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Potentiation by spiperone and other butyrophenones of fluid secretion by isolated salivary glands of ixodid ticks
European Journal of Pharmacology, 73 (1981) 163--173
163
Elsevier/North-Holland Biomedical Press
POTENTIATION BY SPIPERONE AND O T H E R B U T Y R O P H E N O N E S OF F L U I D SECRETION BY ISOLATED S A L I V A R Y GLANDS O F IXODID TICKS DOROTHY L.-P. WONG and WILLIAM R. KAUFMAN *
Department of Zoology, University of Alberta, Edmonton, Canada T6G 2E9 Received 10 March 1981, accepted 7 May 1981
D.L.-P. WONG and W.R. KAUFMAN, Potentiation by spiperone and other butyrophenones of fluid secretion by isolated salivary glands ofixodid ticks, European J. Pharmacol. 73 (1981) 163--173. Isolated salivary glands from the ixodid tick, Amblyomma hebraeum Koch are stimulated to secrete fluid when exposed to dopamine (DA), the maximum response occurring at 10 -6 M. Spiperone, and a number of other butyrophenone derivatives, although lacking intrinsic activity, are able to potentiate the secretion elicited by supramaximal concentrations of DA; this potentiation by spiperone is evident at concentrations in the femtomolar range. Tranylcypromine, a potent, competitive inhibitor of monoamine oxidase (MAO) in tick salivary gland homogenates, has both intrinsic activity and potentiates DA-induced salivation. The fact that spiperone potentiates ergometrine-induced salivation indicates that the prime mechanism of the butyrophenone effect is not by inhibiting catecholamine catabolism. The results also suggest that the receptor for DA and that for butyrophenones are distinct sites. Droperidol, benperidol and bromperidol, all potent neuroleptic drugs, failed (at 10 -9 M) to potentiate salivation. By contrast, R951, R27275 and R l 1 8 7 (all at 10 -9 M) were very effective potentiators on the salivary gland system, despite the fact that they lack the basic structural requirements for neuroleptic activity. These results suggest that the butyrophenone site in tick salivary glands is different from butyrophenone binding sites in mammalian CNS. Salivary fluid secretion in vitro
Dopamine
Butyrophenone receptor
1. Introduction Female ticks (Acari) of the family Ixodidae employ their salivary glands as an osmo- and volume-regulatory system. During the taking of a blood meal, excess fluid is secreted directly back into the host's circulation (Tatchell, 1967; Kaufman and Phillips, 1973a; Kaufman et al., 1980). It is in this way that most of the pathogenic agents transmitted b y ticks gain access to the definitive host. As part of a program to learn more a b o u t the physiology of these important vectors of disease, we have developed an in vitro preparation of the salivary gland which enables one to critically evaluate the effects of drugs on salivary fluid secretion (Kaufman and Phillips, 1973b; Kaufrnan, 1976). These and other studies have taught us that salivation is under nervous * To whom
Spiperone
control, the transmitter substance probably being a catecholamine. The most p o t e n t agonists on this in vitro preparation are dopamine (DA) and some ergot alkaloids (Kaufman, 1977). Other drugs (apomorphine, N-methyldopamine) which often have high affinity for DA-receptors in mammalian systems are also agonists on tick salivary glands (Kaufman, 1 9 7 7 ) s u g g e s t i n g that the post-junctional receptor mediating fluid secretion in this tissue might also be a DA-receptor. It was thus contrary to expectation that a variety of drugs known to be p o t e n t inhibitors at mammalian central nervous system (CNS) and peripheral DA-receptors (chlorpromazine, ~-flupenthixol, spiperone, pimozide) failed to attenuate DA-effects in the tick system at reasonable concentrations; moreover, a number of usually effec-
correspondence should be addressed.
0014-2999/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
164 tive a-adrenolytic (phenoxybenzamine, phentolamine) and fl-adrenolytic (propanolol, dichloroisoprenaline) drugs were similarly unremarkable as putative inhibitors (Kaufman, 1977). Although spiperone and pimozide did n o t attenuate fluid secretion, they did have an unexpected action: both drugs augmented the tissue's response to DA even when the latter was present at supramaximal concentration. Yet neither spiperone nor pimozide" possessed intrinsic activity. In consideration of all the above-mentioned results, it has been proposed that the epithelium possesses a DA-receptor with hitherto undescribed properties (Kaufman, 1977, 1981). Because there appears to be b u t a passing reference in the literature to a b u t y r o p h e n o n e (haloperidol) occasionally eliciting such a potentiation at high concentrations (Woodruff et al., 1970), we considered it appropriate to pursue this phenomenon further. As will be shown below, the property is c o m m o n to a number of butyrophenone analogues and, to a first approximation at least, the property is unrelated to p o t e n c y of the congener as a neuroleptic. Molecular mechanism behind the synergistic action has n o t y e t been fully investigated, b u t it cannot be explained in terms of any efficacy these drugs may have as monoamine oxidase (MAO) inhibitors.
D.L.-P. WONG, W.R. KAUFMAN
2.2. The in vitro preparation Glands were dissected o u t and set up for observation exactly as described by Kaufman (1977). Briefly, partially fed ticks were removed from the host (rabbit) and dissected open in a dish filled with a slightly modified Hanks' balanced saline (composition in g/l: NaC1 11.5; D-glucose 1.6; KC1 0.4; CaCl: 0.14; MgSO4 0.1; KH2PO4 0.06; Na2HPO4 0.05; phenol red 0.01; 360 mosm; pH 7.0). The excised glands were introduced to a second petri dish lined with paraffin wax and filled with liquid paraffin. The system was arranged such that the acinar (secretory) portion of the gland could be bathed in a nutrient medium (TC 199) in isolation from the main duct. Appearance of fluid at the orifice o f the duct indicated that secretion had been stimulated by a drug. Since the secreted droplet assumed a spherical shape under the liquid paraffin, its volume (and hence the rate of secretion) could be determined from a simple calculation of the diameter as measured with an ocular micrometer fitted to a dissection microscope. Under these conditions isolated glands secrete spontaneously only rarely, b u t they are rapidly activated when the medium contains catecholamines. Further details are presented elsewhere (Kaufman and Phillips, 1973b; Kaufman, 1976, 1977).
2.3. Experimental protocol 2. Materials and m e t h o d s
2.1. Ticks The ticks used in this study were taken from a colony established here with specimens originating from the Veterinary Institute, Onderstepoort, Rep. South Africa. Ticks were first partially fed to 150-400 mg (unfed weights were approximately 20-30 mg) as described b y Kaufman and Phillips (1973a). This partial feeding is required to bring the salivary glands to a physiologically c o m p e t e n t state (Kaufman, 1976).
If glands are exposed to a maximal concentration of DA (10 -6 M; Kaufman, 1976) the full response is achieved within several minutes. To assay the influence of butyrophenones we first subjected each gland to three concentrations of DA (2.3 X 10 -8, 9 × 10 -8, 2 . 3 ) < 1 0 -7 M), equivalent respectively to a b o u t 18%, 50% and 80% maximal response (fig. 1), in the following manner. Glands were bathed for 60 sec in the lowest concentration, the medium was changed for a fresh aliquot o f the same solution, and the volume of saliva collected from the orifice over the succeeding 5 min was recorded. In a similar way, the vol-
P O T E N T I A T I O N O F D O P A M I N E BY B U T Y R O P H E N O N E S
100"
8
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== x
40.
20"
I -e
3 x 10 -e
1'0-;'
3 x 10-'
[DOPAMINE](M)
10 -e
3 x 10-s
Fig. !" Log d o s e - r e s p o n s e curve for D A o n isolated tick salivary glands. T h e curve was c o n s t r u c t e d as follows. Glands w e r e e x p o s e d to t h e t h r e e l o w e r conc e n t r a t i o n s o f D A (3 x 10 - s , 9.1 x 10 -8 , 2.3 x 10 -7 M) a c c o r d i n g t o t h e p r o t o c o l d e s c r i b e d in M e t h o d s ; t h e y were t h e n w a s h e d with TC 199 lacking D A until s e c r e t i o n s t o p p e d . T h e glands were t h e n e x p o s e d t o t h e t h r e e u p p e r c o n c e n t r a t i o n s (2.3 X 10 -7, 10 -6, 2.3 x 10 -6 M) in t h e same m a n n e r . Since each gland was e x p o s e d t o 2.3 x 10 -~ M D A first at t h e end o f a c u m u l a t i v e a d d i t i o n o f drug and t h e n as a single a d m i n i s t r a t i o n f o l l o w i n g a p e r i o d o f rest, o n e can estimate t h e degree o f d e s e n s i t i z a t i o n at this c o n c e n t r a t i o n a t t r i b u t a b l e to an e x t e n d e d p e r i o d o f e x p o sure to t h e drug. A b s o l u t e rates o f s e c r e t i o n resulting f r o m t h e t w o e x p o s u r e s were c o m p a r e d by a pairs t-test. R a t e o f s e c r e t i o n resulting f r o m the s e c o n d e x p o s u r e averaged 88 ± 11% ( m e a n +- S.E.M., n = 8) t h a t o f first e x p o s u r e (P > 0.05). In a s e p a r a t e series o f 8 glands, a similar analysis was c o n d u c t e d w i t h 3 x 10 -8 M D A as t h e middle c o n c e n t r a t i o n . R a t e o f s e c r e t i o n o n the s e c o n d e x p o s u r e to 3 x 10 - s M D A was 106+_ 10% (mean-+ S.E.M., n = 8) t h a t o f t h e first e x p o s u r e , this d i f f e r e n c e also being insignificant (P > 0.05 f r o m a pairs t-test o n the a b s o l u t e rates o f s e c r e t i o n ) . In t h e figure, we have p l o t t e d t h e m e a n values o f t h e 2 e x p o s u r e s . Finally, t h e r e is no significant d i f f e r e n c e ( S t u d e n t ' s t-test) b e t w e e n t h e perc e n t a g e s (88 -+ 11 a n d 106 +_ 10). In c o n c l u s i o n , t h e r e is n o e v i d e n c e for a u t o i n h i b i t i o n b y D A in t h e subm i c r o m o l a r range.
ume o f secretion resulting from a 5-min exposure to the middle and then the highest dose was recorded, in each case following a 60-sec pre-exposure. Glands were then washed for 30 min (a total of 6 changes of aliquot) in TC 199 lacking DA, by which time the glands were no longer secreting. Glands were then
165
treated, in the same manner as before, to the three doses of DA prepared in TC 199 containing a given concentration of butyrophenone. To test for reversibility o f butyrophenone action, a third exposure to DA was accomplished following a 30-min wash period in TC 199 lacking DA and butyrophenone. Fig. 2 (see also Kaufman, 1977) shows that there is a decay in response by glands subjected to 3 exposures to DA a l o n e - namely, at 2.3 X 10 -7 M, the second response is only 80% (on average) that of the first one and the third response is only 60%. Thus for statistical analysis, all responses to the butyrophenone exposure were compared to the control exposure normalized for this expected decay. (Incidentally, this decay in response appears to be an effect, direct or indirect, of exposure to drug per se, rather than of poor viability in the nutrient medium. Glands cultured in TC 199, but n o t treated with DA, secrete after 3 days at about 73% of the rate expected for non-cultured glands (Kaufman and Barnett, 1977)).
8
x
100-
l)11
80-
10
c~ vc~
~
9
80-
11 ~
4.0-
_~ 20-
11
x ~
0
lo •
3 ,~ l o - . 1'o, [ DOPAMINE ](M)
3 ~ lO,
Fig. 2. S e q u e n t i a l e x p o s u r e s t o 3 c o n c e n t r a t i o n s o f D A (3 x 10 - s , 9 . 1 x 10 -8 , 2 . 3 x 10 -7 M) w i t h 30m i n w a s h o u t s b e t w e e n each series. (o) first e x p o s u r e , (A) s e c o n d e x p o s u r e , ( a ) t h i r d e x p o s u r e . On average, t h e s e c o n d r e s p o n s e to 2.3 x 10 -7 M DA was 80 -+ 7% ( m e a n + S.E.M.; n = 10) and the t h i r d r e s p o n s e was 61 _+ 8% ( m e a n _+ S.E.M.; n = 9) t h a t o f t h e first r e s p o n s e . In all s u b s e q u e n t e x p e r i m e n t s , a c c o u n t was t a k e n o f this a n t i c i p a t e d d e c a y in r e s p o n s e w h e n perf o r m i n g statistical analysis.
166 2.4. Sources o f drugs and chemicals
All the butyrophenone analogues were the gift of Janssen Pharmaceutica, Beerse, Belgium: spiperone, pimozide, droperidol, benperidol, haloperidol, trifluperidol, bromperidol, penfluridol, R951, Rl187, R2308, and R27275. Chlorpromazine was the gift of SPECIA, Paris. Reserpine was the gift of CibaGeigy AG, Basle, Switzerland, and tranylcypromine the gift of Smith Kline and French Canada Ltd., Montreal. Ergonovine maleate, dopamine HC1, norepinephrine, adenosine 3',5'-cyclic monophosphoric acid (cyclic AMP), N6,O21 -dibutyryladenosine 3',5'-cyclic monophosphoric acid (monosodium salt) dihydrate (dibutyryl cyclic AMP) and tryptamine were purchased from Sigma Chemical Company, and TC 199 (with Hanks' salts and L-glutamine but without NaHCO3) was purchased from Gibco. Osmotic concentration of the TC 199 was increased to 360 mOsm by the addition of NaC1 (4.32 g/l). All other chemicals were of purest reagent grade (Fisher Scientific and BDH). Tryptamine bisuccinate [side chaln-2-14C], 51.5 mCi/mmol was pur: chased from New England Nuclear. 2.5. MA 0 assay
Salivary glands were dissected out of partially-fed ticks under Hanks' balanced saline and weighed as described by Harris and Kaufman (1981). Pairs of salivary glands were homogenized in 300-600 pl 1.45% KC1 (isosmotic with tick haemolymph) in small glass homogenizers (about 20 passes of the piston). After removal of an aliquot for protein determination, the homogenate was transferred to small plastic vials, frozen in ethanol-dry ice and stored at --16°C. The assay procedure was adapted from that of Wurtman and Axelrod (1963). The incubation mixture consisted of equal volumes (10 ~zl) of 4 stock solutions (1) 14C-labelled tryptamine (51.5 mCi/mmol) diluted 1 : 2 0 (v/v) with 0.5 M phosphate buffer, pH 7.5, (2) a solution consisting of variable concentrations
D.L.-P. WONG, W.R. KAUFMAN of non-radiolabeUed tryptamine (0-1000 I~M) in phosphate buffer, (3) as solution 2 with the addition of a given concentration of tranylcypromine, and (4) salivary gland crude homogenate. Thus the final incubation mixture contained 50 nCi tryptamine bisuccinate, total tryptamine concentration ranging from 9.5 to 510 pM and a variable concentration of test drug, all in 375 mM phosphate buffer, pH 7.5. Ten t~l each of solutions 1, 2 and 3 were pre-incubated for 5 min at 37°C in 15-ml glass-stoppered centrifuge tubes. Ten /~l of crude homogenate was added, and after a 15-min incubation, the reaction was stopped with 200 pl 2 N HC1; the reaction was linear for at least 30 min. The 14C-labelled metabolite(s) were extracted into 6 ml toluene by vortexing for 30 sec. After a brief centrifugation to separate the phases, 4 ml of the organic layer was added to 10 ml Econofluor (New England Nuclear), and the radioactivity monitored on a Nuclear Chicago Mark 1 Liquid Scintillation Counter. After correcting the samples for the blanks (parallel tubes stopped at zero time), MAO activity was expressed as nmoles product generated in 15 min. Protein was analyzed by the method of Bradford (1976) using bovine serum albumin as standard. The aliquot of homogenate (generally 30 t~l) was mixed with an equal volume of 1 N NaOH to solubilize the protein. Protein samples were contained in disposable microcentrifuge tubes (Cole-Palmer), frozen in ethanol-dry ice and stored at --16°C until analyzed.
3. Results Fig. 3A demonstrates both the potency and complexity of spiperone's action. A significant potentiation (44% over control) is apparent at 10 -14 M, with no significant change in effect up to 10 -11 M. Then a higher plateau (90% over control) is achieved at 10 -1°10 -9 M, followed thereafter by a significant
POTENTIATION OF DOPAMINE BY BUTYROPHENONES
167
220-
Dopamine • 2.3 x 10 7M
t
200-
'9
180 i
160 140 -
× 120~e 10080"
,ot 't,t'
60-"-r-% , 0
10 "14
I'
10 .12 10 m [ SPIPERONE] (M)
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T
i 160-
~6
'~ 140.
/ 1
~ 12o.
11,
/
1008010-8
10-e
60- ---I"~, 0
-
J.. ,~_~ ~'~-7 10" 10I~0[SPIPERONE 1 (M) during experiment
,'~ 10 -s
Fig. 3. (A) The effect on DA-induced secretion in vitro by spiperone at the indicated concentrations. The complex nature of this log dose-response curve (i.e., distinct plateaus between 10-14--10 -11 M and between 10 - 1 ° 10 -9 M, and the decrease in response at 10 -8 M) suggests multiple mechanisms of potentiation at different concentration ranges. Potentiation by spiperone is statistically significant at all concentrations except for 10 -1 s and 10 -13 M. (B) Reversibility of spiperone's potentiation following a 30-rain washout period according to the protocol described in Methods. Potentiation is reversible at concentrations of spiperone below 10-7 M.
reduction back to 44% potentiation at 10 -8 M. At y e t higher concentrations, there appears the suggestion of a renewed increase in potentiation, although large SE's a t t e s t to far higher variability (10 -s M). Fig. 3B illustrates the reversibility of spiperone's potentiation during the 3rd exposure to DA. Except when glands were exposed to micromolar levels of spiperone, return to control rates was essentially complete following the 30-min washout period. Although we have concentrated our attention on the potentiation b y b u t y r o p h e n o n e s on a near maximal concentration of DA (2.3 × 10 -7 M), in all cases the b u t y r o p h e n o n e s also potentiated secretion elicited by lower concentrations of DA (data n o t shown). Table 1 shows the results for 11 other b u t y r o p h e n o n e analogues assayed at 2 concentrations (10 -9 M and 10 -6 M) and for chlorpromazine (10 -6 M). Many of the drugs showed a potentiating effect at either or both concentrations assayed. In table 2 these drugs are listed in descending order o f magnitude o f response observed at each concentration. There are a variety of possible mechanisms w h e r e b y potentiation could be achieved. The drugs could interact directly with the receptor
to alter the DA-receptor kinetics or they could act on the tissue in any of a variety of ways at points distant from the receptor to amplify the secretory process. For example, tick tissues -- and particularly the salivary glands -- are a rich source o f M A d (Atkinson et al., 1974). We thus considered the possibility that b u t y r o p h e n o n e s might potentiate secretion b y virtue of inhibiting MAd. In fig. 4 we show that tranylcypromine is a potent, competitive inhibitor of M A d in broken cell preparations of salivary glands (Ki < 5 ~M). We further demonstrate that tranylcypromine on its own, at concentrations between 10 -6 and 10 -3 M, stimulates fluid secretion (fig. 5) and potentiates DA-induced fluid secretion (fig. 6). All these results might suggest that M A d plays a significant role in the termination of transmitter action in the tick salivary gland. If the forgoing is so, and if the main mechanism of b u t y r o p h e n o n e potentiation is inhibition of M A d , one should n o t expect spiperone to potentiate the action of any agonist which is n o t a substrate of MAd. Ergometrine is such an agonist on the tick salivary gland (Kaufman, 1977), and, as predicted, tranylcypromine (10 -4 M) did n o t potentiate ergometrine-induced fluid secre-
TABLE 1 E f f e c t o n d o p a m i n e - i n d u c e d fluid s e c r e t i o n b y 12 b u t y r o p h e n o n e c o n g e n e r s a n d c h l o r p r o m a z i n e . % Maximum response a m e a n _+ S.E.M. (n)
x- ~: - ~ - C ~ - C H 2 - % R1
Drug
X
R1
Concentration of butyrophenone
R2
10 -9 M Control (2.3 × 10 -7 M D A ) Spiperone
10 -6 M 80"+7 (10)b
0
F
O
150 "+ 12 (8)
130 "+ 16 (7)
134"+
161+31
V
-
Pimozide
-H
F
9(3)
(4)
¥ 0
Droperidol
F
O
(C)'~
91 "+ 13 (6)
142 -+ 13 (7)
90+21(6)
122-+
0
Benperidol
F
O
Haloperidol
F
O
Trifluperidol
F
O
II
-
"
5(8)
111 + 12 (5)
151 -+ 21 (7)
97 -+ 18 (6)
130 -+ 13 (6)
90 -+ 21 (6)
100 -+ 10 (6)
CF 3
" Bromperidol
F
Penfluridol
F
O
86-+ T
R 27275
F
O
R 1187
H
O
II
3 (3)
73"+
7 (8)
Clr3
172 "+ 17 (4)
-
"
~%c%
165-+10(2)
54 "+ 12 (3)
137-+
8(4)
_:-V COOCH2CH --v. 3 R 951
R 2308
Chlorpromazine
~ ~-/o'
0
t
-
~"~ -~"~ ~ - : ~
t.~
Ca2CH2Cn2N(C~3~ 2
179 -+ 25 (5)
124"+26(4)
91 "+ 10
(4)
138"+
7 (4)
74"+
2(4)
a 100% m a x i m u m is d e f i n e d as t h e r e s p o n s e t o 2.3 X 1 0 - 7 M D A d u r i n g first e x p o s u r e t o drugs (see M e t h o d s for protocol). b See M e t h o d s s e c t i o n a n d fig. 2 for e x p l a n a t i o n o f t h e c o n t r o l s in this e x p e r i m e n t .
POTENTIATION OF DOPAMINE BY BUTYROPHENONES
169
TABLE 2 Relative efficacy of butyrophenones as neuroleptic agents and as potentiators of DA-induced fluid secretion 1 in isolated tick salivary glands. Compounds bounded by brackets were equi-effective. Average clinical dose (rag/day) 2
Drug
Potentiation of DA-induced fluid secretion (data from table 1), descending order of efficacy 3,4
Concentration o f butyrophenone 10 -9 M 10 -6 M R951 Pimozide R27275 Haloperidol Rl187 Droperidol Spiperone [Rl187] Pimozide LR2308" R2308 [Spiperone | Haloperidol LTrifluperidolJ Trifluperidol Benperidol
<1 2 5 6 10 20
Spiperone Benperidol Trifluperidol Pimozide Droperidol Haloperidol Chlorpromazine
>500
Droperidol Benperidol Bromperidol Penfluridol
R951 Penfluridoi R27275
i i D A ] = 2.3 )< 1 0 - 7 M .
2 Data from Seeman (1977). 3 In this table, the term 'efficacy' refers only to the magnitude of response at the assayed concentrations. The true efficacy can only be determined from complete dose-response curves which are not y e t available. 4 Drugs appearing above the dashed line result in a statistically significant potentiation; drugs appearing below the dashed line do not.
15"
/ 10
/ I/V (nmoles/15 min)
~ -2'o
'
/
/
s
J
J 16 ,uM Tranylcypromine
2'o
4b
6'o
8'0
[TRYPTAMINE] 1 (pM) x 1000
Fig. 4. Competitive inhibition by tranylcypromine on MAC activity in crude homogenate of salivary gland. In this experiment, K M for substrate and K i for tranylcypromine were 57 pM and 3.6 #M respectively. In a separate experiment in which the concentration of tranylcypromine was 1.3 pM, the K M and K i were 25 pM and 1.3/2M respectively.
tion (unpublished data). Isolated salivary glands were thus treated to sequential expos u r e s o f e r g o m e t r i n e ( 1 0 -6 M ) i n a m a n n e r already described for previous experiments. During the second exposure to ergometrine, s p i p e r o n e w a s p r e s e n t a t 1 0 -9 M, a n d t h e resulting secretion was 134-+ 21% (mean-+ S . E . ; n = 4) t h a t o f t h e f i r s t e x p o s u r e t o e r g o m e t r i n e . I n t h e c o n t r o l (i.e., s p i p e r o n e n o t present during the second exposure to ergometrine) the resulting secretion was only 64 + 9% (n = 8) t h a t o f t h e f i r s t e x p o s u r e . T h e potentiation observed with spiperone was h i g h l y s i g n i f i c a n t (P = 0 . 0 0 5 ) as e s t a b l i s h e d by a pairs t-test on the absolute values. W e c o n s i d e r e d t h e a d e n y l a t e c y c l a s e syst e m as a p o s s i b l e s i t e o f b u t y r o p h e n o n e a c t i o n in t h i s t i s s u e , s i n c e t h e r e is m o u n t i n g evidence that the fluid secretory response to D A in t i c k s a l i v a r y g l a n d s is m e d i a t e d b y
170
D.L.-P. WONG, W.R. KAUFMAN
60"
120"
8
"X" 4
• Dopamine • D o p a m m e .10~4M
~°Q
Tranylcypromine
1oo
,~ 4 0 : v
,~
80"
¢~ z
60"
o
9* 9
6
z
o
(o ~ 20-
O3 6
x .<
7
40"
x
7
0 10 ~7
10 •
10-5
10-4
lO-a
ITRANYLCYPROMfNEI (M)
Fig. 5. The effect of reserpine on intrinsic activity of tranylcypromine on isolated salivary glands. (e) control glands (A) reserpinized. Ticks were injected with reserpine (140 p m o l / k g b.w.) according to the method of Kaufman et al. (1980) 17 h before dissecting out the glands. Below 10-3 M tranylcypromine, reserpine does not appear to attenuate the response, suggesting that tranylcypromine may act directly on the postjunctional receptor. At 10 -3 M, at least part of tranylcypromine's action appears to be mediated by endogenous catecholamine, since reserpine attenuates the response.
increased intracellular levels of cyclic AMP (Sauer et al., 1976; 1979; Needham and Sauer, 1979a, b; McMuUen and Sauer, 1978; McMuUen et al., 1980). We a t t e m p t e d first to elicit fluid secretion in vitro with exogenous cyclic AMP and to subsequently test whether butyrophenones could potentiate this response. But in 3 cases, the glands were unresponsive to sequential 15-min exposures of 10 -4, 10 -a and 10 -2 M cyclic AMP, and in 4 similar cases, glands were equally unresponsive to the same concentrations of dibutyryl cyclic AMP. In 4 further cases, glands were exposed to 5 × 10 -3 M dibutyryl cyclic AMP for 35 min followed by a 30-min exposure to the same medium plus 10 -9 M spiperone. In all experiments, the medium was changed for a fresh aliquot every 5 min. Although none of these 11 glands secreted saliva in response to the cyclic nucleotides, all of them were subsequently shown to be viable on exposure to 10 -6 M DA.
20-
0
==9
-,
lb-,
IOOPAMINEj(M)
lb-,
Fig. 6. The effect on DA-induced fluid secretion by 10 -4 M tranylcypromine. The potentiation by tranylcypromine was significant at the 3 concentrations of DA. * 0.05 > P > 0.01; ** P < 0.01.
4. Discussion Responses of tick salivary glands to the various drugs e m p l o y e d in this study and earlier (Kaufman, 1977) suggest that the site of action of butyrophenones is distinct from that of DA, and that both these sites have unusual properties when compared to those characteristic of dopaminergic systems from other species. That the b u t y r o p h e n o n e site is a distinct entity is suggested from the lack of competition between spiperone and DA. Despite the fact that spiperone possesses no intrinsic activity, it interacts with a concentration o f DA at least 7 orders o f magnitude greater (fig. 3). Although the concentration of DA employed in most of these experiments was about 80% maximal, we have observed equally impressive potentiations with concentrations of DA up to 10 -4 M (unpublished observations). Since butyrophenones are markedly lipidsoluble, they tend to accumulate in the lipid components of the cell membrane, and so it is important to consider that the drug concentration in the vicinity of the receptor sites may exceed the free concentration in the bathing medium even by many orders of magnitude (Seeman, 1977). There are a number of studies showing that apparent selectivity
POTENTIATION OF DOPAMINE BY BUTYROPHENONES of action among a series of pharmacological agents may be largely determined b y relative surface activities (Seeman and Bialy, 1963; Murphy and Zografi, 1970; Roufogalis, 1975). There is good reason to believe, however, that such effects are probably insignificant in the nanomolar and subnanomolar range (Seeman, 1977). In any case, the latter author presents octanol/water partition coefficients (P) for 5 of the drugs utilized in the present study. In ascending order of P they are: chlorpromazine, haloperidol, trifluperidol, pimozide and penfluridol. This ranking does n o t match with ascending order of b u t y r o p h e n o n e efficacy (at 10 -9 or 10 -6 M) which can be read from table 2. In addition to the likelihood that butyrophenones do n o t interact with the binding site for DA in this tissue, the data in table 2 further suggest that the b u t y r o p h e n o n e site itself is a receptor-type pharmacologically distinct from the site to which butyrophenones bind in the mammalian CNS. There is no correlation between efficacy as a potentiator in the tick salivary gland system and p o t e n c y as a neuroleptic (table 2) (although we recognize that the clinical dose range among these drugs spans only a single order of magnitude). Moreover, we regard it as most significant that R951, R 2 7 2 7 5 and R l 1 8 7 (all at 10 -9 M) were effective at potentiating fluid secretion despite the fact that these c o m p o u n d s all lack the basic structural requirements for neuroleptic activity (see Janssen, 1967). In contrast, droperidol, benperidol and bromperidol, all p o t e n t neuroleptic drugs, failed to potentiate fluid secretion significantly at 1 0 - 9 M (see tables). In summary, we propose that butyrophenones bind at some site in such a way as to amplify the DA-to-receptor interaction. Further research is necessary to elucidate the level at which this amplification occurs, although we propose that the mechanism seems unlikely to be intereference with DA-catabolism. A number of years ago, Barnett and Taber (1968) described a potentiation p h e n o m e n o n which to a certain degree was similar to the
171
one illustrated here. They demonstrated that some tricyclic antidepressants, some antihistamines and cocaine increased the maxim u m response of the isolated rat vas deferens to cumulative doses of noradrenaline. The observed increases in maximum response (up to 37%) were independent of other effects these drugs had on the noradrenaline dose-response curve; some shifted the curve to the left (e.g. cocaine), some to the right (e.g. phentolamine) and some produced no significant shift in the noradrenaline dose response curve (e.g. amitryptiline). Also the effect on the maxim u m response was reversed b y a washout and 10-min recovery time for most of the drugs, whereas the other effect (leftward or rightward shift in the dose-response curve) was not reversed so easily. They presented reasonable evidence that the mechanism o f potentiation was simply a reversal of the desensitization process of auto-inhibition. This explanation probably does not apply to the tick s',divary gland preparation. Although we have observed DA auto-inhibition at concentrations of 10 -s M and above (Wong and Kaufman, in preparation), we did n o t observe it in the submicromolar range (see legend to fig. 1). Bevan and Verity (1967) and Reiffenstein (1968) showed that cocaine similarly increased the maximal responses of, respectively, rabbit aorta strip and cat spleen strip to noradrenaline. Both authors have proposed that this potentiation cannot be due solely to the wellknown presynaptic effect of cocaine (i.e. inhibition of noradrenaline re-uptake), and that some direct action on the muscle cells is likely. Woodruff et al. (1970) demonstrated a marked potentiation (120-240%) b y ergometrine of the maximum response to noradrenaline (but n o t DA) on the guinea-pig isolated vas deferens. Although similar mechanisms may apply to the three abovementioned studies and to the b u t y r o p h e n o n e effect reported here, as yet there is no convincing explanation for the p h e n o m e n o n at the molecular or even cellular level.
172 Note added in proof Since this manuscript went to press, a paper by W. Brandt, M. Reiter and K. Seibel (Naunyn-Schmiedeberg's Arch. Pharmacol. 273 (1972): 2 9 4 - 3 0 6 ) h a s come to our attention. In guinea pig papillary muscle, 3 mM tyramine increases the maximal force of contraction induced by 0.01 mM noradrenaline by about 30%. The authors suggest that under normal circumstances, noradrenaline activates a 'cellular mechanism' which interferes with noradrenaline's inotropic action. Because of this mechanism, the maximal effect of noradrenaline is less than the inherent ability for the muscle to develop force. It was proposed t h a t tyramine's potentiation is a result of inhibiting noradrenaline's action on the aforementioned cellular mechanism.
Acknowledgements We are grateful to the following for generous gifts of drugs: Janssen Pharmaceutica, Beerse, Belgium (all the butyrophenone congenors), Ciba-Geigy AG, Basle (reserpine), Smith Kline and French Canada Ltd., Montreal (tranylcypromine). This study received generous financial support from NSERC Canada.
References Atkinson, P.W., K.C. Binnington and W.J. Roulston, 1974, High monoamine oxidase activity in the tick, Boophilus microplus and inhibition by Chlordimeform and related pesticides, J. Aust. Entomol. Soc. 13,207. Barnett, A.D.D., and R.I. Taber, 1968, A new type of drug enhancement: Increased maximum response to cumulative noradrenaline in the isolated rat vas deferens, Br. J. Pharmacol. Chemother. 3 3 , 1 7 1 . Bevan, J.A., and M.A. Verity, 1967, Sympathetic nerve-free vascular muscle, J. Pharmacol. Exp. Ther. 157, 117. 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. Harris, R.A. and W.R. Kaufman, 1981, Hormonal control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum, J. Insect Physiol. 2 7 , 2 4 1 . Janssen, P.A.J., 1967, Haloperidol and related butyrophenones, in: Psychopharmacological Agents, Vol. II, ed. Maxwell Gordon (Academic Press, New York and London) p. 199.
D.L.-P. WONG, W.R. KAUFMAN Kaufman, W., 1976, The influence of various factors on fluid secretion by in vitro salivary glands of ixodid ticks, J. Exp. Biol. 6 4 , 7 2 7 . Kaufman, W., 1977, The influence of adrenergic agonists and their antagonists on isolated salivary glands of ixodid ticks, Eur. J. Pharmacol. 45, 61. Kaufman, W.R., 1981, Fluid balance in ixodid ticks: Regulation of salivary gland secretion in vivo and in vitro, Presented to European Society for Comparative Physiology and Biochemistry, 2nd Meeting, 23-25 April 1980 (Elsevier, in press). Kaufman, W.R., A.A. Aeschlimann and P.A. Diehl, 1980, Regulation of body volume by salivation in a tick challenged with fluid loads, Am. J. Physiol. 238, R102. Kaufman, W.R. and S.F. Barnett, 1977, Dermacentor andersoni: Culture of whole salivary glands, Exp. Parasitol. 42, 106. Kaufman, W. and J.E. Phillips, 1973a, Ion and water balance in the ixodid tick Dermacentor andersoni. I. Routes of ion and water excretion, J. Exp. Biol. 58,523. Kaufman, W. and J.E. Phillips, 1973b, Ion and water balance in the ixodid tick Dermacentor andersoni. II. Mechanism and control of salivary secretion, J. Exp. Biol. 58,537. McMullen, H.L. and J.R. Sauer, 1978, The relationship of phosphodiesterase and cyclic AMP to the process of fluid secretion in the salivary glands of the ixodid tick Amblyomma americanum, Experientia 34, 1030. McMullen, H.L., J.R. Sauer, J.A. Bantle and R.C. Essenberg, 1980, Regulation of fluid secretion by calcium-dependent modulator proteins of 3':5'cyclic-AMP phosphodiesterase, Biochem. Biophys. Res. Commun. 95, 1555. Murphy, K.S. and Z. Zografi, 1970, Oil-wa~er partitioning of chlorpromazine and other phenothiazinc derivatives using dodecane and n-octanol, J. Pharm. Sci. 59, 1281. Needham, G.R. and J.R. Sauer, 1979a, Dynamics of calcium, catecholamines and cyclic AMP in control of salivary fluid secretion by female ixodid ticks, in: Recent Advances in Acarology, Vol. I., ed. J.G. Rodriguez (Academic Press, New York, San Francisco and London) p. 413. Needham, G.R. and J.R. Sauer, 1979b, Involvement of calcium and cyclic AMP in controlling ixodid tick salivary fluid secretion, J. Parasitol. 65, 531. Reiffenstein, R.J., 1968, Effects of cocaine on the rate of contraction to noradrenaline in the cat spleen strip: Mode of action of cocaine, Br. J. Pharmacol. Chemother. 32, 591. Roufogalis, B.D., 1975, Comparative studies on the membrane actions of depressant drugs: The role of lipophilicity in inhibition of brain sodium and potassium-stimulated ATPase, J. Neurochem. 24, 51.
POTENTIATION OF DOPAMINE BY BUTYROPHENONES Sauer, J., P.M. Mincolla and G.R. Needham, 1976, Adrenaline and cyclic AMP stimulated uptake of chloride and fluid secretion by isolated salivary glands of the lonestar tick, Comp. Biochem. Phys. iol. 53C, 63. Sauer, J.R., G.R. Needham, H.L. McMullen and R.D. Morrison, 1979, Cyclic nucleotides, calcium and salivary fluid secretion in ixodid ticks, in: Recent Advances in Acarology, Vol. I, ed. J.G. Rodriguez (Academic Press, New York, San Francisco and London) p. 365. Seeman, P., 1977, Anti-schizophrenic drags -- membrane receptor sites of action, Biochem. Pharmacol. 26, 1741.
173
Seeman, P.M. and H.S. Bialy, 1963, The surface activity of tranquilizers, Biochem. Pharmacol. 12, 1181. Tatchell, R.J., 1967, Salivary secretion in the cattle tick as a means of water elimination, Nature 213, 940. Woodruff, G.N., R.J. Walker and G.A. Kerkut, 1970, Actions of ergometrine on catecholamine receptors in the guinea-pig vas deferens and in the snail brain, Comp. General Pharmacol. 1, 54. Wurtman, R.J. and J. Axelrod, 1963, A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. Pharmacol. 12, 1439.
163
Elsevier/North-Holland Biomedical Press
POTENTIATION BY SPIPERONE AND O T H E R B U T Y R O P H E N O N E S OF F L U I D SECRETION BY ISOLATED S A L I V A R Y GLANDS O F IXODID TICKS DOROTHY L.-P. WONG and WILLIAM R. KAUFMAN *
Department of Zoology, University of Alberta, Edmonton, Canada T6G 2E9 Received 10 March 1981, accepted 7 May 1981
D.L.-P. WONG and W.R. KAUFMAN, Potentiation by spiperone and other butyrophenones of fluid secretion by isolated salivary glands ofixodid ticks, European J. Pharmacol. 73 (1981) 163--173. Isolated salivary glands from the ixodid tick, Amblyomma hebraeum Koch are stimulated to secrete fluid when exposed to dopamine (DA), the maximum response occurring at 10 -6 M. Spiperone, and a number of other butyrophenone derivatives, although lacking intrinsic activity, are able to potentiate the secretion elicited by supramaximal concentrations of DA; this potentiation by spiperone is evident at concentrations in the femtomolar range. Tranylcypromine, a potent, competitive inhibitor of monoamine oxidase (MAO) in tick salivary gland homogenates, has both intrinsic activity and potentiates DA-induced salivation. The fact that spiperone potentiates ergometrine-induced salivation indicates that the prime mechanism of the butyrophenone effect is not by inhibiting catecholamine catabolism. The results also suggest that the receptor for DA and that for butyrophenones are distinct sites. Droperidol, benperidol and bromperidol, all potent neuroleptic drugs, failed (at 10 -9 M) to potentiate salivation. By contrast, R951, R27275 and R l 1 8 7 (all at 10 -9 M) were very effective potentiators on the salivary gland system, despite the fact that they lack the basic structural requirements for neuroleptic activity. These results suggest that the butyrophenone site in tick salivary glands is different from butyrophenone binding sites in mammalian CNS. Salivary fluid secretion in vitro
Dopamine
Butyrophenone receptor
1. Introduction Female ticks (Acari) of the family Ixodidae employ their salivary glands as an osmo- and volume-regulatory system. During the taking of a blood meal, excess fluid is secreted directly back into the host's circulation (Tatchell, 1967; Kaufman and Phillips, 1973a; Kaufman et al., 1980). It is in this way that most of the pathogenic agents transmitted b y ticks gain access to the definitive host. As part of a program to learn more a b o u t the physiology of these important vectors of disease, we have developed an in vitro preparation of the salivary gland which enables one to critically evaluate the effects of drugs on salivary fluid secretion (Kaufman and Phillips, 1973b; Kaufrnan, 1976). These and other studies have taught us that salivation is under nervous * To whom
Spiperone
control, the transmitter substance probably being a catecholamine. The most p o t e n t agonists on this in vitro preparation are dopamine (DA) and some ergot alkaloids (Kaufman, 1977). Other drugs (apomorphine, N-methyldopamine) which often have high affinity for DA-receptors in mammalian systems are also agonists on tick salivary glands (Kaufman, 1 9 7 7 ) s u g g e s t i n g that the post-junctional receptor mediating fluid secretion in this tissue might also be a DA-receptor. It was thus contrary to expectation that a variety of drugs known to be p o t e n t inhibitors at mammalian central nervous system (CNS) and peripheral DA-receptors (chlorpromazine, ~-flupenthixol, spiperone, pimozide) failed to attenuate DA-effects in the tick system at reasonable concentrations; moreover, a number of usually effec-
correspondence should be addressed.
0014-2999/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
164 tive a-adrenolytic (phenoxybenzamine, phentolamine) and fl-adrenolytic (propanolol, dichloroisoprenaline) drugs were similarly unremarkable as putative inhibitors (Kaufman, 1977). Although spiperone and pimozide did n o t attenuate fluid secretion, they did have an unexpected action: both drugs augmented the tissue's response to DA even when the latter was present at supramaximal concentration. Yet neither spiperone nor pimozide" possessed intrinsic activity. In consideration of all the above-mentioned results, it has been proposed that the epithelium possesses a DA-receptor with hitherto undescribed properties (Kaufman, 1977, 1981). Because there appears to be b u t a passing reference in the literature to a b u t y r o p h e n o n e (haloperidol) occasionally eliciting such a potentiation at high concentrations (Woodruff et al., 1970), we considered it appropriate to pursue this phenomenon further. As will be shown below, the property is c o m m o n to a number of butyrophenone analogues and, to a first approximation at least, the property is unrelated to p o t e n c y of the congener as a neuroleptic. Molecular mechanism behind the synergistic action has n o t y e t been fully investigated, b u t it cannot be explained in terms of any efficacy these drugs may have as monoamine oxidase (MAO) inhibitors.
D.L.-P. WONG, W.R. KAUFMAN
2.2. The in vitro preparation Glands were dissected o u t and set up for observation exactly as described by Kaufman (1977). Briefly, partially fed ticks were removed from the host (rabbit) and dissected open in a dish filled with a slightly modified Hanks' balanced saline (composition in g/l: NaC1 11.5; D-glucose 1.6; KC1 0.4; CaCl: 0.14; MgSO4 0.1; KH2PO4 0.06; Na2HPO4 0.05; phenol red 0.01; 360 mosm; pH 7.0). The excised glands were introduced to a second petri dish lined with paraffin wax and filled with liquid paraffin. The system was arranged such that the acinar (secretory) portion of the gland could be bathed in a nutrient medium (TC 199) in isolation from the main duct. Appearance of fluid at the orifice o f the duct indicated that secretion had been stimulated by a drug. Since the secreted droplet assumed a spherical shape under the liquid paraffin, its volume (and hence the rate of secretion) could be determined from a simple calculation of the diameter as measured with an ocular micrometer fitted to a dissection microscope. Under these conditions isolated glands secrete spontaneously only rarely, b u t they are rapidly activated when the medium contains catecholamines. Further details are presented elsewhere (Kaufman and Phillips, 1973b; Kaufman, 1976, 1977).
2.3. Experimental protocol 2. Materials and m e t h o d s
2.1. Ticks The ticks used in this study were taken from a colony established here with specimens originating from the Veterinary Institute, Onderstepoort, Rep. South Africa. Ticks were first partially fed to 150-400 mg (unfed weights were approximately 20-30 mg) as described b y Kaufman and Phillips (1973a). This partial feeding is required to bring the salivary glands to a physiologically c o m p e t e n t state (Kaufman, 1976).
If glands are exposed to a maximal concentration of DA (10 -6 M; Kaufman, 1976) the full response is achieved within several minutes. To assay the influence of butyrophenones we first subjected each gland to three concentrations of DA (2.3 X 10 -8, 9 × 10 -8, 2 . 3 ) < 1 0 -7 M), equivalent respectively to a b o u t 18%, 50% and 80% maximal response (fig. 1), in the following manner. Glands were bathed for 60 sec in the lowest concentration, the medium was changed for a fresh aliquot o f the same solution, and the volume of saliva collected from the orifice over the succeeding 5 min was recorded. In a similar way, the vol-
P O T E N T I A T I O N O F D O P A M I N E BY B U T Y R O P H E N O N E S
100"
8
o~ S0" Z o 60'
== x
40.
20"
I -e
3 x 10 -e
1'0-;'
3 x 10-'
[DOPAMINE](M)
10 -e
3 x 10-s
Fig. !" Log d o s e - r e s p o n s e curve for D A o n isolated tick salivary glands. T h e curve was c o n s t r u c t e d as follows. Glands w e r e e x p o s e d to t h e t h r e e l o w e r conc e n t r a t i o n s o f D A (3 x 10 - s , 9.1 x 10 -8 , 2.3 x 10 -7 M) a c c o r d i n g t o t h e p r o t o c o l d e s c r i b e d in M e t h o d s ; t h e y were t h e n w a s h e d with TC 199 lacking D A until s e c r e t i o n s t o p p e d . T h e glands were t h e n e x p o s e d t o t h e t h r e e u p p e r c o n c e n t r a t i o n s (2.3 X 10 -7, 10 -6, 2.3 x 10 -6 M) in t h e same m a n n e r . Since each gland was e x p o s e d t o 2.3 x 10 -~ M D A first at t h e end o f a c u m u l a t i v e a d d i t i o n o f drug and t h e n as a single a d m i n i s t r a t i o n f o l l o w i n g a p e r i o d o f rest, o n e can estimate t h e degree o f d e s e n s i t i z a t i o n at this c o n c e n t r a t i o n a t t r i b u t a b l e to an e x t e n d e d p e r i o d o f e x p o sure to t h e drug. A b s o l u t e rates o f s e c r e t i o n resulting f r o m t h e t w o e x p o s u r e s were c o m p a r e d by a pairs t-test. R a t e o f s e c r e t i o n resulting f r o m the s e c o n d e x p o s u r e averaged 88 ± 11% ( m e a n +- S.E.M., n = 8) t h a t o f first e x p o s u r e (P > 0.05). In a s e p a r a t e series o f 8 glands, a similar analysis was c o n d u c t e d w i t h 3 x 10 -8 M D A as t h e middle c o n c e n t r a t i o n . R a t e o f s e c r e t i o n o n the s e c o n d e x p o s u r e to 3 x 10 - s M D A was 106+_ 10% (mean-+ S.E.M., n = 8) t h a t o f t h e first e x p o s u r e , this d i f f e r e n c e also being insignificant (P > 0.05 f r o m a pairs t-test o n the a b s o l u t e rates o f s e c r e t i o n ) . In t h e figure, we have p l o t t e d t h e m e a n values o f t h e 2 e x p o s u r e s . Finally, t h e r e is no significant d i f f e r e n c e ( S t u d e n t ' s t-test) b e t w e e n t h e perc e n t a g e s (88 -+ 11 a n d 106 +_ 10). In c o n c l u s i o n , t h e r e is n o e v i d e n c e for a u t o i n h i b i t i o n b y D A in t h e subm i c r o m o l a r range.
ume o f secretion resulting from a 5-min exposure to the middle and then the highest dose was recorded, in each case following a 60-sec pre-exposure. Glands were then washed for 30 min (a total of 6 changes of aliquot) in TC 199 lacking DA, by which time the glands were no longer secreting. Glands were then
165
treated, in the same manner as before, to the three doses of DA prepared in TC 199 containing a given concentration of butyrophenone. To test for reversibility o f butyrophenone action, a third exposure to DA was accomplished following a 30-min wash period in TC 199 lacking DA and butyrophenone. Fig. 2 (see also Kaufman, 1977) shows that there is a decay in response by glands subjected to 3 exposures to DA a l o n e - namely, at 2.3 X 10 -7 M, the second response is only 80% (on average) that of the first one and the third response is only 60%. Thus for statistical analysis, all responses to the butyrophenone exposure were compared to the control exposure normalized for this expected decay. (Incidentally, this decay in response appears to be an effect, direct or indirect, of exposure to drug per se, rather than of poor viability in the nutrient medium. Glands cultured in TC 199, but n o t treated with DA, secrete after 3 days at about 73% of the rate expected for non-cultured glands (Kaufman and Barnett, 1977)).
8
x
100-
l)11
80-
10
c~ vc~
~
9
80-
11 ~
4.0-
_~ 20-
11
x ~
0
lo •
3 ,~ l o - . 1'o, [ DOPAMINE ](M)
3 ~ lO,
Fig. 2. S e q u e n t i a l e x p o s u r e s t o 3 c o n c e n t r a t i o n s o f D A (3 x 10 - s , 9 . 1 x 10 -8 , 2 . 3 x 10 -7 M) w i t h 30m i n w a s h o u t s b e t w e e n each series. (o) first e x p o s u r e , (A) s e c o n d e x p o s u r e , ( a ) t h i r d e x p o s u r e . On average, t h e s e c o n d r e s p o n s e to 2.3 x 10 -7 M DA was 80 -+ 7% ( m e a n + S.E.M.; n = 10) and the t h i r d r e s p o n s e was 61 _+ 8% ( m e a n _+ S.E.M.; n = 9) t h a t o f t h e first r e s p o n s e . In all s u b s e q u e n t e x p e r i m e n t s , a c c o u n t was t a k e n o f this a n t i c i p a t e d d e c a y in r e s p o n s e w h e n perf o r m i n g statistical analysis.
166 2.4. Sources o f drugs and chemicals
All the butyrophenone analogues were the gift of Janssen Pharmaceutica, Beerse, Belgium: spiperone, pimozide, droperidol, benperidol, haloperidol, trifluperidol, bromperidol, penfluridol, R951, Rl187, R2308, and R27275. Chlorpromazine was the gift of SPECIA, Paris. Reserpine was the gift of CibaGeigy AG, Basle, Switzerland, and tranylcypromine the gift of Smith Kline and French Canada Ltd., Montreal. Ergonovine maleate, dopamine HC1, norepinephrine, adenosine 3',5'-cyclic monophosphoric acid (cyclic AMP), N6,O21 -dibutyryladenosine 3',5'-cyclic monophosphoric acid (monosodium salt) dihydrate (dibutyryl cyclic AMP) and tryptamine were purchased from Sigma Chemical Company, and TC 199 (with Hanks' salts and L-glutamine but without NaHCO3) was purchased from Gibco. Osmotic concentration of the TC 199 was increased to 360 mOsm by the addition of NaC1 (4.32 g/l). All other chemicals were of purest reagent grade (Fisher Scientific and BDH). Tryptamine bisuccinate [side chaln-2-14C], 51.5 mCi/mmol was pur: chased from New England Nuclear. 2.5. MA 0 assay
Salivary glands were dissected out of partially-fed ticks under Hanks' balanced saline and weighed as described by Harris and Kaufman (1981). Pairs of salivary glands were homogenized in 300-600 pl 1.45% KC1 (isosmotic with tick haemolymph) in small glass homogenizers (about 20 passes of the piston). After removal of an aliquot for protein determination, the homogenate was transferred to small plastic vials, frozen in ethanol-dry ice and stored at --16°C. The assay procedure was adapted from that of Wurtman and Axelrod (1963). The incubation mixture consisted of equal volumes (10 ~zl) of 4 stock solutions (1) 14C-labelled tryptamine (51.5 mCi/mmol) diluted 1 : 2 0 (v/v) with 0.5 M phosphate buffer, pH 7.5, (2) a solution consisting of variable concentrations
D.L.-P. WONG, W.R. KAUFMAN of non-radiolabeUed tryptamine (0-1000 I~M) in phosphate buffer, (3) as solution 2 with the addition of a given concentration of tranylcypromine, and (4) salivary gland crude homogenate. Thus the final incubation mixture contained 50 nCi tryptamine bisuccinate, total tryptamine concentration ranging from 9.5 to 510 pM and a variable concentration of test drug, all in 375 mM phosphate buffer, pH 7.5. Ten t~l each of solutions 1, 2 and 3 were pre-incubated for 5 min at 37°C in 15-ml glass-stoppered centrifuge tubes. Ten /~l of crude homogenate was added, and after a 15-min incubation, the reaction was stopped with 200 pl 2 N HC1; the reaction was linear for at least 30 min. The 14C-labelled metabolite(s) were extracted into 6 ml toluene by vortexing for 30 sec. After a brief centrifugation to separate the phases, 4 ml of the organic layer was added to 10 ml Econofluor (New England Nuclear), and the radioactivity monitored on a Nuclear Chicago Mark 1 Liquid Scintillation Counter. After correcting the samples for the blanks (parallel tubes stopped at zero time), MAO activity was expressed as nmoles product generated in 15 min. Protein was analyzed by the method of Bradford (1976) using bovine serum albumin as standard. The aliquot of homogenate (generally 30 t~l) was mixed with an equal volume of 1 N NaOH to solubilize the protein. Protein samples were contained in disposable microcentrifuge tubes (Cole-Palmer), frozen in ethanol-dry ice and stored at --16°C until analyzed.
3. Results Fig. 3A demonstrates both the potency and complexity of spiperone's action. A significant potentiation (44% over control) is apparent at 10 -14 M, with no significant change in effect up to 10 -11 M. Then a higher plateau (90% over control) is achieved at 10 -1°10 -9 M, followed thereafter by a significant
POTENTIATION OF DOPAMINE BY BUTYROPHENONES
167
220-
Dopamine • 2.3 x 10 7M
t
200-
'9
180 i
160 140 -
× 120~e 10080"
,ot 't,t'
60-"-r-% , 0
10 "14
I'
10 .12 10 m [ SPIPERONE] (M)
o "~ 180-
T
i 160-
~6
'~ 140.
/ 1
~ 12o.
11,
/
1008010-8
10-e
60- ---I"~, 0
-
J.. ,~_~ ~'~-7 10" 10I~0[SPIPERONE 1 (M) during experiment
,'~ 10 -s
Fig. 3. (A) The effect on DA-induced secretion in vitro by spiperone at the indicated concentrations. The complex nature of this log dose-response curve (i.e., distinct plateaus between 10-14--10 -11 M and between 10 - 1 ° 10 -9 M, and the decrease in response at 10 -8 M) suggests multiple mechanisms of potentiation at different concentration ranges. Potentiation by spiperone is statistically significant at all concentrations except for 10 -1 s and 10 -13 M. (B) Reversibility of spiperone's potentiation following a 30-rain washout period according to the protocol described in Methods. Potentiation is reversible at concentrations of spiperone below 10-7 M.
reduction back to 44% potentiation at 10 -8 M. At y e t higher concentrations, there appears the suggestion of a renewed increase in potentiation, although large SE's a t t e s t to far higher variability (10 -s M). Fig. 3B illustrates the reversibility of spiperone's potentiation during the 3rd exposure to DA. Except when glands were exposed to micromolar levels of spiperone, return to control rates was essentially complete following the 30-min washout period. Although we have concentrated our attention on the potentiation b y b u t y r o p h e n o n e s on a near maximal concentration of DA (2.3 × 10 -7 M), in all cases the b u t y r o p h e n o n e s also potentiated secretion elicited by lower concentrations of DA (data n o t shown). Table 1 shows the results for 11 other b u t y r o p h e n o n e analogues assayed at 2 concentrations (10 -9 M and 10 -6 M) and for chlorpromazine (10 -6 M). Many of the drugs showed a potentiating effect at either or both concentrations assayed. In table 2 these drugs are listed in descending order o f magnitude o f response observed at each concentration. There are a variety of possible mechanisms w h e r e b y potentiation could be achieved. The drugs could interact directly with the receptor
to alter the DA-receptor kinetics or they could act on the tissue in any of a variety of ways at points distant from the receptor to amplify the secretory process. For example, tick tissues -- and particularly the salivary glands -- are a rich source o f M A d (Atkinson et al., 1974). We thus considered the possibility that b u t y r o p h e n o n e s might potentiate secretion b y virtue of inhibiting MAd. In fig. 4 we show that tranylcypromine is a potent, competitive inhibitor of M A d in broken cell preparations of salivary glands (Ki < 5 ~M). We further demonstrate that tranylcypromine on its own, at concentrations between 10 -6 and 10 -3 M, stimulates fluid secretion (fig. 5) and potentiates DA-induced fluid secretion (fig. 6). All these results might suggest that M A d plays a significant role in the termination of transmitter action in the tick salivary gland. If the forgoing is so, and if the main mechanism of b u t y r o p h e n o n e potentiation is inhibition of M A d , one should n o t expect spiperone to potentiate the action of any agonist which is n o t a substrate of MAd. Ergometrine is such an agonist on the tick salivary gland (Kaufman, 1977), and, as predicted, tranylcypromine (10 -4 M) did n o t potentiate ergometrine-induced fluid secre-
TABLE 1 E f f e c t o n d o p a m i n e - i n d u c e d fluid s e c r e t i o n b y 12 b u t y r o p h e n o n e c o n g e n e r s a n d c h l o r p r o m a z i n e . % Maximum response a m e a n _+ S.E.M. (n)
x- ~: - ~ - C ~ - C H 2 - % R1
Drug
X
R1
Concentration of butyrophenone
R2
10 -9 M Control (2.3 × 10 -7 M D A ) Spiperone
10 -6 M 80"+7 (10)b
0
F
O
150 "+ 12 (8)
130 "+ 16 (7)
134"+
161+31
V
-
Pimozide
-H
F
9(3)
(4)
¥ 0
Droperidol
F
O
(C)'~
91 "+ 13 (6)
142 -+ 13 (7)
90+21(6)
122-+
0
Benperidol
F
O
Haloperidol
F
O
Trifluperidol
F
O
II
-
"
5(8)
111 + 12 (5)
151 -+ 21 (7)
97 -+ 18 (6)
130 -+ 13 (6)
90 -+ 21 (6)
100 -+ 10 (6)
CF 3
" Bromperidol
F
Penfluridol
F
O
86-+ T
R 27275
F
O
R 1187
H
O
II
3 (3)
73"+
7 (8)
Clr3
172 "+ 17 (4)
-
"
~%c%
165-+10(2)
54 "+ 12 (3)
137-+
8(4)
_:-V COOCH2CH --v. 3 R 951
R 2308
Chlorpromazine
~ ~-/o'
0
t
-
~"~ -~"~ ~ - : ~
t.~
Ca2CH2Cn2N(C~3~ 2
179 -+ 25 (5)
124"+26(4)
91 "+ 10
(4)
138"+
7 (4)
74"+
2(4)
a 100% m a x i m u m is d e f i n e d as t h e r e s p o n s e t o 2.3 X 1 0 - 7 M D A d u r i n g first e x p o s u r e t o drugs (see M e t h o d s for protocol). b See M e t h o d s s e c t i o n a n d fig. 2 for e x p l a n a t i o n o f t h e c o n t r o l s in this e x p e r i m e n t .
POTENTIATION OF DOPAMINE BY BUTYROPHENONES
169
TABLE 2 Relative efficacy of butyrophenones as neuroleptic agents and as potentiators of DA-induced fluid secretion 1 in isolated tick salivary glands. Compounds bounded by brackets were equi-effective. Average clinical dose (rag/day) 2
Drug
Potentiation of DA-induced fluid secretion (data from table 1), descending order of efficacy 3,4
Concentration o f butyrophenone 10 -9 M 10 -6 M R951 Pimozide R27275 Haloperidol Rl187 Droperidol Spiperone [Rl187] Pimozide LR2308" R2308 [Spiperone | Haloperidol LTrifluperidolJ Trifluperidol Benperidol
<1 2 5 6 10 20
Spiperone Benperidol Trifluperidol Pimozide Droperidol Haloperidol Chlorpromazine
>500
Droperidol Benperidol Bromperidol Penfluridol
R951 Penfluridoi R27275
i i D A ] = 2.3 )< 1 0 - 7 M .
2 Data from Seeman (1977). 3 In this table, the term 'efficacy' refers only to the magnitude of response at the assayed concentrations. The true efficacy can only be determined from complete dose-response curves which are not y e t available. 4 Drugs appearing above the dashed line result in a statistically significant potentiation; drugs appearing below the dashed line do not.
15"
/ 10
/ I/V (nmoles/15 min)
~ -2'o
'
/
/
s
J
J 16 ,uM Tranylcypromine
2'o
4b
6'o
8'0
[TRYPTAMINE] 1 (pM) x 1000
Fig. 4. Competitive inhibition by tranylcypromine on MAC activity in crude homogenate of salivary gland. In this experiment, K M for substrate and K i for tranylcypromine were 57 pM and 3.6 #M respectively. In a separate experiment in which the concentration of tranylcypromine was 1.3 pM, the K M and K i were 25 pM and 1.3/2M respectively.
tion (unpublished data). Isolated salivary glands were thus treated to sequential expos u r e s o f e r g o m e t r i n e ( 1 0 -6 M ) i n a m a n n e r already described for previous experiments. During the second exposure to ergometrine, s p i p e r o n e w a s p r e s e n t a t 1 0 -9 M, a n d t h e resulting secretion was 134-+ 21% (mean-+ S . E . ; n = 4) t h a t o f t h e f i r s t e x p o s u r e t o e r g o m e t r i n e . I n t h e c o n t r o l (i.e., s p i p e r o n e n o t present during the second exposure to ergometrine) the resulting secretion was only 64 + 9% (n = 8) t h a t o f t h e f i r s t e x p o s u r e . T h e potentiation observed with spiperone was h i g h l y s i g n i f i c a n t (P = 0 . 0 0 5 ) as e s t a b l i s h e d by a pairs t-test on the absolute values. W e c o n s i d e r e d t h e a d e n y l a t e c y c l a s e syst e m as a p o s s i b l e s i t e o f b u t y r o p h e n o n e a c t i o n in t h i s t i s s u e , s i n c e t h e r e is m o u n t i n g evidence that the fluid secretory response to D A in t i c k s a l i v a r y g l a n d s is m e d i a t e d b y
170
D.L.-P. WONG, W.R. KAUFMAN
60"
120"
8
"X" 4
• Dopamine • D o p a m m e .10~4M
~°Q
Tranylcypromine
1oo
,~ 4 0 : v
,~
80"
¢~ z
60"
o
9* 9
6
z
o
(o ~ 20-
O3 6
x .<
7
40"
x
7
0 10 ~7
10 •
10-5
10-4
lO-a
ITRANYLCYPROMfNEI (M)
Fig. 5. The effect of reserpine on intrinsic activity of tranylcypromine on isolated salivary glands. (e) control glands (A) reserpinized. Ticks were injected with reserpine (140 p m o l / k g b.w.) according to the method of Kaufman et al. (1980) 17 h before dissecting out the glands. Below 10-3 M tranylcypromine, reserpine does not appear to attenuate the response, suggesting that tranylcypromine may act directly on the postjunctional receptor. At 10 -3 M, at least part of tranylcypromine's action appears to be mediated by endogenous catecholamine, since reserpine attenuates the response.
increased intracellular levels of cyclic AMP (Sauer et al., 1976; 1979; Needham and Sauer, 1979a, b; McMuUen and Sauer, 1978; McMuUen et al., 1980). We a t t e m p t e d first to elicit fluid secretion in vitro with exogenous cyclic AMP and to subsequently test whether butyrophenones could potentiate this response. But in 3 cases, the glands were unresponsive to sequential 15-min exposures of 10 -4, 10 -a and 10 -2 M cyclic AMP, and in 4 similar cases, glands were equally unresponsive to the same concentrations of dibutyryl cyclic AMP. In 4 further cases, glands were exposed to 5 × 10 -3 M dibutyryl cyclic AMP for 35 min followed by a 30-min exposure to the same medium plus 10 -9 M spiperone. In all experiments, the medium was changed for a fresh aliquot every 5 min. Although none of these 11 glands secreted saliva in response to the cyclic nucleotides, all of them were subsequently shown to be viable on exposure to 10 -6 M DA.
20-
0
==9
-,
lb-,
IOOPAMINEj(M)
lb-,
Fig. 6. The effect on DA-induced fluid secretion by 10 -4 M tranylcypromine. The potentiation by tranylcypromine was significant at the 3 concentrations of DA. * 0.05 > P > 0.01; ** P < 0.01.
4. Discussion Responses of tick salivary glands to the various drugs e m p l o y e d in this study and earlier (Kaufman, 1977) suggest that the site of action of butyrophenones is distinct from that of DA, and that both these sites have unusual properties when compared to those characteristic of dopaminergic systems from other species. That the b u t y r o p h e n o n e site is a distinct entity is suggested from the lack of competition between spiperone and DA. Despite the fact that spiperone possesses no intrinsic activity, it interacts with a concentration o f DA at least 7 orders o f magnitude greater (fig. 3). Although the concentration of DA employed in most of these experiments was about 80% maximal, we have observed equally impressive potentiations with concentrations of DA up to 10 -4 M (unpublished observations). Since butyrophenones are markedly lipidsoluble, they tend to accumulate in the lipid components of the cell membrane, and so it is important to consider that the drug concentration in the vicinity of the receptor sites may exceed the free concentration in the bathing medium even by many orders of magnitude (Seeman, 1977). There are a number of studies showing that apparent selectivity
POTENTIATION OF DOPAMINE BY BUTYROPHENONES of action among a series of pharmacological agents may be largely determined b y relative surface activities (Seeman and Bialy, 1963; Murphy and Zografi, 1970; Roufogalis, 1975). There is good reason to believe, however, that such effects are probably insignificant in the nanomolar and subnanomolar range (Seeman, 1977). In any case, the latter author presents octanol/water partition coefficients (P) for 5 of the drugs utilized in the present study. In ascending order of P they are: chlorpromazine, haloperidol, trifluperidol, pimozide and penfluridol. This ranking does n o t match with ascending order of b u t y r o p h e n o n e efficacy (at 10 -9 or 10 -6 M) which can be read from table 2. In addition to the likelihood that butyrophenones do n o t interact with the binding site for DA in this tissue, the data in table 2 further suggest that the b u t y r o p h e n o n e site itself is a receptor-type pharmacologically distinct from the site to which butyrophenones bind in the mammalian CNS. There is no correlation between efficacy as a potentiator in the tick salivary gland system and p o t e n c y as a neuroleptic (table 2) (although we recognize that the clinical dose range among these drugs spans only a single order of magnitude). Moreover, we regard it as most significant that R951, R 2 7 2 7 5 and R l 1 8 7 (all at 10 -9 M) were effective at potentiating fluid secretion despite the fact that these c o m p o u n d s all lack the basic structural requirements for neuroleptic activity (see Janssen, 1967). In contrast, droperidol, benperidol and bromperidol, all p o t e n t neuroleptic drugs, failed to potentiate fluid secretion significantly at 1 0 - 9 M (see tables). In summary, we propose that butyrophenones bind at some site in such a way as to amplify the DA-to-receptor interaction. Further research is necessary to elucidate the level at which this amplification occurs, although we propose that the mechanism seems unlikely to be intereference with DA-catabolism. A number of years ago, Barnett and Taber (1968) described a potentiation p h e n o m e n o n which to a certain degree was similar to the
171
one illustrated here. They demonstrated that some tricyclic antidepressants, some antihistamines and cocaine increased the maxim u m response of the isolated rat vas deferens to cumulative doses of noradrenaline. The observed increases in maximum response (up to 37%) were independent of other effects these drugs had on the noradrenaline dose-response curve; some shifted the curve to the left (e.g. cocaine), some to the right (e.g. phentolamine) and some produced no significant shift in the noradrenaline dose response curve (e.g. amitryptiline). Also the effect on the maxim u m response was reversed b y a washout and 10-min recovery time for most of the drugs, whereas the other effect (leftward or rightward shift in the dose-response curve) was not reversed so easily. They presented reasonable evidence that the mechanism o f potentiation was simply a reversal of the desensitization process of auto-inhibition. This explanation probably does not apply to the tick s',divary gland preparation. Although we have observed DA auto-inhibition at concentrations of 10 -s M and above (Wong and Kaufman, in preparation), we did n o t observe it in the submicromolar range (see legend to fig. 1). Bevan and Verity (1967) and Reiffenstein (1968) showed that cocaine similarly increased the maximal responses of, respectively, rabbit aorta strip and cat spleen strip to noradrenaline. Both authors have proposed that this potentiation cannot be due solely to the wellknown presynaptic effect of cocaine (i.e. inhibition of noradrenaline re-uptake), and that some direct action on the muscle cells is likely. Woodruff et al. (1970) demonstrated a marked potentiation (120-240%) b y ergometrine of the maximum response to noradrenaline (but n o t DA) on the guinea-pig isolated vas deferens. Although similar mechanisms may apply to the three abovementioned studies and to the b u t y r o p h e n o n e effect reported here, as yet there is no convincing explanation for the p h e n o m e n o n at the molecular or even cellular level.
172 Note added in proof Since this manuscript went to press, a paper by W. Brandt, M. Reiter and K. Seibel (Naunyn-Schmiedeberg's Arch. Pharmacol. 273 (1972): 2 9 4 - 3 0 6 ) h a s come to our attention. In guinea pig papillary muscle, 3 mM tyramine increases the maximal force of contraction induced by 0.01 mM noradrenaline by about 30%. The authors suggest that under normal circumstances, noradrenaline activates a 'cellular mechanism' which interferes with noradrenaline's inotropic action. Because of this mechanism, the maximal effect of noradrenaline is less than the inherent ability for the muscle to develop force. It was proposed t h a t tyramine's potentiation is a result of inhibiting noradrenaline's action on the aforementioned cellular mechanism.
Acknowledgements We are grateful to the following for generous gifts of drugs: Janssen Pharmaceutica, Beerse, Belgium (all the butyrophenone congenors), Ciba-Geigy AG, Basle (reserpine), Smith Kline and French Canada Ltd., Montreal (tranylcypromine). This study received generous financial support from NSERC Canada.
References Atkinson, P.W., K.C. Binnington and W.J. Roulston, 1974, High monoamine oxidase activity in the tick, Boophilus microplus and inhibition by Chlordimeform and related pesticides, J. Aust. Entomol. Soc. 13,207. Barnett, A.D.D., and R.I. Taber, 1968, A new type of drug enhancement: Increased maximum response to cumulative noradrenaline in the isolated rat vas deferens, Br. J. Pharmacol. Chemother. 3 3 , 1 7 1 . Bevan, J.A., and M.A. Verity, 1967, Sympathetic nerve-free vascular muscle, J. Pharmacol. Exp. Ther. 157, 117. 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. Harris, R.A. and W.R. Kaufman, 1981, Hormonal control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum, J. Insect Physiol. 2 7 , 2 4 1 . Janssen, P.A.J., 1967, Haloperidol and related butyrophenones, in: Psychopharmacological Agents, Vol. II, ed. Maxwell Gordon (Academic Press, New York and London) p. 199.
D.L.-P. WONG, W.R. KAUFMAN Kaufman, W., 1976, The influence of various factors on fluid secretion by in vitro salivary glands of ixodid ticks, J. Exp. Biol. 6 4 , 7 2 7 . Kaufman, W., 1977, The influence of adrenergic agonists and their antagonists on isolated salivary glands of ixodid ticks, Eur. J. Pharmacol. 45, 61. Kaufman, W.R., 1981, Fluid balance in ixodid ticks: Regulation of salivary gland secretion in vivo and in vitro, Presented to European Society for Comparative Physiology and Biochemistry, 2nd Meeting, 23-25 April 1980 (Elsevier, in press). Kaufman, W.R., A.A. Aeschlimann and P.A. Diehl, 1980, Regulation of body volume by salivation in a tick challenged with fluid loads, Am. J. Physiol. 238, R102. Kaufman, W.R. and S.F. Barnett, 1977, Dermacentor andersoni: Culture of whole salivary glands, Exp. Parasitol. 42, 106. Kaufman, W. and J.E. Phillips, 1973a, Ion and water balance in the ixodid tick Dermacentor andersoni. I. Routes of ion and water excretion, J. Exp. Biol. 58,523. Kaufman, W. and J.E. Phillips, 1973b, Ion and water balance in the ixodid tick Dermacentor andersoni. II. Mechanism and control of salivary secretion, J. Exp. Biol. 58,537. McMullen, H.L. and J.R. Sauer, 1978, The relationship of phosphodiesterase and cyclic AMP to the process of fluid secretion in the salivary glands of the ixodid tick Amblyomma americanum, Experientia 34, 1030. McMullen, H.L., J.R. Sauer, J.A. Bantle and R.C. Essenberg, 1980, Regulation of fluid secretion by calcium-dependent modulator proteins of 3':5'cyclic-AMP phosphodiesterase, Biochem. Biophys. Res. Commun. 95, 1555. Murphy, K.S. and Z. Zografi, 1970, Oil-wa~er partitioning of chlorpromazine and other phenothiazinc derivatives using dodecane and n-octanol, J. Pharm. Sci. 59, 1281. Needham, G.R. and J.R. Sauer, 1979a, Dynamics of calcium, catecholamines and cyclic AMP in control of salivary fluid secretion by female ixodid ticks, in: Recent Advances in Acarology, Vol. I., ed. J.G. Rodriguez (Academic Press, New York, San Francisco and London) p. 413. Needham, G.R. and J.R. Sauer, 1979b, Involvement of calcium and cyclic AMP in controlling ixodid tick salivary fluid secretion, J. Parasitol. 65, 531. Reiffenstein, R.J., 1968, Effects of cocaine on the rate of contraction to noradrenaline in the cat spleen strip: Mode of action of cocaine, Br. J. Pharmacol. Chemother. 32, 591. Roufogalis, B.D., 1975, Comparative studies on the membrane actions of depressant drugs: The role of lipophilicity in inhibition of brain sodium and potassium-stimulated ATPase, J. Neurochem. 24, 51.
POTENTIATION OF DOPAMINE BY BUTYROPHENONES Sauer, J., P.M. Mincolla and G.R. Needham, 1976, Adrenaline and cyclic AMP stimulated uptake of chloride and fluid secretion by isolated salivary glands of the lonestar tick, Comp. Biochem. Phys. iol. 53C, 63. Sauer, J.R., G.R. Needham, H.L. McMullen and R.D. Morrison, 1979, Cyclic nucleotides, calcium and salivary fluid secretion in ixodid ticks, in: Recent Advances in Acarology, Vol. I, ed. J.G. Rodriguez (Academic Press, New York, San Francisco and London) p. 365. Seeman, P., 1977, Anti-schizophrenic drags -- membrane receptor sites of action, Biochem. Pharmacol. 26, 1741.
173
Seeman, P.M. and H.S. Bialy, 1963, The surface activity of tranquilizers, Biochem. Pharmacol. 12, 1181. Tatchell, R.J., 1967, Salivary secretion in the cattle tick as a means of water elimination, Nature 213, 940. Woodruff, G.N., R.J. Walker and G.A. Kerkut, 1970, Actions of ergometrine on catecholamine receptors in the guinea-pig vas deferens and in the snail brain, Comp. General Pharmacol. 1, 54. Wurtman, R.J. and J. Axelrod, 1963, A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. Pharmacol. 12, 1439.