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Ca2+-antagonist drugs reveal the presence of a new receptor for caerulein in the guinea-pig ileum
54 sity stimulation of A6 and C fibres induced a marked rise in CGRP-LM outflow (+ 193 f 55%). Among the different noxious stimuli used, only noxious heat affected (f 135 * 34%) the release of CGRP-LM from the cervicotrigeminal area suggesting that participation of CGRPcontaining primary afferent fibres could be restricted to the transmission of some type of nociceptive information.
Studies on the Effects of Resiniferatoxin, a Novel Pharmacological Tool for Investigation of Capsaicin-Sensitive Primary Afferents R. Porszlsz, G. Petha and J. Szolcslnyi Department of Pharmacology, University Medical School, P&s, Hungary The acute effects of resiniferatoxin (RTX), a potent analog of capsaicin were studied in different experimental arrangements. The examined actions of RTX were: 1) Alterations in afferent function, 2) Modulation of effects evoked by antidromic stimulation, and 3) Effects on axonal conduction. ad 1) In guinea pigs i.v. administered capsaicin failed to elicit the full pulmonary chemoreflex triad, but caused a short period of tachypnoe (6-9 set), hypotension, without bradycardia. RTX (1 pgikg i.v.) caused an initial tachypnoe followed by slowly developing shallow breathing and after a transient hypotension an increase in blood pressure. RTX inhibited the reflex response elicited by capsaicin for about ten minutes. After bilateral cervical vagotomy neither tachypnoe nor hypotension was observed. These results show that in the guinea-pig capsaicin evokes a vagal pulmonary chemoreflex, which differs from that evoked in other mammalian species (rat, cat, rabbit). This reflex response is inhibited by RTX like in other species, but only for a short period of time. ad 2) In the rat, antidromic excitation of dorsal roots (L4, Ls, S,) caused an increase in blood fhrx in glabrous and hairy skin of the paw as well as in the muscles of hindlimb and in the coverings of testis. These responses were abolished by RTX (1 pg/kg iv.) It is concluded that antidromic vasodilatation mediated by capsaiciniresiniferatoxin-sensitive afferents can be evoked not only in exteroceptive but also in interoceptive areas. ad 3) RTX (0. l-l pgkg i.v.) did not alter the evoked compound action potential of the saphenous nerve segment in the rat indicating that this compound is devoid of local anaesthetic activity up to a dose of 1 pg/‘kg. Higher doses (5 or 10 @kg) caused inhibition of both A and C fiber conduction. It is concluded that at the periphery sensory receptors are the primary site of action of RTX.
Importance of the C-Terminal Amide for the Biological Activity of Neurokinin A Derivatives L. Quartara, R. Patacchini, C. GOSO, P. Rover0 and C. A. Maggi Chemistry and Pharmacology Department A. Menarini Pharmaceuticals, Via Sette Santi 3, 50 13 1 Florence, Italy Peptides of the tachykinin (TK) family are amidated at their C-terminal residue through a specific post-transla-
NEUROPEPTIDES
tional enzymatic process. The C-terminal amide is known to be of importance for the agonist activity of TKs. In this study we have assessed the effect of neurokinin A (NKA) its C-terminal heptapeptide NKA(C 10) and their free acid derivatives, NKA-OH andNKA(4- lO)-OH on the isolated guinea-pig bronchus, as compared to the activity of an NKZ receptor antagonist, MEN 10,376 [Ty?, D-Tt@*.“. Lys’O]NKA(4-10) (1) and its free acid derivative MEN 10,456, in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 pM each). NKA and NKA(C10) behaved as full agonists with ECto values of 3 (l-6) and 0.45 (0.04-0.9) nM, respectively (numbers in brackets are 95% cl.). NKA-OH and NKA(4-lO)-OH behaved as weak agonists, their maximal effect (at 30 @I) being 56 f 0.05 and 67 f 0.06% of that to NKA, respectively. MEN 10,376 displayed a barely detectable agonist activity in the guinea-pig bronchus, up to 30 @4. In sharp contrast, the free acid derivative of MEN 10,376, MEN 10,456, was a nearly full agonist (Emax = 93 + 0.01% of that to NKA), with an EC,, of 647 nM (95% c.1.: 420-875 nM). The response to MEN 10,456 was antagonized by MEN 10,376, but not by the NK, receptor antagonist GR 82,334. These findings confirm the importance of C-terminal amidation for expression of the agonist activity of TKs. However the marked recovery of agonist activity observed with the free acid derivative of MEN 10,376 suggests that the C-terminal part of the peptide may not be important per se but rather influences the conformational preferences of the entire molecule. 1. Maggi, C. A. (1991). J. Phannacol. Exp. Ther. 257: 1172.
Ca*+-Antagonist Drugs Reveal the Presence of a New Receptor for Caerulein in the Guinea-Pig Ileum A. Rakovka*, V. Petkov*, M. C. Adomi Ugolotti, R. de Castiglionet and P. Mantovani Institute of Pharmacological Sciences, University of Siena, Italy. *Institute of Physiology, Sofia, Bulgaria. tFarmitalia, Milano, Italy Caerulein (CK) has been shown to contract the guinea-pig ileum through an increase of acetylcholine (ACh) release from the myenteric plexus (1). In the presence of nifedipine (Nif.), CK produced a significant inhibitory effect on the electrically induced contractions. In order to characterise the responses of the guinea-pig ileum to CK in the presence of Nif. and oxyglumide, the spontaneous and electrically evoked contractions of the ileum as well as the release of 3H-Acetylcholine [‘H]ACh from the myenteric plexus neurons were studied. Ileum or long-strips, were collected from guinea-pigs (250-350 g body weight) and were prepared according to the method of Paton and Vizi (2). r3H]ACh release from ileum longitudinal muscle was studied according to the method of Wikberg (3).
NEUROPEPTIDES
IN NORMAL
AND PATHOLOGICAL
55
FUNCTION
CK (from lo-l1 M to 10m7 M) dose-dependently increased the tone of the muscle and the release of [‘H]ACh. Nif. (10” M) reduced the amplitude of the electrically induced contractions by about 60 x 70% and the following administration of CK ( 1F M) caused a further significant reduction of the twitches. This inhibitory effect of CK was not influenced by yohimbine (l@’ M) or naloxone (10 -5M) and was still present in ilea of guinea-pigs pretreated with reserpine (3mg/Kg i.p.; 24h). This effect was antagonised by oxyglumide ( 1O-sM) which is able to abolish also the concomitant release of [‘H]ACh, indicating to be CCK-ergic in nature. These findings indicated the existence of two CKreceptors in guinea-pig ileum. One is devoted to the contraction of the organ and to the release of ACh; the other evidentiating with Nif. causes relaxation of the twitches and a reduction of release of ACh. Both receptors are specifically antagonised by oxyglumide.
BHSar-SP and INKA were unchanged. However, there was a 22% increase (P < 0.05) in density of silver grains over the epithelium but not blood vessels, in ICGRPlabelled sections. This study demonstrates that the receptors for SP, NKA and CGRP are located on different cell types, even though all three peptides appear to be colocalized in primary afferent sensory fibres. ICGRP binding sites on the epithelium appear to be upregulated after capsaicin.
1. Vizi, E. S., Bertaccini, G., Impicciatore, M. and Knoll, J. (1972). Europ. J. Pharmac. 17: 175-178. 2. Paton, W. D. M. andVizi, E. S. (1969). Br. J. Phannacol., 35: 10-29. 3. Wikberg, J. (1977). Acta Physiol. Stand. 101: 302-317.
D. Regoli, N. Rouissi, L. Cbrttien, R. Couture*, P. Picard* and D. Jukic Department of Pharmacology, Medical School, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J 1H 5N4 and *Department of Physiology, Medical School, Universitt de Montreal, Montreal, Quebec H3C 3J7, Canada
Autoradiographic Binding Sites for Tachykinins and Calcitonin Gene-Related Peptide in Rat Urinary Bladder: Effect of Capsaicin D. Rau and E. Burcher School of Physiology and Pharmacology, University of New South Wales, Kensington, NSW 2033, Australia The neuropeptides substance P (SP), neurokinin A (NKA) and calcitoningene-relatedpeptide (CGRP) coexist in capsaicin-sensitive primary afferent sensory neurons in a number of tissues including rat urinary bladder. Although receptors for these peptides occur in the rat bladder, it is not clear whether they are innervated by or associated with the capsaicin-sensitive nerves. We investigated the autoradiographic distribution of binding sites for these neuropeptides, using male Wistar rats (160-220 g) pretreatedtwo weeks previously with capsaicin (cumulative dose of 150 mg/kg) or vehicle. Adjacent frozen sections were radiolabelled with 25-50 pM [1251]-BoltonHunter [Sar9,Met(OZ)11]-SP (BHSar-SP), [‘251]iodohistidy1 NKA (INKA) or [‘251]-rat CGRP-I (ICGRP). Labelled slides were then dipped in photographic emulsion, exposed for 4-6 days, developed and stained (1,2). The density of autoradiographic silver grains was assessed using computer densitometry (MD20 system). In vehicle-pretreated animals, dense specific binding of BHSar-SP was seen mainly over smooth muscle but also over postcapillary venules and arterioles. INKA labelled smooth muscle only, and ICGRP labelled the epithelium and arterioles in the submucosa. In capsaicin-pretreated animals, the distribution and density of binding sites for
1. Burcher, E. and Buck, S. H. (1986). European Journal of Pharmacology 128: 165-178. 2. Watkins, D. J., Geraghty, D. P. and Burcher, E. (1990). Clinical and Experimental Pharmacology and Physiology Suppl. 16: 196.
Pharmacology Antagonists
of Neurokinin Receptor
New antagonists for NK-1, NK-2 andNK-3 receptors have become available from various industrial and academic laboratories. Peptidic and non-peptidic compounds, some of them active orally and able to cross the blood-brain barrier, have been described and extensively tested on binding assays (rat and guinea pig cerebral membranes) pharmacological preparations (rabbit jugular and cava veins (NK- I), guinea pig ileums adequately treated (NKl), rabbit pulmonary arteries without endothelium (NK2), hamster urinary bladder (NK-2), rat portal vein (NK-3) and in some in vivo assays in the rat (rat blood pressure, rat tail flick against SP applied intrathecally (i.t.), rat cardiovascular parameters induced by SP applied into the cerebral ventricles (i.c.v.)) or the mouse (mouse writhing syndrome induced by phenylbenzoquinone). The various compounds, CP-96345, RP-67580, SR48968, R-396, R-544, R-474, have been characterized in regard to a) their specificity for neurokinins (by comparing with angiotensin II, bradykinin, desArg9-bradykinin, bombesin, neurotensin and other non-peptide agonists), b) their selectivity for one or the other neurokinin receptors or receptor subtypes, c) their activities on the central nervous system (both at the spinal and supraspinal levels) and d) their ability to cross the blood-brain barrier and some compounds to act by the oral route. The results indicate that CP-96345, RP-67580 and R544 act as pure and fairly selective NK- 1 receptor antagonists, while SR-48968 and R-396 are selective NK-2 antagonists and R-486 is a selective NK-3 receptor antagonist. CP-96345, RP-6758 and R-544 reduce SP-induced hyperalgesia and pain evoked by peripheral stimuli, while
Studies on the Effects of Resiniferatoxin, a Novel Pharmacological Tool for Investigation of Capsaicin-Sensitive Primary Afferents R. Porszlsz, G. Petha and J. Szolcslnyi Department of Pharmacology, University Medical School, P&s, Hungary The acute effects of resiniferatoxin (RTX), a potent analog of capsaicin were studied in different experimental arrangements. The examined actions of RTX were: 1) Alterations in afferent function, 2) Modulation of effects evoked by antidromic stimulation, and 3) Effects on axonal conduction. ad 1) In guinea pigs i.v. administered capsaicin failed to elicit the full pulmonary chemoreflex triad, but caused a short period of tachypnoe (6-9 set), hypotension, without bradycardia. RTX (1 pgikg i.v.) caused an initial tachypnoe followed by slowly developing shallow breathing and after a transient hypotension an increase in blood pressure. RTX inhibited the reflex response elicited by capsaicin for about ten minutes. After bilateral cervical vagotomy neither tachypnoe nor hypotension was observed. These results show that in the guinea-pig capsaicin evokes a vagal pulmonary chemoreflex, which differs from that evoked in other mammalian species (rat, cat, rabbit). This reflex response is inhibited by RTX like in other species, but only for a short period of time. ad 2) In the rat, antidromic excitation of dorsal roots (L4, Ls, S,) caused an increase in blood fhrx in glabrous and hairy skin of the paw as well as in the muscles of hindlimb and in the coverings of testis. These responses were abolished by RTX (1 pg/kg iv.) It is concluded that antidromic vasodilatation mediated by capsaiciniresiniferatoxin-sensitive afferents can be evoked not only in exteroceptive but also in interoceptive areas. ad 3) RTX (0. l-l pgkg i.v.) did not alter the evoked compound action potential of the saphenous nerve segment in the rat indicating that this compound is devoid of local anaesthetic activity up to a dose of 1 pg/‘kg. Higher doses (5 or 10 @kg) caused inhibition of both A and C fiber conduction. It is concluded that at the periphery sensory receptors are the primary site of action of RTX.
Importance of the C-Terminal Amide for the Biological Activity of Neurokinin A Derivatives L. Quartara, R. Patacchini, C. GOSO, P. Rover0 and C. A. Maggi Chemistry and Pharmacology Department A. Menarini Pharmaceuticals, Via Sette Santi 3, 50 13 1 Florence, Italy Peptides of the tachykinin (TK) family are amidated at their C-terminal residue through a specific post-transla-
NEUROPEPTIDES
tional enzymatic process. The C-terminal amide is known to be of importance for the agonist activity of TKs. In this study we have assessed the effect of neurokinin A (NKA) its C-terminal heptapeptide NKA(C 10) and their free acid derivatives, NKA-OH andNKA(4- lO)-OH on the isolated guinea-pig bronchus, as compared to the activity of an NKZ receptor antagonist, MEN 10,376 [Ty?, D-Tt@*.“. Lys’O]NKA(4-10) (1) and its free acid derivative MEN 10,456, in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 pM each). NKA and NKA(C10) behaved as full agonists with ECto values of 3 (l-6) and 0.45 (0.04-0.9) nM, respectively (numbers in brackets are 95% cl.). NKA-OH and NKA(4-lO)-OH behaved as weak agonists, their maximal effect (at 30 @I) being 56 f 0.05 and 67 f 0.06% of that to NKA, respectively. MEN 10,376 displayed a barely detectable agonist activity in the guinea-pig bronchus, up to 30 @4. In sharp contrast, the free acid derivative of MEN 10,376, MEN 10,456, was a nearly full agonist (Emax = 93 + 0.01% of that to NKA), with an EC,, of 647 nM (95% c.1.: 420-875 nM). The response to MEN 10,456 was antagonized by MEN 10,376, but not by the NK, receptor antagonist GR 82,334. These findings confirm the importance of C-terminal amidation for expression of the agonist activity of TKs. However the marked recovery of agonist activity observed with the free acid derivative of MEN 10,376 suggests that the C-terminal part of the peptide may not be important per se but rather influences the conformational preferences of the entire molecule. 1. Maggi, C. A. (1991). J. Phannacol. Exp. Ther. 257: 1172.
Ca*+-Antagonist Drugs Reveal the Presence of a New Receptor for Caerulein in the Guinea-Pig Ileum A. Rakovka*, V. Petkov*, M. C. Adomi Ugolotti, R. de Castiglionet and P. Mantovani Institute of Pharmacological Sciences, University of Siena, Italy. *Institute of Physiology, Sofia, Bulgaria. tFarmitalia, Milano, Italy Caerulein (CK) has been shown to contract the guinea-pig ileum through an increase of acetylcholine (ACh) release from the myenteric plexus (1). In the presence of nifedipine (Nif.), CK produced a significant inhibitory effect on the electrically induced contractions. In order to characterise the responses of the guinea-pig ileum to CK in the presence of Nif. and oxyglumide, the spontaneous and electrically evoked contractions of the ileum as well as the release of 3H-Acetylcholine [‘H]ACh from the myenteric plexus neurons were studied. Ileum or long-strips, were collected from guinea-pigs (250-350 g body weight) and were prepared according to the method of Paton and Vizi (2). r3H]ACh release from ileum longitudinal muscle was studied according to the method of Wikberg (3).
NEUROPEPTIDES
IN NORMAL
AND PATHOLOGICAL
55
FUNCTION
CK (from lo-l1 M to 10m7 M) dose-dependently increased the tone of the muscle and the release of [‘H]ACh. Nif. (10” M) reduced the amplitude of the electrically induced contractions by about 60 x 70% and the following administration of CK ( 1F M) caused a further significant reduction of the twitches. This inhibitory effect of CK was not influenced by yohimbine (l@’ M) or naloxone (10 -5M) and was still present in ilea of guinea-pigs pretreated with reserpine (3mg/Kg i.p.; 24h). This effect was antagonised by oxyglumide ( 1O-sM) which is able to abolish also the concomitant release of [‘H]ACh, indicating to be CCK-ergic in nature. These findings indicated the existence of two CKreceptors in guinea-pig ileum. One is devoted to the contraction of the organ and to the release of ACh; the other evidentiating with Nif. causes relaxation of the twitches and a reduction of release of ACh. Both receptors are specifically antagonised by oxyglumide.
BHSar-SP and INKA were unchanged. However, there was a 22% increase (P < 0.05) in density of silver grains over the epithelium but not blood vessels, in ICGRPlabelled sections. This study demonstrates that the receptors for SP, NKA and CGRP are located on different cell types, even though all three peptides appear to be colocalized in primary afferent sensory fibres. ICGRP binding sites on the epithelium appear to be upregulated after capsaicin.
1. Vizi, E. S., Bertaccini, G., Impicciatore, M. and Knoll, J. (1972). Europ. J. Pharmac. 17: 175-178. 2. Paton, W. D. M. andVizi, E. S. (1969). Br. J. Phannacol., 35: 10-29. 3. Wikberg, J. (1977). Acta Physiol. Stand. 101: 302-317.
D. Regoli, N. Rouissi, L. Cbrttien, R. Couture*, P. Picard* and D. Jukic Department of Pharmacology, Medical School, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J 1H 5N4 and *Department of Physiology, Medical School, Universitt de Montreal, Montreal, Quebec H3C 3J7, Canada
Autoradiographic Binding Sites for Tachykinins and Calcitonin Gene-Related Peptide in Rat Urinary Bladder: Effect of Capsaicin D. Rau and E. Burcher School of Physiology and Pharmacology, University of New South Wales, Kensington, NSW 2033, Australia The neuropeptides substance P (SP), neurokinin A (NKA) and calcitoningene-relatedpeptide (CGRP) coexist in capsaicin-sensitive primary afferent sensory neurons in a number of tissues including rat urinary bladder. Although receptors for these peptides occur in the rat bladder, it is not clear whether they are innervated by or associated with the capsaicin-sensitive nerves. We investigated the autoradiographic distribution of binding sites for these neuropeptides, using male Wistar rats (160-220 g) pretreatedtwo weeks previously with capsaicin (cumulative dose of 150 mg/kg) or vehicle. Adjacent frozen sections were radiolabelled with 25-50 pM [1251]-BoltonHunter [Sar9,Met(OZ)11]-SP (BHSar-SP), [‘251]iodohistidy1 NKA (INKA) or [‘251]-rat CGRP-I (ICGRP). Labelled slides were then dipped in photographic emulsion, exposed for 4-6 days, developed and stained (1,2). The density of autoradiographic silver grains was assessed using computer densitometry (MD20 system). In vehicle-pretreated animals, dense specific binding of BHSar-SP was seen mainly over smooth muscle but also over postcapillary venules and arterioles. INKA labelled smooth muscle only, and ICGRP labelled the epithelium and arterioles in the submucosa. In capsaicin-pretreated animals, the distribution and density of binding sites for
1. Burcher, E. and Buck, S. H. (1986). European Journal of Pharmacology 128: 165-178. 2. Watkins, D. J., Geraghty, D. P. and Burcher, E. (1990). Clinical and Experimental Pharmacology and Physiology Suppl. 16: 196.
Pharmacology Antagonists
of Neurokinin Receptor
New antagonists for NK-1, NK-2 andNK-3 receptors have become available from various industrial and academic laboratories. Peptidic and non-peptidic compounds, some of them active orally and able to cross the blood-brain barrier, have been described and extensively tested on binding assays (rat and guinea pig cerebral membranes) pharmacological preparations (rabbit jugular and cava veins (NK- I), guinea pig ileums adequately treated (NKl), rabbit pulmonary arteries without endothelium (NK2), hamster urinary bladder (NK-2), rat portal vein (NK-3) and in some in vivo assays in the rat (rat blood pressure, rat tail flick against SP applied intrathecally (i.t.), rat cardiovascular parameters induced by SP applied into the cerebral ventricles (i.c.v.)) or the mouse (mouse writhing syndrome induced by phenylbenzoquinone). The various compounds, CP-96345, RP-67580, SR48968, R-396, R-544, R-474, have been characterized in regard to a) their specificity for neurokinins (by comparing with angiotensin II, bradykinin, desArg9-bradykinin, bombesin, neurotensin and other non-peptide agonists), b) their selectivity for one or the other neurokinin receptors or receptor subtypes, c) their activities on the central nervous system (both at the spinal and supraspinal levels) and d) their ability to cross the blood-brain barrier and some compounds to act by the oral route. The results indicate that CP-96345, RP-67580 and R544 act as pure and fairly selective NK- 1 receptor antagonists, while SR-48968 and R-396 are selective NK-2 antagonists and R-486 is a selective NK-3 receptor antagonist. CP-96345, RP-6758 and R-544 reduce SP-induced hyperalgesia and pain evoked by peripheral stimuli, while