Naunyn-Schmiedeberg's

Archivesof Pharmacology

Naunyn-Schmiedeberg's Arch Pharmacol (I 986) 333:290- 293

9 Springer-Vedag 1986

Studies on effects of the substance P analogues [i)-Pro 2, D-Trp7,gl-substance P and [i)-Arg D-Trp 7'9, L-Leul ]-substance P not related to their antagonist action Fred Lembeek, Rainer Amann, and Lorfind Barth6 Department of Experimental and Clinical Pharmacology, University of Graz, Universit/itsplatz 4, A-8010 Graz, Austria Summary. 1. Two taehykinin antagonists, [D-Pro 2, D-TrpT'9] substance P (AP-2) and [o-Arg t, D-Trp 7'9, L-Leull]-sub stance P (spantide) were rejected or infused intraarterially into the isolated perfased rabbit ear connected m the body via the nerve only. The effects of these antagonists on venous outflow, release of histamine, and on acetylcholine-induced reflex fall in blood pressure were recorded. The effect of spantide was also investigated on cholinergic "twitch" responses to the isolated field stimulated ileum of the guineapig. 2. Bolus injections o f A P 2 (6.6 nmol and 20 nmol) and spantide 20 nmol and 66 nmol) i.a. caused a dose-dependent reduction in venous outflow, which could mainly be explained by the release of histamine since the histamine H1 receptor blocker mepyramine inhibited this effect; release of histamine was also directly demonstrated. 3. Injections of AP-2 (20 nmol) and spantide (66 nmol) caused nociceptor stimulation which might in part result from the histamine release. 4. The reflex fali in blood pressure due to nociceptor stimulation by acetylcholine was reduced by less than 30% by infusion of the tachykinin antagonists in a concentration of 12 gmol 1-1 but not at 2.4 lamol 1-1. 5. Spantide (up to 100 ~mol 1-1) did not inhibit electrically evoked "twitch" responses of the guinea-pig ileum. The local anaesthetic drug procaine ( 4 . 2 - 4 2 gmol t- i) inhibited these contractions in a concentration-dependent manner. 6. It is concluded that the tachykinin antagonists might show effects which are not related to their specific tachykinin antagonistic action as indicated by the findings in the rabbit ear. However, the present results on the stimulated ileum, along with earlier data obtained at this preparation do not indicate these SP analogues having a strong general neuron suppressive action. Careful testing of specifity of tachykinin antagonists seems advisable when they are used as pharmacoiogical tools in a new preparation. Key words: Tachykinin Antagonists -- Nociceptors -- Histamine release

Introduction Experiments by Karlsson et al. (1984) indicated that tachykinin antagonists might have non-specific neuroSend offprint requests to F, Lembeck at the above address

suppressive properties. This assumption was based on two findings. (1) The compound action potential of the frog sciatic nerve was blocked by two substance P (SP) analogues with tachykmin antagonistic properties in the concentration of 200 lamol 1-~; (2) the antagonist [I>Arg 5, D-TrpT'9] SPs-a~ blocked the contraction of the guinea-pig hilus bronchi due to non-cholinergic nerve stimulation to a larger extent than that induced by SP. These findings were confirmed and completed in a recent publication (Post et al. 1985), Tachykinin antagonists are now being used as pharmacological tools for elucidating the roles of endogenous tachykinins in physiological and pathophysiological processes. A non-specific neurosuppressive effect of tachykinin antagonists would have a considerable impact on the interpretation of data obtained with these substances. A possible neuron-depressing effect of two tachykinin antagonists [D-Pro 2, D-Trp7"9]-SP (AP-2) (H6rig and Schu[theiss 1984) and [D-Arg 1, D-Trp 7'9, L-Leutt]-SP (spantide) (Rosell etal. 1983) was therefore investigated on the reflex blood pressure ~esponse to acety;choline injected intraarterially into the isolated perfused rabbit ear that remained in contact with the body via the nerve only. In this preparation, stimulation of sensory nerve elements by, among others, acetylcholine, bradykinin or histamine induces a reflex fall in blood pressure which enables the quantification of the stimulation (Lembeck 1957; Juan and Lembeck 1974). This response is most likely to involve capsaicin sensitive neurons of the C-fibre type, more specifically, polymodal nociceptors (Juan et al. 1980; Szolcsinyi 1984). This preparation also offers the advantage of effects on vascular tone and histamine release being investigated Etectrtcally-evoked longitudinal "twitch" contractions of the guinea-pig ileum are mediated by cholinergic postganglionic neurons of the myenteric plexus (see Paton 1955; Furness and Costa :~980). These responses were 1eft uninfluenced by [D-Pro 2, D-TrpV'9]-SP (up to 80 lamol 1-~) (Barlh6 el al. 1982). In the present study the possible suppressive action of spantide was also investigated on this type of neuronal response.

Materials and methods Experiments were performed in rabbits of 2.5 to 3_5 kg body weight under pentobarbitone (35 mg k g - , i.v.) anaesthesia according to Lembeck (1957) and Juan and Lembeck (1974). The central artery of the ear was cannulated and the organ

291 was perfused with oxygenated Tyrode's solution (100% 0 2 ) , either under a constant pressure of 60 mbar (Results B and C), which resulted in a flow of about 4 ml rain- 1, or under a constant flow of 3 ml min -1 by a roller pump. SP analogues were dissolved in saline containing 0.1% w/v gelatine; no effect on perfusion rate or blood pressure was observed when vehicle alone was injected in volumes of 0.3 ml. All other drugs were dissolved in saline. For bolus injections a volume of 0.1 ml was used. AP-2 and spantide were applied intraarterially either as bolus injection (Results B and C) or by infusion during 3 min (Results A and D). Before and after the infusion of AP-2 and spantide the same volume of the solvent was infused. Perfusion rate was measured by the venous outflow with an electronic drop recorder. Systemic blood pressure was measured in the carotid artery by a Statham pressure transducer P23Db. Histamine content of the venous outflow was determined by high performance liquid chromatography, according to Skofitsch et al. (1983). During the first 60 rain after completion of surgery, sensitivity of the preparation was tested by injecting acetylcholine in intervals of15 min. When the reflex response to acetylcholine showed to be reproducible experiments were started. Basal median blood pressure varied from 85 to 115 m m Hg between different animals but remained constant during an experiment. Guinea-pigs of 0 . 2 - 0 . 4 kg body mass were killed by a blow to the head. Approximately 1.5 cm segments of middle ileum were suspended in a 2 ml organ bath at 37~ in Tyrode's solution aerated with 02. Longitudinal contractions were recorded via an isotonic transducer (Hugo Sachs Elektronik) on a Rikadenki writer. The tension on the tissue was 5 mN. Electrical field stimulation was applied through a pair of longitudinal electrodes (8 m m apart). Single pulses of supramaximal voltage (40 V), and of 0.2 ms pulse width were delivered at a frequency of 0.05 Hz. In some experiments field stimulation (40 V, 0.1 ms impulse width, 150 impulses at 5 Hz) was carried out in the presence of atropine (1 gmol 1-1).

Substances. [D-Pro 2, D-TrpT'9]-SP ("AP-2") and [D-Arg 1, D-Trp 7'9, L-Leu 11]-SP ("spantide") were a generous gift of Dr. S. Leander, Ferring Pharmaceuticals, Malm6, Sweden. The other durgs used were acetylcholine chloride (Becker, Vienna, Austria); atropine sulfate (Merck, Vienna, Austria), compound 48/80 (Sigma Chemical Co., St. Louis, MO, USA); histamine dihydrochloride (Sigma); mepyramine maleate (Smith, Kline and French, Welwyn Garden City, UK); noradrenaline hydrochloride (Sigma); procaine hydrochloride (Gebro, Fieberbrunn, Austria), tetrodotoxin (Sigma). All doses of the drugs refer to the base. All compounds used were of analytical grade. Statistics. Values are expressed as mean _+ SEM; statistical significance was assessed by the paired t-test. Results

Initial experiments in which AP-2 and spantide were injected i.a. into the ear showed a reduction of the venous outflow which could be explained as the result of histamine release (see Results B, C and D). In the following experiments (Results A) the perfusion fluid contained therefore 3.5 ~tmol 1-1 of mepyramine.

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Fig. 1. Reflex fall in blood pressure of the anaesthetized rabbit with isolated perfused ear, to injection of 61 nmol acetylcholine into the ear artery. Columns represent mean _+ SEM of values obtained before, during and after infusion of the SP antagonists AP-2 (121amol 1-1) and spantide (12 pmol 1-1) in the presence of 3.5 ~mol 1-1 mepyramine. Number of animals n = 6 in both cases. Asterisks indicate statistically significant reduction of the response as compared to preinfusion value; *P < 0.05, **P < 0.01

A. Nociceptor stimulation by acetylcholine during infusion of tachykinin antagonists or procaine Acetylcholine (61 nmol) was injected i.a. at intervals of 5 rain. Values of blood pressure responses obtained during infusion of the tachykinin antagonists were compared to those obtained in pre- and post-infusion periods (ACh was injected 3 min after the start of antagonist infusion). In control periods the aliquot volume of the solvent was infused. The reflex fall was not reduced during infusion of 2.4 gmol 1-~ of AP-2 or spantide. At a concentration of 12 gmol 1-1, the reflex fall in blood pressure was reduced by 24% (AP-2) and by 27% (spantide), (P < 0.05 and P < 0.01, respectively; Fig. 1). The local anaesthetic drug procaine ( 1 0 - 1 5 0 ~tmol 1-1) was infused with the same time course as the SP antagonists, i.e. acetylcholine was injected 3 rain after the infusion of procaine had been started. The reflex fall in blood pressure evoked by acetylcholine was dose-dependently inhibited by procaine. The concentration of procaine causing a 50% reduction of this reflex response was 123 _+ 4 gmol 1-1 (n = 5). In all of the above experiments mepyramine (3.5 lamol 1-1) was present in the perfusion medium.

B. Reduction of perfusion rate by i.a. injection of the tachykinin antagonists When substance P analogues were injected i.a. ( 2 - 2 2 nmol) in intervals of 20 min a dose dependent reduction in the flow rate was observed. AP-2 was more potent in this respect; the threshold dose was 2 nmol, a 50% reduction of venous outflow was caused by 5.4 +_ 0.2 nmol AP-2. For spantide the threshold dose was 6.6 nmol, a 50% reduction of venous outflow was achieved by 22.9 _+ 2.9 nmol (n = 4 each). Immediately after i.a. injection of spantide (66 nmnol) or AP-2 (20 nmol), a dose which caused a more than 90% reduction of venous outflow, a reflex fall in blood pressure was observed. No blood pressure reflex was seen when lower doses were injected, or when the antagonists were infused.

C. Influence of mepyramine on the reduction of flow rate by AP-2 and spantide As a release of histamine by substance P analogues has been shown previously (Hakanson et al. 1982; Skofitsch et al.

292 release eightfold over the preinjection value. The histamine release by spantide was not tested (Fig. 3).

E. Effect of spantide on the isolated, field stimulated guinea-pig ileum Q: - 1 O0

Noradrerlaline 59 p mol

Histamine 9 n mol

AP-2 20 n mol

Spantide 66 nmol

Fig. 2. Reduction in venous outflow from the isolated perfused rabbit ear due to bolus injections ofnoradrenaline (n = 6), histamine (n = 6), and the two SP analogues AP-2 and spantide (n = 3 each), doses as indicated. Values for each substance were obtained before (left-hand-side open columns) during (hatched columns) and 60 rain after the infusion of mepyramine (3.5 gmol 1-~, right hand-side open columns). Means + SEM are given. Significant differences are indicated by asterisks; P < 0.05, ***P < 0.001

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48/80 5 0 0 pug

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Fig. 3. Release of histamine (as determined by high performance liquid chromatography) from the isolated perfused rabbit ear. The effluent was collected in 3-rain periods, perfusion rate was 3 ml rain-1. The tachykinin antagonist AP-2 (12 l~mol 1-1) was infused for 3 rain (horizontal bar); the arrow indicates bolus injection of compound 48/80 (500 gg). Means +_ SEM of 4 experiments, Asterisks indicate statistically significant elevations in histamine release. *P < 0.025, *** P <0.001

1983) mepyramine was used to investigate histamine release by AP-2 and spantide as a possible mechanism of their vasoconstrictor effect. Noradrenaline, histamine and the SP analogues were injected i.a. ; all these drugs caused a reduction of venous outflow. The SP analogues simultaneously caused a reflex fall in blood pressure as described above. In the presence of 3.5 gmol 1-1 mepyramine, the vasoconstrictor response to histamine was nearly abolished whereas that to noradrenaline was unaffected. The effect of 20 nmol AP-2 was reduced to 18%, that of 66 nmol spantide to 34% (Fig. 2). The reflex fall in blood pressure was abolished in 3 out of 6 cases.

D. Histamine release by AP-2 The venous outflow was collected during 11 periods of three minutes each. During the third period AP-2 (12 gmol 1-2) was infused, at the beginning of the eighth period 500 ~tg compound 48/80 was injected to check the viability of the preparation (Schachter 1953). The histamine concentration increased approximately twofold in the fraction collected during the infusion of AP-2 and 4-fold in the subsequent one. Injection of compound 48/80 increased the histamine

Single impulses of electrical stimulation evoked "twitch" responses in isolated ileum; these contractions were abolished by atropine (0.5gmol 1-2) or tetrodotoxin (0.2 pmol 1-1). The effect of spantide and of the local anaesthetic drug procaine was investigated while mepyramine (3.5 l~mol 1-i) was present in the organ bath. Spantide ( 1 - 1 0 0 gmol 1-1) did not reduce "twitch" responses to any extent (n = 5). The substance P antagonistic properties of spantide were confirmed by the findings that 30 gmol 1- 2 of this drug caused an approximately 10-fold shift to the right in SP concentration-response curves (n = 3) and, in the presence of atropine (0.5 gmol l - t ) , suppressed by approximately 70% non-cholinergic contractions due to field stimulation (5 Hz for 30 s; n = 3) cf. Franco et al. 1979; Barth6 and Holzer 1985). On the other hand, "twitch" responses to field stimulation (in the absence of atropine) were readily inhibited by procaine ( 4 . 2 - 4 2 }~mol 1-1), the concentration causing an 50% inhibition being 13.7 + 3.1 pmol 1- t (n = 8).

Discussion The above data point to the necessity of careful testing of specificity of tachykinin antagonists in any experimental system before using them as pharmacological tools. The nerve-mediated cholinergic "twitch" contractions of the guinea-pig ileum were not inhibited by spantide up to 100 lamol 1-1. In a recent publication similar results were obtained with AP-2 (Barth6 et al. 1982). These results exclude a strong neuron-supressive action of AP-2 or spantide on the guinea-pig ileum. Earlier data (Leander et al. 1981 ; Leander and Hakanson 1985; Hakanson et al. 1982; Barth6 et al. 1982) as well as the present experiments indicate that AP-2 and spantide are fairly specific antagonists of SP devoid of general neuronsuppressive properties on intestinal preparations. One should remember that a considerable inhibition of the compound action potential in the frog sciatic nerve was only achieved in the presence of an extremely high concentration of AP-2 (Karlsson et al. 1984). This also seems to hold true for the rat sciatic nerve (Post et al. 1985) where 100 gmol 1-1 of AP-2 was needed to achieve a significant reduction in the action potential for A-alpha fibres and 400 txmol 1- 2 for doing so in C-fibres. In the pain reflex ear preparation 12 gmol 1-1 of either tachykinin antagonist caused a partial inhibition of the pain reflex fall in blood pressure. Because of the vasoconstrictor action of the antagonists which was not completely blocked by mepyramine, effects of higher doses were not investigated. Thus, the nature of the inhibitory effect remains obscure; it may represent a general neuron-suppressive action, however, other explanations may also be possible. SP does not stimulate nociceptors in the rabbit ear preparation (Lembeck and Gamse 1977). On the other hand, etedoisin, another tachykinin, is capable of inducing a reflex fall in blood pressure when injected into the rabbit ear artery; the action of acetylcholine is enhanced by a previous

293 injection o f this peptide (Juan et al. 1984). Thus, it cannot be excluded that the inhibitory action o f the antagonists could result from removing some tonic, tachykinin-mediated potentiation o f drug-induced reflex response. One o f the possible non-specific actions of the tachykinin antagonists is histamine release (see t t a k a n s o n et al. 1982; Skofitsch et al. 1983) which has now been confirmed in the perfused r a b b i t ear preparation. Release o f histamine seems to be responsible for the reduction in the venous outflow due to AP-2 and spantide and might account for the reflex fall in blood pressure seen u p o n the bolus injection o f these drugs; it m a y well be that endogenous histamine, released from mast cells in the close proximity o f sensory nerve endings, could overcome the inhibitory action even o f the high concentration o f mepyramine used. Alternatively, tachykinin antagonists m a y have some algesic action on their own if used as bolus injections resulting in high local concentrations.

Acknowledgements. This investigation was supported by the Austrian Scientific Research Fund (grant No P 5616), the Austrian National Bank (grant No 2216) and the Pain Research Comission of the Austrian Academy of Sciences. The authors wish to thank Dr. S. Leander (Ferring Pharmaceuticals, Malm6, Sweden) for his useful comments to the manuscript. References Barth6 L, Holzer P (1985) Search for a physiological role of substance P in gastrointestinal motility. Neuroscience 16:1 - 3 2 Barth6 L, Holzer P, Donnerer J, Lembeck F (1982) Evidence for the involvement of substance P in the atropine-resistant peristalsis of the guinea-pig ileum. Neurosci Lett 32: 6 9 - 74 Franco R, Costa M, Furness JB (1979) Evidence for the release of endogenous substance P from intestinal nerves. NaunynSchmiedeberg's Arch Pharmacol 306:195 - 201 Furness JB, Costa M (1980) Types of nerves in the enteric nervous system. Neuroscience 5:1 --20 Hakanson R, H6rig J, Leander S (1982) The mechanism of action of a substance P antagonist (D-Proz, D-TrpV'9)-SP. Br J Pharmacol 77: 6 9 7 - 700 H6rig I, Schultheiss H (1984) Structure-activity relationship of C-terminal hexa- and heptapeptide substance P antagonists as studied in the guinea-pig ileum. Eur J Pharmacol 105:65-72

Juan H, Lembeck F (1974) Action of peptides and other algesics on paravascular pain receptors of the isolated perfused rabbit ear. Naunyn-Schmiedeberg's Arch Pharmacol 283:151 - 164 Juan H, Lembeck F, Seewann S, Hack U (1980) Nociceptor stimulation and PGE release by capsaicin. Naunyn-Schmiedeberg's Arch Pharmacol 312:139 - 143 Juan H, Sametz W, Petronijevic S, Lembeck F (1984) Prostaglandin release and nociceptor stimulation by peptides. NaunynSchmiedeberg's Arch Pharmacol 326: 6 4 - 68 Karlsson JA, Finney MJB, Persson CGA, Post C (1984) Substance P antagonists and the role of tachykinins in non-cholinergic bronchoconstriction. Life Sci 35: 268 ~ -- 2691 Leander S, Hakanson R (1985) Are substance P antagonists general ncurosuppressive agents? In: Hakanson K, Sundler F (eds) Tachykinin Antagonists. Elsevier, pp 395-404 Leander S, Hakanson R, Rosell S, Folkers K, Sundler F, Tornqvist K (1981) A specific substance P antagonist blocks smooth muscle contractions induced by non-cholinergic, non-adrenergic nerve stimulation. Nature 294: 467 - 469 Lembeck F (1957) Untersuchungen fiber die Ausl6sung afferenter Impulse. Nauny-Schmiedeberg's Arch Pharmacol 230:1 - 9 Lembeck F, Gamse R (1977) Lack of algesic effect of substance P on paravascular pain receptors. Naunyn-Schmiedeberg's Arch Pharmacol 299: 2 9 5 - 303 Paton WDM (1955) The response of guinea-pig ileum to electrical stimulation by coaxial electrodes. J Physiol 127:40-41 P Post C, Butterworth JF, Strichartz GR, Karlsson JA, Persson CGA (1985) Tachykinin antagonists have potent local anaesthetic actions. Eur J Pharmacol 117 : 3 4 7 - 354 Rosell S, Bj6rkroth U, Xu JC, Folkers K (1983) The pharmacological profile of a substance P (SP) antagonist. Evidence for the existence of subpopulations of SP receptors. Acta Physiol Scand 117:445-449 Schachter M (1953) Anaphylaxis and histamine release in the rabbit. Br J Pharmacol 8:412-419 Skofitsch G, Donnerer J, Petronijevic S, Saria A, Lembeck F (1983) Release of histamine by neuropeptides from the perfused rat hindquarter. Naunyn-Schmiedeberg's Arch Pharmacol 322: 153-157 Szolcsfinyi J (1984) Capsaicin-sensitive chemoceptive neural system with dual sensory-efferent function. In: Chahl LA, Szolcs~nyi J, Lembeck F (eds) 29th IUPS, Satellite Symposium, Australia. Akad6miai Kiad6, Budapest, pp 2 7 - 5 2

Received January 27, 1986/Accepted April 2, 1986