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Blockade of sensory nerve mediated contraction of the rabbit iris sphincter by a series of novel tachykinin antagonists
Regulatory Peptides, 20 (1988) 99-105
Elsevier
99
RPT 00656
Blockade of sensory nerve mediated contraction of the rabbit iris sphincter by a series of novel tachykinin antagonists R. Hfikanson, B. Beding, A. Ljungqvist, J.-Y. C h u , S. Leander, J. T r o j n a r a n d K. Folkers Departments of Pharmacology and Ophthalmology, University"of Lund, Lund (Sweden), Ferring Pharmaceuticals, Malm6 (Sweden), and Institute.for Biomedical Research, University of Texas at Austin, Austin, TX (U.S.A.)
(Received 30 April 1987; revisedversion receivedand accepted 11 September 1987)
Summary Electrical stimulation of the isolated rabbit iris sphincter muscle in the presence of atropine gives rise to a contraction that can be blocked by tachykinin antagonists. The ability of a series of novel tachykinin antagonists to inhibit the contractile effect of SP on the guinea-pig taenia coli and to suppress the electrically evoked contraction of the atropinized rabbit iris sphincter was tested. Several of the novel antagonists were found to be more potent in terms of pA2 and pICso values than the two previously described analogs, [D-Pro 2, D-TrpT'9]SP-(1-11) and [D-Arg 1, D-Trp 7'9, Leu ~qSP-(1 11) (Spantide). Apart from D-Trp in positions 7 and 9 the characteristic features of the potent novel antagonists were D-CI2Phe (or D-Cys(Bzl)) in position 5, Asn in position 6 and Nle in position 11. In addition Pal in position 3 seemed to offer an enhanced potency. Tachykinin; Tachykinin antagonist; Iris sphincter; Sensory nerve; Axon reflex; Ocular injury
Introduction Tachykinins, substance P (SP) in particular, are associated with sensory mechanisms, such as axon reflex-mediated responses to local injury (cf. [1]). There is much Correspondence: R. Hfikanson,Department of Pharmacology,S61vegatan 10, S-223 62 Lund, Sweden.
I00
experimental evidence that local reflexes involving SP in sensory ncrve fibers play a role in the response to injury in the rabbit eye [2]. The iris is supplied with sensory fibers from the ophthalmic branch of the trigeminal nerve. Some of these fibers carry SP [3 6] and they all disappear after trigeminal lesioning [7,8]. Ocular responses to injury include miosis and disruption of the blood aqueous barrier. Both these responses are mimicked to some extent by the injection of SP into the rabbit eye [9-11]. SP is released from nerve endings in the urea in conjunction with stimulation of the trigeminal nerve [9]. Following either trigeminal denervation or exposure to the SP-depleting agent capsaicin [12], ocular responses to injury are greatly inhibited [2,7,13]. Finally, tachykinin antagonists, which represent a useful tool in the study of sensory nerve-mediated responses [14], effectively suppress ocular responses not only to exogenous SP but also to physical injury and to chemical irritants [15]. All SP antagonists examined so far are analogs of SP and their basic feature is D-tryptophan substitutions in positions 7 and 9 [16]. The effects of these competitive antagonists seem to be selectively directed against tachykinin-mediated neurotransmission without affecting other types of neural events [17,18]. Electrical stimulation of the atropinized rabbit iris sphincter produces a slow contraction that cannot be blocked by hexamethonium or guanethidine or by GABAergic, histaminergic Or purinergic blocking agents (Ueda [19] and Beding et al., unpublished observation). However, the contraction is abolished by the neuronal blocker tetrodotoxin and by tachykinin antagonists [20]. In trigeminal-denervated iris sphincter preparations and in capsaicin-exposed preparations electrical stimulation fails to elicit a non-cholinergic non-adrenergic contraction [21 24]. Furthermore, the trigeminally denervated rabbit iris sphincter develops supersensitivity to SP [23]. Together these results suggest that a trigeminal nerve-mediated contraction can be evoked in the isolated rabbit iris sphincter and that this response is tachykininergic in nature. Usually the effectiveness of tachykinin antagonists is assessed by studies testing their ability to counteract the effect of SP (or related tachykinins) on various gut smooth muscle preparations [16]. In the present study we assessed in addition the ability of a series of novel tachykinin antagonists to suppress the sensory nervemediated contraction of the isolated and atropinized rabbit iris sphincter muscle.
Materials and Methods
Studies on the isolated rabbit iris sphincter
Adult pigmented rabbits of either sex, weighing 1.5-3 kg, were killed by a blow to the neck and exsanguinated. The eyes were taken out and opened by an incision 23 mm posteriorly to the limbus, followed by excision of the iris from the ciliary margin. The iris was cut in half and freed from the dilator muscle, leaving approximately a 2-mm wide sphincter muscle. The two halves were mounted vertically on perspex holders in two separate 7-ml tissue baths maintained at 37°C. The bathing fluid was a modified Krebs solution of the following composition (mM): NaCI 133, NaHCO~ 16.3, KCI 4.7, MgCI2 1.0, NaH2PO4 1.4, CaCI/2.5 and glucose 7.8. The
1-01 solution was bubbled with a gas mixture of 93% 0 2 / 7 % C02 giving a pH of 7.2 7.3. Mechanical activity was recorded isometrically using a Grass FT03 force displacement transducer and a Grass model 7 polygraph. Before the start of each experiment the sphincter muscle was allowed to equilibrate for 90 rain under a constant load of 1.5 mN which was maintained throughout the experiments. The preparation is known to respond to exogenous SP with a strong contraction [18,19,21,24]. Electrical field stimulation with square wave pulses (20 Hz, 15 V over the electrodes, 0.3 ms duration, pulse train 10 s) was applied by means of a pair of platinum electrodes connected to a Grass $4C stimulator. Pulse train stimulation of the rabbit iris sphincter muscle in the presence of atropine (10 6 M) and guanethidine (5 x 10 -6 M) evoked slow contractions that were inhibited by tachykinin antagonists and abolished by tetrodotoxin (10 -~ M). Adrenergic or nicotinic cholinergic antagonists are without effect on this response (Beding et al., unpublished observations). The frequency response relationship has been described elsewhere [20]. The peak contraction was reached 20-25 s after the onset of stimulation and tension returned to baseline levels approximately 3~4 min after the end of the pulse train. Although the contractile response to repeated additions of SP is unchanged, repeated electrical stimulation led to a gradual exhaustion of the contractile response [24]. The two iris halves, mounted in separate baths, were therefore stimulated in parallel, one being exposed to increasing concentrations of a tachykinin antagonist, the other not (control preparation). In each experiment the suppression of the electrically evoked contraction in the drug-exposed preparation was directly compared with the equivalent response in the control preparation (set as 100). Dose-response curves were constructed and the pICso values (i.e. the negative logarithm of the molar concentration of the antagonist producing 50% inhibition of the electrically evoked contraction) were calculated. Studies on the isolated guinea pig taenia coli Guinea pig taenia coli preparations, consisting of longitudinal smooth muscle with the attached myenteric plexus [25], were placed in Krebs solution, kept at 4°C for about 1 h and then mounted vertically on a Perspex holder in a 7-ml organ bath maintained at 37°C. One end was attached to a rigid support and the other to a lever connected via a spring to a Grass FT03 force displacement transducer for isotonic registration of mechanical activity. The load on the muscle was set at 0.2 g. The mechanical activity of the preparation was continuously recorded on a Grass model 7 polygraph. The bathing fluid (composed as above) was oxygenated giving a pH of 7.2 7.3. Concentration-response curves were constructed by adding step-wise increasing amounts of SP (200 pl volumes) to the bath. Between applications the bath was rinsed, and the next SP concentration was not tested until the effect of the preceding one had been washed away. The contractile response was expressed in relation to that evoked by a standard concentration of carbachol (10-s M) added by the end of the experiment. All ECso values were calculated by linear regression analysis of the steepest part of each concentration response curve. Three to four concentration-response curves for SP were obtained from each preparation; one with SP alone and 3 with different concentrations of the SP antagonist present. The SP
102
a n t a g o n i s t s were a d d e d in a v o l u m e o f 40/~1. The pA2 values and the slopes o f the Schild plots were calculated as described in [26,27].
Drugs A t r o p i n e (Sigma, St. Louis, M O , U . S . A . ) g u a n e t h i d i n e ( C I B A , Basle, Switzerland), t e t r o d o t o x i n (Sigma). T h e p e p t i d e s were synthesized by the solid phase m e t h o d on a B e c k m a n 990B a u t o m a t i c synthesizer. All peptides except the D-Cys(Bzl) 5 a n a l o g were synthesized on b e n z h y d r y l a m i n e resin ( B e c k m a n B i o p r o d u c t s , Palo Alto, C A ) as described [28]. T h e y were cleaved from the resin by H F ( 1 ) in the presence o f an±sole a n d thioanisole [29] a n d purified by gel filtration a n d p a r t i t i o n c h r o m a t o g r a p h y to 9 7 - 9 9 % p u r i t y as d e t e r m i n e d by H P L C . A m a n u s c r i p t including the details o f these p r o c e d u r e s is in p r e p a r a t i o n ( F o l k e r s et al., to be published). F o r synthesis o f the D-Cys(Bzl) 5 a n a l o g also c h l o r o m e t h y l b e n z y l divinylbenzyl ( 1 % ) p o l y s t y r e n e with theoretical l o a d i n g o f 1.27 m e q / g was used. T h e C - t e r m i n a l a m i n o acid was a n c h o r e d by the p o t a s s i u m f l u o r i d e / d i m e t h y l f o r m a m i d e m e t h o d [30]. Side chain p r o tection was as follows: T r p ( C H O ) , Lys(Cbz), A r g ( H + ) . Boc protective g r o u p was used as ~ - a m i n o p r o t e c t i o n . T h e peptides were cleaved from the carrier by a m m o n olysis in m e t h a n o l . F i n a l d e p r o t e c t i o n was achieved by an overnight t r e a t m e n t with 25% t h i o a n i s o l in trifluoroacetic acid. The p r o d u c t s were purified by ion exchange c h r o m a t o g r a p h y on S e p h a r o s e S-fast flow ( P h a r m a c i a , U p p s a l a , Sweden) a n d prep a r a t i v e reversed p h a s e H P L C . The p u r i t y (usually better than 99.5%) o f the final TABLE i Effectiveness of a series of tachykinin antagonists
Amino acid sequence
pAz and slope (exogenous SP; taenia col±)
ptCs0 (electrical nerve stimulation; iris sphincter)
[D-Pro< D-Trp7'9]SP [D-Argx, D-Trp7'9, Leu I qSP [D-Arg% D-CI2Phe5, o-Trp 7'9, Nle ~qSP [D-Argj , D-CIzPhe5, Asp°, l>Trp 7'9, Nle 1lISP [D-Arg~, o-ClzPhe s, Ash6, o-Trp 7'9, Nle 11]SP [D-Arg~, o-Cys (Bzl)s, Asn 6, D-Trp7'9, Nle 1qSP [D-ArgI , Nle3'l < D-ClzPhe5, Asn6, D-TrpT'9]SP [D-PaU, D-CI2Phe"~,Asn6, D-Trp7'9 Nle 1qSP [D-ArgI , o-ClzPhe s,Asn 6, o-Trp 7, D-Pal9, Nle ~qSP [o-Arg~, Pal< D-CI2Phe5, AsnK D-TrpT'K N1C qSP
6.1 + 0.3 7.1 ± 0.4 7.4 + 0.7 6.9 ±0.1 8.0±0.2 8.1 :~0.1 7.4 ± 0.1 7.4 ± 0.3 6.8 ± 0.4 8.0±0.3
5.1±0.4 5.2±0.4 5.9±0.4 5.4±0.4 6.0±0.7 6.3±0.5 6.2±0.6 5.4~0.5 5.7±0.6 6.5±0.4
-1.00~0.07 -0.98±0.02 -0.84±0.25 -0.80±0.17 -1.01±0.16 -0.97±0.09 0.89±0.13 -0.88±0.20 -0.97±0.18 -1.08±0.09
pA2 and the slope of the Schild plot for each drug were calculated as described in [26]. Means ± S.E.M. (n = 5-12 for the taenia col±; 6-10 for the iris sphincter). D-C12Phe represents D-3,4 dichloroPhe. Cys(Bzl) is benzoylated cysteine and Pal is 3-pyridylalanine. All antagonists presented above are full-length analogs of SP (11 amino acid residues).
103
products was analyzed by reversed phase HPLC. Amino acid analysis and fast atom bombardment mass spectroscopy were used for structure confirmation.
Results
We tested the capacity of a series of newly synthesized D-TrpV'9-substituted SP analogs to inhibit the SP-evoked contraction of the guinea pig taenia coli and to suppress the contractile response of the rabbit iris sphincter evoked by electrical stimulation of sensory nerve fibers. All the analogs tested blocked the contractile effects of exogenous SP in a competitive manner, the slopes of the Schild plots being close to - 1 (Table I). The results, expressed as pA2 and plCso values, respectively, A
mNL I
I
"1. SP 10 .8 M
A t r o p i n e 10 -8 M B
E ~
I
I
,I. SP 1 0 " 8 M
u 100 ~6
S p a n t i d e 10 .4 M
so
c
ooi I
I
"I'SP 10"8 M u
D
A t r o p i n e 10 "6 M + S p a n t i d e 10 .4 M
_/ I
I TTX
10 -6 M
4O-
'7_
~
4" SP 10 "8 M
~
20-
E¢> Z
10' "9
10-8 10-7 10-6 10-5 ' ' Tachykinin antagonist (M)
10-4
Fig. 1. A-D: the response of the isolated rabbit iris sphincter muscle to electrical stimulation (20 Hz, 15 V over the electrodes, 0.3 ms, pulse train 10 s; indicated by black verticals) consists of a rapid twitch that can be blocked by atropine (A) and a slow contraction that can be blocked by tachykinin antagonists (e.g. Spantide) (B). The combination of atropine and Spantide virtually abolishes the response to electrical stimulation (C). Both the atropine-sensitive response and the response that is sensitive to blockade of tachykinin receptors are neuronal as they are both blocked by the neuronal blocker tetrodotoxin (TTX) (D). The contractile response to SP (arrows) can be blocked by Spantide (B) but neither by atropine (A) nor by TTX (D). E: the atropine-resistant contraction is concentration-dependently inhibited by the tachykinin antagonists, 3 of which are illustrated: [D-Arg 1, D-Trp 7"9, Leu11]Sp (Spantide) (I), [D-Arg 1, DCys (Bzl) s, Asn 6, D-Trp 7'9, NIC 1]SP (II) and [D-Arg 1, Pal 3, D-CI2Phe 5, Asn ~, D-Trp 7'9, NIC lISP (II1). Drug-exposed preparations were stimulated in parallel with control preparations (see Materials and Methods). The contraction evoked in the control specimen was set as I00 and the contraction of the drug-exposed preparation was expressed as percentage of control. The plCso values presented in Table I were calculated from experiments such as these.
104
are summarized in Table I. The previously described antagonists, (i)-Pro 2, l)11) and (D-Arg l, D-Trp 7'°, Leu ~~)SP-( 1 11) (Spantide) are included for comparison. The results are illustrated in Fig. 1 which also demonstrates the noncholinergic, presumably tachykinergic nature o f the electrically evoked contraction of the atropinized rabbit iris sphincter [24].
Trp 7'9)8p-( 1
Discussion All the novel D-TrpT'9-substituted analogs o f SP were f o u n d to be competitive antagonists o f SP and several o f them were more potent than (D-Arg 1, D-Trp 7'9, Leu l l ) S P - ( I - 1 1) (Spantide) when tested on the guinea pig taenia coli preparation. Several o f them were also quite effective in blocking the nerve-mediated contraction o f the atropinized rabbit iris sphincter, which is t h o u g h t to reflect the release o f tachykinins [24]. The m a j o r structural features o f the novel potent antagonists, apart from D-Arg ~ and D-Trp ~'9, were D-ClzPhe or Cys(Bzl) in position 5, Asn in position 6 and Nle in position 11. In addition, L-Pal in position 3 offered an increase in potency. The advantage in the use o f the rabbit iris sphincter for assessing tachykinin antagonism lies in the simplicity o f the preparation, the low concentrations o f antagonist required, and the attraction o f working with a response involving an anticipated target in the local response to injury.
Acknowledgements G r a n t support from the Swedish M R C (04X-1007) and Ferring Pharmaceuticals, Malm6, Sweden.
References 1 Pernow, B., Substance P, Pharm. Rev., 35 (1983) 85 141. 2 Bynke, G., H~tkanson, R. and Sundler, F., Substance P and its role in antidromic sensory reflexes in the eye. In L.A. Chahl, J. Szolszanyi and F. Lembeck (Eds.), Antidromic Vasodilatation and Neurogenic Inflammation, Akad6miai Kiad6, Budapest, 1984, pp. 269-280. 3 Cuello, A.C., Del Fiacco, M. and Paxinos, G., The central and peripheral ends of the substance Pcontaining sensory neurons in the rat trigeminal system, Brain Res., 152 (1978) 499-509. 4 Tervo, K., Tervo, T., Er/ink6, L., Er/ink6, O. and Cuello, A.C., lmmunoreactivity for substance P in the Gasserian ganglion, ophthalmic nerve and anterior segment of the rabbit eye, Histochem. J., 13 (1981) 435-443. 5 Tervo, K., Tervo, T., ErS.nk6, L., Er/ink6, O., Valtonen, S. and Cuello, A.C., Effect of sensory and sympathetic denervation on substance P immunoreactivity in nerve fibers of the rabbit eye, Exp. Eye Res., 34 (1982) 577-585. 6 Tornqvist, K., Mandahl, A., Leander, S., Lor~n, I., Hgtkanson, R. and Sundler, F., Substance Pimmunoreactive nerve fibers in the anterior segment of the rabbit eye. Distribution and possible physiological significance, Cell Tissue Res., 222 (1982) 467-477. 7 Butler, J.M., Powell, D. and Unger, W.G., Substance P levels in normal and sensorily denervated rabbit eyes. Exp. Eye Res., 30 (1980) 311 313.
105 8 Miller, A., Costa, M., Furness, J.B. and Chubb, I.W., Substance P immunoreactive sensory nerves supply the rat iris and cornea, Neurosci. Lett., 23 (1981) 243-249. 9 Bill, A., Stjernschantz, J., Mandahl, A., Brodin, E. and Nilsson, G., Substance P: release on trigeminal nerve stimulation, effects in the eye, Acta Physiol. Scand., 106 (1979) 371-373. 10 Holmdahl, G., Hfikanson, R., Leander, S., Rosell, S., Folkers, K. and Sundler, F., A substance P antagonist, [D-Pro:, D-TrpT'9]Sp, inhibits inflammatory responses in the rabbit eye, Science, 214 (1981) 1029-1031. 11 Stjernschantz, J., Sears, M. and Stjernschantz, L., lntraocular effects of substance P in the rabbit, Invest. Ophthalmol. Vis. Sci., 20 (1981) 53-60. 12 Buck, S.H. and Burks, T.F., The neuropharmacology of capsaicin: review of some recent observations, Pharm. Rev., 38 (1986) 179-226. 13 Butler, J.M. and Hammond, B.R., The effects of sensory denervation on the responses of the rabbit eye to prostaglandin El, bradykinin and substance P, Brit. J. Pharmacol., 69 (1980) 495-502. 14 Rosell, S. and Folkers, K., Substance P antagonists: a new type of pharmacological tool, Trends Pharm. Sci., 3 (1982) 211-212. 15 Hfikanson, R., Bynke, G., Beding, B. and Wahlestedt, C., Tachykinin antagonists suppress responses to ocular injury in the rabbit. In R. Hfikanson and F. Sundler (Eds.), Tachykinin Antagonists, Fernstr6m Symp. 6, Elsevier, Amsterdam, 1985, pp. 91 96. 16 Folkers, K., Hfikanson, R., H6rig, J., Jie-Cheng, X. and Leander, S., Biological evaluation of substance P antagonists, Brit. J. Pharmacol., 83 (1984) 449-456. 17 Leander, S., Hftkanson, R., Rosell, S., Folkers, K., Sundler, F. and Tornqvist, K., A specific substance P antagonist blocks smooth muscle contractions induced by non-cholinergic, non-adrenergic nerve stimulatiom Nature (Lond.), 294 (1981) 467-469. 18 Leander, S. and Hfikanson, R., Are substance P antagonists general neurosuppressive agents? In R. Hfikanson and F. Sundler (Eds.), Tachykinin Antagonists, Fernstr6m Symp. 6, Elsevier, Amsterdam, 1985, pp. 395-404. 19 Ueda, N., Muramatsu, 1., Sakakibara, Y. and Fujiwara, M., Noncholinergic, nonadrenergic contraction and substance P in rabbit iris sphincter muscle, Jpn. J. Pharmacol., 31 (1981) 1071-1079. 20 Wahlestedt, C., Bynke, G., Beding, B., yon Leithner, P. and Hfikanson, R., Neurogenic mechanisms in control of the rabbit iris sphincter muscle, Eur. J. Pharmacol., I17 (1985) 303-309. 21 Ueda, N., Muramatsu, I., Hayashi, H. and Fujiwara, M., Trigeminal nerve: the possible origin of substance P-ergic response in isolated rabbit iris sphincter muscle, Life Sci., 31 (1982) 369-375. 22 Zhang, S.Q., Butler, J.M., Ohara, K.J. and Cole, D.F., Sensory neural mechanisms in contraction of the isolated sphincter pupillae: the role for substance P and the effects of sensory denervation on the responses to miotics, Exp. Eye Res., 35 (1982) 43-54. 23 Fujiwara, M., Hayashi, H., Muramatsu, I. and Ueda, N., Supersensitivity of the rabbit iris sphincter muscle induced by trigeminal denervation: the role of substance P, J. Physiol. (Lond.), 350 (1984) 588-597. 24 Hfikanson, R., Beding, B., Ekman, R., Heilig, M., Wahlestedt, C. and Sundler, F., Multiple tachykinin pools in sensory nerve fibers in the rabbit iris, Neuroscience, 21 (1987) 943-950. 25 Burnstock, G., Campbell, G. and Rand, M.J., The inhibitory innervation of the taenia of the guinea-pig caecum, J. Physiol. (Lond.), 182 (1966) 504-526. 26 Arunlakshana, O. and Schild, H.O., Some quantitative uses of drug antagonists, Br. J. Pharmacol. Chemother., 14 (1959) 48 58. 27 Tallarida, K.J., Cowan, A. and Adler, M.W., pAz and receptor differentiation: a statistical analysis of competitive antagonism, Life Sci., 25 (1979) 637-654. 28 Folkers, K., Bowers, C.Y., Momary, F., Friebel, K.J., Kubiak, T. and Maher J., Antiovulatory potency and conformation of an antagonist of the luteinizing hormone-releasing hormone having six D-amino acids, Z. Naturforsch., 37B (1982) 872 876. 29 Wan, Y.-P., Humphries, J., Fisher, G., Folkers, K. and Bowers, C.Y., Inhibitors of the luteinizing hormone-releasing hormone based upon modifications in the 2, 3 and 6 positions, J. Med. Chem., 19 (1976) 199-202. 30 Horiki, K., lgano, K. and Inouye, K,, Synthesis of the Merrifields resin esters of N-protected amino acids with the aid of hydrogen bonding, Chem. Lett., (1978) 165-168.
Elsevier
99
RPT 00656
Blockade of sensory nerve mediated contraction of the rabbit iris sphincter by a series of novel tachykinin antagonists R. Hfikanson, B. Beding, A. Ljungqvist, J.-Y. C h u , S. Leander, J. T r o j n a r a n d K. Folkers Departments of Pharmacology and Ophthalmology, University"of Lund, Lund (Sweden), Ferring Pharmaceuticals, Malm6 (Sweden), and Institute.for Biomedical Research, University of Texas at Austin, Austin, TX (U.S.A.)
(Received 30 April 1987; revisedversion receivedand accepted 11 September 1987)
Summary Electrical stimulation of the isolated rabbit iris sphincter muscle in the presence of atropine gives rise to a contraction that can be blocked by tachykinin antagonists. The ability of a series of novel tachykinin antagonists to inhibit the contractile effect of SP on the guinea-pig taenia coli and to suppress the electrically evoked contraction of the atropinized rabbit iris sphincter was tested. Several of the novel antagonists were found to be more potent in terms of pA2 and pICso values than the two previously described analogs, [D-Pro 2, D-TrpT'9]SP-(1-11) and [D-Arg 1, D-Trp 7'9, Leu ~qSP-(1 11) (Spantide). Apart from D-Trp in positions 7 and 9 the characteristic features of the potent novel antagonists were D-CI2Phe (or D-Cys(Bzl)) in position 5, Asn in position 6 and Nle in position 11. In addition Pal in position 3 seemed to offer an enhanced potency. Tachykinin; Tachykinin antagonist; Iris sphincter; Sensory nerve; Axon reflex; Ocular injury
Introduction Tachykinins, substance P (SP) in particular, are associated with sensory mechanisms, such as axon reflex-mediated responses to local injury (cf. [1]). There is much Correspondence: R. Hfikanson,Department of Pharmacology,S61vegatan 10, S-223 62 Lund, Sweden.
I00
experimental evidence that local reflexes involving SP in sensory ncrve fibers play a role in the response to injury in the rabbit eye [2]. The iris is supplied with sensory fibers from the ophthalmic branch of the trigeminal nerve. Some of these fibers carry SP [3 6] and they all disappear after trigeminal lesioning [7,8]. Ocular responses to injury include miosis and disruption of the blood aqueous barrier. Both these responses are mimicked to some extent by the injection of SP into the rabbit eye [9-11]. SP is released from nerve endings in the urea in conjunction with stimulation of the trigeminal nerve [9]. Following either trigeminal denervation or exposure to the SP-depleting agent capsaicin [12], ocular responses to injury are greatly inhibited [2,7,13]. Finally, tachykinin antagonists, which represent a useful tool in the study of sensory nerve-mediated responses [14], effectively suppress ocular responses not only to exogenous SP but also to physical injury and to chemical irritants [15]. All SP antagonists examined so far are analogs of SP and their basic feature is D-tryptophan substitutions in positions 7 and 9 [16]. The effects of these competitive antagonists seem to be selectively directed against tachykinin-mediated neurotransmission without affecting other types of neural events [17,18]. Electrical stimulation of the atropinized rabbit iris sphincter produces a slow contraction that cannot be blocked by hexamethonium or guanethidine or by GABAergic, histaminergic Or purinergic blocking agents (Ueda [19] and Beding et al., unpublished observation). However, the contraction is abolished by the neuronal blocker tetrodotoxin and by tachykinin antagonists [20]. In trigeminal-denervated iris sphincter preparations and in capsaicin-exposed preparations electrical stimulation fails to elicit a non-cholinergic non-adrenergic contraction [21 24]. Furthermore, the trigeminally denervated rabbit iris sphincter develops supersensitivity to SP [23]. Together these results suggest that a trigeminal nerve-mediated contraction can be evoked in the isolated rabbit iris sphincter and that this response is tachykininergic in nature. Usually the effectiveness of tachykinin antagonists is assessed by studies testing their ability to counteract the effect of SP (or related tachykinins) on various gut smooth muscle preparations [16]. In the present study we assessed in addition the ability of a series of novel tachykinin antagonists to suppress the sensory nervemediated contraction of the isolated and atropinized rabbit iris sphincter muscle.
Materials and Methods
Studies on the isolated rabbit iris sphincter
Adult pigmented rabbits of either sex, weighing 1.5-3 kg, were killed by a blow to the neck and exsanguinated. The eyes were taken out and opened by an incision 23 mm posteriorly to the limbus, followed by excision of the iris from the ciliary margin. The iris was cut in half and freed from the dilator muscle, leaving approximately a 2-mm wide sphincter muscle. The two halves were mounted vertically on perspex holders in two separate 7-ml tissue baths maintained at 37°C. The bathing fluid was a modified Krebs solution of the following composition (mM): NaCI 133, NaHCO~ 16.3, KCI 4.7, MgCI2 1.0, NaH2PO4 1.4, CaCI/2.5 and glucose 7.8. The
1-01 solution was bubbled with a gas mixture of 93% 0 2 / 7 % C02 giving a pH of 7.2 7.3. Mechanical activity was recorded isometrically using a Grass FT03 force displacement transducer and a Grass model 7 polygraph. Before the start of each experiment the sphincter muscle was allowed to equilibrate for 90 rain under a constant load of 1.5 mN which was maintained throughout the experiments. The preparation is known to respond to exogenous SP with a strong contraction [18,19,21,24]. Electrical field stimulation with square wave pulses (20 Hz, 15 V over the electrodes, 0.3 ms duration, pulse train 10 s) was applied by means of a pair of platinum electrodes connected to a Grass $4C stimulator. Pulse train stimulation of the rabbit iris sphincter muscle in the presence of atropine (10 6 M) and guanethidine (5 x 10 -6 M) evoked slow contractions that were inhibited by tachykinin antagonists and abolished by tetrodotoxin (10 -~ M). Adrenergic or nicotinic cholinergic antagonists are without effect on this response (Beding et al., unpublished observations). The frequency response relationship has been described elsewhere [20]. The peak contraction was reached 20-25 s after the onset of stimulation and tension returned to baseline levels approximately 3~4 min after the end of the pulse train. Although the contractile response to repeated additions of SP is unchanged, repeated electrical stimulation led to a gradual exhaustion of the contractile response [24]. The two iris halves, mounted in separate baths, were therefore stimulated in parallel, one being exposed to increasing concentrations of a tachykinin antagonist, the other not (control preparation). In each experiment the suppression of the electrically evoked contraction in the drug-exposed preparation was directly compared with the equivalent response in the control preparation (set as 100). Dose-response curves were constructed and the pICso values (i.e. the negative logarithm of the molar concentration of the antagonist producing 50% inhibition of the electrically evoked contraction) were calculated. Studies on the isolated guinea pig taenia coli Guinea pig taenia coli preparations, consisting of longitudinal smooth muscle with the attached myenteric plexus [25], were placed in Krebs solution, kept at 4°C for about 1 h and then mounted vertically on a Perspex holder in a 7-ml organ bath maintained at 37°C. One end was attached to a rigid support and the other to a lever connected via a spring to a Grass FT03 force displacement transducer for isotonic registration of mechanical activity. The load on the muscle was set at 0.2 g. The mechanical activity of the preparation was continuously recorded on a Grass model 7 polygraph. The bathing fluid (composed as above) was oxygenated giving a pH of 7.2 7.3. Concentration-response curves were constructed by adding step-wise increasing amounts of SP (200 pl volumes) to the bath. Between applications the bath was rinsed, and the next SP concentration was not tested until the effect of the preceding one had been washed away. The contractile response was expressed in relation to that evoked by a standard concentration of carbachol (10-s M) added by the end of the experiment. All ECso values were calculated by linear regression analysis of the steepest part of each concentration response curve. Three to four concentration-response curves for SP were obtained from each preparation; one with SP alone and 3 with different concentrations of the SP antagonist present. The SP
102
a n t a g o n i s t s were a d d e d in a v o l u m e o f 40/~1. The pA2 values and the slopes o f the Schild plots were calculated as described in [26,27].
Drugs A t r o p i n e (Sigma, St. Louis, M O , U . S . A . ) g u a n e t h i d i n e ( C I B A , Basle, Switzerland), t e t r o d o t o x i n (Sigma). T h e p e p t i d e s were synthesized by the solid phase m e t h o d on a B e c k m a n 990B a u t o m a t i c synthesizer. All peptides except the D-Cys(Bzl) 5 a n a l o g were synthesized on b e n z h y d r y l a m i n e resin ( B e c k m a n B i o p r o d u c t s , Palo Alto, C A ) as described [28]. T h e y were cleaved from the resin by H F ( 1 ) in the presence o f an±sole a n d thioanisole [29] a n d purified by gel filtration a n d p a r t i t i o n c h r o m a t o g r a p h y to 9 7 - 9 9 % p u r i t y as d e t e r m i n e d by H P L C . A m a n u s c r i p t including the details o f these p r o c e d u r e s is in p r e p a r a t i o n ( F o l k e r s et al., to be published). F o r synthesis o f the D-Cys(Bzl) 5 a n a l o g also c h l o r o m e t h y l b e n z y l divinylbenzyl ( 1 % ) p o l y s t y r e n e with theoretical l o a d i n g o f 1.27 m e q / g was used. T h e C - t e r m i n a l a m i n o acid was a n c h o r e d by the p o t a s s i u m f l u o r i d e / d i m e t h y l f o r m a m i d e m e t h o d [30]. Side chain p r o tection was as follows: T r p ( C H O ) , Lys(Cbz), A r g ( H + ) . Boc protective g r o u p was used as ~ - a m i n o p r o t e c t i o n . T h e peptides were cleaved from the carrier by a m m o n olysis in m e t h a n o l . F i n a l d e p r o t e c t i o n was achieved by an overnight t r e a t m e n t with 25% t h i o a n i s o l in trifluoroacetic acid. The p r o d u c t s were purified by ion exchange c h r o m a t o g r a p h y on S e p h a r o s e S-fast flow ( P h a r m a c i a , U p p s a l a , Sweden) a n d prep a r a t i v e reversed p h a s e H P L C . The p u r i t y (usually better than 99.5%) o f the final TABLE i Effectiveness of a series of tachykinin antagonists
Amino acid sequence
pAz and slope (exogenous SP; taenia col±)
ptCs0 (electrical nerve stimulation; iris sphincter)
[D-Pro< D-Trp7'9]SP [D-Argx, D-Trp7'9, Leu I qSP [D-Arg% D-CI2Phe5, o-Trp 7'9, Nle ~qSP [D-Argj , D-CIzPhe5, Asp°, l>Trp 7'9, Nle 1lISP [D-Arg~, o-ClzPhe s, Ash6, o-Trp 7'9, Nle 11]SP [D-Arg~, o-Cys (Bzl)s, Asn 6, D-Trp7'9, Nle 1qSP [D-ArgI , Nle3'l < D-ClzPhe5, Asn6, D-TrpT'9]SP [D-PaU, D-CI2Phe"~,Asn6, D-Trp7'9 Nle 1qSP [D-ArgI , o-ClzPhe s,Asn 6, o-Trp 7, D-Pal9, Nle ~qSP [o-Arg~, Pal< D-CI2Phe5, AsnK D-TrpT'K N1C qSP
6.1 + 0.3 7.1 ± 0.4 7.4 + 0.7 6.9 ±0.1 8.0±0.2 8.1 :~0.1 7.4 ± 0.1 7.4 ± 0.3 6.8 ± 0.4 8.0±0.3
5.1±0.4 5.2±0.4 5.9±0.4 5.4±0.4 6.0±0.7 6.3±0.5 6.2±0.6 5.4~0.5 5.7±0.6 6.5±0.4
-1.00~0.07 -0.98±0.02 -0.84±0.25 -0.80±0.17 -1.01±0.16 -0.97±0.09 0.89±0.13 -0.88±0.20 -0.97±0.18 -1.08±0.09
pA2 and the slope of the Schild plot for each drug were calculated as described in [26]. Means ± S.E.M. (n = 5-12 for the taenia col±; 6-10 for the iris sphincter). D-C12Phe represents D-3,4 dichloroPhe. Cys(Bzl) is benzoylated cysteine and Pal is 3-pyridylalanine. All antagonists presented above are full-length analogs of SP (11 amino acid residues).
103
products was analyzed by reversed phase HPLC. Amino acid analysis and fast atom bombardment mass spectroscopy were used for structure confirmation.
Results
We tested the capacity of a series of newly synthesized D-TrpV'9-substituted SP analogs to inhibit the SP-evoked contraction of the guinea pig taenia coli and to suppress the contractile response of the rabbit iris sphincter evoked by electrical stimulation of sensory nerve fibers. All the analogs tested blocked the contractile effects of exogenous SP in a competitive manner, the slopes of the Schild plots being close to - 1 (Table I). The results, expressed as pA2 and plCso values, respectively, A
mNL I
I
"1. SP 10 .8 M
A t r o p i n e 10 -8 M B
E ~
I
I
,I. SP 1 0 " 8 M
u 100 ~6
S p a n t i d e 10 .4 M
so
c
ooi I
I
"I'SP 10"8 M u
D
A t r o p i n e 10 "6 M + S p a n t i d e 10 .4 M
_/ I
I TTX
10 -6 M
4O-
'7_
~
4" SP 10 "8 M
~
20-
E¢> Z
10' "9
10-8 10-7 10-6 10-5 ' ' Tachykinin antagonist (M)
10-4
Fig. 1. A-D: the response of the isolated rabbit iris sphincter muscle to electrical stimulation (20 Hz, 15 V over the electrodes, 0.3 ms, pulse train 10 s; indicated by black verticals) consists of a rapid twitch that can be blocked by atropine (A) and a slow contraction that can be blocked by tachykinin antagonists (e.g. Spantide) (B). The combination of atropine and Spantide virtually abolishes the response to electrical stimulation (C). Both the atropine-sensitive response and the response that is sensitive to blockade of tachykinin receptors are neuronal as they are both blocked by the neuronal blocker tetrodotoxin (TTX) (D). The contractile response to SP (arrows) can be blocked by Spantide (B) but neither by atropine (A) nor by TTX (D). E: the atropine-resistant contraction is concentration-dependently inhibited by the tachykinin antagonists, 3 of which are illustrated: [D-Arg 1, D-Trp 7"9, Leu11]Sp (Spantide) (I), [D-Arg 1, DCys (Bzl) s, Asn 6, D-Trp 7'9, NIC 1]SP (II) and [D-Arg 1, Pal 3, D-CI2Phe 5, Asn ~, D-Trp 7'9, NIC lISP (II1). Drug-exposed preparations were stimulated in parallel with control preparations (see Materials and Methods). The contraction evoked in the control specimen was set as I00 and the contraction of the drug-exposed preparation was expressed as percentage of control. The plCso values presented in Table I were calculated from experiments such as these.
104
are summarized in Table I. The previously described antagonists, (i)-Pro 2, l)11) and (D-Arg l, D-Trp 7'°, Leu ~~)SP-( 1 11) (Spantide) are included for comparison. The results are illustrated in Fig. 1 which also demonstrates the noncholinergic, presumably tachykinergic nature o f the electrically evoked contraction of the atropinized rabbit iris sphincter [24].
Trp 7'9)8p-( 1
Discussion All the novel D-TrpT'9-substituted analogs o f SP were f o u n d to be competitive antagonists o f SP and several o f them were more potent than (D-Arg 1, D-Trp 7'9, Leu l l ) S P - ( I - 1 1) (Spantide) when tested on the guinea pig taenia coli preparation. Several o f them were also quite effective in blocking the nerve-mediated contraction o f the atropinized rabbit iris sphincter, which is t h o u g h t to reflect the release o f tachykinins [24]. The m a j o r structural features o f the novel potent antagonists, apart from D-Arg ~ and D-Trp ~'9, were D-ClzPhe or Cys(Bzl) in position 5, Asn in position 6 and Nle in position 11. In addition, L-Pal in position 3 offered an increase in potency. The advantage in the use o f the rabbit iris sphincter for assessing tachykinin antagonism lies in the simplicity o f the preparation, the low concentrations o f antagonist required, and the attraction o f working with a response involving an anticipated target in the local response to injury.
Acknowledgements G r a n t support from the Swedish M R C (04X-1007) and Ferring Pharmaceuticals, Malm6, Sweden.
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