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Effect of thiorphan on response of the guinea-pig gallbladder to tachykinins
European Journal of Pharmacology, 165 (1989) 51-61
51
Elsevier EJP 50786
Effect of thiorphan on response of the guinea-pig gallbladder to tachykinins Carlo Alberto Maggi *, Riccardo Patacchini, Daniela Renzi 1, Paolo Santicioli, Domenico Regoli 2, Paolo Rovero 3, G u y Drapeau 2, Calogero Surrenti 1 and Alberto Meli Pharmacology Department, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, Florence, 1Department of Clinical Pathophysiology, Gastroenterology Unit, University of Florence, Italy, 2 Department of Physiology and Pharmacology Sherbrooke University, Canada and 3 Chemistry Department, A. Menarini Pharmaceuticals, Florence, Italy
Received 12 September 1988, revised MS received 6 February 1989, accepted 21 February 1989
Tachykinins produced a concentration-related contraction of the isolated guinea-pig gallbladder, with a rank order of potency neurokinin A (NKA) > Arg-neurokinin B = neurokinin B (NKB) > substance P (SP). Only the effect of SP was potentiated by thiorphan (0.1-10 #M). A significant enhancement of the response to SP was also produced by captopril (1 #M). [Nlel°]NKA-(4-10) and [fl-AlaS]NKA-(4-10), selective NK-2 receptor agonists, were active, whereas [Pro9]Sp sulfone (selective NK-1 agonist) was almost ineffective. [MePhe7]NKB (selective NK-3 agonist) had some activity but only at high concentrations. Septide was almost ineffective and DiMeC 7 had an action comparable to that of [MePhe7]NKB. None of the effects induced by these synthetic tachykinin analogs were significantly potentiated by thiorphan. Capsaicin (10 #M) produced a contraction which was unaffected by thiorphan. Both capsaicin and NKA-induced contractions were antagonized by Spantide at concentrations (5-10/~M) which had no effect against the atropine-sensitive contractions produced by electrical field stimulation. Capsaicin (1 #M) produced a consistent release of SP-like immunoreactivity (SP-LI) and a second application of the drug had no further effect, indicating complete desensitization. SP-LI release by capsaicin was almost doubled in the presence of thiorphan. These findings indicate that NK-2 and possibly some NK-3 receptors mediate the contractile response of the guinea-pig gallbladder to tachykinins. Both exogenous and endogenous (released by capsaicin) SP were degraded to a significant extent in this organ via a thiorphan-sensitive mechanism, the identity of which remains to be established. Gallbladder; Tachykinins; Capsaicin; Substance P; Neurokinin A; Tachykinin receptors; Thiorphan; Captopril; (Guinea-pig)
1. Introduction
Tachykinins have a potent contractile effect in various sections of the gastrointestinal tract where
they may play a role as neurotransmitters (see Barth6 and Holzer, 1985, for review). There seem
to be two major sources of these peptides in the gastrointestinal tract of rats and guinea-pigs: intrinsic neurons of the myenteric plexus and pe-
* To whom all correspondence should be addressed at A. Menarini Pharmaceuticals.
ripheral endings of capsaicin-sensitive sensory nerves (Barth6 and Holzer, 1985). Less information is available about the role of tachykinins in regulating the motility of the biliary tract (Meldrum et al., 1987; Shook and Burks, 1987). Both anatomical and biochemical evidence indicates the txistence of substance P-like immunoreactivity (SP-LI) in nerve fibers of the guinea-pig gallbladder (Cai et al., 1983; Bucsics et al., 1983; Franco-Cereceda et al., 1987). As in the intestine, the tachykinin-like immunoreactive material in the biliary tract is, at least in part, stored in capsaicin-sensitive primary afferents
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
52 (Franco-Cereceda et al., 1987; Maggi et al., in press) and the release of endogenous tachykinins from peripheral terminals of primary afferents seems to be involved in capsaicin-induced contractions and plasma extravasation at this level (Lundberg et al., 1984; Maggi et al., in press). The aim of this study was to characterize the tachykinin receptor mediating the contraction of the isolated guinea-pig gallbladder (Shook and Burks, 1987) by using several synthetic agonists which display improved selectivity a n d / o r metabolic stability compared to that of natural tachykinins. Moreover, we looked at the possible influence of catabolism by endopeptidases on the response to exogenous and endogenous (SP-LI release by capsaicin) tachykinins by studying the effect of thiorphan, an inhibitor of 'enkephalinase' (see Schwartz et al., 1985 for review). Thiorphan has been shown to reduce the metabolism of tachykinins (Turner, 1987) and to markedly potentiate the effect of these peptides on the respiratory tract (Sekizawa et al., 1987a,b). Metabolic inactivation by tissue enzymes has been shown to interfere with the characterization of tachykinin receptors in mammalian tissues (Ireland et al., 1988; Stephens-Smith et al., 1988; Fox et al., 1988; Devillier et al., 1988). 2. Materials and methods
2.1. Functional experiments Male albino guinea-pigs weighing 240-260 g were stunned and bled. The whole gallbladder was rapidly removed and placed in oxygenated (96% 0 2 and 4% COz) Krebs solution at 37°C. For functional experiments, each organ was divided in two halves along the longitudinal axis and mounted in 5 ml organ baths to record tension isometrically. Each strip was suspended under a resting tension of 15 mN and tension was recorded by means of an isometric transducer connected to a Basile 7050 Unirecord. The strips were fieldstimulated by means of two platinum wire electrodes placed at the top and bottom of the organ bath and connected to a GRASS S l l stimulator. Each experiment began after a 1 h equilibration period. Cumulative concentration-response curves
for tachykinins were made, each concentration being added when the effect of the preceding one had reached steady state. Preliminary experiments indicated that there was negligible desensitization to the effect of tachykinins since responses of similar amplitude were obtained when 1 ~M neurokinin A was added to the bath before and at the end of a cumulative curve. The contractile response to tachykinins or capsaicin was expressed as a percentage of the response to KCI (80 mM, added to the bath).
2.2. Radioimmunoassay (RIA) To evaluate SP-LI release, the gallbladders were trimmed and placed in 2 ml organ baths at 37 °C and superfused (2 m l / m i n ) with oxygenated (96% 02 and 4% CO2) Krebs solution containing 0.1% bovine albumin and 0.003% bacitracin (Sigma). A 60 min equilibration period was allowed before drug administration. Fractions (4 ml) were collected every 2 min in tubes containing acetic acid to a final concentration of 2 N. At the end of the experiments, the tissues were blotted 2-3 times on filter paper and weighed. The superfusates were freeze-dried, reconstituted in the assay buffer (0.1 M, p H 7.4 phosphate buffer containing 0.9% NaC1, 0.01% N a N 3 and 0.1% bovine serum albumin) and used for RIA. SP-LI was measured as reported previously (Maggi et al., 1988b) with anti SP 144 serum which crossreacts 1% with neurokinin A, 0.5% with neurokinin B and less than 0.1% with physalaemin and eledoisin. SP-LI release by capsaicin (1 ~tM) was studied in the absence or presence of thiorphan (10 ~M).
2.3. Statistical analys& All values in the text, figures and tables are m e a n s _ S.E. Statistical analysis was performed by means of Student's t-test for paired or unpaired data or by an analysis of variance, when applicable. Regression analysis was performed by means of the least-squares method. The ECs0 values and 95% confidence limits (c.1.) were calculated accordingly. Statistical analysis of the differences between the maximum effects produced by the various
53
peptides (expressed as % of the response to KCl) was made with an analysis of variance. NKA was found to produce maximal responses both in the absence and presence of &orphan. For each
GAkPJ
NKA
peptide, lated as response presence
the ED,, values and 95% c.1. were calcuthe concentration producing 50% of the to NKA, either in the absence or the of thiorphan.
(4-10)
NKA
Control
SP Arg - NKB
60-
40-
k?
20-
9 ,o S 5 a c L % 0 2
OI
9 loo-
8
7
6
5
I 4
In the presence of thiorphon (10 J,IM1
80-
60-
40-
20-
o9
8
7 -Log
6 (M)
5
4
Fig. 1. inundation-resins curves showing the contractile response of the isolated guinea-pig gallbladder to tacbylcinins and tachykinin-related peptides in the absence (upper panel) and presence (lower panel) of thiorphan (10 +4) given 15 rnin before the peptides. Each point is the mean f SE. of at least 4 experiments.
54
2.4. Drugs
The drugs used were: capsaicin and thiorphan (Sigma), atropine HC1 and isoprenaline HCI (Serva), neurokinin A (NKA), neurokinin B (NKB), Spantide, substance P and DiMeC 7 (Peninsula), captopril (Squibb), Septide (Bachem). Arginine-NKB (Arg-NKB) and [fl-AlaS]NKA-(410), a newly developed selective NK-2 receptor agonist (Rovero et al., in press), were synthesized at Menarini Labs. by Dr P. Rovero. [Prog]sP sulfone, [Nlel°]NKA-(4-10) and [MePheT]NKB were synthesized at the Department of Physiology and Pharmacology of the University of Sherbrooke, Canada. The anti SP serum was a kind gift of Dr. P. Pradelles, SPI-LERI, Gif-surYvette, France.
3. Results
3.1. Functional experiments: general
Nearly 50% of the gallbladder strips were mechanically quiescent. Spontaneous activity, which had a low amplitude ( < 0.1 mN) and a frequency of 0.3-0.5/min, was observed in the other strips. Exposure to KC1 (80 mM added to the bath) produced a prompt contractile response (18 + 2 mN, n = 36), which reached maximum within 10-20 min, while isoprenaline (3/~M) pro-
duced a prompt relaxation and the disappearance of the spontaneous activity (n = 4). Field stimulation (0.5 ms, 60 V, 1-50 Hz, train duration 5 s) produced frequency-related contractions which were mainly (> 80%) atropine (3 ~M)-sensitive (n = 4). 3.2. Motor response to tachykinins m the absence and presence of thiorphan
NKA was the most potent and effective of the mammalian tachykinins tested (fig. 1, table 1). We studied the effect of NKB dissolved in sulfolane (n = 3) in some experiments. The activity found with the natural peptide was comparable to that found with Arg-NKB, the water soluble derivative developed by Regoli et al. (1986); these authors showed that Arg-NKB had the same activity spectrum as natural NKB. Therefore, data from experiments with Arg-NKB are presented. The following order of potency for the tachykinins was found in the absence of thiorphan: NKA (211) > Arg-NBK (8)> SP (1), a pattern which suggests that NK-2 receptors are involved. It should be noted that, at a high concentration (30 ~M), the response to SP was of nearly the same magnitude as that to N K A (about 70% of the response to KC1) (fig. 1). In contrast, the maximal response to N K A was significantly higher than that to ArgNKB (table 1), which, at 10 /~M, did not exceed 40% of the response to KC1 (fig. 1).
TABLE 1 Contractile effect of some tachykirtins and tachykinin-related peptides on the isolated guinea-pig gallbladder in the absence or presence of thiorphan. Peptide
NKA SP Arg NKB [Nle 1° ]NKA-(4-10) [ fl-Ala 8 ]NKA-(4-10) [Pro 9 ]SP sulfone [MePheV]NKB Septide DiM eC 7
Normal Krebs ECs0 (nM)
95% c.l. (nM)
44 9309 1203 669 38 ND 18454 ND 26269
27-86 4413-26260 131-4463 578-779 25-67 ND 2346-64554 ND 1864-37151
In the presence of thiorphan (10 # M ) R.P. 211 1 8 14 245 ND 0.50 ND 0.3
Em~x (% of KC1)
ECs0 (nM)
95% c.1. (nM)
68 + 13 70+6 37 ± 6 a 48 _+7 70 + 9 3 -+ 3 a 40 + 10 a 4 -+ 3 a 40 5=2 a
66 1164 1368 559 33 NT 26897 ND 31154
46-102 925-1511 929-2596 209-2326 22-57 NT 8079-52870 ND 5680-52720
R.P. 18 1 0.85 2 35 NT 0.04 ND 0.04
Emax (% of KC1) 85 -+ 16 90_+7 42 + 5 a 60 _+12 75 + 8 NT 45 5=8 a 3 -+ 3 a 40 _+2
a Significantly different from the effect of N K A P < 0.05; c.1. = confidence limits; R.P. = relative potency; N D = not determined; N T = not tested.
55
5 min
j
G E
t
0.03
t t
t
t
t t
0.1 0.3 I 3 SuDstonce P (pM)
0.03
10
t
t
t t
O.l 0.3 I 3 SuOstance P (~M)
t
10
J~
Thiorphan 10/JM
E ×
t
0.003
t
tt
t t t
OO1 0.03 O.1 0.3 1 Neurokinin A (IJM)
t
3
t
t
t
t
0.003 0.O1 0.03 O.1 0 3 Neurokinin A (/JM)
t
1
t
3
t
10
/l
Thiorphan 10 ~ M
Fig. 2. Typical tracings showing the effect of thiorphan on the contractile response to substance P and neurokinin A in the isolated guinea-pig gallbladder. The effects of the two tachykinins were investigated on two halves of the gallbladder from the same animal.
To investigate the possible involvement of peptide catabolism by 'enkephalinase', we studied the effects of the tachykinins in the presence of thiorphan (10 /~M, 15 min before, fig. 1 lower panel, table 1). Thiorphan alone produced a small contraction of the isolated guinea-pig gallbladder in about 70% of the preparations. When present, the small contractile effect of thiorphan faded during the incubation period and, in most instances, tension returned to the baseline value before the peptides were added. Of the mammalian tachykinins tested, only the effect of SP was significantly potentiated by thiorphan (figs. 2 and 3, table 1). The potency of SP was increased by thiorphan by about ten-fold (fig. 2, table 1). In contrast, the potency of N K A and Arg-NKB (n = 5) was almost unaffected by thiorphan (fig. 1, table 1) and the same was true for NKB dissolved in sulfolane (n = 3, data not shown). In fig. 3, typical tracings show the effect of thiorphan on
the response to SP and N K A on two halves of the gallbladder from the same animal. The order of potency for the mammalian tachykinins was the same in the presence and absence of thiorphan; however, there was a quantitative difference, N K A was only 18 times more potent than SP (table 1). Further, Arg-NKB was slightly less active than SP in the presence of thiorphan (table 1). To further characterize the receptor responsible for the contractile response of the guinea-pig gallbladder to the tachykinins, we investigated the action of synthetic peptides (fig. 1, table 1) selective for the NK-1, NK-2 and NK-3 receptors, namely [Pro9]Sp sulfone, [Nlel°]NKA-(4-10) and [MePheV]NKB, respectively (Drapeau et al., 1987). [Nlel°]NKA-(4-10) was the most active of these peptides, being about 16 and 9 times less potent than N K A in the absence and presence of thiorphan, respectively. The maximum response to [Nlel°]NKA-(4-10) was slightly lower than that to
56
100
100,
8O
80
6O
6o
t hiorphan 10 n M / /
4O
/~/
3.3. Effect of Septide and DiMeC:, *"
/. ~/
th]orphan 0.1 ~ , ~ /
40-
¢
20 ~ _ _ ~ :
control
OJ
ntrol
0 8
7
6
5
4
8
7
6
5
4
o
$ lOO
100
m 80
80 60
captopril 1 pM
7
40
40
*
/
20
20
thiorphan . / 10 FM 7 / / ¢
~d 6o
0 8
•
6
5
0 4 8 7 Substance P (- Log M)
/o
ol 6
5
4
Fig. 3. Enhancement by thiorphan (10 nM to 10 /~M, 15 min before) or captopril (1 ixM, 15 rain before) of the responses induced by SP. In these experiments a control curve for SP was made before (empty circles) and after application of thiorphan or captopril (filled circles). Each point is the mean_+S.E. of 5 experiments. * Significantly different from control, P < 0.05.
N K A (both in the presence and absence of thiorphan), but this difference did not reach the level of significance (table 1). [Prog]sP sulfone was virtually ineffective up to 10 /~M (table 1, fig. 1). [MePheV]NKB displayed some activity, being 1/419 and 1/15 as potent as N K A and Arg-NKB, respectively. The maximal effect of [MePhe7]NKB was similar to that of Arg-NKB and was significantly less than that of N K A . The maximal effect and potency of [MePheV]NKB were almost unaffected by thiorphan (table 1, fig. 1). To further assess the involvement of N K - 2 receptors, we studied the effect of [fl-Ala 8] NKA(4-10), a newly developed peptide which displays high selectivity for N K - 2 as compared to NK-1 or NK-3 receptors (Rovero et al., in press). As shown in fig. 1 and table 1, [fl-AlaS]NKA-(4-10) had potent contractile activity in this bioassay organ, and was slightly more potent than N K A in the presence and absence of thiorphan.
Septide had almost no effect up to 10 ffM (n = 4). On the other hand, DiMeC7 had some activity, but only in the ffM range (n = 5). DiMeC 7 was about 1/597 and 1 / 2 3 as potent as N K A and Arg-NKB, respectively, and about as potent as [MePheV]NKB. Its maximal effect was significantly lower than that of N K A , but it was not significantly different from that of Arg-NKB or [MePheT]NKB. Neither the activity of Septide nor that of DiMeC v was significantly modified in the presence of thiorphan (fig. 1, table 1).
3.4. Effect of low concentrations of thiorphan or captopril on the response to SP As described above, SP was the only mammalian tachykinin whose activity was significantly enhanced by 10 t~M thiorphan. This pattern is in contrast with the notion that 'enkephalinase' (endopeptidase 24.11) cleaves not only SP but also N K A and N K B (see Turner, 1987). Therefore the question was raised as to whether the potentiating action of 10 # M thiorphan might involve inhibition of other peptidases. To assess this point, we determined the effect of lower concentrations of thiorphan (10 nM to 0.1 #M) or captopril (0.1-1 /~M) on the response to SP. Thiorphan (10 nM) had no significant effect whereas 0.1 ftM thiorphan significantly potentiated the response to SP (fig. 3). At 0.1 #M, captopril failed to significantly affect the response to SP (n = 4 data not shown) but significantly enhanced the response at 1 # M (fig. 3).
3.5, Motor response to capsaicin in the absence and presence of thiorphan Capsaicin (1 /~M) had inconsistent motor effects, as it produced a small contraction only in 3 out of 6 cases. At 10 /~M, capsaicin produced a p r o m p t contractile response in all cases (n = 10), which peaked 1-3 min after application and averaged 23 + 2% of the effect of KC1. A second application of capsaicin (10 /~M, 30 min later) failed to induce any contraction. The vehicle for 10 ffM capsaicin (5/~1 of absolute ethanol) did not
57
affect the tone of the preparations (n = 3). No significant potentiation was observed when the contractile effect of capsaicin was studied in the presence of thiorphan. In fact, the capsaicin-induced contraction averaged 31 + 6% of the response to KC1 (n = 6, n.s. as compared to the response of controls). Likewise, the response to 1 /~M capsaicin was not significantly affected by thiorphan (10/~M, n = 4 data not shown) as compared to the control response.
Guinea - plg gallbladder 100.
80-
911spantlde 10 UM
/ 60,
~ 40J 20-
ONKA - Log ( M )
Fig. 4. Concentration-response curves showing the contractile effect of neurokinin A in the isolated guinea-pig gallbladder in the absence and presence of Spantide. Each point of the curve made in the presence of Spantide, except that at 3 /~M, is significantly (P < 0.05) different from the corresponding control. Each point is the mean +__S.E. of 4 experiments.
respectively. The total evoked release was 281 + 24 and 550 ± 76 pg in the absence and presence of thiorphan, respectively (n = 4 each, P < 0.05). Guinea - pig gallbladder
250
3. 7. Release of SP-LI by capsaicin in the absence and presence of thiorphan The basal outflow of SP-LI was 16.3 + 1.3 and 14.6 + 1.6 p g / g per fraction in the absence and presence of thiorphan (n = 4 each, n.s.). SP-LI was promptly increased by superfusion of gallbladder strips with capsaicin (1/~M, fig. 5). The release of peptide reached a peak 4-5 rain after administration of capsaicin and then declined to baseline levels despite the continuous presence of the drug. A second application of capsaicin 60 rain later had no further effect, indicating complete desensitization. Capsaicin (1 /~M)-induced SP-LI release was almost doubled as compared to control release in the presence of thiorphan (10/~M) (fig. 5). Indeed, the peak increase in SP-LI outflow was 9.1- and 18-fold larger than the basal values measured in the absence and presence of thiorphan,
//CI~
c
3.6. Effect of spantide on the capsaicin- or NKA-induced contraction The tachykinin antagonist, Spantide (5-10 ~tM), produced a small ( < 10% of the response to KCI) transient (3-5 min) contraction of the guinea-pig gallbladder. The response to field stimulation (1020 Hz for 5 s, 0.5 ms pulse width, 60 V) was unaffected by Spantide (10 min before). However, Spantide produced a marked inhibition (74%) of the capsaicin (10/~M)-induced contraction. In fact, the response to capsaicin averaged 23 + 2 and 6 ± 2% of the response to KC1 in controls and in the presence of Spantide (n = 10 and 4, P < 0.01 as compared to controls). Spantide (10 ~tM) produced a rightward shift of the concentration-response curve for N K A (fig. 4).
0 control
200
0 control ~ thlOrphan 10 ~JM
Q
"6 150
100 0
5O
L 0
Fraction number
Fig. 5. Effect of thiorphan on the capsaicln (1 /~M, applied at the arrow)-induced release of substance P-like immunoreactivity from isolated guinea-pig gallbladders. Each point is the mean + S.E. of 4 experiments. Fractions (4 ml) were collected every 2 rain. * Significantly different from controls, P < 0.05.
58 4. Discussion
4.1. NK-2 receptors in the guinea-pig gallbladder Much evidence has been presented in recent years to support the idea that multiple tachykinin receptors exist in mammalian tissues. Both binding (Buck et al., 1984; Lee et al., 1986) and functional experiments (Regoli et al., 1986; 1987; Maggi et al., 1987) have indicated that three distinct patterns of activity of tachykinins and tachykinin-related peptides exist, which correspond to activation of distinct receptors. These tachykinin receptors have been termed NK-1, NK-2 and NK-3, respectively. Correlation analysis has shown that there is a good correlation between functional and binding experiments when selected bioassays with organs containing only one type of tachykinin receptor are used (Dion et al., 1987b; Lee et al., 1986). The contractile response of the isolated guinea-pig gallbladder to tachykinins is apparently due to direct activation of smooth muscle cells since it was unaffected by atropine, tetrodotoxin, chlorpheniramine or dbcGMP, which in this organ behaves as a cholecystokinin antagonist (Meldrum et al., 1987; Shook and Burks, 1987). Shook and Burks (1987) reported the effect of SP, N K A and three non-mammalian tachykinins (physalaemin, eledoisin and kassinin) in this tissue and proposed the presence of NK-2 receptors. We thought it worthwhile to readdress this question for the following reasons: (a) no information was given in previous reports about the response to NKB; (b) the rank order of potency for non-mammalian tachykinins (kassinin > eledoisin > physalaemin), as reported by Shook and Burks (1987), is typical for NK-3 rather than for NK-2 receptors (see Regoli et al., 1987); (c) the availability of selective agonists for NK-1, NK-2 and NK-3 receptors allows a more accurate characterization of receptors than natural peptides do (Dion et al., 1987a; Devillier et al., 1988; Giuliani et al., 1988a,b; Maggi et al., 1988a); (d) metabolic factors are acknowledged to be a major problem in tachykinin receptor classification (Watson, 1983; Sekizawa et al., 1987a,b; Devillier et al., 1988).
Overall, the present findings indicate that NK-2 receptors are the main mediators of the contractile response of the guinea-pig gallbladder to tachykinins. The rank order of potency for the mammalian tachykinins, even those measured in the presence of thiorphan, is consistent with this hypothesis. The use of the selective agonists for the three receptors indicated that [NIe~°]NKA-(410) was only slightly less potent that NKA. This peptide was shown to have improved selectivity for NK-2 receptors as compared to NKA or NKA-(4-10) (Drapeau et al., 1987). Further evidence for the involvement of NK-2 receptors was provided by the experiments with [/3-AlaS]NKA(4-10), a newly developed heptapeptide with high selectivity for NK-2 receptors (Rovero et al., in press). Two NK-1 receptor agonists ([Pro9]Sp sulfone and Septide) (Drapeau et al., 1987; Wormser et al., 1986) were ineffective. We found that [MePheV]NKB and DiMeC v consistently had some contractile activity. These effects can easily be explained if a population of NK-3 receptors takes part to the response. This would also explain why kassinin is more potent than eledoisin in this tissue (Shook and Burks, 1987). Binding studies are required to verify the hypothesis that there is a mixed population of NK-2 and NK-3 sites in the guinea-pig gallbladder. It has been proposed recently that a fourth type of tachykinin receptor (NK-4) exists (Mc Knight et al., 1987; 1988). This proposal was put forward after it was observed that certain agonists which are almost devoid of activity at NK-2 receptors have potent contractile activity in the guinea-pig trachea, a preparation classified previously as being endowed with NK-2 receptors. Septide was found to be the most potent of these agonists (Mc Knight et al., 1987; 1988). Our findings indicate that the putative NK-4 receptors are unlikely to be involved in the action of the tachykinins on the guinea-pig gallbladder since Septide was almost ineffective, even in the presence of thiorphan. 4.2. 'Enkephalinase' and tachykinin metabolism in the guinea-pig gallbladder An important advance in the field of tachykinin receptor classification was made with the dis-
59
covery that 'enkephalinase' (see Turner, 1987 and Littlewood et al., 1988 for reviews) plays a major role in inactivating tachykinins in mammalian peripheral organs in such a way that the biological responses to exogenous tachykinins are potentiated (Sekizawa et al., 1987a,b); Iwamoto et al., 1988; Devillier et al., 1988). Further, metabolic factors have been shown to be important for tachykinin receptor classification in functional assays (Fox et al., 1988; Ireland et al., 1988; Stephens-Smith et al., 1988; Devillier et al., 1988). In this study we used thiorphan, a well-known enkephalinase (endopeptidase-24.11) inhibitor (Schwartz et al., 1985), to investigate the possible role of this catabolic system in the contractile responses to exogenous and endogenous (released by capsaicin) tachykinins. Our findings show a differential susceptibility of exogenous tachykinins to potentiation by thiorphan. In fact, only the activity of SP was potentiated by thiorphan while that of NKB or N K A was unaffected. This pattern of activity is in marked contrast to that which one would expect from inhibition of enkephalinase. In fact, both biochemical (see Turner, 1987) and functional (Sekizawa et al., 1987a,b) studies indicate that all mammalian tachykinins are sensitive to the action of this endopeptidase with the following order of sensitivity: NKB > SP > NKA. Sekizawa et al. (1987a) reported that the activities of these peptides were potentiated by leu-thiorphan (10 /~M) in isolated ferret trachea, the effect of NKB being more affected than that of SP and the effect of SP being more affected than that of NKA. It is therefore conceivable that the action of thiorphan observed in this study might involve blockade or inhibition of some enzymatic system different from enkephalinase. Indeed, thiorphan inhibits enkephalinase at nM concentrations while it is about 40 times less potent on angiotensin-converting enzyme (ACE) (Schwartz et al., 1981). ACE has been shown to metabolize SP but not N K A or NKB (see Skidgel et al., 1987), and ACE inhibitors have been reported to potentiate certain biological response to exogenous SP (Cascieri et al., 1984; Shore et al., 1988). As the threshold concentration for the potentiating effect of thiorphan in the guinea-pig gallbladder was 0.1 ~M and captopril mimicked
the effect of thiorphan on the response to exogenous SP, it is conceivable that the effects of thiorphan described in this study might have involved inhibition of ACE. Direct biochemical determination of the enzymatic activity of this tissue for tachykinins is needed to solve this question. Since NK-1 receptors are apparently not involved in the response studied, we think that SP acts in this tissue by activating NK-2 and possibly some NK-3 receptors. As the effects of N K A or NKB were not potentiated by thiorphan, it is unlikely that the effect of thiorphan occurred at the level of tachykinin receptors (see Iwamoto et al., 1988) a n d / o r second messengers which mediate the contractile effect of tachykinins. We also found that thiorphan markedly increased the yield of SP-LI released by capsaicin, indicating that tachykinins released from sensory nerves are also susceptible to breakdown by thiorphan-sensitive mechanism(s). It could be that a peptide(s) is produced during the degradation of endogenous SP by this thiorphan-sensitive mechanism that is not recognized by the SP antiserum in our RIA. Moreover, the capsaicin-induced contraction of the guinea-pig gallbladder is possibly mediated by endogenous tachykinins in view of its antagonism by Spantide. However, our data suggest that SP may not play a major role in this response because thiorphan did not significantly affect the amplitude of the capsaicin-induced contraction. As NKA-LI is co-stored and co-released with SP-LI from capsaicin-sensitive nerves (Hua et al., 1985; Saria et al., 1986), it is conceivable that this might occur in the guinea-pig gallbladder, where N K A is a much more potent spasmogen that SP and its action is unaffected by thiorphan.
References Barthr, L. and P. Holzer, 1985, Search for a physiological role of substance P in gastrointestinal motility, Neuroscience 16, 1. Bucks, S.H., E. Burcher, C.W. Schultz, W. Lovenberg and T.L. O'Donahue, 1984, Novel pharmacology of substance K binding sites: a third type of tachykinin receptor, Science 226, 987. Bucsics, A., P. Holzer and F. Lembeck, 1983, The substance P content of peripheral tissues in several mammals, Peptides 4, 451.
60 Cai, W., J. Gu, W. Huang, G.P. Mcgregor, M.A. Ghatei, S.R. Bloom and J.M. Polak, 1983, Peptide immunoreactive nerves and cells of the guinea-pig gallbladder and biliary pathways, Gut 24, 1186. Cascieri, M.A., H.G. Bull, R.A. Mumford, A.A. Patchett, N.A. Thornberry and T. Liang, 1984, Carboxyl terminal tripeptidyl hydrolysis of substance P by purified rabbit lung ACE and the potentiation of substance P activity by captopril and MK-422, Mol. Pharmacol. 25, 287. Devillier, P., C. Advenier, G. Drapeau, J. Marsac and D. Regoli, 1988, Comparison of the effects of epithelium removal and of an enkephalinase inhibitor on the neurokinin-induced contractions of guinea-pig isolated trachea, Br. J. Pharmacol. 94, 675. Dion, S., P. D'Orleans-Juste, G. Drapeau, N.E. Rhaleb, N. Rouissi, C. Tousignant and D. Regoli, 1987a, Characterization of neurokinin receptors in various isolated organs by the use of selective agonists, Life Sci. 41, 2269. Dion, S., G. Drapeau, N.E. Rhaleb, P. D'Orleans-Juste and D. Regoli, 1987b, Receptors for substance P and neurokinins: correlation between binding and biological activities, European J. Pharmacol. 138, 125. Drapeau, G., P. D'Orleans-Juste, S. Dion, N.E. Rhaleb, N.E. Rouissi and D. Regoli, 1987, Selective agonists for substance P and neurokinin receptors, Neuropeptides 10, 43. Fox, A.J., J. Hall and I.K.M. Morton, 1988, Tissue peptidase activity as a possible determinant of relative activity of tachykinins and other peptides, Regul. Pept. 22, 68. Franco-Cereceda, A., H. Henke, J.M. Lundberg, J.B. Petermann, T. H/Skfelt and J.A. Fischer, 1987, Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin, Peptides 8, 399. Giuliani, S., C.A. Maggi and A. Meli, 1988a, NK-1 receptors mediate the tachykinin stimulation of salivary secretion: selective agonists provide further evidence, European J. Pharmacol. 150. 377. Giuliani, S., C.A. Maggi, P. Rovero and A. Meli, 1988b, Neurokinins induce a relaxation of the rat duodenum in vivo by activating postganglionic sympathetic elements in prevertebral ganglia - Involvement of an NK-2 type of neurokinin receptor, J. Pharmacol. Exp. Ther. 246, 322. Hua, X.Y., E. Theodorsson-Norheim, E. Brodin, J.M. Lundberg and T. H6kfelt, 1985, Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig, Regul. Pept. 13, 1. Irelan& S.J., C.C. Jordan, M.L. Stephens-Smith and P. Ward, 1988, Receptors mediating the contractile response to neurokinin agonists in the guinea-pig trachea, Regul. Pept. 22, 93, Iwamoto, I., I. Ueki and J.A. Nadel, 1988, Effect of neutral endopeptidase inhibitors on [3H]substance P binding in rat ileum, Neuropeptides 11,185. Lee, C.M., N.J. Campbell, B.J. Williams and L.L. Iversen, 1986, Multiple tachykinin binding sites in peripheral tissue and brain, European J. Pharmacol. 130, 209. Littlewood, G.M., L.L. Iversen and A.J. Turner, 1988, Neu-
ropeptides and their peptidases: functional considerations Neurochem. Internat. 12, 383. Lundberg, J.M., E. Brodin. X. Hua and A. Saria, 1984, Vascular permeability changes and smooth muscle contraction ir relation to capsaicin-sensitive afferents in the guinea pig Acta Physiol. Scand. 120, 217. Maggi, C.A., S. Giuliani, P. Santicioli, D. Regoli and A. Meli. 1987, Peripheral effects of neurokinins: functional evidence for the existence of multiple receptors, J. Autonom Pharmacol. 7, 11. Maggi, C.A., M. Partani, M. Astolfi, P. Santicioli, P. Rovero. L. Abelli, V. Somma, S. Giuliani, D. Regoli, R. Patacchini and A. Meli, 1988a, Neurokinin receptors in the rat loweT urinary tract, J. Pharmacol. Exp. Ther. 246, 308. Maggi, C.A., P. Santicioli, P. Geppetti, R. Patacchini, S. Frilli, M. Astolfi, B. Fusco and A. Meli, 1988b, Simultaneous release of substance P and calcitonin gene-related peptidelike immunoreactivity from isolated muscle of the guinea-pi~ urinary bladder, Neurosci. Lett. 87, 163. Maggi, C.A., P. Santicioli, D. Renzi, R. Patacchini, C. Surrenti and A. Meli, Simultaneous release of substance P- and CGRP-Iike immunoreactivity and motor response of the isolated guinea-pig gallbladder to capsaicin, Gastroenterology (in press). Mc Knight, A.T., J.J. Maguire and M.A. Varney, 1987, Characterization of receptors for tachykinin in guinea-pig isolated trachea, Br. J. Pharmacol. 91, 360P. Mc Knight, A.T., J.J. Maguire, M.A. Varney, B.J. Williams and L.L. Iversen, 1988, Characterization of tachykinin receptors using selectivity of agonists and antagonists: evidence for an NK-4 type, Regul. Pept. 22, 126. Meldrum, L.A., J. Bojarski and J. Calam, 1987, Effects of substance P and other neuropeptides on guinea-pig gallbladder muscle, Digestion 37, 193. Regoli, D., G. Drapeau, S. Dion and P. D'Orleans-Juste, 1986, Receptors for neurokinins in peripheral organs, in: Substance P and Neurokinins, Montreal 1986, July 10-23, eds. J.L. Henry, R. Couture, A.C. Cuello. G. Pelletier, R. Quirion and D. Regoli (Springer Verlag, New York) p. 99. Regoli, D., G. Drapeau, S. Dion and P. D'Orleans-Juste, 1987, Pharmacological receptors for substance P and neurokinins, Life Sci. 40, 109. Rovero, P., N.E. Rhaleb, S. Dion, N. Rouissi, C. Tousignant, S. Telemaque, G. Drapeau and D. Regoli, Structure activity studies of neurokinin A, Neuropeptides (in press). Saria, A., R. Gamse, J. Petermann, J.A. Fischer, E. Theodorsson-Norheim and J.M. Lundberg, 1986, Simultaneous release of several tachykinins and calcitonin gene-related peptide from rat spinal cord slices, Neurosci. Lett. 63, 310. Schwartz, J.C., J. Costentin and J.M. Lecomte, 1985, Pharmacology of enkephalinase inhibitors, Trends Pharmacol. Sci. 6, 472. Schwartz, J.C., B. Malfroy and S. De La Baume, 1981, Biological inactivation of enkephalins and the role of enkephalindipeptidyl-carboxypeptidase ('enkephalinase') as neuropeptidase, Life Sci. 29, 1715. Sekizawa. K., J. Tamaoki, P.D. Graf, C.B. Basbaum, D.B.
61 Borson and J.A. Nadel, 1987a, Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction in ferret trachea, J. Pharmacol. Exp. Ther. 243, 1211. Sekizawa, K., J. Tamaoki, J.A. Nadel and D.B. Borson, 1987b, Enkephalinase inhibitor potentiates substance P and electrically induced contraction in ferret trachea, J. Appl. Physiol. 63, 1401. Shook, J.E. and T.F. Burks, 1987, A novel bioassay for the NK-2 neurokinin receptor: the guinea-pig gallbladder, Life Sci. 39, 2533. Shore, S.A., N.P. Stimler Gerard, S.R. Coats and J.M. Drazen, 1988, Substance P induced bronchoconstriction in the guinea-pig. Enhancement by inhibitors of neutral metalloendopeptidase and ACE, Am. Rev. Resp. Dis. 137, 331. Skidgel, A., R. Defendini and E.G. Erdos, 1987, Angiotensin I
converting enzyme and its role in neuropeptide metabolism, in: Neuropeptides and their Peptidases, ed. A.J. Turner (Ellis Horwood Ltd. Chichester, England) p. 165. Stephens-Smith, M., S.J. Ireland and C.C. Jordan, 1988, Influence of peptidase inhibitors to neurokinin receptor agonists in the guinea-pig trachea, Regul. Pept. 22, 177. Turner, A.J., 1987, Endopeptidase-24.11, in: Neuropeptides And Their Peptidases, ed. A.J. Turner (Ellis Horwood Ltd., Chichester, England) p. 183. Watson, S.P., 1983, Rapid degradation of [3H]substance P in guinea-pig ileum and rat vas deferens in vitro, Br. J. Pharmacol. 79, 543. Wormser, U., R. Laufer, Y. Hart, M. Chorev, C. Gilon and Z. Selinger, 1986, Highly selective agonists for substance P receptor subtypes, EMBO J. 5, 2805.
51
Elsevier EJP 50786
Effect of thiorphan on response of the guinea-pig gallbladder to tachykinins Carlo Alberto Maggi *, Riccardo Patacchini, Daniela Renzi 1, Paolo Santicioli, Domenico Regoli 2, Paolo Rovero 3, G u y Drapeau 2, Calogero Surrenti 1 and Alberto Meli Pharmacology Department, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, Florence, 1Department of Clinical Pathophysiology, Gastroenterology Unit, University of Florence, Italy, 2 Department of Physiology and Pharmacology Sherbrooke University, Canada and 3 Chemistry Department, A. Menarini Pharmaceuticals, Florence, Italy
Received 12 September 1988, revised MS received 6 February 1989, accepted 21 February 1989
Tachykinins produced a concentration-related contraction of the isolated guinea-pig gallbladder, with a rank order of potency neurokinin A (NKA) > Arg-neurokinin B = neurokinin B (NKB) > substance P (SP). Only the effect of SP was potentiated by thiorphan (0.1-10 #M). A significant enhancement of the response to SP was also produced by captopril (1 #M). [Nlel°]NKA-(4-10) and [fl-AlaS]NKA-(4-10), selective NK-2 receptor agonists, were active, whereas [Pro9]Sp sulfone (selective NK-1 agonist) was almost ineffective. [MePhe7]NKB (selective NK-3 agonist) had some activity but only at high concentrations. Septide was almost ineffective and DiMeC 7 had an action comparable to that of [MePhe7]NKB. None of the effects induced by these synthetic tachykinin analogs were significantly potentiated by thiorphan. Capsaicin (10 #M) produced a contraction which was unaffected by thiorphan. Both capsaicin and NKA-induced contractions were antagonized by Spantide at concentrations (5-10/~M) which had no effect against the atropine-sensitive contractions produced by electrical field stimulation. Capsaicin (1 #M) produced a consistent release of SP-like immunoreactivity (SP-LI) and a second application of the drug had no further effect, indicating complete desensitization. SP-LI release by capsaicin was almost doubled in the presence of thiorphan. These findings indicate that NK-2 and possibly some NK-3 receptors mediate the contractile response of the guinea-pig gallbladder to tachykinins. Both exogenous and endogenous (released by capsaicin) SP were degraded to a significant extent in this organ via a thiorphan-sensitive mechanism, the identity of which remains to be established. Gallbladder; Tachykinins; Capsaicin; Substance P; Neurokinin A; Tachykinin receptors; Thiorphan; Captopril; (Guinea-pig)
1. Introduction
Tachykinins have a potent contractile effect in various sections of the gastrointestinal tract where
they may play a role as neurotransmitters (see Barth6 and Holzer, 1985, for review). There seem
to be two major sources of these peptides in the gastrointestinal tract of rats and guinea-pigs: intrinsic neurons of the myenteric plexus and pe-
* To whom all correspondence should be addressed at A. Menarini Pharmaceuticals.
ripheral endings of capsaicin-sensitive sensory nerves (Barth6 and Holzer, 1985). Less information is available about the role of tachykinins in regulating the motility of the biliary tract (Meldrum et al., 1987; Shook and Burks, 1987). Both anatomical and biochemical evidence indicates the txistence of substance P-like immunoreactivity (SP-LI) in nerve fibers of the guinea-pig gallbladder (Cai et al., 1983; Bucsics et al., 1983; Franco-Cereceda et al., 1987). As in the intestine, the tachykinin-like immunoreactive material in the biliary tract is, at least in part, stored in capsaicin-sensitive primary afferents
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
52 (Franco-Cereceda et al., 1987; Maggi et al., in press) and the release of endogenous tachykinins from peripheral terminals of primary afferents seems to be involved in capsaicin-induced contractions and plasma extravasation at this level (Lundberg et al., 1984; Maggi et al., in press). The aim of this study was to characterize the tachykinin receptor mediating the contraction of the isolated guinea-pig gallbladder (Shook and Burks, 1987) by using several synthetic agonists which display improved selectivity a n d / o r metabolic stability compared to that of natural tachykinins. Moreover, we looked at the possible influence of catabolism by endopeptidases on the response to exogenous and endogenous (SP-LI release by capsaicin) tachykinins by studying the effect of thiorphan, an inhibitor of 'enkephalinase' (see Schwartz et al., 1985 for review). Thiorphan has been shown to reduce the metabolism of tachykinins (Turner, 1987) and to markedly potentiate the effect of these peptides on the respiratory tract (Sekizawa et al., 1987a,b). Metabolic inactivation by tissue enzymes has been shown to interfere with the characterization of tachykinin receptors in mammalian tissues (Ireland et al., 1988; Stephens-Smith et al., 1988; Fox et al., 1988; Devillier et al., 1988). 2. Materials and methods
2.1. Functional experiments Male albino guinea-pigs weighing 240-260 g were stunned and bled. The whole gallbladder was rapidly removed and placed in oxygenated (96% 0 2 and 4% COz) Krebs solution at 37°C. For functional experiments, each organ was divided in two halves along the longitudinal axis and mounted in 5 ml organ baths to record tension isometrically. Each strip was suspended under a resting tension of 15 mN and tension was recorded by means of an isometric transducer connected to a Basile 7050 Unirecord. The strips were fieldstimulated by means of two platinum wire electrodes placed at the top and bottom of the organ bath and connected to a GRASS S l l stimulator. Each experiment began after a 1 h equilibration period. Cumulative concentration-response curves
for tachykinins were made, each concentration being added when the effect of the preceding one had reached steady state. Preliminary experiments indicated that there was negligible desensitization to the effect of tachykinins since responses of similar amplitude were obtained when 1 ~M neurokinin A was added to the bath before and at the end of a cumulative curve. The contractile response to tachykinins or capsaicin was expressed as a percentage of the response to KCI (80 mM, added to the bath).
2.2. Radioimmunoassay (RIA) To evaluate SP-LI release, the gallbladders were trimmed and placed in 2 ml organ baths at 37 °C and superfused (2 m l / m i n ) with oxygenated (96% 02 and 4% CO2) Krebs solution containing 0.1% bovine albumin and 0.003% bacitracin (Sigma). A 60 min equilibration period was allowed before drug administration. Fractions (4 ml) were collected every 2 min in tubes containing acetic acid to a final concentration of 2 N. At the end of the experiments, the tissues were blotted 2-3 times on filter paper and weighed. The superfusates were freeze-dried, reconstituted in the assay buffer (0.1 M, p H 7.4 phosphate buffer containing 0.9% NaC1, 0.01% N a N 3 and 0.1% bovine serum albumin) and used for RIA. SP-LI was measured as reported previously (Maggi et al., 1988b) with anti SP 144 serum which crossreacts 1% with neurokinin A, 0.5% with neurokinin B and less than 0.1% with physalaemin and eledoisin. SP-LI release by capsaicin (1 ~tM) was studied in the absence or presence of thiorphan (10 ~M).
2.3. Statistical analys& All values in the text, figures and tables are m e a n s _ S.E. Statistical analysis was performed by means of Student's t-test for paired or unpaired data or by an analysis of variance, when applicable. Regression analysis was performed by means of the least-squares method. The ECs0 values and 95% confidence limits (c.1.) were calculated accordingly. Statistical analysis of the differences between the maximum effects produced by the various
53
peptides (expressed as % of the response to KCl) was made with an analysis of variance. NKA was found to produce maximal responses both in the absence and presence of &orphan. For each
GAkPJ
NKA
peptide, lated as response presence
the ED,, values and 95% c.1. were calcuthe concentration producing 50% of the to NKA, either in the absence or the of thiorphan.
(4-10)
NKA
Control
SP Arg - NKB
60-
40-
k?
20-
9 ,o S 5 a c L % 0 2
OI
9 loo-
8
7
6
5
I 4
In the presence of thiorphon (10 J,IM1
80-
60-
40-
20-
o9
8
7 -Log
6 (M)
5
4
Fig. 1. inundation-resins curves showing the contractile response of the isolated guinea-pig gallbladder to tacbylcinins and tachykinin-related peptides in the absence (upper panel) and presence (lower panel) of thiorphan (10 +4) given 15 rnin before the peptides. Each point is the mean f SE. of at least 4 experiments.
54
2.4. Drugs
The drugs used were: capsaicin and thiorphan (Sigma), atropine HC1 and isoprenaline HCI (Serva), neurokinin A (NKA), neurokinin B (NKB), Spantide, substance P and DiMeC 7 (Peninsula), captopril (Squibb), Septide (Bachem). Arginine-NKB (Arg-NKB) and [fl-AlaS]NKA-(410), a newly developed selective NK-2 receptor agonist (Rovero et al., in press), were synthesized at Menarini Labs. by Dr P. Rovero. [Prog]sP sulfone, [Nlel°]NKA-(4-10) and [MePheT]NKB were synthesized at the Department of Physiology and Pharmacology of the University of Sherbrooke, Canada. The anti SP serum was a kind gift of Dr. P. Pradelles, SPI-LERI, Gif-surYvette, France.
3. Results
3.1. Functional experiments: general
Nearly 50% of the gallbladder strips were mechanically quiescent. Spontaneous activity, which had a low amplitude ( < 0.1 mN) and a frequency of 0.3-0.5/min, was observed in the other strips. Exposure to KC1 (80 mM added to the bath) produced a prompt contractile response (18 + 2 mN, n = 36), which reached maximum within 10-20 min, while isoprenaline (3/~M) pro-
duced a prompt relaxation and the disappearance of the spontaneous activity (n = 4). Field stimulation (0.5 ms, 60 V, 1-50 Hz, train duration 5 s) produced frequency-related contractions which were mainly (> 80%) atropine (3 ~M)-sensitive (n = 4). 3.2. Motor response to tachykinins m the absence and presence of thiorphan
NKA was the most potent and effective of the mammalian tachykinins tested (fig. 1, table 1). We studied the effect of NKB dissolved in sulfolane (n = 3) in some experiments. The activity found with the natural peptide was comparable to that found with Arg-NKB, the water soluble derivative developed by Regoli et al. (1986); these authors showed that Arg-NKB had the same activity spectrum as natural NKB. Therefore, data from experiments with Arg-NKB are presented. The following order of potency for the tachykinins was found in the absence of thiorphan: NKA (211) > Arg-NBK (8)> SP (1), a pattern which suggests that NK-2 receptors are involved. It should be noted that, at a high concentration (30 ~M), the response to SP was of nearly the same magnitude as that to N K A (about 70% of the response to KC1) (fig. 1). In contrast, the maximal response to N K A was significantly higher than that to ArgNKB (table 1), which, at 10 /~M, did not exceed 40% of the response to KC1 (fig. 1).
TABLE 1 Contractile effect of some tachykirtins and tachykinin-related peptides on the isolated guinea-pig gallbladder in the absence or presence of thiorphan. Peptide
NKA SP Arg NKB [Nle 1° ]NKA-(4-10) [ fl-Ala 8 ]NKA-(4-10) [Pro 9 ]SP sulfone [MePheV]NKB Septide DiM eC 7
Normal Krebs ECs0 (nM)
95% c.l. (nM)
44 9309 1203 669 38 ND 18454 ND 26269
27-86 4413-26260 131-4463 578-779 25-67 ND 2346-64554 ND 1864-37151
In the presence of thiorphan (10 # M ) R.P. 211 1 8 14 245 ND 0.50 ND 0.3
Em~x (% of KC1)
ECs0 (nM)
95% c.1. (nM)
68 + 13 70+6 37 ± 6 a 48 _+7 70 + 9 3 -+ 3 a 40 + 10 a 4 -+ 3 a 40 5=2 a
66 1164 1368 559 33 NT 26897 ND 31154
46-102 925-1511 929-2596 209-2326 22-57 NT 8079-52870 ND 5680-52720
R.P. 18 1 0.85 2 35 NT 0.04 ND 0.04
Emax (% of KC1) 85 -+ 16 90_+7 42 + 5 a 60 _+12 75 + 8 NT 45 5=8 a 3 -+ 3 a 40 _+2
a Significantly different from the effect of N K A P < 0.05; c.1. = confidence limits; R.P. = relative potency; N D = not determined; N T = not tested.
55
5 min
j
G E
t
0.03
t t
t
t
t t
0.1 0.3 I 3 SuDstonce P (pM)
0.03
10
t
t
t t
O.l 0.3 I 3 SuOstance P (~M)
t
10
J~
Thiorphan 10/JM
E ×
t
0.003
t
tt
t t t
OO1 0.03 O.1 0.3 1 Neurokinin A (IJM)
t
3
t
t
t
t
0.003 0.O1 0.03 O.1 0 3 Neurokinin A (/JM)
t
1
t
3
t
10
/l
Thiorphan 10 ~ M
Fig. 2. Typical tracings showing the effect of thiorphan on the contractile response to substance P and neurokinin A in the isolated guinea-pig gallbladder. The effects of the two tachykinins were investigated on two halves of the gallbladder from the same animal.
To investigate the possible involvement of peptide catabolism by 'enkephalinase', we studied the effects of the tachykinins in the presence of thiorphan (10 /~M, 15 min before, fig. 1 lower panel, table 1). Thiorphan alone produced a small contraction of the isolated guinea-pig gallbladder in about 70% of the preparations. When present, the small contractile effect of thiorphan faded during the incubation period and, in most instances, tension returned to the baseline value before the peptides were added. Of the mammalian tachykinins tested, only the effect of SP was significantly potentiated by thiorphan (figs. 2 and 3, table 1). The potency of SP was increased by thiorphan by about ten-fold (fig. 2, table 1). In contrast, the potency of N K A and Arg-NKB (n = 5) was almost unaffected by thiorphan (fig. 1, table 1) and the same was true for NKB dissolved in sulfolane (n = 3, data not shown). In fig. 3, typical tracings show the effect of thiorphan on
the response to SP and N K A on two halves of the gallbladder from the same animal. The order of potency for the mammalian tachykinins was the same in the presence and absence of thiorphan; however, there was a quantitative difference, N K A was only 18 times more potent than SP (table 1). Further, Arg-NKB was slightly less active than SP in the presence of thiorphan (table 1). To further characterize the receptor responsible for the contractile response of the guinea-pig gallbladder to the tachykinins, we investigated the action of synthetic peptides (fig. 1, table 1) selective for the NK-1, NK-2 and NK-3 receptors, namely [Pro9]Sp sulfone, [Nlel°]NKA-(4-10) and [MePheV]NKB, respectively (Drapeau et al., 1987). [Nlel°]NKA-(4-10) was the most active of these peptides, being about 16 and 9 times less potent than N K A in the absence and presence of thiorphan, respectively. The maximum response to [Nlel°]NKA-(4-10) was slightly lower than that to
56
100
100,
8O
80
6O
6o
t hiorphan 10 n M / /
4O
/~/
3.3. Effect of Septide and DiMeC:, *"
/. ~/
th]orphan 0.1 ~ , ~ /
40-
¢
20 ~ _ _ ~ :
control
OJ
ntrol
0 8
7
6
5
4
8
7
6
5
4
o
$ lOO
100
m 80
80 60
captopril 1 pM
7
40
40
*
/
20
20
thiorphan . / 10 FM 7 / / ¢
~d 6o
0 8
•
6
5
0 4 8 7 Substance P (- Log M)
/o
ol 6
5
4
Fig. 3. Enhancement by thiorphan (10 nM to 10 /~M, 15 min before) or captopril (1 ixM, 15 rain before) of the responses induced by SP. In these experiments a control curve for SP was made before (empty circles) and after application of thiorphan or captopril (filled circles). Each point is the mean_+S.E. of 5 experiments. * Significantly different from control, P < 0.05.
N K A (both in the presence and absence of thiorphan), but this difference did not reach the level of significance (table 1). [Prog]sP sulfone was virtually ineffective up to 10 /~M (table 1, fig. 1). [MePheV]NKB displayed some activity, being 1/419 and 1/15 as potent as N K A and Arg-NKB, respectively. The maximal effect of [MePhe7]NKB was similar to that of Arg-NKB and was significantly less than that of N K A . The maximal effect and potency of [MePheV]NKB were almost unaffected by thiorphan (table 1, fig. 1). To further assess the involvement of N K - 2 receptors, we studied the effect of [fl-Ala 8] NKA(4-10), a newly developed peptide which displays high selectivity for N K - 2 as compared to NK-1 or NK-3 receptors (Rovero et al., in press). As shown in fig. 1 and table 1, [fl-AlaS]NKA-(4-10) had potent contractile activity in this bioassay organ, and was slightly more potent than N K A in the presence and absence of thiorphan.
Septide had almost no effect up to 10 ffM (n = 4). On the other hand, DiMeC7 had some activity, but only in the ffM range (n = 5). DiMeC 7 was about 1/597 and 1 / 2 3 as potent as N K A and Arg-NKB, respectively, and about as potent as [MePheV]NKB. Its maximal effect was significantly lower than that of N K A , but it was not significantly different from that of Arg-NKB or [MePheT]NKB. Neither the activity of Septide nor that of DiMeC v was significantly modified in the presence of thiorphan (fig. 1, table 1).
3.4. Effect of low concentrations of thiorphan or captopril on the response to SP As described above, SP was the only mammalian tachykinin whose activity was significantly enhanced by 10 t~M thiorphan. This pattern is in contrast with the notion that 'enkephalinase' (endopeptidase 24.11) cleaves not only SP but also N K A and N K B (see Turner, 1987). Therefore the question was raised as to whether the potentiating action of 10 # M thiorphan might involve inhibition of other peptidases. To assess this point, we determined the effect of lower concentrations of thiorphan (10 nM to 0.1 #M) or captopril (0.1-1 /~M) on the response to SP. Thiorphan (10 nM) had no significant effect whereas 0.1 ftM thiorphan significantly potentiated the response to SP (fig. 3). At 0.1 #M, captopril failed to significantly affect the response to SP (n = 4 data not shown) but significantly enhanced the response at 1 # M (fig. 3).
3.5, Motor response to capsaicin in the absence and presence of thiorphan Capsaicin (1 /~M) had inconsistent motor effects, as it produced a small contraction only in 3 out of 6 cases. At 10 /~M, capsaicin produced a p r o m p t contractile response in all cases (n = 10), which peaked 1-3 min after application and averaged 23 + 2% of the effect of KC1. A second application of capsaicin (10 /~M, 30 min later) failed to induce any contraction. The vehicle for 10 ffM capsaicin (5/~1 of absolute ethanol) did not
57
affect the tone of the preparations (n = 3). No significant potentiation was observed when the contractile effect of capsaicin was studied in the presence of thiorphan. In fact, the capsaicin-induced contraction averaged 31 + 6% of the response to KC1 (n = 6, n.s. as compared to the response of controls). Likewise, the response to 1 /~M capsaicin was not significantly affected by thiorphan (10/~M, n = 4 data not shown) as compared to the control response.
Guinea - plg gallbladder 100.
80-
911spantlde 10 UM
/ 60,
~ 40J 20-
ONKA - Log ( M )
Fig. 4. Concentration-response curves showing the contractile effect of neurokinin A in the isolated guinea-pig gallbladder in the absence and presence of Spantide. Each point of the curve made in the presence of Spantide, except that at 3 /~M, is significantly (P < 0.05) different from the corresponding control. Each point is the mean +__S.E. of 4 experiments.
respectively. The total evoked release was 281 + 24 and 550 ± 76 pg in the absence and presence of thiorphan, respectively (n = 4 each, P < 0.05). Guinea - pig gallbladder
250
3. 7. Release of SP-LI by capsaicin in the absence and presence of thiorphan The basal outflow of SP-LI was 16.3 + 1.3 and 14.6 + 1.6 p g / g per fraction in the absence and presence of thiorphan (n = 4 each, n.s.). SP-LI was promptly increased by superfusion of gallbladder strips with capsaicin (1/~M, fig. 5). The release of peptide reached a peak 4-5 rain after administration of capsaicin and then declined to baseline levels despite the continuous presence of the drug. A second application of capsaicin 60 rain later had no further effect, indicating complete desensitization. Capsaicin (1 /~M)-induced SP-LI release was almost doubled as compared to control release in the presence of thiorphan (10/~M) (fig. 5). Indeed, the peak increase in SP-LI outflow was 9.1- and 18-fold larger than the basal values measured in the absence and presence of thiorphan,
//CI~
c
3.6. Effect of spantide on the capsaicin- or NKA-induced contraction The tachykinin antagonist, Spantide (5-10 ~tM), produced a small ( < 10% of the response to KCI) transient (3-5 min) contraction of the guinea-pig gallbladder. The response to field stimulation (1020 Hz for 5 s, 0.5 ms pulse width, 60 V) was unaffected by Spantide (10 min before). However, Spantide produced a marked inhibition (74%) of the capsaicin (10/~M)-induced contraction. In fact, the response to capsaicin averaged 23 + 2 and 6 ± 2% of the response to KC1 in controls and in the presence of Spantide (n = 10 and 4, P < 0.01 as compared to controls). Spantide (10 ~tM) produced a rightward shift of the concentration-response curve for N K A (fig. 4).
0 control
200
0 control ~ thlOrphan 10 ~JM
Q
"6 150
100 0
5O
L 0
Fraction number
Fig. 5. Effect of thiorphan on the capsaicln (1 /~M, applied at the arrow)-induced release of substance P-like immunoreactivity from isolated guinea-pig gallbladders. Each point is the mean + S.E. of 4 experiments. Fractions (4 ml) were collected every 2 rain. * Significantly different from controls, P < 0.05.
58 4. Discussion
4.1. NK-2 receptors in the guinea-pig gallbladder Much evidence has been presented in recent years to support the idea that multiple tachykinin receptors exist in mammalian tissues. Both binding (Buck et al., 1984; Lee et al., 1986) and functional experiments (Regoli et al., 1986; 1987; Maggi et al., 1987) have indicated that three distinct patterns of activity of tachykinins and tachykinin-related peptides exist, which correspond to activation of distinct receptors. These tachykinin receptors have been termed NK-1, NK-2 and NK-3, respectively. Correlation analysis has shown that there is a good correlation between functional and binding experiments when selected bioassays with organs containing only one type of tachykinin receptor are used (Dion et al., 1987b; Lee et al., 1986). The contractile response of the isolated guinea-pig gallbladder to tachykinins is apparently due to direct activation of smooth muscle cells since it was unaffected by atropine, tetrodotoxin, chlorpheniramine or dbcGMP, which in this organ behaves as a cholecystokinin antagonist (Meldrum et al., 1987; Shook and Burks, 1987). Shook and Burks (1987) reported the effect of SP, N K A and three non-mammalian tachykinins (physalaemin, eledoisin and kassinin) in this tissue and proposed the presence of NK-2 receptors. We thought it worthwhile to readdress this question for the following reasons: (a) no information was given in previous reports about the response to NKB; (b) the rank order of potency for non-mammalian tachykinins (kassinin > eledoisin > physalaemin), as reported by Shook and Burks (1987), is typical for NK-3 rather than for NK-2 receptors (see Regoli et al., 1987); (c) the availability of selective agonists for NK-1, NK-2 and NK-3 receptors allows a more accurate characterization of receptors than natural peptides do (Dion et al., 1987a; Devillier et al., 1988; Giuliani et al., 1988a,b; Maggi et al., 1988a); (d) metabolic factors are acknowledged to be a major problem in tachykinin receptor classification (Watson, 1983; Sekizawa et al., 1987a,b; Devillier et al., 1988).
Overall, the present findings indicate that NK-2 receptors are the main mediators of the contractile response of the guinea-pig gallbladder to tachykinins. The rank order of potency for the mammalian tachykinins, even those measured in the presence of thiorphan, is consistent with this hypothesis. The use of the selective agonists for the three receptors indicated that [NIe~°]NKA-(410) was only slightly less potent that NKA. This peptide was shown to have improved selectivity for NK-2 receptors as compared to NKA or NKA-(4-10) (Drapeau et al., 1987). Further evidence for the involvement of NK-2 receptors was provided by the experiments with [/3-AlaS]NKA(4-10), a newly developed heptapeptide with high selectivity for NK-2 receptors (Rovero et al., in press). Two NK-1 receptor agonists ([Pro9]Sp sulfone and Septide) (Drapeau et al., 1987; Wormser et al., 1986) were ineffective. We found that [MePheV]NKB and DiMeC v consistently had some contractile activity. These effects can easily be explained if a population of NK-3 receptors takes part to the response. This would also explain why kassinin is more potent than eledoisin in this tissue (Shook and Burks, 1987). Binding studies are required to verify the hypothesis that there is a mixed population of NK-2 and NK-3 sites in the guinea-pig gallbladder. It has been proposed recently that a fourth type of tachykinin receptor (NK-4) exists (Mc Knight et al., 1987; 1988). This proposal was put forward after it was observed that certain agonists which are almost devoid of activity at NK-2 receptors have potent contractile activity in the guinea-pig trachea, a preparation classified previously as being endowed with NK-2 receptors. Septide was found to be the most potent of these agonists (Mc Knight et al., 1987; 1988). Our findings indicate that the putative NK-4 receptors are unlikely to be involved in the action of the tachykinins on the guinea-pig gallbladder since Septide was almost ineffective, even in the presence of thiorphan. 4.2. 'Enkephalinase' and tachykinin metabolism in the guinea-pig gallbladder An important advance in the field of tachykinin receptor classification was made with the dis-
59
covery that 'enkephalinase' (see Turner, 1987 and Littlewood et al., 1988 for reviews) plays a major role in inactivating tachykinins in mammalian peripheral organs in such a way that the biological responses to exogenous tachykinins are potentiated (Sekizawa et al., 1987a,b); Iwamoto et al., 1988; Devillier et al., 1988). Further, metabolic factors have been shown to be important for tachykinin receptor classification in functional assays (Fox et al., 1988; Ireland et al., 1988; Stephens-Smith et al., 1988; Devillier et al., 1988). In this study we used thiorphan, a well-known enkephalinase (endopeptidase-24.11) inhibitor (Schwartz et al., 1985), to investigate the possible role of this catabolic system in the contractile responses to exogenous and endogenous (released by capsaicin) tachykinins. Our findings show a differential susceptibility of exogenous tachykinins to potentiation by thiorphan. In fact, only the activity of SP was potentiated by thiorphan while that of NKB or N K A was unaffected. This pattern of activity is in marked contrast to that which one would expect from inhibition of enkephalinase. In fact, both biochemical (see Turner, 1987) and functional (Sekizawa et al., 1987a,b) studies indicate that all mammalian tachykinins are sensitive to the action of this endopeptidase with the following order of sensitivity: NKB > SP > NKA. Sekizawa et al. (1987a) reported that the activities of these peptides were potentiated by leu-thiorphan (10 /~M) in isolated ferret trachea, the effect of NKB being more affected than that of SP and the effect of SP being more affected than that of NKA. It is therefore conceivable that the action of thiorphan observed in this study might involve blockade or inhibition of some enzymatic system different from enkephalinase. Indeed, thiorphan inhibits enkephalinase at nM concentrations while it is about 40 times less potent on angiotensin-converting enzyme (ACE) (Schwartz et al., 1981). ACE has been shown to metabolize SP but not N K A or NKB (see Skidgel et al., 1987), and ACE inhibitors have been reported to potentiate certain biological response to exogenous SP (Cascieri et al., 1984; Shore et al., 1988). As the threshold concentration for the potentiating effect of thiorphan in the guinea-pig gallbladder was 0.1 ~M and captopril mimicked
the effect of thiorphan on the response to exogenous SP, it is conceivable that the effects of thiorphan described in this study might have involved inhibition of ACE. Direct biochemical determination of the enzymatic activity of this tissue for tachykinins is needed to solve this question. Since NK-1 receptors are apparently not involved in the response studied, we think that SP acts in this tissue by activating NK-2 and possibly some NK-3 receptors. As the effects of N K A or NKB were not potentiated by thiorphan, it is unlikely that the effect of thiorphan occurred at the level of tachykinin receptors (see Iwamoto et al., 1988) a n d / o r second messengers which mediate the contractile effect of tachykinins. We also found that thiorphan markedly increased the yield of SP-LI released by capsaicin, indicating that tachykinins released from sensory nerves are also susceptible to breakdown by thiorphan-sensitive mechanism(s). It could be that a peptide(s) is produced during the degradation of endogenous SP by this thiorphan-sensitive mechanism that is not recognized by the SP antiserum in our RIA. Moreover, the capsaicin-induced contraction of the guinea-pig gallbladder is possibly mediated by endogenous tachykinins in view of its antagonism by Spantide. However, our data suggest that SP may not play a major role in this response because thiorphan did not significantly affect the amplitude of the capsaicin-induced contraction. As NKA-LI is co-stored and co-released with SP-LI from capsaicin-sensitive nerves (Hua et al., 1985; Saria et al., 1986), it is conceivable that this might occur in the guinea-pig gallbladder, where N K A is a much more potent spasmogen that SP and its action is unaffected by thiorphan.
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