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Tachykinin antagonists inhibit nerve-mediated contractions in the circular muscle of the human ileum
GASTROENTEROLOGY
1992:102:88-98
Tachykinin Antagonists Inhibit NerveMediated Contractions in the Circular Muscle of the Human Ileum Involvement
of Neurokinin-2
Receptors
CARLO ALBERT0 MAGGI, SANDRO GIULIANI, RICCARDO PATACCHINI, PAOLO SANTICIOLI, ELVAR THEODORSSON, GABRIELE BARBANTI, DAMIANO TURINI, and ANTON10 GIACHETTI Pharmacology Department, A. Menarini Pharmaceuticals, Karolinska Hospital, Stockholm, Sweden; and Department
The effects of some newly developed tachykinin antagonists that are selective for the neurokinin (NK)1 (L 668,169) or the NK-2 (MEN 10,207, L 659,877 and R 396) tachykinin receptor on the cholinergic and noncholinergic contraction and on the nonadrenergit noncholinergic relaxation produced by electrical field stimulation (50 Hz) were investigated in mucosa-free circular strips of the human ileum. The strips were contracted by substance P and neurokinin A as wel1 as by selective NK-2-receptor ligands, [fiAla*]neurokinin A(4-101, and MDL 28,564, the latter peptide being capable of discriminating between NK-2-receptor subtypes. The selectivity of the antagonists for NK-1 or NK-2 receptors was confirmed in pharmacological experiments using substance P, neurokinin A, and [bAla8]neurokinin A(4-10) as stimulants. Among the NK-2-selective antagonists, MEN 10,207 displayed the highest affinity, followed by L 659,877 and R 396. The antagonists MEN 10,207 and L 659,877 inhibited the noncholinergic contraction to electrical stimulation in a concentration-dependent manner; L 668,169 and R 396 were poorly effective. Thus the potency of antagonists toward the noncholinergic response closely paralleled their rank order of potency at NK-2 receptors. The cholinergic contraction and nonadrenergic noncholinergic relaxation were not inhibited by the antagonists. Both substance P- and neurokinin A-like immunoreactivities were detected in extra& of the human ileum, and the identity of the corresponding peptides was confirmed by reverse-phase high-performance liquid chromatography. It was concluded that in addition to NK-1 receptors, the circular muscle of the human ileum also contains NK-2 receptors. Activation of the latter is chiefly responsi-
Florence, Italy; Department of Clinical Chemistry, of Urology, University of Ferrara, Ferrara, Italy
ble for the noncholinergic ulation.
contraction
to nerve stim-
achykinins are a family of peptides that share the common C-terminal sequence Phe-X-GlyLeu-Met NH,. In mammals, three peptides of this family-substance P (SP) neurokinin A, and neurokinin B-are believed to play a role as transmitters, either in the peripheral or the centra1 nervous system.‘v2 Tachykinins act as excitatory transmitters in the mammalian gut, where they exert potent spasmogenic effects via specific receptors located on muscle cells or indirectly by activating intramural neurons.3-7 A physiological role for tachykinins as excitatory transmitters in the rodent smal1 intestine has been proposed, and they are the most likely mediators of the atropine-resistant peristalsis that can easily be shown in the guinea pig ileum.3 Tachykinins are also present in the human gut.8-‘o In the smal1 intestine, tachykininlike immunoreactivity is localized into nerve fibers distributing to the longitudinal and circular muscle layers, and specific tachykinin receptors are present in both muscular layers of the human ileum as shown by autoradiography.” From the functional point of view, tachykinins are potent spasmogens on both the longitudinal and circular muscle of the human ileum’2*‘3 and appendix.14 In the circular muscle of the human ileum, both NK-1 and NK-2 tachykinin receptors mediate the contractile effect of these peptides, which act without any apparent contribution of cholinergic
T
0 1992 by the American
Gastroenterological
OOlE-5085/92/$3.00
Association
January 1992
TACHYKININERGIC
nerves.13 In a previous study,13 we reported that a consistent fraction of the contractile response of the circular muscle of the human ileum to electrical field stimulation (EFS) is atropine-resistant but tetrodotoxin-sensitive, indicating the existente of noncholinergic excitatory nerves. It has been proposed that in addition to acetylcholine, the tachykinins SP and neurokinin A (also known as substance KJ play a role in the ascending contractile component of the peristaltic reflex in the human ileum.15 Evidente for this was obtained in two ways: (a) by using [D-PrO’,DTrp7vg]SP, a tachykinin antagonist; and (b) by measuring release of SP- and neurokinin A-like immunoreactivity during the ascending contraction.’ It should be noted that the previously used” tachykinin antagonist suffers from a number of drawbacks:‘6*‘7 (a ) p oor, if any, ability to discriminate between NK-1 and NK-2 receptors;17 (b) local anesthetic properties; and (c) ability to antagonize the action of other peptides such as bombesin.” Inasmuch as both NK-1 and NK-2 receptors are present in the circular muscle of the human ileum,“s’3 the data obtained with [D-Pro’,D-Trp7”]SP do not allow determination of the relative contribution of SP (which possesses greater affinity for NK-1 receptors) compared with neurokinin A (which possesses greater affinity for NK-2 receptors) in the overall response. The aim of this study was to address the question of whether endogenous tachykinins might be responsible for the atropine-resistant excitatory neurogenic response of the circular muscle of the human ileum to EFS. With this aim, we investigated the effect of a number of newly developed, receptor-selective tachykinin antagonists’8-20 on the contractile response produced by EFS or exogenous peptides. In addition, we measured content of SP- and neurokinin A-like immunoreactivity of the human ileum by radioimmunoassay coupled with identification of the peptides by reverse-phase high-performance liquid chromatography. Finally, in view of the recent evidente for the existente of NK-2-receptor subtypes,21-24 we also investigated the effect of MDL 28,564, a newly developed NK-2-selective ligand that appears extremely useful to distinguish between NK-2-receptor subtypes.
duced by sodium thiopenthal(500 mg IV) and maintained with N,O/O, (1/2) and halothane (0.6%-1%). Patients received pancuronium bromide (6 mg IV) during induction of anesthesia. Al1 specimens appeared macroscopically normal without signs of tumor or inflammation. The tissues were placed in ice-cold Krebs’ solution within 2-3 minutes after surgical removal. The specimens were pinned flat on a Petri dish containing Krebs’ solution and the mucosa was carefully dissected off. Smal1 strips (0.5-0.8 cm long, 2-3 mm wide) of muscle were cut along the circular axis. Al1 experiments started within 3 hours of surgical excision of the samples. Composition of Krebs’ solution was as follows (mmol/L): NaCl, 119; NaHCO,, 25; KH,PO,, 1.2; MgSO,, 1.5; KCl, 4.7; CaCl,, 2.5; and glucose, ll. Krebs’ solution was gassed with 96% 0, and 4% CO, (pH 7.4). Guanethidine (3 pmol/L) was present throughout the experiments. Atropine (3 pmol/L) was also added to the Krebs’ solution in some experiments. This concentration of atropine was chosen on the basis of previous experimentsI to produce full blockade of contraction produced by a maximally effective concentration of carbachol, thereby allowing investigation of the noncholinergic responses. The strips were suspended under a resting tension of 5 mN in organ baths (5 mL) containing gassed Krebs’ solution at 37°C. Mechanica1 activity was recorded by means of isotonic transducers (Basile no. 7006; Comerio, VA, Italy) and recorded on a Basile 7050 Unirecord. A resting load of 5 mN was applied to strips; on the basis of previous experiments,13 this load allows maxima1 shortening of the preparation in response to stimulants. The strips were electrically stimulated (50 Hz; pulse width 0.5 milliseconds; trains of 5 seconds every 60-90 seconds; maxima1 voltage) by means of two platinum wire electrodes placed at the top and the bottom of the organ bath using a GRASS Sll stimulator (Quincy, MA). Al1 experiments began after an equilibration period of 60 minutes, during which the bathing solution was renewed every 10-15 minutes. Contractile responses were expressed as percents of the maxima1 contractile response to carbachol(l0 pmol/L). At least two comparable responses to carbachol were recorded before studying the responses to EFS or neuropeptides. Concentration-response curves to peptides were constructed in a noncumulative manner for SP and in a cumulative manner for neurokinin A, [BAla’]neurokinin A(4lO), MDL28,564, and carbachol. [BAla’]Neurokinin A(4-10) is a highly selective ligand for NK-2 receptors that behaves as a full agonist at NK-2 receptors (both subtypes).25,26 MDL 28,564 is a new analog of neurokinin A(410) endowed with high selectivity for NK-2 receptors: MDL 28,564 behaves as a full agonist and a competitive antagonist at the two subtypes of NK-2 receptor, respectively.22~24 Curves to SP were constructed in a noncumulative manner because of marked tachyphylaxis, whereas no tachyphylaxis was evident for the response to the other peptides or carbachol. For noncumulative curves, increasing concentrations of SP were added to the bath at lo-zo-minute intervals and left in contact until maxima1 responses had developed. A thorough washing out was made three to
Materials
and Methods
The motor responses to EFS and neuropeptides were investigated on 102 ileal strips from 24 patients (age, 51-85 years) who underwent abdominal surgery for carcinoma of the bladder base (enterocystoplasty). The study was approved by the Ethica1 Committee of the Faculty of Medicine of the University of Ferrara. NO patient received radiotherapy or chemotherapy before intervention. In al1 patients, preanesthetic medication was intramuscular atropine (1 mg) and diazepam (10 mg). Anesthesia was in-
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MAGGI ET AL.
GASTROENTEROLOGY
four times at 2-minute intervals between doses. Cumulative curves were constructed by addition of increasing concentrations of the spasmogen, the next concentration being added when the effect of the preceding one had reached a steady state. In experiments aiming to evaluate the effect of antagonists, these were added to the bath 15 minutes before the start of the dose-response curve to the agonist.
Tissue Content of Substance P- and Neurokinin A-Like lmmunoreactivity Tissue content of SP- and neurokinin A-like immunoreactivity was determined by radioimmunoassay on seven samples of human ileum from different patients. From each sample the mucosa was separated from the muscle, and the two specimens were extracted as follows: the specimens were weighed and extracted in 2N acetic acid (1:lO wt/vol) at 95’C for 10 minutes. The samples were homogenized and centrifuged at 4000g for 30 minutes at 4”C, and the supernatant was collected. The pellet was extracted again in bidistilled water at 95’C for 10 minutes (1:lO wt/vol) and centrifuged as described above. The supernatant was collected, added to that obtained after acid extraction, and lyophilized until used for radioimmunoassay. Substance P-like immunoreactivity was analyzed using antiserum SP2,27 which reacts with SP and SP sulfoxide but not with other tachykinins. The detection limit was 10 pmol/L. Intraassay and interassay coefficients of variation were 7% and ll%, respectively. [Tyr’lSubstance P was labeled with lz51 using the chloramine T technique. Neurokinin A-like immunoreactivity was analyzed using antiserum K12, which reacts with neurokinin A (lOO%),neurokinin A(3-10) (48%), neurokinin A(4-10) (45%), neurokinin B (26%), neuropeptide K (61%), and eledoisin (30%) but not with SP.** The detection limit of the assay was 12 pmol/L. Intraassay and interassay coefficients of variation were 7% and 12%, respectively. Neurokinin A was labeled using the Bolton-Hunter reagent.
Reverse-Phase High-Performance Chromatography
Liquid
Reverse-phase high-performance liquidchromatography was performed using LKB Ultropac TSK ODST (LKB Produkter, Bromma, Sweden), 5 Frn, 4.6 X 250 mm eluted with a 40-minute linear gradient of 20%-40% acetonitrile in water containing 0.1% trifluoroacetic acid. Two Pharmacia P~OO pumps (Pharmacia, Uppsala, Sweden) were controlled by a GP250 gradient programmer (Pharmacia). Fractions of 0.5 mL were collected at an elution rate of 1 mL/min. Each fraction was lyophilized and redissolved in 100 pL of distilled water before analysis. The fractions were assayed for immunoreactivity in the tubes used for their collection.
Statistical Analysis Al1 data in the text are mean _t SEM. Statistical analysis was performed using Student’s t test for paired or unpaired data or analysis of variante when applicable. Af-
Vol. 102, No. 1
finity of the antagonists in the pharmacological experiments was evaluated as described by Van Rossumzg and expressed as PA, values. Drugs Drugs used were atropine HCl (Serva, Heidelberg, Germany), guanethidine sulphate (ICFI, Milan, Italy) tetrodotoxin (Sankyo, Tokyo, Japan). The NK-l-selective antagonist L 668,169 and the NK-2-selective antagonist L 659,877 were purchased from Cambridge Research Biochemicals (Cambridge, England). [BAla’]Neurokinin A(4lO), SP, neurokinin A, and MEN 10,207 were synthesized by Dr. P. Rovero, Chemistry Department, Menarini Pharmaceuticals, by conventional solid-phase methods. R 396, an NK-2 antagonist, was kindly provided by Professor D. Regoli, Department of Pharmacology, University of Sherbrooke, Canada. MDL 28,564 was a kind gift of Dr. S. H. Buck, Merrell Dow, Cincinnati, Ohio. Concentrated solutions (1-10 mmol/L) of L 668,169, L 659,877, [BAla’]neurokinin A(4-lO), and MDL 28,564 were prepared in dimethyl sulfoxide (DMSO) and diluted in Krebs’ solution. Control experiments showed that DMSO alone (O.l%0.3% final concentration) had no effect on the responses studied. The other peptides were dissolved in Krebs’ solution. Concentrations of L 668,169 and L 659,877 >3 and 10 pmol/L, respectively, were not tested because of precipitation in the bath. The amino acid sequences of peptides tested are shown in Table 1. Results Response to Exogenous Tachykinins and Synthetic Tachykinin Agonists: Effect of Antagonists Concentration-response curves to SP (n = 16), neurokinin A (n = 16), and the selective NK-2-receptor ligands [BAla*]neurokinin A(4-10) (n = lg), and MDL 28,564 (n = 4) are shown in Figure 1 as compared with carbachol. Curves to tachykinins and synthetic agonists were obtained in the presence of atropine. Threshold concentrations were 1 nmol/L for neurokinin A and [BAlas]neurokinin A(4-10) and 10 nmol/L for the other agonists. The maxima1 effect of tachykinins and synthetic agonists, expressed as percent of the maxima1 response to carbachol was as follows: SP, 71% + 3%; neurokinin A, 70% f 3%; [BAla’]neurokinin A(4-lO), 68% f 4%; and MDL 28,564, 61% f 2%. Data in Table 2 show the affinity values (expressed as PA,) of the various tachykinin antagonists against SP, neurokinin A, and [BAlas]neurokinin A(4-10). MEN 10,207, L 659,877, and R 396 antagonized the response to neurokinin A or the selective NK-2-receptor agonist with the following rank order of potency: MEN 10,207 > L 659,877 > R 396, while L 668,169 was inactive up to 3 pmol/L. On the other hand, L 668,169 was the only antagonist tested to produce a significant antagonism toward SP, with an
TACHYKININERGIC
January 1992
Table
1. Amino Acid
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH~ H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NHz H-Asp-Ser-Phe-Val-BAla-Leu-Met-NH, H-Asp-Ser-Phe-Val-Gly-LeuY(CH,NH)Leu-NH* H-Asp-Tyr-DTrp-Val-DTrp-DTrp-Arg-NH, cyclo(Leu-Met-Gln-Trp-Phe-Gly) cyclo(GIn-DTrp-(NMe)Phe(R)Gly[ANC-2]Leu-Met), Ac-Leu-Asp-Gin-Trp-Phe-Gly-NH,
apparent PA, of 6.31 (Table 2). Each antagonist was also tested (n = 3 for each peptide) against carbachol without any significant effect. Response to Electrical Field Stimulation: Effect of Atropine and Tetrodotoxin We previously reportedX3 that the response of the circular muscle of the human isolated ileum to EFS has three distinct components, i.e., a cholinergic and noncholinergic contraction and a nonadrenergit, noncholinergic (NANC) relaxation. These components are more or less evident depending on the frequency of stimulation. For the purposes of this
lOO-
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0
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IN HUMAN ILEUM 91
Sequences of Peptides Used in This Study
Agonists SP Neurokinin A [BAla*]Neurokinin A (4-10) MDL 28,564 Antagonists MEN 10,207 L 659,877 L 668,169 R 396
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Figure 1. Concentration-response curves for contraction of the circular muscle of human ileum produced by natura1 tachykinins SP, (0) and neurokinin A (0) and by the receptor-selective NK-2 agonists [@Alaqneurokinin A (4-10) (0)and MDL 28,564 (ml compared with carbachol (A). Each value is mean f SE of 4-18 experiments. Responses are expressed as percents of the maximal response to each agonist.
study, we concentrated our analysis on the response produced by EFS at 50 Hz. Electrical field stimulation (50 Hz; trains of 5 seconds every 60-90 seconds; 0.5-millisecond pulse width; maxima1 voltage) produced regular phasic contractions of mucosa-free circular strips of the human ileum. In the absente of atropine, the predominant pattern of response (about 80% of cases) was a primary contraction (solid arrows in Figure 2) that faded toward baseline at cessation of the train of stimuli and was followed by a more sustained and delayed tonic-phasic contraction (hollow arrows in Figure 2). In the remainder, the response to EFS with the above-mentioned parameters was monophasic. These latter preparations were studied only in the presence of atropine to rule out the cholinergic response (Figure 3). At a concentration (3 pmol/L) that abolished the response to carbachol (10 pmol/L), atropine also inhibited (>85%) the early component of the biphasic response to EFS. This latter response wil1 be hereafter referred to as the cholinergic response to EFS. In the absente of atropine, the cholinergic response to EFS averaged 23% + 5% of the response to carbachol (range, lO%-71%), while the delayed response averaged 56% t 6% (range, 36%89%) (n = 28). The effect of tachykinin antagonists was tested on strips showing distinct cholinergic and noncholinergit responses to EFS, either in the absente or in the presence of atropine. As reported previously,‘3 a relaxant response to EFS became evident in the presence of atropine and was followed by a delayed, NANC contraction (Figure 3). Atropine-treated strips were used to test the effects of tachykinin antagonists on the NANC contraction and relaxation. Al1 the responses to field stimulation, either in the absence or in the presence of atropine, were abolished (1 pmol/L; n = 6), indicating that by tetrodotoxin they depend on activation of intramural nerves. The only exception, observed in three of six strips tested, was a smal1 immediate phasic contraction to field stimulation which was stil1 present after tetrodotoxin, probably involving direct activation of muscle
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MAGGI ET AL.
TabJe 2.
GASTROENTEROLOGY
Vol. 102, No. 1
VaJues of Tachykinin Antagonists Toward Contractions Produced by Substance P, Neurokinin A, or [PAJa’jNeurokinin A(4-10) in the Circular MuscJe of Human Isolated IJeum
pA2
[BAla*]Neurokinin Antagonist
SP
MEN 10,207 L 659, 877 R 396 L 668,169
Inactive up to 10 pmol/L
Neurokinin A
A(4-10)
7.25 + 0.11 6.64 + 0.16 6.10 + 0.09 Inactive up to 3 pmol/L
Inactive up to 10 pmol/L Inactive up to 30 pmol/L 6.31 + 0.10
7.29 + 0.13 6.83 + 0.12 6.11 + 0.08 Inactive up to 3 pmol/L
NOTE. Each value is mean f SEM of at least four determinations.
cells. In those strips in which atropine failed to completely abolish the early component of the biphasic response to EFS (e.g., Figure 2), the residual response (1 kmol/L). was also resistant to tetrodotoxin Effect of Tachykinin Antagonists on the Responses to Electrical Field Stimulation Data in Figure 4 show the effect of MEN 10,207, L 659,877, R 396, and L 668,169 (0.1-3 pmol/ L) on the delayed noncholinergic response to EFS (n = at least 4 for each antagonist). In the same experiments, performed in the absente of atropine, none of the antagonists produced a significant depression of the cholinergic response (see Figure 2 for a typical experiment with MEN 10,207). MEN 10,207 was the most effective antagonist tested, followed by L 659,877;R 396 and L 668,169 were barely effective at 3-10 pmol/L (Figure 4). Thus the rank order of potency of tachykinin antagonist closely resembled that found in pharmacological experiments against neurokinin A or the selective NK-2 agonist [PAla*]neurokinin A(4-10). In other experiments, the effect of antagonists was investigated on the NANC contraction and NANC
oe
t’ CONTROL
0.1 $4
relaxation in the presence of atropine and guanethidine (3 pmol/L) (Figures 3 and 5). MEN 10,207 and L 659,877 produced a concentration-dependent inhibition of the NANC contraction. The NANC relaxation was unaffected by MEN 10,207 (Figure 3) and slightly reduced by L 659,877 at 3 kmol/L (Figure 5). L 668,169 and R 396 had no significant effect up to 3 pmol/L (n = 5 strips for each antagonist). Substance P- and Neurokinin A-Like Immunoreactivity in the Human Ileum Both SP- and neurokinin A-like immunoreactivities were detected in extracts of the human ileum as follows: muscle layers, 11.7 f 4 and 4.9 + 1.7 pmol/g of wet weight; mucosal layer, 5.9 f 2 and 3.8 f 1.4 pmol/g of wet weight for SP- and neurokinin A-like immunoreactivity, respectively (n = 7 in each case). Reverse-phase high-performance liquid chromatography showed the presence of authentic SP and neurokinin A and their oxidized forms in both the muscular (Figure 6A and B) and mucosal (Figure SC and D) layers of the ileum. A smal1 but distinct peak coeluting in the same position of neuropeptide K was also evident (Figure 6B and D).
t’
t’
t’
0.3 pM
1 PM
3 PM
,otpMe
t’
ATROPINE 3w
MEN 10207 Figure 2. Effect of increasing concentrations of MEN 10,207 on the contraction produced by EFS (dot& In the majority of the strips tested (such as the example shown here), the response to field stimulation was characterized by an early phasic contraction (solid arrow) that faded toward baseline after cessation of the stimulus and was followed by a complex and delayed phasic-tonic contraction (bollow urrow). Note that MEN 10,207 decreased the delayed component of the response in a concentration-dependent manner while leaving the early phasic contraction unaffected. The latter is markedly inhibited by atropine, indicating its cholinergic nature.
TACHYKININERGIC
January 1992
TRANSMISSION
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5 min
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1 ATROPINE
3 pCLM 2 min
Figure 3. (Upperpanek) Example of monophasic response to EFS and its modification by atropine, which unmasked a primary relaxation followed by a rebound, atropine-resistant contraction. (Lower panels) MEN 10,207 inhibited the noncholinergic contraction, leaving the NANC primary relaxation unaffected.
i 1 I
\
z I I
0.1
/LM MEN ATROPINE
Discussion Tachykinins as Excitatory Transmitters Circular Muscle of the Human Ileum
Rt
\
in the
Beside confirming the presence of SP, the present findings show for the first time that authentic neurokinin A and neuropeptide K are present in both mucosal and muscular layers of human ileum. The ratio of SP to neurokinin A found in tissue extracts (2.3 in the muscle, 1.6 in the mucosa) is consistent with the idea of a common origin of the two peptides from the B-preprotachykinin gene.3os31 Along with the data of release of SP- and neurokinin A-like immunoreactivity during the ascending contraction component of the peristaltic reflex in the human ileum,” these data raise the question of a possible physiological role of tachykinins in the regulation of human ileal motility. Such a role was first suggested by the data of Grider15 using an SP analogue endowed with tachykinin-antagonist properties. However, for the reasons exposed in the Introduction, such a tool cannot provide definitive and unequivocal proof for a participation of endogenous tachykinins, nor can it give information about the relative contribution of NK-1 and NK-2 receptors. In the present experimental conditions, contrac-
0.3 @vl 10207
1 PM
3 ,uM
tions produced by EFS of circular muscle strips of the human ileum appear to have three components, one determined by activation of cholinergic nerves; one, putatively tachykininergic, sustained by NANC excitatory nerves; and one, stil1 evident in some strips in the presence of atropine and tachykinin antagonists, also tetrodotoxin resistant and probably originating from direct activation of smooth muscle cells. The present findings provide strong experimental support for the proposal’ that tachykinins play a role as excitatory transmitters in the human ileum and show that these peptides are likely to account for the major part of the noncholinergic excitatory transmission in the circular muscle. Among the antagonists tested, MEN 10,207 was the most effective in antagonizing the NANC excitatory transmission. It appears unlikely that the inhibitory action of MEN 10,207 on the NANC excitatory response to EFS might involve local anesthetic properties. In fact, the compound had negligible if any inhibitory action toward the atropine-sensitive contraction or the NANC relaxation, which were tetrodotoxin sensitive. This cannot be ruled for L 659,877, which at 3 pmol/L exerted a slight inhibition of NANC relaxation. It should be noted that the concentrations of
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MAGGI ET AL.
GASTROENTEROLOGY
100 -
800 F 60. z i z
40-
% 20-
O1
7 - LOG
6
5
M CONCENTRATION
Figure 4. Effect of various tachykinin antagonists on the delayed noncholinergic contractile response to electrical stimulation. Each value is mean ?r:SE of at least four experiments. 0, MEN 10,207; 0, L 659,677; 0, L 666,169; H, R 396.
tachykinin antagonists required to inhibit the NANC excitatory response were somewhat higher than those expected to be active on the basis of the affinity measured against exogenously administered tachykinins. This finding has also been observed in other examples of tachykininergic transmission [e.g., the NANC contraction of the guinea pig isolated bronchi (Maggi et al., submitted)] and may be related to problems of diffusion of the antagonists to sites of tachykinin release where high local concentrations of the agonist can be reached during nerve stimulation. Certain tachykinin antagonists have been reported to antagonize responses to bombesin.” Thus the specificity of the inhibitory effect of MEN 10,207 on the NANC excitatory innervation might reflect antagonism toward endogenous bombesinlike peptides rather than toward endogenous tachykinins. However, this possibility can be ruled out for two reasons: (a) bombesin (3 pmol/L) has negligible if any contractile effect on the circular muscle of the human ileum (n = 4 strips from four patients) (unpublished data); and (b) MEN 10,207 (3 pmol/L) does not affect the contractile response of the rat isolated bladder to bombesin (Maggi A, Giuliani S, 1990, unpublished data). The circular muscle of human ileum is a multireceptorial preparation for tachykinins. Autoradiographic and functional experiments have provided clear evidente that both NK-1 and NK-2 receptors are present in the circular muscle of the human
Vol. 102, No. 1
smal1 intestine.“*13 Such a conclusion is further supported by the present data. Thus, L 668,169, which has been characterized as a selective NK-l-receptor antagonist,@ antagonized the contractions produced by SP (the putative endogenous ligand for NK-1 receptors) while being inactive, up to its solubility limit (3 pmol/L) against NK-2-receptor-mediated response. By contrast, the NK-2-receptor antagonists MEN 10,207, L 659,877 and R 396(18-20) antagonized with similar potency the response to neurokinin A (the putative endogenous ligand for NK-2 receptors) and the NK-2-selective agonist and were ineffective against SP. The results of these pharmacological experiments indicate that the pharmacological tools used in these studies maintain their characteristics of receptor selectivity even when assayed in a multireceptorial bioassay and validate conclusions about the relative role of SP and neurokinin A in the overall noncholinergic contraction of the human ileum. As observed in a previous study,13 the contraction produced by SP or neurokinin A as wel1 as the response to NK-l- and NK-2-receptor selective agonists is atropine resistant. However, we cannot exclude an indirect component in the responses to tachykinins, possibly mediated through release of other transmitters. Of the antagonists tested, only MEN 10,207 and L 659,877 exerted a significant antagonism toward the
O-
20.
z 0 F =
40.
60.
i 80. s lOOi
r
s -LOG
.
5
M
Figure 5. Effect of MEN 10,207 (circles) and L 659,677 (squares) on the NANC relaxation (PHed symbols) and the noncholinergic contraction (empty symbok) of the circular muscle of human ileum in response to electrical stimulation. Al1 experiments were conducted in the presence of atropine and guanethidine. Each value is mean + SE of five experiments. *Significantly different from baseline (P < 6.65).
TACHYKININERGIC
January 1992
TRANSMISSION
IN HUMAN
UKA t
1 (~2
0
10
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30
Fraction
40
50
70
30
.
20
.
00
Receptor Subtypes in the Human
The development of new, highly selective ligands for NK-2 receptors has allowed a pharmacological differentiation of NK-2-receptor subtypes, provisorily termed NK-2, and NK-2,, respectively2*-24 as follows: NK-2, receptors are characterized by (a) antagonists with the rank order of potency of MEN 10,207 > L 659,877 > R 396 and (b) MDL 28,564 behaving as a full agonist at these sites; NK-2, receptors are characterized by (a) the rank order of potency of antagonists of L 659,877 > R 396 > MEN 10,207 and
0
10
20
30
Fraction
number
NANC contraction produced by field stimulation. The inactivity of R 396 might be explained by its poor affinity for NK-2-receptor subtype present in the human ileum, which wil1 be discussed below. The inactivity of L 668,169 suggests that under the present experimental conditions, the contribution of SP in NANC excitation is minor compared with that played by neurokinin A or putative other endogenous ligands endowed with high affinity for NK-2 receptors. The latter conclusion is strictly related to the experimental conditions used in this study: we cannot exclude that NK-1 receptors play a more important role when different preparations are used (e.g., whole ileum instead of strips). Furthermore, the response to SP- and NK-1-selective agonists showed desensitization, and we cannot exclude the possibility that NK-1 receptors were desensitized during repetitive activation of NANC excitatory nerves. The availability of more potent NK-l-receptor antagonists also seems important to assess the exact role played by SP as compared with neurokinin A in the regulation of intestinal motility. Tachykinin Ileum
60
95
NPK 1
D
40.
Figure 6. Reverse-phase highperformance liquid chromatography characterization of SP- and neurokinin A-like immunoreactivity (LI) in extracts of mucosa (A and B) and muscle (C and D) samples from the human ileum.
ILEUM
40
50
60
70
9
60
number
(b) MDL 28,564 inactive or poorly active as an agonist but acting as a competitive antagonist.‘*-24 Examples of preparations containing the NK-2, receptor are the endothelium-deprived rabbit pulmonary artery’* and the SKLK B82#3 fibroblast cel1 line transfected with complementary DNA encoding the NK-2 receptor originally isolated from bovine stomachz and guinea pig isolated trachea and bronchi.22,24Examples of preparations in which NK-2, receptor is present are the hamster isolated trachea” and urinary bladderz and the rat vas deferens and urinary bladder [Buck et al.” and Maggi A, Pattachini R, 1991, unpublished data]. On the basis of the above-mentioned criteria, it appears that the NK-2-receptor-mediating contraction of the circular muscle to both exogenous and endogenous tachykinins is of the NK-2, subtype. Conclusions This study has provided strong pharmacological evidente for a role of tachykinins as excitatory NANC transmitters in the human ileum. Both NK-1 and NK-2 receptors mediate contraction of the circular muscle of the human ileum to peptides of this family, the latter belonging to the NK-2, subtype. Under the present experimental conditions, the relative role of neurokinin A (or other peptides of this family with high affinity for NK-2 receptors) seems to be more important than that of SP, although both types of tachykinins are present in human ileum. The new generation of receptor-selective tachykinin antagonists allows the definition of discrete events mediated by endogenous tachykinins on specific targets in the gut and might represent a new class of drugs for controlling intestinal motility.
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References 1. Maggio JE. Tachykinins. Annu Rev Neurosci 1988;11:13-28. 2. Pernow B. Substance P. Pharmacol Rev 1983;35:85-141. 3. Bartho L, Holzer P. Search for a physiological role of substance P in gastrointestinal motility. Neuroscience 1987;16:132. 4. Holzer P. Different contractile effects of substance P on the intestine of mammals. Naunyn Schmiedebergs Arch Pharmacol 1982;320:217-220. 5. Katayama Y, North RA, Williams JT. The action of substance P on neurons of the myenteric plexus of the guinea-pig smal1 intestine. Proc R Sec London B 1979;206:191-208. 6. Maggi CA, Patacchini R, Giachetti A, Meli A. Tachykinin receptors in the circular muscle of the guinea-pig ileum. Br J Pharmacol1990;101:996-1000. 7. Costa M, Furness JB, Pullin CO, Bornstein J. Substance P enteric neurons mediate non-cholinergic transmission to the circular muscle of the guinea-pig intestine. Naunyn Schmiedebergs Arch Pharmacol 1985;328:446-453. 8. Ferri GL, Adrian TE, Allen JM, Soimero L, Cancellieri A, Yeats JC, Blank M, Polak JM, Bloom SR, Intramural distribution of regulatory peptides in the sigmoid-recto-anal region of the human gut. Gut 1988;29:762-768. 9. Llewellyn-Smith IJ, Furness JB, Murphy R, O’Brien PE, Costa M. Substance P containing nerves in the human smal1 intestine. Distribution ultrastructure and characterization of the immunoreactive peptide. Gastroenterology 1984;86:421-435. 10. Wattchow DA, Furness JB, Costa M. Distribution and coexistence of peptides in nerve fibers of the external muscle of human gastrointestinal tract. Gastroenterology 1988;95:3241. 11. Gates TS, Zimmermann RP, Mantyh CR, Vigna SR, Maggio JE, Welton ML, Passaro EP, Mantyh PW. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography. Peptides 1989;9:12071219. 12. Maggi CA, Patacchini R, Santicioli P, Giuliani S, Turini D, Barbanti G, Beneforti P, Misuri D, Meli A. Human isolated smal1 intestine: motor responses of the longitudinal muscle to held stimulation and exogenous neuropeptides. Naunyn Schmiedebergs Arch Pharmacol1989;339:415-423. 13. Maggi CA, Patacchini R, Santicioli P, Giuliani S, Turini D, Barbanti G, Giachetti A, Meli A. Human isolated ileum: motor responses of the circular muscle to electrical field stimulation and exogenous neuropeptides. Naunyn Schmiedebergs Arch Pharmacol 1990;341:256-261. 14. Ekblad E, Arnbjornsson E, Ekman R, Hakanson R, Sundler F. Neuropeptides in the human appendix. Distribution and motor effects. Dig Dis Sci 1989;34:1217-1230. regulating in15. Grider JR. Identification of neurotransmitters testinal peristaltic reflex in humans. Gastroenterology 1989; 97:1414-1419.
16 Regoli D. Peptide antagonists. Trends Pharmacol Sci 1985; 6:481-484. 17. Buck SH, Shatzer SA. Agonist and antagonist binding to tachykinin peptide NK-2 receptors. Life Sci 1988;42:27012706. 18. Williams BJ, Curtis NR, McKnight AT, Maguire J, Foster A,
GASTROENTEROLOGY Vol. 102, No. 1
Tridgett T. Development of NK-2 selective antagonists (abstr). Regul Pept 1988;22:189. 19. Rovero P, Pestellini V, Maggi CA, Patacchini R, Regoli D, Giachetti A. A highly selective NK-2 tachykinin receptor antagonist containing n-tryptophan. Eur J Pharmacol 1990;175:113115. 20. Dion S, Rouissi N, Nantel F, Drapeau G, Regoli D, Naline E, Advenier C. Receptors for neurokinins in human bronchus and urinary bladder are of the NK-2 type. Eur J Pharmacol 1990;178:215-219. 21. Maggi CA, Patacchini R, Giuliani S, Rovero P, Dion S, Regoli 0, Giachetti A, Meli A. Competitive antagonists discriminate between NK-2 receptor subtypes. Br J Pharmacol 1990; 100:588-594. 22. Buck SH, Harbeson SL, Hassmann CF BI, Shatzer SA, Rouissi N, Nantel F, Van Giersbergen PLM. [Leug(CH,NH)Leu”]neurokinin A(4-10) (MDL 28,564) distinguishes tissue tachykinin peptide NK-2 receptor% Life Sci Pharmacol Lett 1990;47:PL37-PL41. 23. Van Giesbergen PLM, Shatzer SA, Henderson AK, Lai J, Nakanishi S, Yamamura HI, Buck SH. Characterization of a novel tachykinin peptide NK-2 receptor transfected into murine fibroblast B82 cells. Proc Nat1 Acad Sci USA 1991; 88:1661-1665. 24 Maggi CA, Patacchini R, Astolfi M, Rovero P, Giuliani S, Giachetti A. NK-2 receptor agonists and antagonists. Ann N Y Acad Sci 1991 (in press). 25. Rovero P, Rhaleb NE, Dion S, Rouissi N, Tousignant C, Telemaque S, Drapeau G, Regoli D. Structure activity studies of neurokinin A. Neuropeptides 1989;13:263-270. 26. Maggi CA, Giuliani S, Ballati L, Rovero P, Abelli L, Manzini S, Giachetti A, Meli A. In vivo pharmacology of [BAla’]-NKA(4lO), a selective NK-2 tachykinin receptor agonist. Eur J Pharmacol 1990;177:81-87. E, Rose11 S. 27. Brodin E, Lindefors N, Theodorsson-Norheim Tachykinins multiplicity in rat centra1 nervous system as studied using antisera raised against substance P and neurokinin A. Regul Pept 1986;13:253-272. E, Norheim 1, Oberg K, Lundberg JM, 28. Theodorsson-Norheim Tatemoto K, Lindgren PG. Neuropeptide K: a major tachykinin in plasma and tumor tissue from carcinoid patients. Biochem Biophys Res Comm 1985;131:77-83. 29. Van Rossum JM. Cumulative dose-response curves. 11.Technique for making the dose-response curves in isolated organs and the evaluation of drug parameters. Arch Int Pharmacodyn Ther 1963;143:299-320. 30. Nawa H, Kotani H, Nakanishi S. Tissue specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 1984;312:729-734. 31. Krause JE, Chirgwin JM, Carter MS, Xu ZS, Hershey AD. Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A. Proc Nat1 Acad Sci USA 1987;84:881-885.
Received November 26, 1990. Accepted May 10,199l. Address requests for reprints to: Carlo Alberto Maggi, M.D., Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131, Florence, Italy.
1992:102:88-98
Tachykinin Antagonists Inhibit NerveMediated Contractions in the Circular Muscle of the Human Ileum Involvement
of Neurokinin-2
Receptors
CARLO ALBERT0 MAGGI, SANDRO GIULIANI, RICCARDO PATACCHINI, PAOLO SANTICIOLI, ELVAR THEODORSSON, GABRIELE BARBANTI, DAMIANO TURINI, and ANTON10 GIACHETTI Pharmacology Department, A. Menarini Pharmaceuticals, Karolinska Hospital, Stockholm, Sweden; and Department
The effects of some newly developed tachykinin antagonists that are selective for the neurokinin (NK)1 (L 668,169) or the NK-2 (MEN 10,207, L 659,877 and R 396) tachykinin receptor on the cholinergic and noncholinergic contraction and on the nonadrenergit noncholinergic relaxation produced by electrical field stimulation (50 Hz) were investigated in mucosa-free circular strips of the human ileum. The strips were contracted by substance P and neurokinin A as wel1 as by selective NK-2-receptor ligands, [fiAla*]neurokinin A(4-101, and MDL 28,564, the latter peptide being capable of discriminating between NK-2-receptor subtypes. The selectivity of the antagonists for NK-1 or NK-2 receptors was confirmed in pharmacological experiments using substance P, neurokinin A, and [bAla8]neurokinin A(4-10) as stimulants. Among the NK-2-selective antagonists, MEN 10,207 displayed the highest affinity, followed by L 659,877 and R 396. The antagonists MEN 10,207 and L 659,877 inhibited the noncholinergic contraction to electrical stimulation in a concentration-dependent manner; L 668,169 and R 396 were poorly effective. Thus the potency of antagonists toward the noncholinergic response closely paralleled their rank order of potency at NK-2 receptors. The cholinergic contraction and nonadrenergic noncholinergic relaxation were not inhibited by the antagonists. Both substance P- and neurokinin A-like immunoreactivities were detected in extra& of the human ileum, and the identity of the corresponding peptides was confirmed by reverse-phase high-performance liquid chromatography. It was concluded that in addition to NK-1 receptors, the circular muscle of the human ileum also contains NK-2 receptors. Activation of the latter is chiefly responsi-
Florence, Italy; Department of Clinical Chemistry, of Urology, University of Ferrara, Ferrara, Italy
ble for the noncholinergic ulation.
contraction
to nerve stim-
achykinins are a family of peptides that share the common C-terminal sequence Phe-X-GlyLeu-Met NH,. In mammals, three peptides of this family-substance P (SP) neurokinin A, and neurokinin B-are believed to play a role as transmitters, either in the peripheral or the centra1 nervous system.‘v2 Tachykinins act as excitatory transmitters in the mammalian gut, where they exert potent spasmogenic effects via specific receptors located on muscle cells or indirectly by activating intramural neurons.3-7 A physiological role for tachykinins as excitatory transmitters in the rodent smal1 intestine has been proposed, and they are the most likely mediators of the atropine-resistant peristalsis that can easily be shown in the guinea pig ileum.3 Tachykinins are also present in the human gut.8-‘o In the smal1 intestine, tachykininlike immunoreactivity is localized into nerve fibers distributing to the longitudinal and circular muscle layers, and specific tachykinin receptors are present in both muscular layers of the human ileum as shown by autoradiography.” From the functional point of view, tachykinins are potent spasmogens on both the longitudinal and circular muscle of the human ileum’2*‘3 and appendix.14 In the circular muscle of the human ileum, both NK-1 and NK-2 tachykinin receptors mediate the contractile effect of these peptides, which act without any apparent contribution of cholinergic
T
0 1992 by the American
Gastroenterological
OOlE-5085/92/$3.00
Association
January 1992
TACHYKININERGIC
nerves.13 In a previous study,13 we reported that a consistent fraction of the contractile response of the circular muscle of the human ileum to electrical field stimulation (EFS) is atropine-resistant but tetrodotoxin-sensitive, indicating the existente of noncholinergic excitatory nerves. It has been proposed that in addition to acetylcholine, the tachykinins SP and neurokinin A (also known as substance KJ play a role in the ascending contractile component of the peristaltic reflex in the human ileum.15 Evidente for this was obtained in two ways: (a) by using [D-PrO’,DTrp7vg]SP, a tachykinin antagonist; and (b) by measuring release of SP- and neurokinin A-like immunoreactivity during the ascending contraction.’ It should be noted that the previously used” tachykinin antagonist suffers from a number of drawbacks:‘6*‘7 (a ) p oor, if any, ability to discriminate between NK-1 and NK-2 receptors;17 (b) local anesthetic properties; and (c) ability to antagonize the action of other peptides such as bombesin.” Inasmuch as both NK-1 and NK-2 receptors are present in the circular muscle of the human ileum,“s’3 the data obtained with [D-Pro’,D-Trp7”]SP do not allow determination of the relative contribution of SP (which possesses greater affinity for NK-1 receptors) compared with neurokinin A (which possesses greater affinity for NK-2 receptors) in the overall response. The aim of this study was to address the question of whether endogenous tachykinins might be responsible for the atropine-resistant excitatory neurogenic response of the circular muscle of the human ileum to EFS. With this aim, we investigated the effect of a number of newly developed, receptor-selective tachykinin antagonists’8-20 on the contractile response produced by EFS or exogenous peptides. In addition, we measured content of SP- and neurokinin A-like immunoreactivity of the human ileum by radioimmunoassay coupled with identification of the peptides by reverse-phase high-performance liquid chromatography. Finally, in view of the recent evidente for the existente of NK-2-receptor subtypes,21-24 we also investigated the effect of MDL 28,564, a newly developed NK-2-selective ligand that appears extremely useful to distinguish between NK-2-receptor subtypes.
duced by sodium thiopenthal(500 mg IV) and maintained with N,O/O, (1/2) and halothane (0.6%-1%). Patients received pancuronium bromide (6 mg IV) during induction of anesthesia. Al1 specimens appeared macroscopically normal without signs of tumor or inflammation. The tissues were placed in ice-cold Krebs’ solution within 2-3 minutes after surgical removal. The specimens were pinned flat on a Petri dish containing Krebs’ solution and the mucosa was carefully dissected off. Smal1 strips (0.5-0.8 cm long, 2-3 mm wide) of muscle were cut along the circular axis. Al1 experiments started within 3 hours of surgical excision of the samples. Composition of Krebs’ solution was as follows (mmol/L): NaCl, 119; NaHCO,, 25; KH,PO,, 1.2; MgSO,, 1.5; KCl, 4.7; CaCl,, 2.5; and glucose, ll. Krebs’ solution was gassed with 96% 0, and 4% CO, (pH 7.4). Guanethidine (3 pmol/L) was present throughout the experiments. Atropine (3 pmol/L) was also added to the Krebs’ solution in some experiments. This concentration of atropine was chosen on the basis of previous experimentsI to produce full blockade of contraction produced by a maximally effective concentration of carbachol, thereby allowing investigation of the noncholinergic responses. The strips were suspended under a resting tension of 5 mN in organ baths (5 mL) containing gassed Krebs’ solution at 37°C. Mechanica1 activity was recorded by means of isotonic transducers (Basile no. 7006; Comerio, VA, Italy) and recorded on a Basile 7050 Unirecord. A resting load of 5 mN was applied to strips; on the basis of previous experiments,13 this load allows maxima1 shortening of the preparation in response to stimulants. The strips were electrically stimulated (50 Hz; pulse width 0.5 milliseconds; trains of 5 seconds every 60-90 seconds; maxima1 voltage) by means of two platinum wire electrodes placed at the top and the bottom of the organ bath using a GRASS Sll stimulator (Quincy, MA). Al1 experiments began after an equilibration period of 60 minutes, during which the bathing solution was renewed every 10-15 minutes. Contractile responses were expressed as percents of the maxima1 contractile response to carbachol(l0 pmol/L). At least two comparable responses to carbachol were recorded before studying the responses to EFS or neuropeptides. Concentration-response curves to peptides were constructed in a noncumulative manner for SP and in a cumulative manner for neurokinin A, [BAla’]neurokinin A(4lO), MDL28,564, and carbachol. [BAla’]Neurokinin A(4-10) is a highly selective ligand for NK-2 receptors that behaves as a full agonist at NK-2 receptors (both subtypes).25,26 MDL 28,564 is a new analog of neurokinin A(410) endowed with high selectivity for NK-2 receptors: MDL 28,564 behaves as a full agonist and a competitive antagonist at the two subtypes of NK-2 receptor, respectively.22~24 Curves to SP were constructed in a noncumulative manner because of marked tachyphylaxis, whereas no tachyphylaxis was evident for the response to the other peptides or carbachol. For noncumulative curves, increasing concentrations of SP were added to the bath at lo-zo-minute intervals and left in contact until maxima1 responses had developed. A thorough washing out was made three to
Materials
and Methods
The motor responses to EFS and neuropeptides were investigated on 102 ileal strips from 24 patients (age, 51-85 years) who underwent abdominal surgery for carcinoma of the bladder base (enterocystoplasty). The study was approved by the Ethica1 Committee of the Faculty of Medicine of the University of Ferrara. NO patient received radiotherapy or chemotherapy before intervention. In al1 patients, preanesthetic medication was intramuscular atropine (1 mg) and diazepam (10 mg). Anesthesia was in-
TRANSMISSION
IN HUMAN
ILEUM
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MAGGI ET AL.
GASTROENTEROLOGY
four times at 2-minute intervals between doses. Cumulative curves were constructed by addition of increasing concentrations of the spasmogen, the next concentration being added when the effect of the preceding one had reached a steady state. In experiments aiming to evaluate the effect of antagonists, these were added to the bath 15 minutes before the start of the dose-response curve to the agonist.
Tissue Content of Substance P- and Neurokinin A-Like lmmunoreactivity Tissue content of SP- and neurokinin A-like immunoreactivity was determined by radioimmunoassay on seven samples of human ileum from different patients. From each sample the mucosa was separated from the muscle, and the two specimens were extracted as follows: the specimens were weighed and extracted in 2N acetic acid (1:lO wt/vol) at 95’C for 10 minutes. The samples were homogenized and centrifuged at 4000g for 30 minutes at 4”C, and the supernatant was collected. The pellet was extracted again in bidistilled water at 95’C for 10 minutes (1:lO wt/vol) and centrifuged as described above. The supernatant was collected, added to that obtained after acid extraction, and lyophilized until used for radioimmunoassay. Substance P-like immunoreactivity was analyzed using antiserum SP2,27 which reacts with SP and SP sulfoxide but not with other tachykinins. The detection limit was 10 pmol/L. Intraassay and interassay coefficients of variation were 7% and ll%, respectively. [Tyr’lSubstance P was labeled with lz51 using the chloramine T technique. Neurokinin A-like immunoreactivity was analyzed using antiserum K12, which reacts with neurokinin A (lOO%),neurokinin A(3-10) (48%), neurokinin A(4-10) (45%), neurokinin B (26%), neuropeptide K (61%), and eledoisin (30%) but not with SP.** The detection limit of the assay was 12 pmol/L. Intraassay and interassay coefficients of variation were 7% and 12%, respectively. Neurokinin A was labeled using the Bolton-Hunter reagent.
Reverse-Phase High-Performance Chromatography
Liquid
Reverse-phase high-performance liquidchromatography was performed using LKB Ultropac TSK ODST (LKB Produkter, Bromma, Sweden), 5 Frn, 4.6 X 250 mm eluted with a 40-minute linear gradient of 20%-40% acetonitrile in water containing 0.1% trifluoroacetic acid. Two Pharmacia P~OO pumps (Pharmacia, Uppsala, Sweden) were controlled by a GP250 gradient programmer (Pharmacia). Fractions of 0.5 mL were collected at an elution rate of 1 mL/min. Each fraction was lyophilized and redissolved in 100 pL of distilled water before analysis. The fractions were assayed for immunoreactivity in the tubes used for their collection.
Statistical Analysis Al1 data in the text are mean _t SEM. Statistical analysis was performed using Student’s t test for paired or unpaired data or analysis of variante when applicable. Af-
Vol. 102, No. 1
finity of the antagonists in the pharmacological experiments was evaluated as described by Van Rossumzg and expressed as PA, values. Drugs Drugs used were atropine HCl (Serva, Heidelberg, Germany), guanethidine sulphate (ICFI, Milan, Italy) tetrodotoxin (Sankyo, Tokyo, Japan). The NK-l-selective antagonist L 668,169 and the NK-2-selective antagonist L 659,877 were purchased from Cambridge Research Biochemicals (Cambridge, England). [BAla’]Neurokinin A(4lO), SP, neurokinin A, and MEN 10,207 were synthesized by Dr. P. Rovero, Chemistry Department, Menarini Pharmaceuticals, by conventional solid-phase methods. R 396, an NK-2 antagonist, was kindly provided by Professor D. Regoli, Department of Pharmacology, University of Sherbrooke, Canada. MDL 28,564 was a kind gift of Dr. S. H. Buck, Merrell Dow, Cincinnati, Ohio. Concentrated solutions (1-10 mmol/L) of L 668,169, L 659,877, [BAla’]neurokinin A(4-lO), and MDL 28,564 were prepared in dimethyl sulfoxide (DMSO) and diluted in Krebs’ solution. Control experiments showed that DMSO alone (O.l%0.3% final concentration) had no effect on the responses studied. The other peptides were dissolved in Krebs’ solution. Concentrations of L 668,169 and L 659,877 >3 and 10 pmol/L, respectively, were not tested because of precipitation in the bath. The amino acid sequences of peptides tested are shown in Table 1. Results Response to Exogenous Tachykinins and Synthetic Tachykinin Agonists: Effect of Antagonists Concentration-response curves to SP (n = 16), neurokinin A (n = 16), and the selective NK-2-receptor ligands [BAla*]neurokinin A(4-10) (n = lg), and MDL 28,564 (n = 4) are shown in Figure 1 as compared with carbachol. Curves to tachykinins and synthetic agonists were obtained in the presence of atropine. Threshold concentrations were 1 nmol/L for neurokinin A and [BAlas]neurokinin A(4-10) and 10 nmol/L for the other agonists. The maxima1 effect of tachykinins and synthetic agonists, expressed as percent of the maxima1 response to carbachol was as follows: SP, 71% + 3%; neurokinin A, 70% f 3%; [BAla’]neurokinin A(4-lO), 68% f 4%; and MDL 28,564, 61% f 2%. Data in Table 2 show the affinity values (expressed as PA,) of the various tachykinin antagonists against SP, neurokinin A, and [BAlas]neurokinin A(4-10). MEN 10,207, L 659,877, and R 396 antagonized the response to neurokinin A or the selective NK-2-receptor agonist with the following rank order of potency: MEN 10,207 > L 659,877 > R 396, while L 668,169 was inactive up to 3 pmol/L. On the other hand, L 668,169 was the only antagonist tested to produce a significant antagonism toward SP, with an
TACHYKININERGIC
January 1992
Table
1. Amino Acid
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH~ H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NHz H-Asp-Ser-Phe-Val-BAla-Leu-Met-NH, H-Asp-Ser-Phe-Val-Gly-LeuY(CH,NH)Leu-NH* H-Asp-Tyr-DTrp-Val-DTrp-DTrp-Arg-NH, cyclo(Leu-Met-Gln-Trp-Phe-Gly) cyclo(GIn-DTrp-(NMe)Phe(R)Gly[ANC-2]Leu-Met), Ac-Leu-Asp-Gin-Trp-Phe-Gly-NH,
apparent PA, of 6.31 (Table 2). Each antagonist was also tested (n = 3 for each peptide) against carbachol without any significant effect. Response to Electrical Field Stimulation: Effect of Atropine and Tetrodotoxin We previously reportedX3 that the response of the circular muscle of the human isolated ileum to EFS has three distinct components, i.e., a cholinergic and noncholinergic contraction and a nonadrenergit, noncholinergic (NANC) relaxation. These components are more or less evident depending on the frequency of stimulation. For the purposes of this
lOO-
80. z : 60
: >i
40s
I 8
20-
0
O9
IN HUMAN ILEUM 91
Sequences of Peptides Used in This Study
Agonists SP Neurokinin A [BAla*]Neurokinin A (4-10) MDL 28,564 Antagonists MEN 10,207 L 659,877 L 668,169 R 396
i
TRANSMISSION
a - LOG
7
M
s
.
5
CONCENTRATION
Figure 1. Concentration-response curves for contraction of the circular muscle of human ileum produced by natura1 tachykinins SP, (0) and neurokinin A (0) and by the receptor-selective NK-2 agonists [@Alaqneurokinin A (4-10) (0)and MDL 28,564 (ml compared with carbachol (A). Each value is mean f SE of 4-18 experiments. Responses are expressed as percents of the maximal response to each agonist.
study, we concentrated our analysis on the response produced by EFS at 50 Hz. Electrical field stimulation (50 Hz; trains of 5 seconds every 60-90 seconds; 0.5-millisecond pulse width; maxima1 voltage) produced regular phasic contractions of mucosa-free circular strips of the human ileum. In the absente of atropine, the predominant pattern of response (about 80% of cases) was a primary contraction (solid arrows in Figure 2) that faded toward baseline at cessation of the train of stimuli and was followed by a more sustained and delayed tonic-phasic contraction (hollow arrows in Figure 2). In the remainder, the response to EFS with the above-mentioned parameters was monophasic. These latter preparations were studied only in the presence of atropine to rule out the cholinergic response (Figure 3). At a concentration (3 pmol/L) that abolished the response to carbachol (10 pmol/L), atropine also inhibited (>85%) the early component of the biphasic response to EFS. This latter response wil1 be hereafter referred to as the cholinergic response to EFS. In the absente of atropine, the cholinergic response to EFS averaged 23% + 5% of the response to carbachol (range, lO%-71%), while the delayed response averaged 56% t 6% (range, 36%89%) (n = 28). The effect of tachykinin antagonists was tested on strips showing distinct cholinergic and noncholinergit responses to EFS, either in the absente or in the presence of atropine. As reported previously,‘3 a relaxant response to EFS became evident in the presence of atropine and was followed by a delayed, NANC contraction (Figure 3). Atropine-treated strips were used to test the effects of tachykinin antagonists on the NANC contraction and relaxation. Al1 the responses to field stimulation, either in the absence or in the presence of atropine, were abolished (1 pmol/L; n = 6), indicating that by tetrodotoxin they depend on activation of intramural nerves. The only exception, observed in three of six strips tested, was a smal1 immediate phasic contraction to field stimulation which was stil1 present after tetrodotoxin, probably involving direct activation of muscle
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MAGGI ET AL.
TabJe 2.
GASTROENTEROLOGY
Vol. 102, No. 1
VaJues of Tachykinin Antagonists Toward Contractions Produced by Substance P, Neurokinin A, or [PAJa’jNeurokinin A(4-10) in the Circular MuscJe of Human Isolated IJeum
pA2
[BAla*]Neurokinin Antagonist
SP
MEN 10,207 L 659, 877 R 396 L 668,169
Inactive up to 10 pmol/L
Neurokinin A
A(4-10)
7.25 + 0.11 6.64 + 0.16 6.10 + 0.09 Inactive up to 3 pmol/L
Inactive up to 10 pmol/L Inactive up to 30 pmol/L 6.31 + 0.10
7.29 + 0.13 6.83 + 0.12 6.11 + 0.08 Inactive up to 3 pmol/L
NOTE. Each value is mean f SEM of at least four determinations.
cells. In those strips in which atropine failed to completely abolish the early component of the biphasic response to EFS (e.g., Figure 2), the residual response (1 kmol/L). was also resistant to tetrodotoxin Effect of Tachykinin Antagonists on the Responses to Electrical Field Stimulation Data in Figure 4 show the effect of MEN 10,207, L 659,877, R 396, and L 668,169 (0.1-3 pmol/ L) on the delayed noncholinergic response to EFS (n = at least 4 for each antagonist). In the same experiments, performed in the absente of atropine, none of the antagonists produced a significant depression of the cholinergic response (see Figure 2 for a typical experiment with MEN 10,207). MEN 10,207 was the most effective antagonist tested, followed by L 659,877;R 396 and L 668,169 were barely effective at 3-10 pmol/L (Figure 4). Thus the rank order of potency of tachykinin antagonist closely resembled that found in pharmacological experiments against neurokinin A or the selective NK-2 agonist [PAla*]neurokinin A(4-10). In other experiments, the effect of antagonists was investigated on the NANC contraction and NANC
oe
t’ CONTROL
0.1 $4
relaxation in the presence of atropine and guanethidine (3 pmol/L) (Figures 3 and 5). MEN 10,207 and L 659,877 produced a concentration-dependent inhibition of the NANC contraction. The NANC relaxation was unaffected by MEN 10,207 (Figure 3) and slightly reduced by L 659,877 at 3 kmol/L (Figure 5). L 668,169 and R 396 had no significant effect up to 3 pmol/L (n = 5 strips for each antagonist). Substance P- and Neurokinin A-Like Immunoreactivity in the Human Ileum Both SP- and neurokinin A-like immunoreactivities were detected in extracts of the human ileum as follows: muscle layers, 11.7 f 4 and 4.9 + 1.7 pmol/g of wet weight; mucosal layer, 5.9 f 2 and 3.8 f 1.4 pmol/g of wet weight for SP- and neurokinin A-like immunoreactivity, respectively (n = 7 in each case). Reverse-phase high-performance liquid chromatography showed the presence of authentic SP and neurokinin A and their oxidized forms in both the muscular (Figure 6A and B) and mucosal (Figure SC and D) layers of the ileum. A smal1 but distinct peak coeluting in the same position of neuropeptide K was also evident (Figure 6B and D).
t’
t’
t’
0.3 pM
1 PM
3 PM
,otpMe
t’
ATROPINE 3w
MEN 10207 Figure 2. Effect of increasing concentrations of MEN 10,207 on the contraction produced by EFS (dot& In the majority of the strips tested (such as the example shown here), the response to field stimulation was characterized by an early phasic contraction (solid arrow) that faded toward baseline after cessation of the stimulus and was followed by a complex and delayed phasic-tonic contraction (bollow urrow). Note that MEN 10,207 decreased the delayed component of the response in a concentration-dependent manner while leaving the early phasic contraction unaffected. The latter is markedly inhibited by atropine, indicating its cholinergic nature.
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TRANSMISSION
IN HUMAN
ILEUM
93
5 min
CONTROL
1 ATROPINE
3 pCLM 2 min
Figure 3. (Upperpanek) Example of monophasic response to EFS and its modification by atropine, which unmasked a primary relaxation followed by a rebound, atropine-resistant contraction. (Lower panels) MEN 10,207 inhibited the noncholinergic contraction, leaving the NANC primary relaxation unaffected.
i 1 I
\
z I I
0.1
/LM MEN ATROPINE
Discussion Tachykinins as Excitatory Transmitters Circular Muscle of the Human Ileum
Rt
\
in the
Beside confirming the presence of SP, the present findings show for the first time that authentic neurokinin A and neuropeptide K are present in both mucosal and muscular layers of human ileum. The ratio of SP to neurokinin A found in tissue extracts (2.3 in the muscle, 1.6 in the mucosa) is consistent with the idea of a common origin of the two peptides from the B-preprotachykinin gene.3os31 Along with the data of release of SP- and neurokinin A-like immunoreactivity during the ascending contraction component of the peristaltic reflex in the human ileum,” these data raise the question of a possible physiological role of tachykinins in the regulation of human ileal motility. Such a role was first suggested by the data of Grider15 using an SP analogue endowed with tachykinin-antagonist properties. However, for the reasons exposed in the Introduction, such a tool cannot provide definitive and unequivocal proof for a participation of endogenous tachykinins, nor can it give information about the relative contribution of NK-1 and NK-2 receptors. In the present experimental conditions, contrac-
0.3 @vl 10207
1 PM
3 ,uM
tions produced by EFS of circular muscle strips of the human ileum appear to have three components, one determined by activation of cholinergic nerves; one, putatively tachykininergic, sustained by NANC excitatory nerves; and one, stil1 evident in some strips in the presence of atropine and tachykinin antagonists, also tetrodotoxin resistant and probably originating from direct activation of smooth muscle cells. The present findings provide strong experimental support for the proposal’ that tachykinins play a role as excitatory transmitters in the human ileum and show that these peptides are likely to account for the major part of the noncholinergic excitatory transmission in the circular muscle. Among the antagonists tested, MEN 10,207 was the most effective in antagonizing the NANC excitatory transmission. It appears unlikely that the inhibitory action of MEN 10,207 on the NANC excitatory response to EFS might involve local anesthetic properties. In fact, the compound had negligible if any inhibitory action toward the atropine-sensitive contraction or the NANC relaxation, which were tetrodotoxin sensitive. This cannot be ruled for L 659,877, which at 3 pmol/L exerted a slight inhibition of NANC relaxation. It should be noted that the concentrations of
94
MAGGI ET AL.
GASTROENTEROLOGY
100 -
800 F 60. z i z
40-
% 20-
O1
7 - LOG
6
5
M CONCENTRATION
Figure 4. Effect of various tachykinin antagonists on the delayed noncholinergic contractile response to electrical stimulation. Each value is mean ?r:SE of at least four experiments. 0, MEN 10,207; 0, L 659,677; 0, L 666,169; H, R 396.
tachykinin antagonists required to inhibit the NANC excitatory response were somewhat higher than those expected to be active on the basis of the affinity measured against exogenously administered tachykinins. This finding has also been observed in other examples of tachykininergic transmission [e.g., the NANC contraction of the guinea pig isolated bronchi (Maggi et al., submitted)] and may be related to problems of diffusion of the antagonists to sites of tachykinin release where high local concentrations of the agonist can be reached during nerve stimulation. Certain tachykinin antagonists have been reported to antagonize responses to bombesin.” Thus the specificity of the inhibitory effect of MEN 10,207 on the NANC excitatory innervation might reflect antagonism toward endogenous bombesinlike peptides rather than toward endogenous tachykinins. However, this possibility can be ruled out for two reasons: (a) bombesin (3 pmol/L) has negligible if any contractile effect on the circular muscle of the human ileum (n = 4 strips from four patients) (unpublished data); and (b) MEN 10,207 (3 pmol/L) does not affect the contractile response of the rat isolated bladder to bombesin (Maggi A, Giuliani S, 1990, unpublished data). The circular muscle of human ileum is a multireceptorial preparation for tachykinins. Autoradiographic and functional experiments have provided clear evidente that both NK-1 and NK-2 receptors are present in the circular muscle of the human
Vol. 102, No. 1
smal1 intestine.“*13 Such a conclusion is further supported by the present data. Thus, L 668,169, which has been characterized as a selective NK-l-receptor antagonist,@ antagonized the contractions produced by SP (the putative endogenous ligand for NK-1 receptors) while being inactive, up to its solubility limit (3 pmol/L) against NK-2-receptor-mediated response. By contrast, the NK-2-receptor antagonists MEN 10,207, L 659,877 and R 396(18-20) antagonized with similar potency the response to neurokinin A (the putative endogenous ligand for NK-2 receptors) and the NK-2-selective agonist and were ineffective against SP. The results of these pharmacological experiments indicate that the pharmacological tools used in these studies maintain their characteristics of receptor selectivity even when assayed in a multireceptorial bioassay and validate conclusions about the relative role of SP and neurokinin A in the overall noncholinergic contraction of the human ileum. As observed in a previous study,13 the contraction produced by SP or neurokinin A as wel1 as the response to NK-l- and NK-2-receptor selective agonists is atropine resistant. However, we cannot exclude an indirect component in the responses to tachykinins, possibly mediated through release of other transmitters. Of the antagonists tested, only MEN 10,207 and L 659,877 exerted a significant antagonism toward the
O-
20.
z 0 F =
40.
60.
i 80. s lOOi
r
s -LOG
.
5
M
Figure 5. Effect of MEN 10,207 (circles) and L 659,677 (squares) on the NANC relaxation (PHed symbols) and the noncholinergic contraction (empty symbok) of the circular muscle of human ileum in response to electrical stimulation. Al1 experiments were conducted in the presence of atropine and guanethidine. Each value is mean + SE of five experiments. *Significantly different from baseline (P < 6.65).
TACHYKININERGIC
January 1992
TRANSMISSION
IN HUMAN
UKA t
1 (~2
0
10
20
30
Fraction
40
50
70
30
.
20
.
00
Receptor Subtypes in the Human
The development of new, highly selective ligands for NK-2 receptors has allowed a pharmacological differentiation of NK-2-receptor subtypes, provisorily termed NK-2, and NK-2,, respectively2*-24 as follows: NK-2, receptors are characterized by (a) antagonists with the rank order of potency of MEN 10,207 > L 659,877 > R 396 and (b) MDL 28,564 behaving as a full agonist at these sites; NK-2, receptors are characterized by (a) the rank order of potency of antagonists of L 659,877 > R 396 > MEN 10,207 and
0
10
20
30
Fraction
number
NANC contraction produced by field stimulation. The inactivity of R 396 might be explained by its poor affinity for NK-2-receptor subtype present in the human ileum, which wil1 be discussed below. The inactivity of L 668,169 suggests that under the present experimental conditions, the contribution of SP in NANC excitation is minor compared with that played by neurokinin A or putative other endogenous ligands endowed with high affinity for NK-2 receptors. The latter conclusion is strictly related to the experimental conditions used in this study: we cannot exclude that NK-1 receptors play a more important role when different preparations are used (e.g., whole ileum instead of strips). Furthermore, the response to SP- and NK-1-selective agonists showed desensitization, and we cannot exclude the possibility that NK-1 receptors were desensitized during repetitive activation of NANC excitatory nerves. The availability of more potent NK-l-receptor antagonists also seems important to assess the exact role played by SP as compared with neurokinin A in the regulation of intestinal motility. Tachykinin Ileum
60
95
NPK 1
D
40.
Figure 6. Reverse-phase highperformance liquid chromatography characterization of SP- and neurokinin A-like immunoreactivity (LI) in extracts of mucosa (A and B) and muscle (C and D) samples from the human ileum.
ILEUM
40
50
60
70
9
60
number
(b) MDL 28,564 inactive or poorly active as an agonist but acting as a competitive antagonist.‘*-24 Examples of preparations containing the NK-2, receptor are the endothelium-deprived rabbit pulmonary artery’* and the SKLK B82#3 fibroblast cel1 line transfected with complementary DNA encoding the NK-2 receptor originally isolated from bovine stomachz and guinea pig isolated trachea and bronchi.22,24Examples of preparations in which NK-2, receptor is present are the hamster isolated trachea” and urinary bladderz and the rat vas deferens and urinary bladder [Buck et al.” and Maggi A, Pattachini R, 1991, unpublished data]. On the basis of the above-mentioned criteria, it appears that the NK-2-receptor-mediating contraction of the circular muscle to both exogenous and endogenous tachykinins is of the NK-2, subtype. Conclusions This study has provided strong pharmacological evidente for a role of tachykinins as excitatory NANC transmitters in the human ileum. Both NK-1 and NK-2 receptors mediate contraction of the circular muscle of the human ileum to peptides of this family, the latter belonging to the NK-2, subtype. Under the present experimental conditions, the relative role of neurokinin A (or other peptides of this family with high affinity for NK-2 receptors) seems to be more important than that of SP, although both types of tachykinins are present in human ileum. The new generation of receptor-selective tachykinin antagonists allows the definition of discrete events mediated by endogenous tachykinins on specific targets in the gut and might represent a new class of drugs for controlling intestinal motility.
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References 1. Maggio JE. Tachykinins. Annu Rev Neurosci 1988;11:13-28. 2. Pernow B. Substance P. Pharmacol Rev 1983;35:85-141. 3. Bartho L, Holzer P. Search for a physiological role of substance P in gastrointestinal motility. Neuroscience 1987;16:132. 4. Holzer P. Different contractile effects of substance P on the intestine of mammals. Naunyn Schmiedebergs Arch Pharmacol 1982;320:217-220. 5. Katayama Y, North RA, Williams JT. The action of substance P on neurons of the myenteric plexus of the guinea-pig smal1 intestine. Proc R Sec London B 1979;206:191-208. 6. Maggi CA, Patacchini R, Giachetti A, Meli A. Tachykinin receptors in the circular muscle of the guinea-pig ileum. Br J Pharmacol1990;101:996-1000. 7. Costa M, Furness JB, Pullin CO, Bornstein J. Substance P enteric neurons mediate non-cholinergic transmission to the circular muscle of the guinea-pig intestine. Naunyn Schmiedebergs Arch Pharmacol 1985;328:446-453. 8. Ferri GL, Adrian TE, Allen JM, Soimero L, Cancellieri A, Yeats JC, Blank M, Polak JM, Bloom SR, Intramural distribution of regulatory peptides in the sigmoid-recto-anal region of the human gut. Gut 1988;29:762-768. 9. Llewellyn-Smith IJ, Furness JB, Murphy R, O’Brien PE, Costa M. Substance P containing nerves in the human smal1 intestine. Distribution ultrastructure and characterization of the immunoreactive peptide. Gastroenterology 1984;86:421-435. 10. Wattchow DA, Furness JB, Costa M. Distribution and coexistence of peptides in nerve fibers of the external muscle of human gastrointestinal tract. Gastroenterology 1988;95:3241. 11. Gates TS, Zimmermann RP, Mantyh CR, Vigna SR, Maggio JE, Welton ML, Passaro EP, Mantyh PW. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography. Peptides 1989;9:12071219. 12. Maggi CA, Patacchini R, Santicioli P, Giuliani S, Turini D, Barbanti G, Beneforti P, Misuri D, Meli A. Human isolated smal1 intestine: motor responses of the longitudinal muscle to held stimulation and exogenous neuropeptides. Naunyn Schmiedebergs Arch Pharmacol1989;339:415-423. 13. Maggi CA, Patacchini R, Santicioli P, Giuliani S, Turini D, Barbanti G, Giachetti A, Meli A. Human isolated ileum: motor responses of the circular muscle to electrical field stimulation and exogenous neuropeptides. Naunyn Schmiedebergs Arch Pharmacol 1990;341:256-261. 14. Ekblad E, Arnbjornsson E, Ekman R, Hakanson R, Sundler F. Neuropeptides in the human appendix. Distribution and motor effects. Dig Dis Sci 1989;34:1217-1230. regulating in15. Grider JR. Identification of neurotransmitters testinal peristaltic reflex in humans. Gastroenterology 1989; 97:1414-1419.
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Tridgett T. Development of NK-2 selective antagonists (abstr). Regul Pept 1988;22:189. 19. Rovero P, Pestellini V, Maggi CA, Patacchini R, Regoli D, Giachetti A. A highly selective NK-2 tachykinin receptor antagonist containing n-tryptophan. Eur J Pharmacol 1990;175:113115. 20. Dion S, Rouissi N, Nantel F, Drapeau G, Regoli D, Naline E, Advenier C. Receptors for neurokinins in human bronchus and urinary bladder are of the NK-2 type. Eur J Pharmacol 1990;178:215-219. 21. Maggi CA, Patacchini R, Giuliani S, Rovero P, Dion S, Regoli 0, Giachetti A, Meli A. Competitive antagonists discriminate between NK-2 receptor subtypes. Br J Pharmacol 1990; 100:588-594. 22. Buck SH, Harbeson SL, Hassmann CF BI, Shatzer SA, Rouissi N, Nantel F, Van Giersbergen PLM. [Leug(CH,NH)Leu”]neurokinin A(4-10) (MDL 28,564) distinguishes tissue tachykinin peptide NK-2 receptor% Life Sci Pharmacol Lett 1990;47:PL37-PL41. 23. Van Giesbergen PLM, Shatzer SA, Henderson AK, Lai J, Nakanishi S, Yamamura HI, Buck SH. Characterization of a novel tachykinin peptide NK-2 receptor transfected into murine fibroblast B82 cells. Proc Nat1 Acad Sci USA 1991; 88:1661-1665. 24 Maggi CA, Patacchini R, Astolfi M, Rovero P, Giuliani S, Giachetti A. NK-2 receptor agonists and antagonists. Ann N Y Acad Sci 1991 (in press). 25. Rovero P, Rhaleb NE, Dion S, Rouissi N, Tousignant C, Telemaque S, Drapeau G, Regoli D. Structure activity studies of neurokinin A. Neuropeptides 1989;13:263-270. 26. Maggi CA, Giuliani S, Ballati L, Rovero P, Abelli L, Manzini S, Giachetti A, Meli A. In vivo pharmacology of [BAla’]-NKA(4lO), a selective NK-2 tachykinin receptor agonist. Eur J Pharmacol 1990;177:81-87. E, Rose11 S. 27. Brodin E, Lindefors N, Theodorsson-Norheim Tachykinins multiplicity in rat centra1 nervous system as studied using antisera raised against substance P and neurokinin A. Regul Pept 1986;13:253-272. E, Norheim 1, Oberg K, Lundberg JM, 28. Theodorsson-Norheim Tatemoto K, Lindgren PG. Neuropeptide K: a major tachykinin in plasma and tumor tissue from carcinoid patients. Biochem Biophys Res Comm 1985;131:77-83. 29. Van Rossum JM. Cumulative dose-response curves. 11.Technique for making the dose-response curves in isolated organs and the evaluation of drug parameters. Arch Int Pharmacodyn Ther 1963;143:299-320. 30. Nawa H, Kotani H, Nakanishi S. Tissue specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 1984;312:729-734. 31. Krause JE, Chirgwin JM, Carter MS, Xu ZS, Hershey AD. Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A. Proc Nat1 Acad Sci USA 1987;84:881-885.
Received November 26, 1990. Accepted May 10,199l. Address requests for reprints to: Carlo Alberto Maggi, M.D., Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131, Florence, Italy.