- Email: [email protected]
Prejunctional actions of N-ethyl-maleimide and phenoxybenzamine in rat vas deferens
ELSEVIER
European Journal of Pharmacology 265 (1994) 125 132
Prejunctional actions of N-ethyl-maleimide and phenoxybenzamine in rat vas deferens Ingrid Browne, George Thomas, Katherine Gavin, James R. D o c h e r t y * Department of Physiolo,w, Royal College of Surgeons in Irehmd, 123 St. Stephen's Greeen, Dublin 2, Ireland Received 23 June 1994; revised MS received 17 August 1994; accepted 19 August 1994
Abstract
In studies of electrically evoked isometric contractions of rat vas deferens, N-ethyl-maleimide (30 #M) prctrcatment significantly reduced the prejunctional inhibitory potencies of xylazine and 5-hydroxytryptamine but failed to affect the potency of the a t-adrenoceptor agonist amidephrine. Phenoxybenzamine (1 ~M) or N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ) (10 #M) produced significant shifts in the potency of xylazine and significantly reduced the maximum inhibition, but the combination of phenoxybenzamine or EEDQ and N-ethyl-maleimide (30 ~tM) produced no further alteration in the effects of xylazine. In displacement studies, N-ethyl-maleimide displaced the binding of [3H]MK 912 ((2S,12bS)l',3'-dimethylspiro(~3A~5'~6~6'~7~2b-~ctahydr~-2H-benz~[b]fur~[2~3-a]quinaz~ine)-2~4'-pyrimidin-2'~ne) to rat renal cortex membranes with a K i of 466 _+ 133 #M (n = 5), and so does not bind to ae-adrenoceptors in the concentration range in which it affects prejunctional receptor mediated responses. This may suggest that N-ethyl-maleimide has actions other than inactivation of G-proteins or that the irreversible ae-adrenoceptor antagonists phenoxybenzamine and EEDQ inactivate G-proteins sensitive to N-ethyl-maleimide in concentrations at which they bind to ae-adrenoceptors. Keywords: Vas deferens, rat; az-Adrenoceptor; N-Ethyl-maleimide; Phenoxybenzamine; EEDQ (N-ethoxycarbonyl-2-cthoxy1,2-dihydroquinoline)
1. Introduction
N-Ethylmaleimide alkylates proteins and has been demonstrated to inactivate certain G-proteins (Ueda et al., 19901, and to attenuate prejunctional cQ-adrenoceptor mediated inhibition of neurotransmitter release in a number of tissues including rat cerebral cortex (Kitamura and Nomura, 1987), mouse vas deferens (Kaschube and Brasch, 1990), rat tail artery (Weber, 1989), rabbit renal arteries ( R u m p et al., 19921 and mouse atria (Murphy et al., 1992). Furthermore, R u m p et al. (1992) have shown that N-ethyl-maleimide attenuates a 2- but not a l - a d r e n o c e p t o r mediated inhibition of noradrenaline release in rabbit renal arteries. The present study began with an investigation of the interaction between N-ethyl-maleimide and prejunc-
* Corresponding author. Tel. (353) 1-4780 20(1, fax (353) 1-4780
018. 0014-2999/94/$(17.(1(I ~ 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 5 1 9 - 2
tional a2-adrenoceptors in rat kidney cortex which produced results consistent with previous reports that N-ethyl-maleimide may inactivate a G-protein involved in ae-adrenoceptor mediated inhibition. We investigated further the o~2-adrenoceptor antagonist actions of N-ethyl-maleimide in a system in which interaction between N-ethyl-maleimide and the irreversible a tadrenoceptor antagonists phenoxybenzamine and Nethoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline ( E E D Q ) could be examined under controlled conditions: inhibition by the oe2-adrenoceptor agonist xylazine of the rat vas dcferens isometric twitch response. E E D Q , like phenoxybenzamine, irreversibly inactivates a , - a d r e n o ceptors as well as other receptors such as dopamine receptors (see Adler et al., 19851. It might have been expected that N-ethyl-maleimide and irreversible ee,adrenoceptor antagonists would produce additive effects against responses to xylazine. This proved not to be the case. In further studies, the affinity of N-ethylmaleimide for c~:-adrenoceptors was investigated in
126
1. Browne et al. / Europea, Journal o1"Pharmacolo,~y 265 (1~)~)4)125 132
In experiments examining the actions of irreversible antagonists, tissues were exposed to phenoxybenzamine (1 p,M for 10 min), E E D Q (0.2 /,tM for 30 min) or N-ethyl-maleimide (30 or 100 /,tM for 60 min) before washing out for at least 60 min before beginning the main experiment. When combinations of drugs were administered, exposure to phenoxybenzamine or E E D Q was carried out before exposure to N-ethylmaleimide.
with [3H]noradrenaline-free Krebs-Henseleit solution at a rate of 0.33 m l / m i n . The Krebs-Henseleit solution had the composition stated above, with additionally corticosterone (30 p.M) and propranolot (1 p,M). Cocaine (3 # M ) was present after pre-incubation with [-~H]noradrenaline. In all experiments, tissues were stimulated with high KCI (70 raM) 3 times (Sc>-S~) for 3 min at intervals of 36 rain, beginning after 90 min of superfusion. Aftcr 90 min of superfusion the tissues were stimulated with high KC1 to remove excess counts (So). Effluent san> pies were collected in 1 ml aliquots beginning after 120 min of superfusion so that sampling began f~ rain before the control stimulation period S~. Antagonist drugs or an equiwdent w~lumc (1%) of distilled water (vehicle) were superfused in the Krebs-Hcnseleit solution beginning 24 min before S,. At the end of the experiment, tissues were made solublc in {].5 ml of tissue solubiliser (Soluene, Packard). A volumc of 1 ml superfusate or dissolved tissue was added to 4 ml of liquid scintillant (Ecoscint A) and the radioactivity was measured by liquid scintillation spectroscopy in a LKB 1214 RackBeta counter with on average 48% counting efficiency for tritium and automatic quench correction. The basal efflux of tritium was expressed as a percentage rate. i.e., the efflux of total tritiated compounds per min was expressed as a percentage of the tritium content of the tissue at the time of collection. To quantify the effects of a drug on the basal cfflux, the percentage ratc of efflux in the 3 rain before stimulation in thc presence of the drug (S~) was divided by the percentage rate of efflux in the 3 min betorc S I. No drug concentration employed in this study caused a significant incrcase in the basal cfflux (as compared with effects of vehicle). The stimulation-evokcd overflow of total tritium was calculated by subtraction of the basal overflow and was expressed as a percentage of the tritium content of the tissue at the onset of the respective stimulation periods (see Smith et al., 1992). To quantify the effects of a drug on stimulation-evoked overflow of tritium, the cvoked overflow in the presence of the drug (,S,) was divided by the overflow ew)ked by S~ and compared with the equivalent ratio obtained in control cxperiments without the test drug.
2.3. Radioactit~e ol:erflow experiments
2.4. Ligand binding studies
Rat renal cortex slices were used, 6 slices from each animal, at least one slice serving as a control. Isolated tissues were pre-incubated for 1 h in 1 ml of KrebsHenseleit medium containing [3H]noradrenaline (0.5 /,tM, specific activity 39 C i / m m o l ) , followed by incubation for 1 h in 1 ml of Krebs-Henseleit medium containing N-ethyl-maleimide or vehicle, before being placed in a Brande[ Superfusor and superfused at 37°C
Membrane binding was carried out by a method used by Connaughton and Docherty (1990), adapted from that of Neylon and Summers (1¢)85). Male rats were anaesthetised with ether and killed by cervical dislocation and exsanguination and the kidneys were dissected out. The renal capsule and medulla were dissected, discarded, and thc renal cortex was homogcnized and resuspended in 10 volumes of buffer, then
ligand binding studies employing the radioligand [~H]MK912, which has relatively high affinity for all subtypes of cee-adrenoceptor (see Uhlen et al., 1992).
2. Materials and methods
Male Wistar rats (200-300 g) were obtained from Trinity College Dublin, and renal cortex and vas deferens were employed as outlined below. 2.1. Rat ~'as defemns
Prostatic or epididymal portions of rat vas deferens were placed between platinum electrodes and attached to myograph transducers under 1 g tension in organ baths at 37°C in Krebs-Henseleit solution of the following composition: (mM): NaCI 119; NaHCO~ 25; l>glucose 11.1; KCI 4.7; CaCI, 2.5; K H 2 P O 4 1.2; MgSO 4 1.0; E D T A 0.03, ascorbic acid 0.28. Tissues were stimulated every 5 min with a single stimulus (0.5 ms pulses, supramaximal voltage) to produce isometric contractions, and, once consistent control responses had been obtained, a cumulative concentration-response curve was obtained to the o~2-adrenoceptor agonist xylazine, to the o~-adrenoceptor agonist amidephrine or to 5-hydroxytryptamine (5-HT) administered in 0.5 log unit increments beginning with 1 nM (xylazine) or 10 nM (amidephrine and 5-HT). In experiments employing epididymal portions, nifedipine ( 1 0 / , M ) was present to block the non-noradrenergic component of the twitch and to prevent postjunctional contractions mediated by c~-adrenoceptor agonists or 5-HT. 2.2. Exposure to irrecersible antagonists
I. Browne et al. / European Journal ~4#Pharmacology 265 (1904) 125 - 132
centrifuged at 40000 x g for 10 min at 4°C. The supernatant was discarded, the pellet resuspended in fresh buffer and the centrifugation step repeated. The pellet was resuspended in 5 volumes of incubation buffer (Tris-HC1 50 raM, E D T A 5 raM, pH 7.4 at 25°C). Homogenates were filtered through two layers of 43 T-mesh to remove connective tissue. Saturation and drug competition studies were conducted in 3 ml polypropylene tubes. In saturation experiments, 100 ml aliquots of membrane suspension were incubated with various concentrations of [~H]MK912 (specific activity: 82.9 C i / m m o l , NEN) at 25°C (0.(11-20 nM) in a final volume of 0.3 ml incubation buffer for 30 rain. In competition studies, 100 ml aliquots of membrane suspension and 100 ml aliquots of [3H]MK912 (final concentration 1 nM) were incubated with 100 ml aliquots of displacing ligands in concentrations from 0.1 nM to 100/~M in 0.5 log unit increments for 30 min. Specific binding was defined as that displaced by 10 # M phentolamine and represents approximately 90% of total binding in rat kidney cortex membranes. Assays were terminated by the addition of a 5 ml aliquot of ice-cold wash buffer followed by rapid vacuum filtration through Whatman G F / C glass filters using a Brandel Cell Harvester. This was followed by four 5 ml washes of the filters using the same buffer. Radioactivity retained on the filters was determined by liquid scintillation spectroscopy using a LKB 1214 RackBeta counter. Ligand 1Cs, values (i.e. concentration of drug producing 50% inhibition of displaceable [-~H]MK912 binding) were converted to the ligand inhibition constant K i using the Cheng-Prusoff (1973) equation: K i = ICs0/(l + L / K D ) w h e r e L and K D are the concentration and the equilibrium dissociation constant of the radioligand, respectively. K D and B ...... values were obtained from saturation experiments. Rat kidney cortex membranes were assayed for protein using the Coomassie Brilliant Blue G-250 method of Bradford (1976). Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 50 ml 95% ethanol. To this solution 100 ml phosphoric acid (85% w / v ) was added. The resulting solution was diluted to a final volume of 1 litre. Final concentrations in the reagent were: Coomassie Brilliant Blue G-250 (0.01% w/v), ethanol (4.7% w / v ) and phosphoric acid (8.5% w/v). Standard solutions of bovine serum albumin were analysed together with analytical samples. Absorbance was read at 595 nm in a SP6-500 UV Spectrophotometer (Pye Unlearn). A range of protein standards (10-100 rag) and 100 ml aliquots of analytical samples were added to 12 × 100 mm test tubes. All protein standards and analytical samples were made up to a final volume with the appropriate incubation buffer. 5 ml of protein reagent was added to each test tube and the absorbance at 595 nm was measured after 2 rain and
127
before 1 h in 3 ml cuvettes against a reagent blank prepared from 100 ml of the appropriate buffer and 5 ml of protein reagent. A standard curve was constructed and the protein content of the analytical samples was determined using this curve,
2.5. Drugs Amidephrine bitartrate (gift: Bristol-Myers); Nethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ: Aldrich, UK); 5-hydroxytryptamine hydrochloride (Research Biochemicals International, USA); [3H]MK912 ((2 S, 12 bS ) 1',3'-dimethylspiro-( 1,3,4,5', 6,6', 7,12 b-octahydro-2 H-benzo[ b ]furo[2,3-a ]quinazoline )-2,4'-pyrimidin-2'one: New England Nuclear) nifedipine (Sigma); N-ethyl-ma[eimide (Sigma); noradrenaline bitartrate (RBI); phenoxybenzamine hydrochloride (Sigma); xylazine hydrochloridc (gift: Bayer). Drugs were dissolved in distilled water, except for phenoxybenzamine (tartaric acid 1 raM)
2.6. Statistics Values are arithmetic mean _+ S.E.M. Agonist or antagonist pD 2, maximum inhibition, B .... or K~ values were compared with the effects of vehicle using a Student's t-test for unpaired or paired data, where appropriate, and by analysis of variance (ANOVAR).
3. Results
3.1. Rat renal cortex In rat renal cortex, the basal outflow of tritium before S~ was 0.113_+ 0.008%, 0.100_+ 0.007% and 0.125 +0.006% of tissue tritium/min, and KCI (70 raM) produced a stimulation evoked release of 0.70 _+ 0.11% (n = 22), 0.79 + 0.17% (n = 9) and 0.49_+ 0.07% (n = 17) of tissue tritium at SI in tissues pre-exposed to vehicle, N-ethyl-maleimide (10 /~M) and N-ethylmaleimide (30 #M), respectively (no significant differences).
In tissues pre-exposed to vehicle, xylazine (10 ~M) significantly reduced, and yohimbine (1 ~zM) significantly increased, the stimulation evoked release of tritium, but in tissues pre-exposed to N-ethyl-maleimide (30 p~M) xylazine did not significantly reduce evoked release of tritium (see Fig. 1). Pretreatment of the rat kidney cortex slices with N-ethyl-maleimide (10 ~ M ) failed to alter the inhibitory effect of xylazine: xylazine (10 # M ) significantly reduced the KCI stimulation evoked release of tritium at S 2 to 42 _+ 18% of Sj (n = 4) (not significantly different from effect of xylazine in vehicle experiments).
I. Browne et al. / European Journal of Pharmacolo~o' 265 (I994) 125-132
128
2oo ] Evoked overflow at $2 (% o t $1)
Table 2 Effects of the test agents phenoxyhcnzamme (PBA), N-ethyl-malcimide (NEM) and E E D Q on the potency and maximum effect of xyhtzinc at inhibiting isometric contraction to a single stimulus in prostatic and epididymal poritons of rat wts dcferens
7
~00
Testagcnt
0
.
.
.
.
.
con xyl yoh VEHICLE
.
.
Xylazincpl),
Maximum inhibition ( ' : )
7.34+1).00 6.67±0.(18 ~ 6.76+0.22h' t~.00 +l).0b; ' 0.8(1+0.12 ;~ 6.88+(t.13 ~
88.3+ 2.[ 48.5+ 3.9 " 57.3~ S.5' ,S3.3 *7 2.S 82.h+ I1.1 54.h+ 6 / ) '
6.55 +IL3h "
61L4+ 13.2 ~
7.57+0.12 7.09 + 0.14 "
t)3.(l+ 23) Sh.3+ 4.3
Prostatic portion
.
con xyl y o l l NEM (30/~M)
Fig. 1. Effects if the a2-adrenoceptor agonist xylazine (xyl)(10 /,M) and the ~2-adrenoceptor antagonist yohimbine (yoh) (1 # M ) on the KCI (70 raM) stimulation-evoked release of tritium in tissues pre-incubated with vehicle or N-ethyl-maleimide (30 /,M). Responses obtained in the presence of xylazine, yohimbine or distilled water vehicle (con) at S 2 were expressed as a percentage of responses obtained at S t in the absence of drug. Asterisks denote effects of drug significantly different from effects of vehicle (* P < 0.05, Student's t-test).
3.2. Prostatic portions o f rat ras deferens: nerremediated responses
In prostatic portions, single pulse electrical stimulation produced a contraction of 1.20 + 0.11 g (n = 26), consisting mainly of the first non-noradrenergic phase. Xylazine produced a concentration-dependent inhibition of the stimulation-evoked contraction, with a pD 2 ( - l o g ICs0) of 7.34 + 0.09, and a maximum inhibition of 88.3 _+ 2.1% (n = 32) (see Tables 1 and 2, and Fig. 2). Vehicle administration caused a small but significant increase in the contractile response to a single stimulus, which was presumably a time-dependent increase ( P < 0.05, paired t-test) (Table 1). Pre-exposure to N-ethyl-maleimide (100 p,M) but not N-ethylmaleimide (30/,tM) significantly reduced the isometric
Vehicle (n = 32) Phenoxybenzamine(l # M ) ( n = 8 ) EEDQ(0.2 #M)(n=8) NEM (30 # M ) ( n = 18) NEM (100/.tM) (n -= h) PBA(1 # M ) + N E M ( 3 ( I , t t M ) (n = g)
E E D Q (0.2 p,M)+ NEM (30 ~ M ) (/i - 5)
EpMid.vmal portion Vehicle (n = 6) NEM ( 3 0 / , M ) (n - ~)
Values are xylazine pD 2 ( - l o g l(',l~) and maximum inhibition of stimulation evoked contraction, mean + S.E.M.. with H, ll~e ntlmber of experiments. Responses significantly different from effects of vehicle (Student's t-test): a p < 0.05, ~' P < 0.(ll. c 1, < (1.0(}1.
contraction (Table 1). Other t e s t agents and combinations either produced no significant effect or increased the contractile response (Table I). Pre-exposure to N-ethyl-maleimide (30 or 100 ttM) significantly shifted the potency of xylazine without altering the maximum inhibitory response (Fig. 2 and Table 2), Pre-exposure to phenoxybenzamine (1 /,M) or EEDQ (0.2 /.tM) significantly shifted the potency of xylazinc and reduced the maximum inhibitory response (Figs. 3 and 4. and Table 2). Exposure to N-ethyl-maleimide (30 p,M) following pre-exposure to phenoxybenzamine or EEDQ produced no further effect than phenoxybenzamine or E E D Q alone (Figs. 3 and 4, and Table 2).
Table I Effects of the test agents phenoxybenzamine (PBA), N-ethyl-maleimide (NEM) and E E D Q on isometric contraction to a single stimulus in prostatic and epididymal portions of rat vas deferens Test agent
Control (g) Test (g)
Prostatic portion Vehicle (n = 26) Phenoxybenzamine (1 ~,M) (n = 6) E E D Q (0.2 /.tM) (n = 12) NEM (30 ~ M ) (n - 16) N E M (100/xM) (n = 5) PBA(I p,M)+NEM(30>M)(n 8) EEDQ(0.2/,tM)+NEM(30p~M)(n=7)
~-
1.20_+0.11 1.00_+ 0.17 1.54_+0.16 1.17.+0.11 1.22_+0.38 1.26+0.23 1.16+0.08
1.40+0.13 '' 1.24+0.15 2.18+0.14c 1.05-+0,19 0.38+0.10 t' 0.89-+(L12 1.46+0,19
0.80+0.10 1.28+0.17
1.32+0.14 c 0.82 +(I,10 c
Epididyrnal portion Vehicle (n = 19) NEM (30 # M ) (n - 18)
Values are isometric tension (g), mean + S.E.M.. with n, the number of experiments. Responses significantly different from control response (Student's t-test for paired data): a P < 0.05, h P < 0.01. " f < 0.001.
6o
u
"8
4o
veh l ~
~
~L~.
~
20
xylazine concentration
(log
M)
Fig. 2. Effects of the ~r2-adrenoceptor agonist xylazine fl~llowing pre-exposure to vehicle ( o ) or N-ethyl-maleimide (30/~M) (e) on the isometric contraction produced by single pulse electrical stimulation in prostatic portions of rat vas deferens. Responses in the presence of xylazine are expressed as a percenlage of Ihe response in lhe absence of xylazine. Vertical bars represent S.E.M. from at leasl IS experiments.
10c
L Browne et al./European Journal of Pharmacolo,~,~, 205 (19941 125-132
80 PBA
'~
4o
4o
N 2O
0 -9
129
2C -'8 -'7 -'6 xylazine concentration (log M)
-'5
Fig. 3. Effects of the c~2-adrenoceptor agonist xylazine following pre-exposure to vehicle (o), phenoxybenzamine (1 #M) (1~) or phenoxybenzamine (1 /,~M) followed by N-ethyl-maleimide (30 #M) (el on the isometric contraction produced by single pulse electrical stimulation in prostatic portions of rat vas deferens. Responses in the presence of xylazine are expressed as a percentage of the response in the absence of xylazine. Vertical bars represent S.E.M. from at least 6 experiments.
3.3. Epididymal portions of rat uas deferens: nervemediated responses In epididymal portions of rat vas deferens, the initial biphasic twitch to a single electrical stimulus was reduced to a monophasic response by nifedipine (10 /zM), which eliminates the first (non-noradrenergic) phase, leaving the second (noradrenergic) phase (see Brown et al., 1983). Under these conditions, the twitch to a single stimulus was 0 . 8 0 _ 0.10 g (n = 19), and xylazine inhibited this response with a pD 2 of 7.57 _+ 0.12 and a maximum inhibition of 93.0 _+ 2.9% (n -- 6). Vehicle significantly increased the contractile response to a single stimulus, whereas N-ethyl-maleimide (30 p,M) significantly reduced the response (Table 1). Preexposure to N-ethyl-maleimide (30 /~M) significantly shifted the potency of xylazine without altering the maximum inhibition (see Fig. 5 and Table 2). In vehicle experiments, amidephrine inhibited the
xylazine concentration (log M)
Fig. 5. Effects of the ~2-adrenoceptor agonist ~lazine on isometric contractions produced by single pulse electrical stimulation in epididymal portions of rat vas deferens in the presence of nifedipine (10 ~M). Symbols: vehicle pre-treated (©), N-ethyl-maleimide (30 #M) pretreated (el. Vertical bars represent S.E.M. from at least 4 experiments.
contractile response to a single stimulus with a pD 2 of 6.60 _+ 0.08 and a maximum inhibition of 87.5 _+ 2.2% (n = 7), and the amidephrine pD 2 and maximum inhibition were not significantly altered by pre-exposure to N-ethyl-maleimide (30 p,M) (6.75 _+ (I.12 and 86.6 _+ 1.4%, respectively) (see Fig. 6). Pre-exposure to N-ethyl-maleimidc (30 p,M) significantly reduced the inhibitory potency of 5-HT (5-HT pD2: vehicle 5.76_+ 0.19, n = 11; N-ethyl-maleimide 5.25 _+ 0.21, n = 11; P < 0.01, Student's t-test for paired data) without significantly altering the maximum inhibition by 5-HT (vehicle 87.5 _+ 1.7%; N-ethyl-maleimide 82.4 _+ 2.9%) (see Fig. 7). 3.4. Ligand binding studies: rat renal cortex In rat renal cortex membranes, analysis indicated that [-~H]MK912 bound to a single population of a 2adrenoceptors with a K o of 5.73 + 1.64 nM and a B ...... of 854 ___191 f m o l / m g protein (n = 5). In tissues preexposed to N-ethyl-maleimide (300 /~M), the B ..... of MK912 was significantly reduced ( P < 0.05) to 313 _+ 32
1 {:(:80
100 60 80 60 2C
~
h NEM
40 20 xylazine concentration (log M) 0 9
Fig. 4. Effects of the ~2-adrenoceptor agonist xylazine following pre-exposure to vehicle (o), EEDQ (0.2 #M) ([]) or EEDQ (0.2 /zM) followed by N-ethyl-maleimide (30 p,M) (e) on the isometric contraction produced by single pulse electrical stimulation in prostatic portions of rat vas deferens. Responses in the presence of xylazine are expressed as a percentage of the response in the absence of xylazine. Vertical bars represent S.E.M. from at least 7 experiments.
-'8 "7 -'6 amidephrine concentration 009 M)
~5
Fig, 6. Effects of the ~1-adrenoceptor agonist amidephrine on isometric contractions produced by single pulse electrical stimulation in epididymal portions of rat vas deferens in the presence of nifedipine (10 /~M). Symbols: vehicle pro-treated (o); N-ethyl-maleimide (30 /zM) pretreated (e). Vertical bars represent S.E.M. from at least 4 experiments,
I. Browne et al. / European Journal of PharmacolojG 265 ~1994) 125-132
130 1 oo
u
vehI
6O
~
2o o -8
-;
6
5
5-hydroxytryptamine concentration
-74
(logM)
Fig. 7. Effects of 5-HT on isometric contractions produced by single pulse elcclrical stimulation in epididymal portions of rat vas deferens in the presence of nifcdipine (10 # M). Symbols: vehicle pro-treated (~); N-ethyl-maleimide(31)btM) pretrcated (o). Vertical bars represent S.EM. from at least 4 experiments.
f m o l / m g protein (n = 5) with no change in K D (3.91 _+ 0.26 nM), as compared to the effects of incubation with vehicle. In displacement studies, yohimbine and N-ethylmaleimide had K~ values of 80.8 + 10.7 nM in = 3) and 466 _+ 133 >M (n = 5), respectively.
4. Discussion
The effects of N-ethyl-maleimide on oc~-adrenoceptor mediated responses have been examined in a number of tissues. Before discussing the overall implications of these results, responses obtained in each tissue will be discussed separately. 4.1. Radioactit'e overflow experiments in rat renal corter
In rat renal cortex slices pre-incubated with [3H]noradrenaline, KCI (70 raM) evoked release of tritium was taken as a measure of transmitter release. Stimulation-evoked release of tritium was significantly reduced by xylazine (10 /xM) and significantly increased by yohimbine (1 # M ) i n veh~icle pre-incubated tissues, but in tissues incubated with N-ethyl-ma[eimide (30 /xM) the inhibitory effects of xylazine were abolished. These results are consistent with previous findings in the literature (see Introduction) and can be interpreted in terms of inactivation of G-proteins (or indeed in terms of c~,-adrenoceptor antagonism, but see below). The fact that the effects of xylazine were abolished by N-ethyl-maleimide whereas the effects of yohimbine were not significantly reduced by N-ethyl-maleimide may bc explained by the fact that xylazine, being a synthetic agonist, may act as a partial agonist under these experimental conditions. In contrast, yohimbine increases the stimulation-ew)ked release by blocking the actions of the endogenous agonist noradrenaline, and noradrenalinc acts as a full agonist. A partial
agonist may produce less efficient coupling of rcceptor to G-protein so that its actions may be more critically dependent on the availability of G-protein. Although N-ethyl-maleimidc might have been expected to increase stimulation-evoked release by its presumed action to uncouple G-proteins involved in ~c~-adrenoccptot" mediated inhibition. N-ethyl-malcimidc (30 # M ) actually tended to decrease stimulation-ew~kcd release of noradrenaline. However, this effect of N-cthylmaleimide has been previously reported (Murphy ct al., 1992) and may represent a non-specific action of N-cthyl-malcimide. 4.2. L ~ a n d binding studies
Ligand binding studies were carried out using [-~H]MK912, a selective c~_-adrenoccptor antagonist (Uhlen ct al., 1992), in rat renal cortex membrancs which contain c~2B-adrenoceptors (see Smith and Docherty, 1992), although a2a-adrenoceptors have also been reportcd (Uhlcn et al., 1992). The prcscnt results fitted best a one-site model: ae~cadrenoceptors (see Smith and Docherty, 1992). In displacement studies, N-ethyl-maleimidc displaccd binding of [3H]MKgI2 with a K~ of 466 p.M, a concentration much higher than that used in most functional studies of the interaction of N-ethyl-maleimide with G-proteins (present results: 30 /xM; Foucart et al., 1990:3 ~M; Kaschube and Brasch, 1990:60 p.M; Murphy el al., 1992:3 # M : Rump ct al., 1992:10 /xM). In saturation studies, N-ethy[-maleimide (300 # M ) pretreatment produced approximately a 6(1~ reduction in the maximum nttmbet of binding sites for MK912. Other authors have reported that N-ethyl-maleimide (100-101)0 # M ) reduced the maximum binding of the c~2-adrcnoccptor ligand [3H]UK 143(14 (Garcia-Sevilla ct a[, 1988). The receptor examined in ligand binding studics was an e~2t~-adrenoceptor, whereas the prejunctiona[ reccptor in rat vas deferens is probably an {C,D-subtypc as found in the rat submandibular gland (sec Smith and Docherty, 1992). However, we also have limited data from ligand binding studies of the rat submandibular gland in which N-ethyl-maleimide shows similar low affinity in displacement studics (authors, unpublished observations). Hence, wc have no reason to think that N-ethyl-malcimide has high affinity for a subtype of Q,-adrenoccptor. 4.3. Electrical stimulation-eroked isometric contracuons in rat vas deferens
In rat whole vas deferens, the electrical stimulation-evoked contraction to a single stimulus consists of a biphasic response, the first phase of which is nonadrenoceptor mediated (probably ATP) and predominates in prostatic portions, and the second phase of
1. Browne et al. / European Journal
which is al-adrenoceptor mediated, and predominates in the epididymal portion (see Brown et al., 1983; Aboud et al., 1993). The second oq-noradrenergic phase can be examined in isolation in the epididymal portion in the presence of nifedipine which eliminates the first non-noradrenergic response, and also prevents postjunctional contractile actions of exogenous agonists at oei-adrenoceptors. In prostatic portions, the twitch response to a single stimulus is mainly nonadrenergic, and can be inhibited by o~2-adrenoceptor agonists by a prejunctional action, whereas ~ r a d r e n o c e p t o r agonists increase stimulation-evoked contractions by a postjunctional action (see Brown et al., 1983). N-Ethyl-maleimide (30 # M for 60 min) reduced the potency of xylazine by a factor of 3-5 in both prostatic and epididymal portions, and reduced the potency of 5-HT in epididymal portions, although it failed to shift the potency of the al-adrenoceptor agonist amidephrine in epididymal portions. N-Ethyl-maleimide (100 # M ) produced no further effect against the inhibition by xylazine. This action of N-ethyl-maleimide to reduce the potency of xylazine and 5-HT may be by inactivation of G-proteins involved in prejunctional o~2-adrenoceptor and 5 - H T r r e c e p t o r mediated responses (see Introduction). In previous studies of rat vas deferens, pertussis toxin failed to modulate the a2-adrenoceptor (Docherty, 1988) or 5-HTl-receptor (Borton and Docherty, 1990) mediated inhibition. Hence, these responses can be described as N-ethylmaleimide-sensitive, pertussis toxin-insensitive, and this may reflect the sensitivity of a G-protein involved (in contrast, note that prejunctional o~2-adrenoceptor mediated responses in rabbit hippocampus are N-ethylmaleimide-sensitive and pertussis toxin-sensitive: Allgaier et al., 1985, 1986). However, assuming that Nethyl-maleimide acts at a G-protein, the fact that Nethyl-maleimide shifted the potency of xylazine and 5-HT without reducing the maximum inhibition might suggest that there is spare G-protein capacity allowing xylazine and 5-HT to produce a maximum effect even though a proportion of the G-proteins have been inactivated. It can be demonstrated theoretically that the concentration of G-proteins will affect apparent affinity of the agonist for the receptor (MacKay, 1990). If this were the case, reduction in the number of ~eadrenoceptors by irreversible alkylation with phenoxybenzamine or E E D Q should allow further effects of N-ethyl-maleimide. Phenoxybenzamine (1 >M) and E E D Q (0.2 p,M) reduced the maximum inhibition by xylazine as well as shifting the potency of xylazine, suggesting that no spare receptors for xylazine remained after this pre-treatment. However, N-ethylmaleimide was not additive with phenoxybcnzamine or EEDQ: the combination produced no further effect than phenoxybenzamine or E E D Q alone. Although N-ethyl-maleimide acts as an irreversible oe,-adreno-
of Pharmacolog~v265
(1994) 125-132
131
ceptor antagonist, this action only occurs at concentrations of 100-300 /,tM and above in ligand binding studies. Hence, the fact that N-ethyl-maleimide was not additive with phenoxybenzamine and E E D Q suggests that, either N-ethyl-maleimide produces nonspecific effects, as has been reported in terms of signal transduction in mouse atria (Foucart et al., 1990), or that phenoxybenzamine and E E D Q have similar actions to N-ethyl-maleimide in addition to o~-adrenoceptor antagonism. Another possibility is that there are two populations of receptors, one of which is linked to an N-ethyl-maleimide-sensitive G-protein and is selectively inactivated by phenoxybenzamine and EEDQ, but we have no evidence to support this: the prejunctional receptors of rat vas deferens appear to bc a homogeneous population of oem-adrenoceptors (see Smith and Docherty, 1992). The shift in the prejunctional potency of xylazine produced by phenoxybenzaminc and E E D Q may not be indicative of the presence of spare receptors, but rather be due to inactivation of G-proteins. This would be consistent with the fact that, in vehicle experiments, xylazine produces an approximately 95% inhibition of the stimulation-evoked contraction but does not totally abolish the contraction. If the maximum effect of xylazine involved activation of most of the prejunctional receptors, then an irreversible antagonist should reduce the maximum inhibition by xylazinc without affecting the potency. The fact that phenoxybcnzamine and E E D Q reduced the potency and the maximum inhibition by xylazine implies either spare receptors or additional actions of these agents, such as G-protein inactivation. Any additional actions of phenoxybenzamine might affect interpretation of agonist affinity calculations from partial receptor inactivation studies (see Docherty, 1989). Although N-ethyl-maleimide did not significantly affect the inhibitory potency of the c~-adrenoceptor agonist amidephrine against isometric contractions of epididymal portions in the presence of nifedipine, it has not been fully established that this action of amidephrine is prejunctional (see Docherty, 1989). If the actions of amidephrine are indeed prejunctional, we have evidence for a prejunctional inhibitory response involving a G-protein insensitvc to N-ethyl-maleimide, in agreement with the findings of Rump ct al. (1992). In summary, N-ethyl-maleimide is not an a~-adrenoceptor antagonist in the range of concentrations at which it prevents the inhibitory action of xylazine on nerve stimulation evoked responses. However, the lack of interaction between N-ethyl-maleimidc and phcnoxybenzamine or E E D Q is not consistent with the assumed actions of these agents. It must be concluded that either N-ethyl-maleimidc acts non-specifically, or that it has similar actions to phenoxybenzamine or EEDQ, or vice versa. This may suggest that phenoxy-
1. Browne et al. / European Journal (~]"Pharmacolo,e,y 265 (19941 125-132
132
benzamine and EEDQ have additional actions to inactivate G-proteins.
Acknowledgements Supported by The Health Research Board (Ireland), the Irish Heart Foundation and Royal College of Surgeons in Ireland.
References Aboud, R.W., M. Shafii and J.R. Doeherty, 1993, Investigations ol the subtypes of ¢tl-adrenoceptor mediating contractions of rat aorta, vas deferens and spleen, Br. J. Pharmacol. 1(}9, 80. Adler, C.t[., E. Meller and M. Goldstein, 1985, Recovery of ~,adrenoceptor binding and function after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), Eur. J, Pharmacol. 116, 175. Allgaicr, C., T.J. Feuerstein, R. Jackisch and G. Hertting, 1985, Islet-activating protein (perlussis toxin) diminishes c~e-adrenoce ptor mediated effects on noradrenaline release, Naunyn-Schmied. Arch. Pharmacol. 331, 235. Allgaier, C,, T.J. Feuerstein and G. Hertting, 1986, N-Ethyl-maleimide (NEM) diminishes er2-adrenoceptor mediated effects on noradrenaline release, Naunyn-Schmied. Arch. Pharmacol. 333, 104.
Borton, M. and J.R. Docherty, 1990, The effects of ageing on prejunctional 5-hydroxytryptamine receptors in the rat vas deferens, Naunyn-Schmied. Arch. Pharmacol. 342, 130. Bradford, M.M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248. Brown, D.A., J.R. Docherly. A.M. French, A. MacDonald, J.C. McGrath and N.C. Scott, 1983, Separation of noradrenergic and non-norudrenergic contractions to field stimulation in the rat vas deferens, Br. J. Pharmacol. 79, 379. ('heng, Y. and W.H. Prusoff, 1973, Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 5()(~: inhibition (1(-%,u) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Connaughton, S. and J.R. Docherly, 1990, Functional evidence for heterogeneity of peripheral prejunctional n'2-adrenoceptors, Br. J. Pharmacol. 101, 285. Docherty, J.R., 1988, Pertussis toxin and prejunctional o~-adrenoceptors in rat heart and w~s deferens, J. Autonom. Pharmacol. 8, 1 g7.
Docherty, J.R., 1989, The pharmacology of ~l- and ¢~2-adrenoceptors: evidence for and against a further subdivision. Pharmacol. "Fher. 44, 241. Foucart, S., T.V. Murphy and 11. Majewski, 1990, Prejunctional /4-adrenoceptors, angiotensin 11 and neuropeptide Y receptors on sympathetic nerves in mouse atria arc linked to N ethylmaleimidc-susceptible G-proteins, J. Auton. N e t < Sysl. 30. 221. Garcia-Sevilla, J.A., P. Areso, M.T. Giralt and 1. Uliharri, 1988, Uncoupling of the platelet c~e-adrenoceptor adenylatc cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity+ Eur. J. Pharmacol. 145, 91. Kaschubc, M. and It. Brasch, 1990+ Pre+ and postjunctional effects o1 N-ethylmalcirnide in the isolated mouse vas deferens, ['~ur. J. Pharmacol. 178, 151. Kitamura, Y. and Y. Nomura, 1987, Uncoupling of rat cerebra[ cortical ~+2-adrenoceptors from GTP-binding proteins by N-ethylmaleimide, J. Neurochem. 49. 1894. MacKay, L)., 1990, Interpretation of relative potencies, relalive efficacies and apparent affinity constants of agonist drugs cstimated lrom concentration-response curves, J. Theor. Biol. 142, 415+ Murphy, T.V., S. Foucart and It. Majewski, 1992, Preiurlctional ~r2-adrenoceptors in mouse atria function through (;-proteins which are sensitive to N-ethylmaleimidc, but not pertussis toxin, Br. J. Pharmacol. 106, 871. Neylon, C.B. and R.J. Summers, 1985, [~H]-Rauwolscine binding to er2-adrenoceptors in the mammalian kidney: apparent receptor heterogeneity between species. Br. J. Pharmacol. 85, 349. Rump, L.C., V. Volk, G. Ruff and P. Schollmeyer, 1992, Activation of ~r 1- and ¢~2-adrenoceptors inhibits noradrenaline release in rabbit renal arteries: effects of pertussis toxin and N-ethylmaleimide+ J. Auton. Pharmacol. 12, 97. Smith, K. and J.R. l)ocherty, 1992, Arc the prcjunctional ¢~-a¢lren oceptors of the rat vas deferens and submandibular gland of the ~2,\- or er2i)-subtypc?, Eur. J. Pharmacol. 219, 203. Smith, K., S, ('onnaughton and J.R. Docherty, 1992, Investigations o] prejunctional ~r2-adrenoceptors in rat atrium, wls deferens and submandibular gland, Eur. J. Pharmacol. 211, 251. Ueda, 1t., If. Misawa, T. Katada, M. ill, 1t. Takagi and M. Satoh, 1990, Functional reconstitution of purified G i and G , with # opioid receptors in guinea-pig striatal m e m b r a n e s pretrealcd with micromolar concentrations of N-cthylnvaleimide, J. Neurochem. 54, 84 I. Uhlcn, S., Y. Xia, V. Chhajlani, (2('. [:elder and J.E.S. Wikberg, 1902, [ ~[ t]-M K 912 binding delineates two ~ 2-adrenoceptor subtypes in rat CNS one of which is identical with the cloned pA2d ~2-adrenoceptor, Br. J. Pharmacol. 1(16, 986. Weber, H.D., 1989, Presynaptic (r2-adrenoccptors at the terminals of perivascular sympathetic nerves arc coupled to an N-ethylmaleimide-sensitivc G-protein. Naunyn-Schmied. Arch. Pharmacol. 339, R35.
European Journal of Pharmacology 265 (1994) 125 132
Prejunctional actions of N-ethyl-maleimide and phenoxybenzamine in rat vas deferens Ingrid Browne, George Thomas, Katherine Gavin, James R. D o c h e r t y * Department of Physiolo,w, Royal College of Surgeons in Irehmd, 123 St. Stephen's Greeen, Dublin 2, Ireland Received 23 June 1994; revised MS received 17 August 1994; accepted 19 August 1994
Abstract
In studies of electrically evoked isometric contractions of rat vas deferens, N-ethyl-maleimide (30 #M) prctrcatment significantly reduced the prejunctional inhibitory potencies of xylazine and 5-hydroxytryptamine but failed to affect the potency of the a t-adrenoceptor agonist amidephrine. Phenoxybenzamine (1 ~M) or N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ) (10 #M) produced significant shifts in the potency of xylazine and significantly reduced the maximum inhibition, but the combination of phenoxybenzamine or EEDQ and N-ethyl-maleimide (30 ~tM) produced no further alteration in the effects of xylazine. In displacement studies, N-ethyl-maleimide displaced the binding of [3H]MK 912 ((2S,12bS)l',3'-dimethylspiro(~3A~5'~6~6'~7~2b-~ctahydr~-2H-benz~[b]fur~[2~3-a]quinaz~ine)-2~4'-pyrimidin-2'~ne) to rat renal cortex membranes with a K i of 466 _+ 133 #M (n = 5), and so does not bind to ae-adrenoceptors in the concentration range in which it affects prejunctional receptor mediated responses. This may suggest that N-ethyl-maleimide has actions other than inactivation of G-proteins or that the irreversible ae-adrenoceptor antagonists phenoxybenzamine and EEDQ inactivate G-proteins sensitive to N-ethyl-maleimide in concentrations at which they bind to ae-adrenoceptors. Keywords: Vas deferens, rat; az-Adrenoceptor; N-Ethyl-maleimide; Phenoxybenzamine; EEDQ (N-ethoxycarbonyl-2-cthoxy1,2-dihydroquinoline)
1. Introduction
N-Ethylmaleimide alkylates proteins and has been demonstrated to inactivate certain G-proteins (Ueda et al., 19901, and to attenuate prejunctional cQ-adrenoceptor mediated inhibition of neurotransmitter release in a number of tissues including rat cerebral cortex (Kitamura and Nomura, 1987), mouse vas deferens (Kaschube and Brasch, 1990), rat tail artery (Weber, 1989), rabbit renal arteries ( R u m p et al., 19921 and mouse atria (Murphy et al., 1992). Furthermore, R u m p et al. (1992) have shown that N-ethyl-maleimide attenuates a 2- but not a l - a d r e n o c e p t o r mediated inhibition of noradrenaline release in rabbit renal arteries. The present study began with an investigation of the interaction between N-ethyl-maleimide and prejunc-
* Corresponding author. Tel. (353) 1-4780 20(1, fax (353) 1-4780
018. 0014-2999/94/$(17.(1(I ~ 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 5 1 9 - 2
tional a2-adrenoceptors in rat kidney cortex which produced results consistent with previous reports that N-ethyl-maleimide may inactivate a G-protein involved in ae-adrenoceptor mediated inhibition. We investigated further the o~2-adrenoceptor antagonist actions of N-ethyl-maleimide in a system in which interaction between N-ethyl-maleimide and the irreversible a tadrenoceptor antagonists phenoxybenzamine and Nethoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline ( E E D Q ) could be examined under controlled conditions: inhibition by the oe2-adrenoceptor agonist xylazine of the rat vas dcferens isometric twitch response. E E D Q , like phenoxybenzamine, irreversibly inactivates a , - a d r e n o ceptors as well as other receptors such as dopamine receptors (see Adler et al., 19851. It might have been expected that N-ethyl-maleimide and irreversible ee,adrenoceptor antagonists would produce additive effects against responses to xylazine. This proved not to be the case. In further studies, the affinity of N-ethylmaleimide for c~:-adrenoceptors was investigated in
126
1. Browne et al. / Europea, Journal o1"Pharmacolo,~y 265 (1~)~)4)125 132
In experiments examining the actions of irreversible antagonists, tissues were exposed to phenoxybenzamine (1 p,M for 10 min), E E D Q (0.2 /,tM for 30 min) or N-ethyl-maleimide (30 or 100 /,tM for 60 min) before washing out for at least 60 min before beginning the main experiment. When combinations of drugs were administered, exposure to phenoxybenzamine or E E D Q was carried out before exposure to N-ethylmaleimide.
with [3H]noradrenaline-free Krebs-Henseleit solution at a rate of 0.33 m l / m i n . The Krebs-Henseleit solution had the composition stated above, with additionally corticosterone (30 p.M) and propranolot (1 p,M). Cocaine (3 # M ) was present after pre-incubation with [-~H]noradrenaline. In all experiments, tissues were stimulated with high KCI (70 raM) 3 times (Sc>-S~) for 3 min at intervals of 36 rain, beginning after 90 min of superfusion. Aftcr 90 min of superfusion the tissues were stimulated with high KC1 to remove excess counts (So). Effluent san> pies were collected in 1 ml aliquots beginning after 120 min of superfusion so that sampling began f~ rain before the control stimulation period S~. Antagonist drugs or an equiwdent w~lumc (1%) of distilled water (vehicle) were superfused in the Krebs-Hcnseleit solution beginning 24 min before S,. At the end of the experiment, tissues were made solublc in {].5 ml of tissue solubiliser (Soluene, Packard). A volumc of 1 ml superfusate or dissolved tissue was added to 4 ml of liquid scintillant (Ecoscint A) and the radioactivity was measured by liquid scintillation spectroscopy in a LKB 1214 RackBeta counter with on average 48% counting efficiency for tritium and automatic quench correction. The basal efflux of tritium was expressed as a percentage rate. i.e., the efflux of total tritiated compounds per min was expressed as a percentage of the tritium content of the tissue at the time of collection. To quantify the effects of a drug on the basal cfflux, the percentage ratc of efflux in the 3 rain before stimulation in thc presence of the drug (S~) was divided by the percentage rate of efflux in the 3 min betorc S I. No drug concentration employed in this study caused a significant incrcase in the basal cfflux (as compared with effects of vehicle). The stimulation-evokcd overflow of total tritium was calculated by subtraction of the basal overflow and was expressed as a percentage of the tritium content of the tissue at the onset of the respective stimulation periods (see Smith et al., 1992). To quantify the effects of a drug on stimulation-evoked overflow of tritium, the cvoked overflow in the presence of the drug (,S,) was divided by the overflow ew)ked by S~ and compared with the equivalent ratio obtained in control cxperiments without the test drug.
2.3. Radioactit~e ol:erflow experiments
2.4. Ligand binding studies
Rat renal cortex slices were used, 6 slices from each animal, at least one slice serving as a control. Isolated tissues were pre-incubated for 1 h in 1 ml of KrebsHenseleit medium containing [3H]noradrenaline (0.5 /,tM, specific activity 39 C i / m m o l ) , followed by incubation for 1 h in 1 ml of Krebs-Henseleit medium containing N-ethyl-maleimide or vehicle, before being placed in a Brande[ Superfusor and superfused at 37°C
Membrane binding was carried out by a method used by Connaughton and Docherty (1990), adapted from that of Neylon and Summers (1¢)85). Male rats were anaesthetised with ether and killed by cervical dislocation and exsanguination and the kidneys were dissected out. The renal capsule and medulla were dissected, discarded, and thc renal cortex was homogcnized and resuspended in 10 volumes of buffer, then
ligand binding studies employing the radioligand [~H]MK912, which has relatively high affinity for all subtypes of cee-adrenoceptor (see Uhlen et al., 1992).
2. Materials and methods
Male Wistar rats (200-300 g) were obtained from Trinity College Dublin, and renal cortex and vas deferens were employed as outlined below. 2.1. Rat ~'as defemns
Prostatic or epididymal portions of rat vas deferens were placed between platinum electrodes and attached to myograph transducers under 1 g tension in organ baths at 37°C in Krebs-Henseleit solution of the following composition: (mM): NaCI 119; NaHCO~ 25; l>glucose 11.1; KCI 4.7; CaCI, 2.5; K H 2 P O 4 1.2; MgSO 4 1.0; E D T A 0.03, ascorbic acid 0.28. Tissues were stimulated every 5 min with a single stimulus (0.5 ms pulses, supramaximal voltage) to produce isometric contractions, and, once consistent control responses had been obtained, a cumulative concentration-response curve was obtained to the o~2-adrenoceptor agonist xylazine, to the o~-adrenoceptor agonist amidephrine or to 5-hydroxytryptamine (5-HT) administered in 0.5 log unit increments beginning with 1 nM (xylazine) or 10 nM (amidephrine and 5-HT). In experiments employing epididymal portions, nifedipine ( 1 0 / , M ) was present to block the non-noradrenergic component of the twitch and to prevent postjunctional contractions mediated by c~-adrenoceptor agonists or 5-HT. 2.2. Exposure to irrecersible antagonists
I. Browne et al. / European Journal ~4#Pharmacology 265 (1904) 125 - 132
centrifuged at 40000 x g for 10 min at 4°C. The supernatant was discarded, the pellet resuspended in fresh buffer and the centrifugation step repeated. The pellet was resuspended in 5 volumes of incubation buffer (Tris-HC1 50 raM, E D T A 5 raM, pH 7.4 at 25°C). Homogenates were filtered through two layers of 43 T-mesh to remove connective tissue. Saturation and drug competition studies were conducted in 3 ml polypropylene tubes. In saturation experiments, 100 ml aliquots of membrane suspension were incubated with various concentrations of [~H]MK912 (specific activity: 82.9 C i / m m o l , NEN) at 25°C (0.(11-20 nM) in a final volume of 0.3 ml incubation buffer for 30 rain. In competition studies, 100 ml aliquots of membrane suspension and 100 ml aliquots of [3H]MK912 (final concentration 1 nM) were incubated with 100 ml aliquots of displacing ligands in concentrations from 0.1 nM to 100/~M in 0.5 log unit increments for 30 min. Specific binding was defined as that displaced by 10 # M phentolamine and represents approximately 90% of total binding in rat kidney cortex membranes. Assays were terminated by the addition of a 5 ml aliquot of ice-cold wash buffer followed by rapid vacuum filtration through Whatman G F / C glass filters using a Brandel Cell Harvester. This was followed by four 5 ml washes of the filters using the same buffer. Radioactivity retained on the filters was determined by liquid scintillation spectroscopy using a LKB 1214 RackBeta counter. Ligand 1Cs, values (i.e. concentration of drug producing 50% inhibition of displaceable [-~H]MK912 binding) were converted to the ligand inhibition constant K i using the Cheng-Prusoff (1973) equation: K i = ICs0/(l + L / K D ) w h e r e L and K D are the concentration and the equilibrium dissociation constant of the radioligand, respectively. K D and B ...... values were obtained from saturation experiments. Rat kidney cortex membranes were assayed for protein using the Coomassie Brilliant Blue G-250 method of Bradford (1976). Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 50 ml 95% ethanol. To this solution 100 ml phosphoric acid (85% w / v ) was added. The resulting solution was diluted to a final volume of 1 litre. Final concentrations in the reagent were: Coomassie Brilliant Blue G-250 (0.01% w/v), ethanol (4.7% w / v ) and phosphoric acid (8.5% w/v). Standard solutions of bovine serum albumin were analysed together with analytical samples. Absorbance was read at 595 nm in a SP6-500 UV Spectrophotometer (Pye Unlearn). A range of protein standards (10-100 rag) and 100 ml aliquots of analytical samples were added to 12 × 100 mm test tubes. All protein standards and analytical samples were made up to a final volume with the appropriate incubation buffer. 5 ml of protein reagent was added to each test tube and the absorbance at 595 nm was measured after 2 rain and
127
before 1 h in 3 ml cuvettes against a reagent blank prepared from 100 ml of the appropriate buffer and 5 ml of protein reagent. A standard curve was constructed and the protein content of the analytical samples was determined using this curve,
2.5. Drugs Amidephrine bitartrate (gift: Bristol-Myers); Nethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ: Aldrich, UK); 5-hydroxytryptamine hydrochloride (Research Biochemicals International, USA); [3H]MK912 ((2 S, 12 bS ) 1',3'-dimethylspiro-( 1,3,4,5', 6,6', 7,12 b-octahydro-2 H-benzo[ b ]furo[2,3-a ]quinazoline )-2,4'-pyrimidin-2'one: New England Nuclear) nifedipine (Sigma); N-ethyl-ma[eimide (Sigma); noradrenaline bitartrate (RBI); phenoxybenzamine hydrochloride (Sigma); xylazine hydrochloridc (gift: Bayer). Drugs were dissolved in distilled water, except for phenoxybenzamine (tartaric acid 1 raM)
2.6. Statistics Values are arithmetic mean _+ S.E.M. Agonist or antagonist pD 2, maximum inhibition, B .... or K~ values were compared with the effects of vehicle using a Student's t-test for unpaired or paired data, where appropriate, and by analysis of variance (ANOVAR).
3. Results
3.1. Rat renal cortex In rat renal cortex, the basal outflow of tritium before S~ was 0.113_+ 0.008%, 0.100_+ 0.007% and 0.125 +0.006% of tissue tritium/min, and KCI (70 raM) produced a stimulation evoked release of 0.70 _+ 0.11% (n = 22), 0.79 + 0.17% (n = 9) and 0.49_+ 0.07% (n = 17) of tissue tritium at SI in tissues pre-exposed to vehicle, N-ethyl-maleimide (10 /~M) and N-ethylmaleimide (30 #M), respectively (no significant differences).
In tissues pre-exposed to vehicle, xylazine (10 ~M) significantly reduced, and yohimbine (1 ~zM) significantly increased, the stimulation evoked release of tritium, but in tissues pre-exposed to N-ethyl-maleimide (30 p~M) xylazine did not significantly reduce evoked release of tritium (see Fig. 1). Pretreatment of the rat kidney cortex slices with N-ethyl-maleimide (10 ~ M ) failed to alter the inhibitory effect of xylazine: xylazine (10 # M ) significantly reduced the KCI stimulation evoked release of tritium at S 2 to 42 _+ 18% of Sj (n = 4) (not significantly different from effect of xylazine in vehicle experiments).
I. Browne et al. / European Journal of Pharmacolo~o' 265 (I994) 125-132
128
2oo ] Evoked overflow at $2 (% o t $1)
Table 2 Effects of the test agents phenoxyhcnzamme (PBA), N-ethyl-malcimide (NEM) and E E D Q on the potency and maximum effect of xyhtzinc at inhibiting isometric contraction to a single stimulus in prostatic and epididymal poritons of rat wts dcferens
7
~00
Testagcnt
0
.
.
.
.
.
con xyl yoh VEHICLE
.
.
Xylazincpl),
Maximum inhibition ( ' : )
7.34+1).00 6.67±0.(18 ~ 6.76+0.22h' t~.00 +l).0b; ' 0.8(1+0.12 ;~ 6.88+(t.13 ~
88.3+ 2.[ 48.5+ 3.9 " 57.3~ S.5' ,S3.3 *7 2.S 82.h+ I1.1 54.h+ 6 / ) '
6.55 +IL3h "
61L4+ 13.2 ~
7.57+0.12 7.09 + 0.14 "
t)3.(l+ 23) Sh.3+ 4.3
Prostatic portion
.
con xyl y o l l NEM (30/~M)
Fig. 1. Effects if the a2-adrenoceptor agonist xylazine (xyl)(10 /,M) and the ~2-adrenoceptor antagonist yohimbine (yoh) (1 # M ) on the KCI (70 raM) stimulation-evoked release of tritium in tissues pre-incubated with vehicle or N-ethyl-maleimide (30 /,M). Responses obtained in the presence of xylazine, yohimbine or distilled water vehicle (con) at S 2 were expressed as a percentage of responses obtained at S t in the absence of drug. Asterisks denote effects of drug significantly different from effects of vehicle (* P < 0.05, Student's t-test).
3.2. Prostatic portions o f rat ras deferens: nerremediated responses
In prostatic portions, single pulse electrical stimulation produced a contraction of 1.20 + 0.11 g (n = 26), consisting mainly of the first non-noradrenergic phase. Xylazine produced a concentration-dependent inhibition of the stimulation-evoked contraction, with a pD 2 ( - l o g ICs0) of 7.34 + 0.09, and a maximum inhibition of 88.3 _+ 2.1% (n = 32) (see Tables 1 and 2, and Fig. 2). Vehicle administration caused a small but significant increase in the contractile response to a single stimulus, which was presumably a time-dependent increase ( P < 0.05, paired t-test) (Table 1). Pre-exposure to N-ethyl-maleimide (100 p,M) but not N-ethylmaleimide (30/,tM) significantly reduced the isometric
Vehicle (n = 32) Phenoxybenzamine(l # M ) ( n = 8 ) EEDQ(0.2 #M)(n=8) NEM (30 # M ) ( n = 18) NEM (100/.tM) (n -= h) PBA(1 # M ) + N E M ( 3 ( I , t t M ) (n = g)
E E D Q (0.2 p,M)+ NEM (30 ~ M ) (/i - 5)
EpMid.vmal portion Vehicle (n = 6) NEM ( 3 0 / , M ) (n - ~)
Values are xylazine pD 2 ( - l o g l(',l~) and maximum inhibition of stimulation evoked contraction, mean + S.E.M.. with H, ll~e ntlmber of experiments. Responses significantly different from effects of vehicle (Student's t-test): a p < 0.05, ~' P < 0.(ll. c 1, < (1.0(}1.
contraction (Table 1). Other t e s t agents and combinations either produced no significant effect or increased the contractile response (Table I). Pre-exposure to N-ethyl-maleimide (30 or 100 ttM) significantly shifted the potency of xylazine without altering the maximum inhibitory response (Fig. 2 and Table 2), Pre-exposure to phenoxybenzamine (1 /,M) or EEDQ (0.2 /.tM) significantly shifted the potency of xylazinc and reduced the maximum inhibitory response (Figs. 3 and 4. and Table 2). Exposure to N-ethyl-maleimide (30 p,M) following pre-exposure to phenoxybenzamine or EEDQ produced no further effect than phenoxybenzamine or E E D Q alone (Figs. 3 and 4, and Table 2).
Table I Effects of the test agents phenoxybenzamine (PBA), N-ethyl-maleimide (NEM) and E E D Q on isometric contraction to a single stimulus in prostatic and epididymal portions of rat vas deferens Test agent
Control (g) Test (g)
Prostatic portion Vehicle (n = 26) Phenoxybenzamine (1 ~,M) (n = 6) E E D Q (0.2 /.tM) (n = 12) NEM (30 ~ M ) (n - 16) N E M (100/xM) (n = 5) PBA(I p,M)+NEM(30>M)(n 8) EEDQ(0.2/,tM)+NEM(30p~M)(n=7)
~-
1.20_+0.11 1.00_+ 0.17 1.54_+0.16 1.17.+0.11 1.22_+0.38 1.26+0.23 1.16+0.08
1.40+0.13 '' 1.24+0.15 2.18+0.14c 1.05-+0,19 0.38+0.10 t' 0.89-+(L12 1.46+0,19
0.80+0.10 1.28+0.17
1.32+0.14 c 0.82 +(I,10 c
Epididyrnal portion Vehicle (n = 19) NEM (30 # M ) (n - 18)
Values are isometric tension (g), mean + S.E.M.. with n, the number of experiments. Responses significantly different from control response (Student's t-test for paired data): a P < 0.05, h P < 0.01. " f < 0.001.
6o
u
"8
4o
veh l ~
~
~L~.
~
20
xylazine concentration
(log
M)
Fig. 2. Effects of the ~r2-adrenoceptor agonist xylazine fl~llowing pre-exposure to vehicle ( o ) or N-ethyl-maleimide (30/~M) (e) on the isometric contraction produced by single pulse electrical stimulation in prostatic portions of rat vas deferens. Responses in the presence of xylazine are expressed as a percenlage of Ihe response in lhe absence of xylazine. Vertical bars represent S.E.M. from at leasl IS experiments.
10c
L Browne et al./European Journal of Pharmacolo,~,~, 205 (19941 125-132
80 PBA
'~
4o
4o
N 2O
0 -9
129
2C -'8 -'7 -'6 xylazine concentration (log M)
-'5
Fig. 3. Effects of the c~2-adrenoceptor agonist xylazine following pre-exposure to vehicle (o), phenoxybenzamine (1 #M) (1~) or phenoxybenzamine (1 /,~M) followed by N-ethyl-maleimide (30 #M) (el on the isometric contraction produced by single pulse electrical stimulation in prostatic portions of rat vas deferens. Responses in the presence of xylazine are expressed as a percentage of the response in the absence of xylazine. Vertical bars represent S.E.M. from at least 6 experiments.
3.3. Epididymal portions of rat uas deferens: nervemediated responses In epididymal portions of rat vas deferens, the initial biphasic twitch to a single electrical stimulus was reduced to a monophasic response by nifedipine (10 /zM), which eliminates the first (non-noradrenergic) phase, leaving the second (noradrenergic) phase (see Brown et al., 1983). Under these conditions, the twitch to a single stimulus was 0 . 8 0 _ 0.10 g (n = 19), and xylazine inhibited this response with a pD 2 of 7.57 _+ 0.12 and a maximum inhibition of 93.0 _+ 2.9% (n -- 6). Vehicle significantly increased the contractile response to a single stimulus, whereas N-ethyl-maleimide (30 p,M) significantly reduced the response (Table 1). Preexposure to N-ethyl-maleimide (30 /~M) significantly shifted the potency of xylazine without altering the maximum inhibition (see Fig. 5 and Table 2). In vehicle experiments, amidephrine inhibited the
xylazine concentration (log M)
Fig. 5. Effects of the ~2-adrenoceptor agonist ~lazine on isometric contractions produced by single pulse electrical stimulation in epididymal portions of rat vas deferens in the presence of nifedipine (10 ~M). Symbols: vehicle pre-treated (©), N-ethyl-maleimide (30 #M) pretreated (el. Vertical bars represent S.E.M. from at least 4 experiments.
contractile response to a single stimulus with a pD 2 of 6.60 _+ 0.08 and a maximum inhibition of 87.5 _+ 2.2% (n = 7), and the amidephrine pD 2 and maximum inhibition were not significantly altered by pre-exposure to N-ethyl-maleimide (30 p,M) (6.75 _+ (I.12 and 86.6 _+ 1.4%, respectively) (see Fig. 6). Pre-exposure to N-ethyl-maleimidc (30 p,M) significantly reduced the inhibitory potency of 5-HT (5-HT pD2: vehicle 5.76_+ 0.19, n = 11; N-ethyl-maleimide 5.25 _+ 0.21, n = 11; P < 0.01, Student's t-test for paired data) without significantly altering the maximum inhibition by 5-HT (vehicle 87.5 _+ 1.7%; N-ethyl-maleimide 82.4 _+ 2.9%) (see Fig. 7). 3.4. Ligand binding studies: rat renal cortex In rat renal cortex membranes, analysis indicated that [-~H]MK912 bound to a single population of a 2adrenoceptors with a K o of 5.73 + 1.64 nM and a B ...... of 854 ___191 f m o l / m g protein (n = 5). In tissues preexposed to N-ethyl-maleimide (300 /~M), the B ..... of MK912 was significantly reduced ( P < 0.05) to 313 _+ 32
1 {:(:80
100 60 80 60 2C
~
h NEM
40 20 xylazine concentration (log M) 0 9
Fig. 4. Effects of the ~2-adrenoceptor agonist xylazine following pre-exposure to vehicle (o), EEDQ (0.2 #M) ([]) or EEDQ (0.2 /zM) followed by N-ethyl-maleimide (30 p,M) (e) on the isometric contraction produced by single pulse electrical stimulation in prostatic portions of rat vas deferens. Responses in the presence of xylazine are expressed as a percentage of the response in the absence of xylazine. Vertical bars represent S.E.M. from at least 7 experiments.
-'8 "7 -'6 amidephrine concentration 009 M)
~5
Fig, 6. Effects of the ~1-adrenoceptor agonist amidephrine on isometric contractions produced by single pulse electrical stimulation in epididymal portions of rat vas deferens in the presence of nifedipine (10 /~M). Symbols: vehicle pro-treated (o); N-ethyl-maleimide (30 /zM) pretreated (e). Vertical bars represent S.E.M. from at least 4 experiments,
I. Browne et al. / European Journal of PharmacolojG 265 ~1994) 125-132
130 1 oo
u
vehI
6O
~
2o o -8
-;
6
5
5-hydroxytryptamine concentration
-74
(logM)
Fig. 7. Effects of 5-HT on isometric contractions produced by single pulse elcclrical stimulation in epididymal portions of rat vas deferens in the presence of nifcdipine (10 # M). Symbols: vehicle pro-treated (~); N-ethyl-maleimide(31)btM) pretrcated (o). Vertical bars represent S.EM. from at least 4 experiments.
f m o l / m g protein (n = 5) with no change in K D (3.91 _+ 0.26 nM), as compared to the effects of incubation with vehicle. In displacement studies, yohimbine and N-ethylmaleimide had K~ values of 80.8 + 10.7 nM in = 3) and 466 _+ 133 >M (n = 5), respectively.
4. Discussion
The effects of N-ethyl-maleimide on oc~-adrenoceptor mediated responses have been examined in a number of tissues. Before discussing the overall implications of these results, responses obtained in each tissue will be discussed separately. 4.1. Radioactit'e overflow experiments in rat renal corter
In rat renal cortex slices pre-incubated with [3H]noradrenaline, KCI (70 raM) evoked release of tritium was taken as a measure of transmitter release. Stimulation-evoked release of tritium was significantly reduced by xylazine (10 /xM) and significantly increased by yohimbine (1 # M ) i n veh~icle pre-incubated tissues, but in tissues incubated with N-ethyl-ma[eimide (30 /xM) the inhibitory effects of xylazine were abolished. These results are consistent with previous findings in the literature (see Introduction) and can be interpreted in terms of inactivation of G-proteins (or indeed in terms of c~,-adrenoceptor antagonism, but see below). The fact that the effects of xylazine were abolished by N-ethyl-maleimide whereas the effects of yohimbine were not significantly reduced by N-ethyl-maleimide may bc explained by the fact that xylazine, being a synthetic agonist, may act as a partial agonist under these experimental conditions. In contrast, yohimbine increases the stimulation-ew)ked release by blocking the actions of the endogenous agonist noradrenaline, and noradrenalinc acts as a full agonist. A partial
agonist may produce less efficient coupling of rcceptor to G-protein so that its actions may be more critically dependent on the availability of G-protein. Although N-ethyl-maleimidc might have been expected to increase stimulation-evoked release by its presumed action to uncouple G-proteins involved in ~c~-adrenoccptot" mediated inhibition. N-ethyl-malcimidc (30 # M ) actually tended to decrease stimulation-ew~kcd release of noradrenaline. However, this effect of N-cthylmaleimide has been previously reported (Murphy ct al., 1992) and may represent a non-specific action of N-cthyl-malcimide. 4.2. L ~ a n d binding studies
Ligand binding studies were carried out using [-~H]MK912, a selective c~_-adrenoccptor antagonist (Uhlen ct al., 1992), in rat renal cortex membrancs which contain c~2B-adrenoceptors (see Smith and Docherty, 1992), although a2a-adrenoceptors have also been reportcd (Uhlcn et al., 1992). The prcscnt results fitted best a one-site model: ae~cadrenoceptors (see Smith and Docherty, 1992). In displacement studies, N-ethyl-maleimidc displaccd binding of [3H]MKgI2 with a K~ of 466 p.M, a concentration much higher than that used in most functional studies of the interaction of N-ethyl-maleimide with G-proteins (present results: 30 /xM; Foucart et al., 1990:3 ~M; Kaschube and Brasch, 1990:60 p.M; Murphy el al., 1992:3 # M : Rump ct al., 1992:10 /xM). In saturation studies, N-ethy[-maleimide (300 # M ) pretreatment produced approximately a 6(1~ reduction in the maximum nttmbet of binding sites for MK912. Other authors have reported that N-ethyl-maleimide (100-101)0 # M ) reduced the maximum binding of the c~2-adrcnoccptor ligand [3H]UK 143(14 (Garcia-Sevilla ct a[, 1988). The receptor examined in ligand binding studics was an e~2t~-adrenoceptor, whereas the prejunctiona[ reccptor in rat vas deferens is probably an {C,D-subtypc as found in the rat submandibular gland (sec Smith and Docherty, 1992). However, we also have limited data from ligand binding studies of the rat submandibular gland in which N-ethyl-maleimide shows similar low affinity in displacement studics (authors, unpublished observations). Hence, wc have no reason to think that N-ethyl-malcimide has high affinity for a subtype of Q,-adrenoccptor. 4.3. Electrical stimulation-eroked isometric contracuons in rat vas deferens
In rat whole vas deferens, the electrical stimulation-evoked contraction to a single stimulus consists of a biphasic response, the first phase of which is nonadrenoceptor mediated (probably ATP) and predominates in prostatic portions, and the second phase of
1. Browne et al. / European Journal
which is al-adrenoceptor mediated, and predominates in the epididymal portion (see Brown et al., 1983; Aboud et al., 1993). The second oq-noradrenergic phase can be examined in isolation in the epididymal portion in the presence of nifedipine which eliminates the first non-noradrenergic response, and also prevents postjunctional contractile actions of exogenous agonists at oei-adrenoceptors. In prostatic portions, the twitch response to a single stimulus is mainly nonadrenergic, and can be inhibited by o~2-adrenoceptor agonists by a prejunctional action, whereas ~ r a d r e n o c e p t o r agonists increase stimulation-evoked contractions by a postjunctional action (see Brown et al., 1983). N-Ethyl-maleimide (30 # M for 60 min) reduced the potency of xylazine by a factor of 3-5 in both prostatic and epididymal portions, and reduced the potency of 5-HT in epididymal portions, although it failed to shift the potency of the al-adrenoceptor agonist amidephrine in epididymal portions. N-Ethyl-maleimide (100 # M ) produced no further effect against the inhibition by xylazine. This action of N-ethyl-maleimide to reduce the potency of xylazine and 5-HT may be by inactivation of G-proteins involved in prejunctional o~2-adrenoceptor and 5 - H T r r e c e p t o r mediated responses (see Introduction). In previous studies of rat vas deferens, pertussis toxin failed to modulate the a2-adrenoceptor (Docherty, 1988) or 5-HTl-receptor (Borton and Docherty, 1990) mediated inhibition. Hence, these responses can be described as N-ethylmaleimide-sensitive, pertussis toxin-insensitive, and this may reflect the sensitivity of a G-protein involved (in contrast, note that prejunctional o~2-adrenoceptor mediated responses in rabbit hippocampus are N-ethylmaleimide-sensitive and pertussis toxin-sensitive: Allgaier et al., 1985, 1986). However, assuming that Nethyl-maleimide acts at a G-protein, the fact that Nethyl-maleimide shifted the potency of xylazine and 5-HT without reducing the maximum inhibition might suggest that there is spare G-protein capacity allowing xylazine and 5-HT to produce a maximum effect even though a proportion of the G-proteins have been inactivated. It can be demonstrated theoretically that the concentration of G-proteins will affect apparent affinity of the agonist for the receptor (MacKay, 1990). If this were the case, reduction in the number of ~eadrenoceptors by irreversible alkylation with phenoxybenzamine or E E D Q should allow further effects of N-ethyl-maleimide. Phenoxybenzamine (1 >M) and E E D Q (0.2 p,M) reduced the maximum inhibition by xylazine as well as shifting the potency of xylazine, suggesting that no spare receptors for xylazine remained after this pre-treatment. However, N-ethylmaleimide was not additive with phenoxybcnzamine or EEDQ: the combination produced no further effect than phenoxybenzamine or E E D Q alone. Although N-ethyl-maleimide acts as an irreversible oe,-adreno-
of Pharmacolog~v265
(1994) 125-132
131
ceptor antagonist, this action only occurs at concentrations of 100-300 /,tM and above in ligand binding studies. Hence, the fact that N-ethyl-maleimide was not additive with phenoxybenzamine and E E D Q suggests that, either N-ethyl-maleimide produces nonspecific effects, as has been reported in terms of signal transduction in mouse atria (Foucart et al., 1990), or that phenoxybenzamine and E E D Q have similar actions to N-ethyl-maleimide in addition to o~-adrenoceptor antagonism. Another possibility is that there are two populations of receptors, one of which is linked to an N-ethyl-maleimide-sensitive G-protein and is selectively inactivated by phenoxybenzamine and EEDQ, but we have no evidence to support this: the prejunctional receptors of rat vas deferens appear to bc a homogeneous population of oem-adrenoceptors (see Smith and Docherty, 1992). The shift in the prejunctional potency of xylazine produced by phenoxybenzaminc and E E D Q may not be indicative of the presence of spare receptors, but rather be due to inactivation of G-proteins. This would be consistent with the fact that, in vehicle experiments, xylazine produces an approximately 95% inhibition of the stimulation-evoked contraction but does not totally abolish the contraction. If the maximum effect of xylazine involved activation of most of the prejunctional receptors, then an irreversible antagonist should reduce the maximum inhibition by xylazinc without affecting the potency. The fact that phenoxybcnzamine and E E D Q reduced the potency and the maximum inhibition by xylazine implies either spare receptors or additional actions of these agents, such as G-protein inactivation. Any additional actions of phenoxybenzamine might affect interpretation of agonist affinity calculations from partial receptor inactivation studies (see Docherty, 1989). Although N-ethyl-maleimide did not significantly affect the inhibitory potency of the c~-adrenoceptor agonist amidephrine against isometric contractions of epididymal portions in the presence of nifedipine, it has not been fully established that this action of amidephrine is prejunctional (see Docherty, 1989). If the actions of amidephrine are indeed prejunctional, we have evidence for a prejunctional inhibitory response involving a G-protein insensitvc to N-ethyl-maleimide, in agreement with the findings of Rump ct al. (1992). In summary, N-ethyl-maleimide is not an a~-adrenoceptor antagonist in the range of concentrations at which it prevents the inhibitory action of xylazine on nerve stimulation evoked responses. However, the lack of interaction between N-ethyl-maleimidc and phcnoxybenzamine or E E D Q is not consistent with the assumed actions of these agents. It must be concluded that either N-ethyl-maleimidc acts non-specifically, or that it has similar actions to phenoxybenzamine or EEDQ, or vice versa. This may suggest that phenoxy-
1. Browne et al. / European Journal (~]"Pharmacolo,e,y 265 (19941 125-132
132
benzamine and EEDQ have additional actions to inactivate G-proteins.
Acknowledgements Supported by The Health Research Board (Ireland), the Irish Heart Foundation and Royal College of Surgeons in Ireland.
References Aboud, R.W., M. Shafii and J.R. Doeherty, 1993, Investigations ol the subtypes of ¢tl-adrenoceptor mediating contractions of rat aorta, vas deferens and spleen, Br. J. Pharmacol. 1(}9, 80. Adler, C.t[., E. Meller and M. Goldstein, 1985, Recovery of ~,adrenoceptor binding and function after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), Eur. J, Pharmacol. 116, 175. Allgaicr, C., T.J. Feuerstein, R. Jackisch and G. Hertting, 1985, Islet-activating protein (perlussis toxin) diminishes c~e-adrenoce ptor mediated effects on noradrenaline release, Naunyn-Schmied. Arch. Pharmacol. 331, 235. Allgaier, C,, T.J. Feuerstein and G. Hertting, 1986, N-Ethyl-maleimide (NEM) diminishes er2-adrenoceptor mediated effects on noradrenaline release, Naunyn-Schmied. Arch. Pharmacol. 333, 104.
Borton, M. and J.R. Docherty, 1990, The effects of ageing on prejunctional 5-hydroxytryptamine receptors in the rat vas deferens, Naunyn-Schmied. Arch. Pharmacol. 342, 130. Bradford, M.M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248. Brown, D.A., J.R. Docherly. A.M. French, A. MacDonald, J.C. McGrath and N.C. Scott, 1983, Separation of noradrenergic and non-norudrenergic contractions to field stimulation in the rat vas deferens, Br. J. Pharmacol. 79, 379. ('heng, Y. and W.H. Prusoff, 1973, Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 5()(~: inhibition (1(-%,u) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Connaughton, S. and J.R. Docherly, 1990, Functional evidence for heterogeneity of peripheral prejunctional n'2-adrenoceptors, Br. J. Pharmacol. 101, 285. Docherty, J.R., 1988, Pertussis toxin and prejunctional o~-adrenoceptors in rat heart and w~s deferens, J. Autonom. Pharmacol. 8, 1 g7.
Docherty, J.R., 1989, The pharmacology of ~l- and ¢~2-adrenoceptors: evidence for and against a further subdivision. Pharmacol. "Fher. 44, 241. Foucart, S., T.V. Murphy and 11. Majewski, 1990, Prejunctional /4-adrenoceptors, angiotensin 11 and neuropeptide Y receptors on sympathetic nerves in mouse atria arc linked to N ethylmaleimidc-susceptible G-proteins, J. Auton. N e t < Sysl. 30. 221. Garcia-Sevilla, J.A., P. Areso, M.T. Giralt and 1. Uliharri, 1988, Uncoupling of the platelet c~e-adrenoceptor adenylatc cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity+ Eur. J. Pharmacol. 145, 91. Kaschubc, M. and It. Brasch, 1990+ Pre+ and postjunctional effects o1 N-ethylmalcirnide in the isolated mouse vas deferens, ['~ur. J. Pharmacol. 178, 151. Kitamura, Y. and Y. Nomura, 1987, Uncoupling of rat cerebra[ cortical ~+2-adrenoceptors from GTP-binding proteins by N-ethylmaleimide, J. Neurochem. 49. 1894. MacKay, L)., 1990, Interpretation of relative potencies, relalive efficacies and apparent affinity constants of agonist drugs cstimated lrom concentration-response curves, J. Theor. Biol. 142, 415+ Murphy, T.V., S. Foucart and It. Majewski, 1992, Preiurlctional ~r2-adrenoceptors in mouse atria function through (;-proteins which are sensitive to N-ethylmaleimidc, but not pertussis toxin, Br. J. Pharmacol. 106, 871. Neylon, C.B. and R.J. Summers, 1985, [~H]-Rauwolscine binding to er2-adrenoceptors in the mammalian kidney: apparent receptor heterogeneity between species. Br. J. Pharmacol. 85, 349. Rump, L.C., V. Volk, G. Ruff and P. Schollmeyer, 1992, Activation of ~r 1- and ¢~2-adrenoceptors inhibits noradrenaline release in rabbit renal arteries: effects of pertussis toxin and N-ethylmaleimide+ J. Auton. Pharmacol. 12, 97. Smith, K. and J.R. l)ocherty, 1992, Arc the prcjunctional ¢~-a¢lren oceptors of the rat vas deferens and submandibular gland of the ~2,\- or er2i)-subtypc?, Eur. J. Pharmacol. 219, 203. Smith, K., S, ('onnaughton and J.R. Docherty, 1992, Investigations o] prejunctional ~r2-adrenoceptors in rat atrium, wls deferens and submandibular gland, Eur. J. Pharmacol. 211, 251. Ueda, 1t., If. Misawa, T. Katada, M. ill, 1t. Takagi and M. Satoh, 1990, Functional reconstitution of purified G i and G , with # opioid receptors in guinea-pig striatal m e m b r a n e s pretrealcd with micromolar concentrations of N-cthylnvaleimide, J. Neurochem. 54, 84 I. Uhlcn, S., Y. Xia, V. Chhajlani, (2('. [:elder and J.E.S. Wikberg, 1902, [ ~[ t]-M K 912 binding delineates two ~ 2-adrenoceptor subtypes in rat CNS one of which is identical with the cloned pA2d ~2-adrenoceptor, Br. J. Pharmacol. 1(16, 986. Weber, H.D., 1989, Presynaptic (r2-adrenoccptors at the terminals of perivascular sympathetic nerves arc coupled to an N-ethylmaleimide-sensitivc G-protein. Naunyn-Schmied. Arch. Pharmacol. 339, R35.