acetylcholine, serotonin and barium chloride were studied following substitution of a medium without calcium for the normal nutrient solution. Except for the last agonist, the effects fall to about 10% within the first 3 min with prompt return to normal value upon reintroduction of regular fluid. This recovery is, however, slower if the previous incubation in Ca-free solution is prolonged. When barium choride is used in a calcium-free medium, the maximum height of contractions falls exponentially at a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 180 min. This decay can be accelerated by giving successive 5-min doses of the agonist or by using EDTA. It is hypothesised that excitation—contraction coupling in rat vas deferens depends on at least two different calcium sources: a deep site associated with the effects of barium, and a superficial one, related to the other agonists. To explain the slow recovery after prolonged calcium lack, a third compartment in series with the latter is suggested. No indication is found that the biphasic effects of barium depend on two different calcium pools.     

Nitric oxide synthase/guanylate cyclase pathway modulates the rat vas deferens contractility induced by phenylephrine

The involvement of the nitric oxide synthase/soluble guanylate cyclase pathway on the modulation of phenylephrine-induced contractility in the rat vas deferens was investigated. Phenlylephrine-concentration response curves were obtained in absence and in presence of inhibitors, N(G)-Nitro-L-arginine (L-NOARG), NG-Nitro-L-arginine methyl esther (L-NAME) or N(G)-monomethyl-L-arginine (L-NMMA) or GC inhibitior, 1H-(1,2,4)-oxadiaziol-(4,3-a)quinoxalin-1-one (ODQ) or nitric oxide donor, 3-morpholinosydnonimine hydrochloride (SIN-1) alone or together with L-NMMA or ODQ. Both nitric oxide synthase and GC inhibitors reduced the Phe-Emax. SIN-1 alone did not change phenylephrine-induced responses and it could reverse the L-NMMA effect but not ODQ effect. The reduction of the phenylephrine-induced contractility obtained in consequence of the inhibition of the nitric oxide/GC pathway suggest that, in the rat vas deferens, despite its well identified relaxant properties, nitric oxide potentiates the contractility induced by adrenergic stimulation.     

Formation of dopamine and noradrenaline in rat vas deferens: comparison with guinea-pig vas deferens.

1 The formation of [14C]-3,4-dihydroxyphenylalanine (DOPA) from [14C]-tyrosine, in the presence of the amino acid decarboxylase inhibitor, brocresine (3-hydroxy-4-bromobenzyloxyamine dihydrogen phosphate), was greatly enhanced in rat vasa deferentia depolarized by a KCl-enriched Krebs-Henseleit solution (52 mM KCl) compared with tissues maintained in unmodified Krebs-Henseleit solution. 2 When the conversion of tyrosine was allowed to proceed as far as catecholamine (brocresine absent) no significant difference was observed between the accumulation of [14C]-catecholamines (CA) in depolarized rat vasa deferentia and the accumulation in control (non-depolarized) tissues. 3 Endogenous CA levels in the depolarized rat vasa deferentia fell to 67% of the controls after a 1 h incubation period and to 53% at the end of 2 hours. 4 Chromatographic separation on Amberlite CG-120 columns of the newly synthesized CA and catechol metabolites from the rat vas deferens revealed that a very high proportion was present as dopamine. The percentage distribution after 1 h incubation in control Krebs-Henseleit was: noradrenaline (NA): 30.6 +/- 5.2; dopamine 56.9 +/- 5.9; acid metabolites: 12.8 +/- 1.1; and in KCl-rich Krebs-Henseleit, NA: 32; dopamine: 44.7 and acid metabolites 23.3. In contrast to the newly synthesized (14C-labelled) CA, endogenous dopamine comprises only 10% of the endogenous CA stores in rat vas deferens. 5 The distribution of newly synthesized NA and dopamine in rat vas deferens is strikingly different from that of guinea-pig vas deferens where more than 80% of newly formed amine is present as NA. In the latter tissue depolarization with K+ causes a striking increase in CA biosynthesis.     

Uptake of desipramine by the rat vas deferens

1. Determinations of desipramine in the isolated rat vas deferens were carried out in order to study the uptake of the drug and to evaluate how this may correlate with the effect on noradrenaline (NA)-induced contraction.2. Vasa deferentia, in contact with concentrations of desipramine of 1 ng-25 mug/ml for 10 min accumulated the drug about 6-fold in respect to the medium. When the time of contact was prolonged to 4 h, desipramine (200 ng/ml) was concentrated about 100-fold. The uptake was not saturable and it was not affected by the presence of imipramine, cocaine, ouabain, dinitrophenol and iodoacetate.3. No metabolic process is involved although the accumulation of desipramine is temperature dependent. The release of desipramine from the vas deferens is exponential and it is not affected by the presence of plasma in the medium.4. No clear correlation was found between tissue concentration of the drug and the potentiation of noradrenaline responses, probably because of the high non-specific binding of the drug to tissue components which may mask specific binding sites for NA resulting in potentiation. However, the concentration of desipramine seems to correlate with the inhibition of NA effect which occurs at high doses of desipramine.     

Changes in neuromuscular transmission of guinea pig vas deferens produced by decamethrin treatment

Responses of the isolated vas deferens of guinea pig to clonidine (inhibition of contractions to field stimulation at 2.5 Hz), tyramine (inhibition of contractions to field stimulation at 10 Hz), prostaglandin E2 (inhibition of contractions to field stimulation at 10 Hz), and noradrenaline (contraction of longitudinal muscle) were determined after administration of decamethrin (18 mg kg-1, ip) once a day for 3 consecutive days. Treatment with decamethrin produced a subsensitivity of the prejunctional alpha 2-adrenoceptor system as evidenced by the fact that the alpha 2-agonist clonidine was less effective in decreasing nerve-stimulated induced contractions of the vas deferens. In addition, the presynaptic action of tyramine on postganglionic motor transmission was impaired. However, no detectable changes in the inhibition by prostaglandin E2 of twitch responses were produced by decamethrin. Decamethrin treatment had a significant effect on noradrenaline responsiveness, causing an increase in the maximum contractile response, indicative of an enhanced postreceptor mechanism. The present results suggest that decamethrin treatment reduces peripheral presynaptic adrenoceptor sensitivity. This reduction will lead subsequently to increased noradrenaline release and postsynaptic adrenoceptor upregulation.     

Responsiveness of rat vas deferens and stomach smooth muscles after treatment with indomethacin

The contractile responses of rat vas deferens and longitudinal stomach fundus strips to stimulants and relaxants were studied after treatment with indomethacin (Ind) injected i.p. at doses of 15 mg/kg for 24 hr and 25 mg/kg for 48 hr. The treatment with Ind reduced the amount of both spontaneously and electrically-evoked release of a prostaglandin (PG)-like substance from the isolated n. hypogastricus-vas deferens preparations. A considerable decrease of the responsiveness to exogeneous noradrenaline occurred in vas deferens isolated from Ind-pretreated rats. The EC50 values of noradrenaline increased as compared to the control value. The contractile responses of the isolated longitudinal stomach fundus strips to acetylcholine, serotonin, PGE2, PGF2 and arachidonic acid were reduced while the relaxant action of noradrenaline and papaverine was potentiated after Ind treatment. It is assumed that endogeneous PGs participate in the regulation of smooth muscle activity through potentiation of the postjunctional excitatory action of noradrenaline in vas deferens and through interaction with the transmembrane ion transport in stomach fundus smooth muscles.     

Release of ATP in rat vas deferens: origin and role of calcium

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.In tissues preincubated with [³H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 M and even more so by prazosin 0.3 M combined with suramin 300 M. They were also reduced by nifedipine 10 M and even more so by nifedipine 10 M combined with ryanodine 20 M (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [³H]-noradrenaline, exogenous noradrenaline 10 M and ,-methylene ATP 10 M elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 M but not suramin 300 M and were greatly reduced by nifedipine 10 M and in Ca²⁺-free medium. Responses to ,-methylene ATP were blocked by suramin 300 M but not prazosin 0.3 M were reduced by nifedipine 10 M (effect on ATP overflow not significant) and were reduced even more in Ca²⁺-free medium. Neuropeptide Y 0.3 M caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both ₁-adrenoceptor and P₂-purinoceptor activation. Ca²⁺ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A purely neural overflow of ATP was not isolated under the conditions of the experiments. Correspondence to: R. Bültmann at the above address     

Inhibition of rat vas deferens contractions by flavonoids in-vitro

Flavonoids are a large heterogeneous group of benzo-gamma-pyrone derivatives, which are abundantly present in our diet. In this study we investigated the effect of ten flavonoids (quercetin, kaempferol, morin, galangin, rutin, apigenin, flavone, naringenin, hesperitin and silybin) on the contractile response elicited by electrical field stimulation in the rat isolated vas deferens. All flavonoids tested inhibited vas deferens contractions. The relative order of potency of the tested flavonoids was naringenin > hesperitin > morin > kaempferol > apigenin > silybin > flavone > rutin > quercetin > galangin. Analysis of the chemical structures showed that the saturation of C-2-C-3 double bond and the presence of hydroxyl groups on the flavonoidic scaffold play an important role in the activity of flavonoids.     

Effects of T-type calcium channel blockers and thalidomide on contractions of rat vas deferens

Background and purpose:

In rat vas deferens, nerve mediated-contractions to a single electrical stimulus consist of an early purinergic and a later adrenergic component with differing sensitivities to L-type calcium channel blockers. We have investigated the effects of the T-type calcium channel blockers mibefradil and (1S, 2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylic dihydrochloride (NNC 55-0396) against contractions in rat vas deferens. In addition, the actions of thalidomide were examined.

Experimental approach:

Prostatic and epididymal portions of rat vas deferens were stimulated with a single electrical stimulus every 5 min, and mouse whole vas deferens was stimulated with 40 pulses at 10 Hz every 5 min.

Key results:

Both mibefradil and NNC 55-0396 (100 µM) produced inhibition of contractions of epididymal portions (42 ± 13%, n= 7, and 43 ± 4%, n= 15, of control respectively). However, both agents produced small inhibitions of responses in prostatic portions, presumably by L-type calcium channel block. Thalidomide (100 µM) inhibited contractions in epididymal (55 ± 4% of control, n= 17) but not in prostatic portions of rat vas deferens. Thalidomide (10–100 µM) also inhibited contractions in mouse vas deferens.

Conclusions and implications:

The T-type calcium channel blockers mibefradil and NNC 55-0396 block particularly the adrenoceptor-mediated, nifedipine-resistant response to nerve stimulation in rat vas deferens, and this may suggest that this component involves T-type calcium channels. In addition, thalidomide has actions that resemble those of the T-type calcium channel blockers, in that it blocks nifedipine-resistant contractions in epididymal portions.     

Inhibitory action of propranolol on the contractions induced by nerve stimulations or calcium in the smooth muscle of rat vas deferens

Inhibitory effects of propranolol on the contractions to various treatments were investigated in the epididymal half of the rat vas deferens. Reportedly, 10(-5)-3 X 10(-4) M propranolol inhibited 150 mM K-induced contractions dose-dependently; 3 X 10(-4) M propranolol abolished the contractions. The present results showed that propranolol at concentrations up to 10(-4) M did not inhibit the maximal contractions to 10(-3) M norepinephrine (NE) or 10(-2) M methacholine (MCh). Propranolol at 3 X 10(-4) M slightly inhibited contractions to NE and MCh by 11% and 12%, respectively. In contrast, propranolol inhibited twitch components of the contractions induced by nerve stimulations at similar doses to those reported for high K contractions. Propranolol also inhibited contractions to Ca in high K-containing solution and shifted the dose-response curve to the right. Propranolol did not affect the depolarizations by high K measured by microelectrodes. Propranolol at concentrations of 10(-5)-3 X 10(-5) M diminished the magnitude of spikes dose-dependently. Spikes were rarely observed in the presence of 10(-4) M propranolol in spite of generation of e.j.p.s with amplitudes that would be sufficient to induce spikes in the absence of propranolol. These results suggest that propranolol inhibits contractions by decreasing Ca-influx through the potential-operated Ca-channels in the smooth muscle cells of rat vas deferens.     

Prevention of supersensitivity-like phenomena in rat vas deferens by colchicine

Rat vas deferens has been shown to become supersensitive to α-adrenergic agonists on brief treatment with epinephrine (Epi-treatment). Epi-treatment increased the contractile response and the number of α-adrenoceptors in the tissue. The application of colchicine (2.5 mM) during Epi-treatment prevented the supersensitivity-like phenomena and increase in number of α-adrenoceptors. Vinblastine (10 μM) also counteracted the effect of Epi-treatment but cytochalasin B (100 μM) and strychnine (2.5 mM) did not. It is suggested that the supersensitivity-like phenomena induced in rat vas deferens by Epi-treatment were due to a change in microtubular components of the membrane.