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Prejunctional Facilitatory Effect of a Thiol-Alkylating Agent N-Ethylmaleimide on Neurogenic Contractions in Rat Prostate Smooth Muscle

Aims: The effect of a non-speciﬁc thiol-alkylating agent N-ethylmaleimide (NEM) was studied on neurogenic contrac- tile mechanisms in rat ventral prostate gland. Methods: Male Wistar albino rats were used. The rats were killed by cervical dislocation under sevoﬂurane anesthesia and ventral prostate gland was removed. Two preparations were obtained from each lobe. Neurally evoked isometric contractions were induced using trains of electrical ﬁeld stimula- tion (EFS; 0.5, 1, 4, or 8 Hz). The effect of NEM on the contractions to EFS was examined in the absence or presence of adrenergic and/or purinergic antagonists. Results: NEM enhanced the EFS-evoked contractions without altering the basal tone. These effects were signiﬁcantly suppressed by an a1-adrenergic receptor antagonist (prazosin), a P2- purinergic antagonist (suramin), a speciﬁc P2X-receptor antagonist pyridoxal-phosphate-6-azophenyl-20,40-disulfonate (PPADS), an ATP analog (a,b-methylene ATP), or a calcium channel blocker (verapamil). This facilitating effect of NEM did not occur following the administration of L-cysteine or glutathione which saturated NEM with excess thiols. However, a thiol-oxidant diamide failed to affect the contractions to EFS. An adrenergic neuron blocker (guanethidine) completely suppressed the responses to NEM. On the other hand, an a2-adrenergic receptor blocker (yohimbine), a nitric oxide synthase inhibitor (Nv-nitro-L-arginine) or a cholinergic muscarinic receptor antagonist (atropine) did not signiﬁcantly affect the facilitatory response of NEM. Conclusions: These ﬁndings suggest that NEM has a prejunctional facilitatory action on the adrenergic nerves in rat prostate tissue to enhance release of transmitters, noradrenaline, and ATP. NEM sensitive proteins involved in transmitter release mechanisms can play a role in this effect.

Key words: N-ethylmaleimide; neurogenic contractions; prostate; rat

INTRODUCTION

Previous studies have revealed that thiol reagents modulate smooth muscle contractile activity in some tissues by control- ling the gating of Ca2þ channels.1–6 It is known that there are sites on the Ca2þ channels that are subject to direct modiﬁca- tion by thiol reagents.7 It has also been suggested that these reagents modulate neuronal Ca2þ channels on nerve terminals and thereby alter the release of neurotransmitters and indi- rectly inﬂuence contractile mechanisms.2,8 Similar presynap- tic effects of a thiol alkylating agent, N-ethylmaleimide (NEM)9,10 have been demonstrated at many sites in the central and peripheral nervous system. For example, NEM produced a series of changes in the responses of the superior cervical ganglion to stimulation of the preganglionic nerve,11 including as a cotransmitter from sympathetic nerve terminals within the prostate gland.14 Although there are many studies of the contractions induced by electrical ﬁeld stimulation (EFS) in the prostate tissue, to our knowledge, the effects of thiol reagents on neurogenic contractions have not been studied in the prostate tissue of any species. We thus compared the ef- fect of NEM on the basal tonus of the isolated rat prostate smooth muscle with the effect on neurogenic contractions induced by trains of EFS. To investigate whether NEM has a prejunctional facilitatory action on neurotransmission mediated by ATP, noradrenaline, or acetylcholine we studied the effect of NEM in the presence or absence of adrenergic, cholinergic, and purinergic receptor antagonists.

MATERIALS AND METHODS

Male Wistar albino rats weighing 250–300 g were used throughout the experiments. The experimental procedure was approved by the animal care committee of the University of C¸ ukurova (TIBDAM) and the experiments were carried out in accordance with the Principles of Laboratory Animal Care (National Institute of Health guideline; publication no. 86-23, revised 1984). All animals were kept under laboratory condi- tions (12 h dark; 12 h light) and allowed access to food and drink ad libitum.

Rats were killed by cervical dislocation under sevoﬂurane anesthesia. After an abdominal incision, the left and right lobes of ventral prostate gland were removed and placed in a petri dish containing Krebs solution (mM: NaCl 118, KCl 4.7, CaCl2 1.5, MgCl2 1.2, NaHCO3 15, NaHPO4 1.2, glucose 11). The capsules of the prostatic lobes were removed along with con- nective and adipose tissue. Two preparations were obtained from each lobe. The ventral prostate tissues were mounted under 9.8 mN tension between two platinum electrodes em- bedded in perspex in 5 ml jacketed organ baths containing Krebs solution. The organ baths were maintained at 378C and aerated with a mixture of 95% O2 and 5% CO2. Tissues were then allowed to equilibrate for 1 hr and washed with fresh Krebs solution every 15 min during this period. Tissue responses were recorded via isometric force transducer (MAY, FDT 10-A). Data were recorded (sampling rate: 1,000 Hz) and stored using data acquisition software (BIOPAC mp30 Sys- tems, Inc., Ankara, Turkey). After the equilibrium period, neurally evoked isometric contractions were induced using trains of supramaximal EFS (0.5, 1, 4, or 8 Hz, 50 V/0.3 cm, 0.5 msec duration, 10-sec trains) delivered from a Grass S88 stimulator (Grass Instruments, Quincy, MA) at 1 min intervals throughout the experiments. In control experiments, EFS- evoked responses were recorded for a 4 hr time period (no drug was added) to examine the stability of the nerve-evoked responses.
In other experiments after the control responses to EFS were recorded for approximately 1 hr, a non-speciﬁc thiol-alkylat- ing agent NEM was applied at various concentrations (25– 200 mM) to the same tissue consecutively for 10 min at inter- vals ranging from 80 to 90 min in an attempt to identify a concentration of drug which inﬂuenced EFS evoked responses without affecting basal tone. These experiments revealed that 100 mM NEM did not affect the basal tone; but higher concen- trations produced tonic contractions. Thus we used this con- centration in all experiments.

In some experiments, the effects of various agents includ- ing, thiols (L-cysteine, 1 mM and glutathione, 1 mM), a nitric oxide synthase inhibitor (Nv-nitro-L-arginine, L-NOARG, 100 mM), a calcium channel antagonist (verapamil, 10 mM), a cholinergic muscarinic receptor antagonist (atropine sulfate, 1 mM), an a1-adrenergic receptor antagonist (prazosin, 1 mM), an a2-adrenergic receptor antagonist (yohimbine, 1-10 mM), an adrenergic neuron blocker (guanethidine, 2 mM), a P2 purinergic antagonist (suramin, 100 mM), or a speciﬁc P2X receptor antagonist (pyridoxal-phosphate-6-azophenyl-20,40- disulfonate, PPADS; 200 mM) were examined on prostate tis- sue contractions induced by EFS in the presence or absence of NEM (100 mM). Each drug was added to the organ bath 20 min before the second NEM application. In another series of experiments we studied the effect of a thiol oxidant di- amide (diazenedicarboxylic acid bis 5 N,N-dimethylamide, 200 mM) on prostate tissue contractions induced by EFS to ex- amine whether its effect is similar to NEM. In some experi- ments, NEM (100 mM) was applied to the prostate strips after prolonged exposure (30 min) to an ATP analog (a,b-methylene ATP treatment, 20 mM) to examine the effect of this agent on the EFS-evoked contractions in preparations in which the puri- nergic receptors were desensitized.14 In another experiment series, we examined the effects of NEM on the contractions induced by an a1-adrenergic receptor agonist phenylephrine (1 mM) or ATP (50 mM).

Drugs

Stock solutions of NEM, L-NOARG, suramin, verapamil, a,b-methylene ATP, atropine sulfate, guanethidine, diamide, L-cysteine, glutathione, PPADS, prazosin, phenylephrine, and ATP were dissolved in distilled water. All drugs were obtained from Sigma Chemical Co., St. Louis, MO.

Statistical Analysis

The data (mean SE) were expressed as the mean contrac- tile response (mN) to EFS in the presence or absence of NEM. All of the data were evaluated with the Bonferroni corrected t-test that was used in analysis of variance (ANOVA). P values of less than 0.05 were considered signiﬁcant. Statistical analysis was performed with GraphPad Prism software (San Diego, CA).

RESULTS

Effect of NEM on the EFS-Induced Contractions of Rat Prostate Tissue Strips

The amplitude of the EFS-evoked contractions was constant for a 4 hr time period (0.19 0.02 mN at the beginning of the experiment and 0.18 0.02 mN at the end of the experiment, n 6). The amplitude of the contractions induced by EFS (50 V, 0.5 msec) at lower frequencies (0.5, 1, and 4 Hz) was signiﬁcantly enhanced by 100 mM NEM (from 0.17 0.02 to 0.38. 0.03 mN at 0.5 Hz; from 0.23 0.02 to 0.37 0.03 mN at 1 Hz; and from 0.36 0.04 to 0.47 0.03 mN at 4 Hz; Figures 1A and B and 2, n 12 strips at each frequency). How- ever, NEM did not affect the amplitude of the contractions at a higher frequency (8 Hz) of EFS (from 1.16 0.06 to
1.17. 0.06 mN, Fig. 1C). NEM did not affect the basal tone of the strips at 100 mM. However in some strips (nearly 15% of total strips), it caused a small increase in the basal tone (14.5 4.6 as % increase). This facilitating effect of NEM was reversible in 45–50 min after washout of the drug. Also the repeated application of 100 mM NEM produced a reproducible effect on the contractions evoked by EFS (Figs. 1A and 2). Higher concentrations (over 100 mM) of NEM elicited a large increase (nearly 20–65% increase depending on concentration of NEM) in basal tone and also at these higher concentrations, NEM increased EFS-induced contractions (data not shown). On the other hand, a thiol oxidant diamide (200 mM) did not elicit any signiﬁcant effect on the control EFS (0.5 Hz)-evoked contractions (Fig. 1D) (n ¼ 6).

Effects of L-Cysteine or Glutathione on the Facilitating Effect of NEM in Rat Prostate Tissue

The facilitating effect of 100 mM NEM on the EFS (0.5 Hz) evoked contractions did not occur following the application of the 1 mM L-cysteine or 1 mM glutathione (Fig. 3A and B). L-Cysteine or glutathione alone did not affect the amplitude of the EFS-evoked contractions or basal tone.

Fig. 1. Representative tracings showing (A–C) the facilitatory effects of 100 mM N-ethylmaleimide (NEM) and (D) the ineffectiviness of 200 mM diamide on the contractions to EFS (0.5, 4, and 8 Hz, 50 V, 0.5 msec) in rat prostate tissue. ‘‘w’’ represents washout.

Effects of Prazosin, Yohimbine, or Guanethedine on the Facilitating Effect of NEM in Rat Prostate Tissue

An a1-adrenergic receptor blocker (prazosin, 1 mM) caused a signiﬁcant suppression on the control contractions to EFS (0.5 Hz) (from 0.2 0.02 to 0.1 0.01 mN) whereas an a2-adrenergic receptor blocker (yohimbine, 1–10 mM) did not elicit any effect on the contractions to EFS (data not shown). NEM (100 mM) facilitated the contractions to EFS in the presence of 1 mM prazosin (Fig. 4A). In the presence of prazo- sin the facilitatory effect of NEM expressed as a percent of the amplitude of the EFS contraction was signiﬁcantly lower (150.7 17.7%, P < 0.05) compared to the effect in the absence of prazosin (203.3 20.6%). On the other hand, the facilitation due to NEM was not signiﬁcantly affected in the presence of yohimbine (data not shown). An adrenergic neuron blocker (guanethidine, 2 mM) completely suppressed the control contractions to EFS (0.5 Hz) (Fig. 4B) and the facili- tating effect of 100 mM NEM on the EFS-evoked contractions (0.5 Hz) did not occur following the application of the guanethidine (Fig. 4B). Fig. 2. The facilitatory effects of 100 mM N-ethylmaleimide (NEM) on the contractions to EFS (0.5 Hz, 50 V, 0.5 msec) in rat prostate tissue. Each col- umn represents the mean contractile response (mN) to EFS in the presence or absence of NEM. Results are expressed as mean SE (n ¼ 12). ωP < 0.05 shows signiﬁcant difference from control. Effects of Suramin, PPADS, or a,b-Methylene ATP on the Facilitating Effect of NEM in Rat Prostate Tissue The P2 purinergic antagonist (suramin, 100 mM), a speciﬁc P2X receptor antagonist (PPADS, 200 mM), or an ATP analog (a,b-methylene ATP, 20 mM) caused a marked suppression of the control contractions to EFS (0.5 Hz) (Fig. 5A–C). NEM (100 mM) still facilitated the remaining contractions in the presence of these purinergic antagonists (Fig. 5A–C). However, the amount of facilitation caused by NEM in the presence of suramin, PPADS, or a,b-methylene ATP was markedly less than that in the absence of these agents. While NEM caused 245.3 25.9% increase in the amplitude of the contractions in the absence of suramin, it elicited only a 128.7 24.1% increase in the presence of this antagonist. The facilitation ratios in the presence of PPADS or a,b-methylene ATP were 131.2 21.4% and 131.7 17.2%, respectively. In some strips,the combination of suramin (100 mM) with prazosin (1 mM) completely suppressed control contractions (from 0.17 0.02 to 0.004 0.0 mN); and the facilitating effect of 100 mM NEM on the EFS-evoked contractions (0.5 Hz) did not occur following the application of suramin prazosin (data not shown). Fig. 3. The suppressive effect of (A) L-cysteine (L-cys; 1 mM) or (B) glutathi- one (Glu; 1 mM) on the facilitatory effects of 100 mM N-ethylmaleimide (NEM) on the contractions to EFS (0.5 Hz, 50 V, 0.5 msec) in rat prostate tissue. Each column represents the mean contractile response (mN) to EFS in the presence or absence of NEM. Results are expressed as mean SE (n ¼ 6–8). ωP < 0.05 shows signiﬁcant difference from control. ‘‘w’’ represents washout. Fig. 4. The suppressive effect of (A) prazosin (PRA; 1 mM) or (B) guanethi- dine (GUA; 2 mM) on the facilitating effects of 100 mM N-ethylmaleimide (NEM) on the contractions to EFS (0.5 Hz, 50 V, 0.5 msec) in rat prostate tissue Each column represents the mean contractile response (mN) to EFS in the presence or absence of NEM. Results are expressed as mean SE (n ¼ 6–8). ωP < 0.05 shows signiﬁcant difference from control. yP < 0.05 shows signiﬁcant difference from NEM. ‘‘w’’ represents washout. Effects of L-NOARG, Atropine, or Verapamil on the Facilitating Effect of NEM in Rat Prostate Tissue The NOS inhibitor, L-NOARG (100 mM) did not affect the control contractions (not shown) or the responses to 100 mM NEM (Table I, n 6). A cholinergic muscarinic receptor antagonist (atropine, 1 mM) caused some decrease in the amplitude of the control contractions to EFS (0.5 Hz) (from 0.19 0.04 to 0.13 0.03 mN) and in the response to 100 mM NEM (Table I, n 8). But, these decreases were not statistically signiﬁcant from control responses. On the other hand, a calcium channel blocker (verapamil, 10 mM) signiﬁ- cantly inhibited the responses to 100 mM NEM (Table I, n 6). This agent caused some decrease of the control con- tractions induced by EFS (0.5 Hz) (from 0.13 0.04 to 0.073 0.01 mN) but this decrease was not statistically signiﬁcant. Effects of NEM on the Contractions Induced by Phenylephrine or ATP in Rat Prostate Tissue Phenylephrine (1 mM) or ATP (50 mM) elicited reproducible and reversible contractions in rat prostate tissue (not shown). 100 mM NEM slightly, but not signiﬁcantly reduced the con- tractile responses to phenylephrine or ATP (from 4.0 0.9 to 2.4 0.7 mN for phenylephrine, n ¼ 4; from 0.9 0.2 to 0.5 0.3 mN for ATP, n ¼ 4). DISCUSSION The present experiments revealed that a non-speciﬁc thiol- alkylating agent NEM enhanced the EFS-induced contractions without altering the basal tone in rat prostate strips. These effects were signiﬁcantly reduced by P2 purinergic antagonist (suramin), a speciﬁc P2X receptor antagonist (PPADS), an ATP analog (a,b-methylene ATP), or an a1-adrenergic receptor blocker (prazosin) and did not occur following the administra- tion of L-cysteine or glutathione in which NEM was saturated with excess thiols. Also, an adrenergic neuron blocker, guanethidine completely suppressed the responses to NEM. These ﬁndings suggest that NEM has a prejunctional facilita- tory action on the adrenergic nerves in rat prostate tissue and enhances release of noradrenaline and ATP. Thus NEM sensitive proteins must be involved in regulating transmitter release mechanisms in the prostate. Fig. 5. The suppressive effect of (A) suramin (SUR; 100 mM), (B) PPADS (PPA; 200 mM), or (C) a,b-methylene ATP (ATP; 20 mM) on the facilitatory effects of 100 mM N-ethylmaleimide (NEM) on the contractions to EFS (0.5 Hz, 50 V, 0.5 msec) in rat prostate tissue. Each column represents the mean contractile response (mN) to EFS in the presence or absence of NEM. Results are expressed as mean SE (n ¼ 6–8). ωP < 0.05 shows signiﬁcant difference from control. ‘‘w’’ represents washout. In the present study, NEM caused a signiﬁcant increase in the amplitude of EFS-evoked contractions without affecting the basal tone of the tissue. In our previous studies in rat urinary bladder, NEM and the other thiol reagents, ethacrynic acid or diamide, induced increases in basal tone which were suppressed by L-cysteine, glutathione, verapamil, and Ca2þ free bathing solution.4,6 However, in these studies, the thiol reagents did not affect the neurogenic contractions to EFS. The ineffectiveness of low concentrations of NEM to alter basal tone in the prostatic smooth muscle suggests that the effect of this agent on neurotransmission is due primarily to a presyn- aptic action rather than a postsynaptic action. Similar presyn- aptic effects of NEM have been demonstrated at many sites in the central and peripheral nervous system. It is well accepted that both noradrenaline and ATP are released from sympathetic nerve terminals within the prostate gland in response to electrical-ﬁeld stimulation.14 The ﬁnding that an adrenergic neuron blocker, guanethidine completely suppressed the control contractions to EFS (0.5 Hz) and also eliminated the facilitating effect of NEM indicate that adrenergic nerves are the major target of NEM in the prostate. Although, cholinergic transmission may also play a partial role in the contractions induced by EFS in the prostate tissue,15 atropine failed to cause a signiﬁcant change in the responses to NEM, suggesting that the facilitating effect of NEM is due primarily to enhancement of the adrenergic responses in this tissue. Since an a1-adrenergic receptor blocker prazosin or P2 purinergic antagonist suramin caused a signiﬁcant reduction in the response to NEM it is reasonable to conclude that the drug enhances the release of both noradrenaline and ATP. This conclusion is supported by the ﬁnding that a combination of suramin and prazosin complete- ly inhibited the responses to NEM. Also, a speciﬁc P2X receptor antagonist PPADS or an ATP analog a,b-methylene ATP caused a marked suppression of both control contractions to EFS and the responses to NEM at 0.5 Hz. Furthermore the magnitude of the inhibition caused by these purinergic antagonists was approximately the same as that induced by prazosin, suggest- ing that ATP and noradrenaline released from adrenergic nerves are of equal importance as mediators of excitatory transmission to the prostatic smooth. Although the mechanism underlying the excitatory effect of NEM in the prostate has not been examined in the present experiments, studies conducted in other laboratories that focused on the actions of this agent on G protein mediated mechanisms in various neurons raise the possibility that same mechanisms may be involved in the presynaptic effect of NEM on the adrenergic nerves. For example, the actions of NEM on several types of G-protein-mediated inhibitions of N-type Ca2þ channels in adult rat superior cervical ganglion neurons were revealed using whole-cell voltage clamp techniques.16 In that study, NEM selectively inactivated pertussis toxin-sensitive Gi-proteins which are known to play a role in inhibition of transmitter release by suppressing calcium channels. In another study, it was shown that NEM blocks the effect of G protein coupled receptors acting via per- tussis toxin-sensitive Gi proteins and abolished the inhibitory effect of baclofen on the GABA release in substantia nigra pars reticulata.17 Thus, NEM can cause an increase in GABA transmission indirectly by down-regulating mechanisms that inhibit transmitter release. In the present study, the effect of NEM may be due to a similar inhibition of a G protein coupled receptor-pertussis toxin-sensitive Gi mechanism that tonically inhibits of noradrenaline and ATP release by suppressing cal- cium channels. Studies in the rat urinary bladder revealed that L-type rather than N-type calcium channels are involved in frequency dependent release of acetylcholine and NA.18 The ﬁnding that an L-type calcium channel blocker, verapamil, inhibited the facilitatory effect of NEM in the present experi- ments is consistent with the possibility that prejunctional L-type channels are important for the facilitation, although a postjunctional action of verapamil to suppress smooth muscle contractions may also play a role. On the other hand, an action of NEM on autofeedback inhibitory mechanisms controlling neurotransmitter release must also be considered. For example, it is possible that autofeedback inhibition mediated by noradrenaline acting on presynaptic a2-adrenergic receptors could be suppressed by NEM, thereby enhancing transmitter release. However, this seems unlikely because an a2-adrenergic agonist, yohimbine, did not elicit any effect on the contractions to EFS or on the response to NEM. There also could be presynaptic muscarinic receptor mediated inhibition, but the ﬁnding that a non- selective muscarinic receptor antagonist, atropine, caused a small inhibition rather than facilitation of the EFS-induced contractions and did not alter the response to NEM excludes this possibility. In the present study, NEM exhibited a frequency dependent effect on contractions to EFS, characterized by less facilitation at the higher frequencies. Previous studies have shown that the inhibition of calcium channels by Gi mechanisms is fre- quency dependent, that is, at higher frequencies calcium channels are less sensitive to Gi inhibition.19,20 If this occurred in our study, then it would be expected that there was less ongoing inhibition at higher frequencies, and thus there would be less inhibition for NEM to block and consequently less facilitation of transmission. This could explain the frequency dependence that we observed in the present study. On the other hand, in a previous study it was shown that ATP release is proportionately greater at lower frequencies stimu- lation in the rat prostate gland.14 Since the facilitatory effect of NEM was less pronounced at higher frequencies, it can be suggested that presynaptic inhibition is stronger on puriner- gic transmission than on adrenergic transmission at 0.5 Hz. The suppressant effects of L-cysteine or glutathione on the facilitatory effect of NEM are predictable because they bind to this agent and prevent it from interacting with –SH groups on tissue proteins. However this result does show that there is a thiol dependent mechanism underlying the effect of NEM. Furthermore, another thiol reagent, diamide did not elicit any effect on the contractions to EFS. The ineffectiveness of diamide supports the suggestion that the facilitatory effect of NEM is due to inhibition of NEM sensitive proteins involved in transmitter release in this tissue. Also, the ﬁnding that a NOS inhibitor, L-NOARG failed to affect the responses excludes a possible role of nitrergic mechanisms in the facilitatory effect. On the other hand, NEM at low concentrations (100 mM) did not enhance but slightly, although not signiﬁcantly, reduced the contractile responses to phenylephrine or ATP. These ﬁnd- ings support our speculation that NEM at low concentrations has a selective and reversible prejunctional facilitatory action on the adrenergic nerves in rat prostate tissue and does not enhance transmission by altering the action of neurotransmit- ters on the smooth muscle. However higher concentrations of NEM seem to have an additional postjunctional excitatory effect that induces an increase in baseline muscle tone. Other studies have revealed various postjunctional effects of NEM. A delayed (30–120 min) postsynaptic excitatory effect of NEM was observed at cholinergic synapses in sympathetic ganglia.11 On the other hand, NEM (30 mM) caused a 30% re- duction in the maximal contraction elicited by norepinephrine in the isolated mouse vas deferens.21 Also, in our a previous study, NEM (25 mM) had no inﬂuence on phenylephrine-induced contraction whereas it signiﬁcantly enhanced 4,7-dimethyl- 1,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide (FPTO)-induced relaxation in mouse corpus cavernosum.22 The mechanisms underlying these postjunctional effects are not known. More detailed information about neurotransmission in the prostate could inﬂuence the development of new treatments for diseases of the prostate gland. Currently, a1-adrenergic receptor blocking agents are used to treat lower urinary tract symptoms (LUTS) in patients with BPH. These agents are thought to act postjunctionally in the prostate to suppress the responses to noradrenaline. An alternate approach to treat- ment could target prejunctional mechanisms using drugs to suppress neurotransmitter release. Our study evaluated for the ﬁrst time the function of NEM-sensitive proteins and/or thiol mechanisms in the control of transmitter release in the prostate. Drugs that enhance NEM-sensitive, Gi-mediated prejunctional inhibition at adrenergic nerve terminals in the prostate might be effective in treating LUTS. CONCLUSIONS NEM may have a prejunctional facilitatory action on the ad- renergic nerves in rat prostate tissue. Thus the enhancement of EFS-induced contractions can be due to enhanced release of transmitters, noradrenaline, and ATP, after NEM treatment. NEM sensitive proteins involved in transmitter release mecha- nisms can play a role in this effect. NEM probably inhibits pertussis toxin-sensitive Gi mediated inhibition of calcium channels and thereby removes tonic inhibition of noradrena- line and ATP release. ACKNOWLEDGMENTS The authors wish to thank Ahmet Kantur and Zeynep Akˇilˇˇli from the same department for their technical assistance. The authors are indebted to C¸ ukurova University Experimental Research Center (TIBDAM) for the supply of rats. This work was supported by the C¸ ukurova University Research Foundation (TF 2009 BAP3). REFERENCES 1. Neering IR, Glover WE. The role of sulfhydryl groups in contraction of vascular smooth muscle. J Pharmacol Exp Ther 1979;208:335–40. 2. Salimi M, Khoyi MA, Dibai MA. Effect of ethacrynic acid on guinea pig ileum. Jpn J Pharmacol 1979;29:151–60. 3. Iesaki T, Wolin MS. Thiol oxidation activates a novel redox-regulated coro- nary vasodilator mechanism involving inhibition of Ca2þ inﬂux. Arterioscler Thromb Vasc Biol 2000;20:2359–65. 4. Resim S, Bu¨yu¨knacar HS, Go¨c¸men C, et al. A possible effect of sulfhydryl reagents on the contractile activity of the rat detrusor muscle. Eur J Pharma- col 2002;442:295–9. 5. Schach C, Xu M, Platoshyn O, et al. 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