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Effect of SP and selective NK1 receptor agonist on inositol phosphates and cytosolic calcium levels in cultured human fibroblasts
Neuropeptides (1993) 2423 l-239
0 Longman Group UK Ltd 1993
Session 5: Peptide Receptors (I) Cl8 SR 140333, a Non-Peptide Antagonist of Substance-P (NK1) Receptor X. Emends-Alt, J. D. Doutremepuich, M. Jung, E. Proietto, V. Santucci, D. Van Broeck, P. Vilain, Ph. Soubri& G. Le Fur and J. C. Breliere Sanofi Recherche, F-34184 Montpellier, France SR 140333 inhibited the binding of [‘zsI]-SP to its receptor from rat cortex membranes and human IM9 cells with Ki of 0.02 nMand 0.01 nM, respectively. In classical binding assays for rat NK;! and NK3 receptors, SR 140333 did not show significant affinity o 1000). In vitro, SR 140333 antagonized [Sar,gMet(O~)lL]-SP-induced endothelium-dependent relaxation of the rabbit pulmonaryarterywithpAz= 10.5. SR 140333 (1 mdidnot antagonize [13Alas]-NKA(4-lo)-induced contraction of the rabbit pulmonary artery and [MePhe’]-NKB-induced contraction of the rat portal vein. In vivo, SR 140333 potently antagonized [Sar?Met(O#l]-SP-induced hypotension (ED50 = 0.5 pgkg iv.) in dogs, [Sti, Met(O$“]SP-induced or antidromic sciatic nerve-stimulated skin plasma extravasation (ED50= 7 and 2.5 pgkg i.v., respectively) in rats, [Sar,gMet(O$*]-SP-induced salivation (ED50= 180 ccgflrgi.p.) in rats and septide-induced scratching (EDJo = 53 ClBflrgi.p.) in mice. Moreover, SR 140333 was active per OS (1-5 mgkg). Finally, SR 140333 (0.125-l &kg iv.) potently blocked the electrophysiological response evoked in ventrobasal thalamic neurons by noxious pinch of rat hindpaw.
in the presence of 10 @l indomethacin (see Maggi et al., this meeting). Both agonists produced concentrationdependent contractions with ECsot of 36 and 84 pM for septide and [S&]SP sulfone, respectively. All the antagonists tested exerted competitive antagonism (slope of Schild plots not significantly different from unity) against both agonists, as follows (pKe values and 95% c.1. in brackets. n = 8-15): AMZgOtZiSt
[Sary/SP sulfone
Septide
(k)CP 96,345 GR 82,334
8.17 (7.95-8.38) 7.17 (6.97-7.37)
9.24 (9.07-9.41) 7.52 (7.30-7.74)
FK 888
7.53 (7.39-7.67)
8.30 (8.08-8.52)
(*)CP 96,345 and FK 888 were significantly more potent against septide than against [Sar9]SP sulfone. A tendency to a similar behaviour was also found for GR 82,334 but did not reach statistical significance. Neither (zt)CP 96,345 (up to 1 clM>nor GR 82,334 (up to 10 @4) or FK 888 (up to 3 pM) had any significant effect on the response to the NK-2 receptor selective agonist, [SAla”] NKA(4-10). These findings support the ideal of the existence of a ‘septide-sensitive’ receptor in the circular muscle of GPI, which is recognized with high afXnity by NK- 1 receptor selective antagonists like (rt>CP 96,345 or FK 888. 1. Petitet, F., SatTroy,M., Totrens, Y., Lavielle, S., Chassaing, G., Loeuillet, D., Glowinski, J. and J. C. Beaujouan (1992). Possible existence of a new tachykinin receptor subtype in the guinea-pig ileum. Peptides, 13,383-398.
Cl9 Evidence for the Presence of a Septidesensitive Tachykinin Receptor in the Circular Muscle of the Guinea-pig Ileum
C20 Effect of SP and Selective NKl Receptor Agonist on Inoritol Phosphates and Cytosolic Calcium Levels in Cultured Human Fibroblasts
R. Pa&chin& S. Meini, S. Giuliani and C. A. Maggi Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3 5013 1, Florence, Italy We have investigated the ability of the tachykinin NK-1 receptor antagonists (*)CP 96,345, GR 82,334 and FK 888 to antagonize contractions produced by the NK-1 receptor agonists septide and [Sa?]substance P (SP) sulfone in the circular muscle of the guinea-pig ileum (GPI) 231
M. Ziche, A. Parenti, L. Morbidelli, S. Atnerini, F. Ledda, E. Baldi* and C. A. Maggit Departments of Pharmacology and *Clinical Physiopathology, University of Florence, Viale Morgagni 65, Firenze, Italy and; ~Pharmacology Department, Menarini S.r.1. Firenze, Italy We previously reported that SP sustains proliferation of cultured human fibroblasts (HF) and that this effect is
232 mediated by the interaction with NKl receptors. In this study we have investigated the intracellular mechanisms involved in this receptor-mediated activity. Subconfluent HF labelled with [3H]-myoinositol were exposed to SP and to the selective NKl receptor agonist [Sar9]-SP sulphone. IP3 levels were measured at 3, 6, 10 s following exposure to the peptides and IPl at 15,30 and 60 min. IP3 levels were increased at 3 s after exposure to both peptides and peaked at 6 s (130% and 122% over basal for SP and [S&l-SP sulphone respectively). IPl levels peaked at 15 min in presence of both peptides declining toward basal levels at 60 min. The effect of SP and of the NKl receptor agonist were also investigated on cytosoiic calcium. I-IF were grown on plastic coverslips and loaded with the fluorescent dye FURA-2. Cytosolic calcium was calculated on the base of fluorescence emission recorded with a spettrofluorimetric apparatus. Both peptides almost doubled intracellular calcium levels (243 and 223 r&l for SP and the NKl receptor agonist respectively; 125.2 nM basal). We conclude that binding of SP to its NKl receptor on human skin fibroblasts generates inositol 1,4,5trisphosphate which releases Ca++ ions into the cytosol.
C21 Differences in the Pharmacological Binding Profile of Rat and Human NK2 Tachykinin Receptors V. Fardin, A. Jolly, F. Bellot*, A. Crespo* and F. Vaissade Rhi$e-Poulenc Rorer S.A., Departments of Biology and Biotechnology*, Centre de Recherche de VitryAlfortville, 94403 Vitry-sur-Seine, Cedex, France The binding of *Z510do-hystidylNKA (lz51-NKA) was studied in rat duodenum and urinary bladder and compared to that observed in HSKR-2 cells, a mouse 3T3 fibroblast cell line which expresses the human NK2 receptor cloned from jejunum. 12’1~NKAbinds with high affinity to a single population of sites in all tissues with similar Kd values. The total number of sites was more than double in urinary bladder than in duodenum and HSKR-2 membrane preparations. The binding (at 100 pM ‘*9+JKA) had the same pharmacological characteristics as that expected for NKz sites in all tissues: high inhibitory potency of NKA, (Lys5, MeLe&‘, NorLeu’O)NKA.+.loand SR 48968 and little or no inhibition of NKI [(Pro9) SP, septide, RP 67580 and CP96,345] or m (senktide) selective compounds. However, the rank order of potency for the two NKz peptide antagonists, L-659,877 and MEN-10,207, clearly differed between the two species, being in rat tissues: L-659,877 > MEN-lo,207 and in HSKR-2: MEN-lo,207 > L-659,877. These data reveal differences between rat and human NI& receptors, but not between rat duodenum and urinary bladder and emphasize the importance of developing a human model to search for NKz antagonists with potent activity in humans.
NEUROPEPTIDES
We are grateful to Dr. R. Kris and Dr. J. Schlessinger for the gift of HSKR-2.
C22 A Novel Peptidic Tachykinin Antagonist Which is Potent at NK3 Receptors J. M. Stables, S. Arkinstall*, I. J. M. Beresford, P. Sealet, P. Ward? and R. M. Hagan Glaxo Group Research, Ware, UK, *Glaxo Institute of Molecular Biology, Geneva and tMedicina1 Chemistry, Glaxo Group Research, Greenford, UK While selective and potent NKI and NKz antagonists have been reported, no potent NK, antagonists have been developed. Here we report on a novel, potent NI& antagonist, GR138676, a conformationally constrained analogue of neurokinin B.
In the m receptor-containing rat portal vein preparation, GR138676 caused a concentration-related (3 nM-1 pM) antagonism of se&tide-induced contractions (PKB = 8.24 f 0.09, n = 15, slope = 1.3 (l&.6)). GR138676 (10-200 &I) antagonised se&tide-evoked release of preloaded arachidonic acid from Chinese hamster ovary cells transfected with a human m receptor gene’@& = 8.27 f 0.04, n = 15, slope = 1.2 (1.0-1.3)). However, GR138676 also antagonised the increase in intracellular calcium evoked by the selective NKI agonist, GR73632, in U373MG cells, @I(8 = 8.26 f 0.04, n = 10, slope = 1.1 (0.9-I .3)). It had little activity at NKz receptors, inhibiting binding of the NK, antagonist ligand [3H]-GR100679 to rat colon membranes with a pKi of 5.8. GR138676 has high affinity for the NKLl receptor. Although it is equiactive at NK receptors, will be a usell tool for characterisation of N& receptors in vitro and investigation of behaviours induced by the selective N& agonist senktide in vivo. 1. Buell, G., Schulz, M. F., Arkinstall, S., Maury, K., Missotten, N., Adami, N., Talabot, F. and Kawashima, E. (1992). Molecular characterisation, expression and localisation of human neurokinin-3 receptor. FEBS Lett. 299: 90-95.
P105 Neurokinin Receptor Antagonists D. Regoli* and R. Couturef *Department of Pharmacology, Medical School, Universiti de Sherbrooke, Sherbrooke (Quebec) Canada, JlH 5N4 and TDepartment of Physiology, Medical School, Universitk de Mont&al, Mont&al (Qukbec) Canada, H3C 3J7
0 Longman Group UK Ltd 1993
Session 5: Peptide Receptors (I) Cl8 SR 140333, a Non-Peptide Antagonist of Substance-P (NK1) Receptor X. Emends-Alt, J. D. Doutremepuich, M. Jung, E. Proietto, V. Santucci, D. Van Broeck, P. Vilain, Ph. Soubri& G. Le Fur and J. C. Breliere Sanofi Recherche, F-34184 Montpellier, France SR 140333 inhibited the binding of [‘zsI]-SP to its receptor from rat cortex membranes and human IM9 cells with Ki of 0.02 nMand 0.01 nM, respectively. In classical binding assays for rat NK;! and NK3 receptors, SR 140333 did not show significant affinity o 1000). In vitro, SR 140333 antagonized [Sar,gMet(O~)lL]-SP-induced endothelium-dependent relaxation of the rabbit pulmonaryarterywithpAz= 10.5. SR 140333 (1 mdidnot antagonize [13Alas]-NKA(4-lo)-induced contraction of the rabbit pulmonary artery and [MePhe’]-NKB-induced contraction of the rat portal vein. In vivo, SR 140333 potently antagonized [Sar?Met(O#l]-SP-induced hypotension (ED50 = 0.5 pgkg iv.) in dogs, [Sti, Met(O$“]SP-induced or antidromic sciatic nerve-stimulated skin plasma extravasation (ED50= 7 and 2.5 pgkg i.v., respectively) in rats, [Sar,gMet(O$*]-SP-induced salivation (ED50= 180 ccgflrgi.p.) in rats and septide-induced scratching (EDJo = 53 ClBflrgi.p.) in mice. Moreover, SR 140333 was active per OS (1-5 mgkg). Finally, SR 140333 (0.125-l &kg iv.) potently blocked the electrophysiological response evoked in ventrobasal thalamic neurons by noxious pinch of rat hindpaw.
in the presence of 10 @l indomethacin (see Maggi et al., this meeting). Both agonists produced concentrationdependent contractions with ECsot of 36 and 84 pM for septide and [S&]SP sulfone, respectively. All the antagonists tested exerted competitive antagonism (slope of Schild plots not significantly different from unity) against both agonists, as follows (pKe values and 95% c.1. in brackets. n = 8-15): AMZgOtZiSt
[Sary/SP sulfone
Septide
(k)CP 96,345 GR 82,334
8.17 (7.95-8.38) 7.17 (6.97-7.37)
9.24 (9.07-9.41) 7.52 (7.30-7.74)
FK 888
7.53 (7.39-7.67)
8.30 (8.08-8.52)
(*)CP 96,345 and FK 888 were significantly more potent against septide than against [Sar9]SP sulfone. A tendency to a similar behaviour was also found for GR 82,334 but did not reach statistical significance. Neither (zt)CP 96,345 (up to 1 clM>nor GR 82,334 (up to 10 @4) or FK 888 (up to 3 pM) had any significant effect on the response to the NK-2 receptor selective agonist, [SAla”] NKA(4-10). These findings support the ideal of the existence of a ‘septide-sensitive’ receptor in the circular muscle of GPI, which is recognized with high afXnity by NK- 1 receptor selective antagonists like (rt>CP 96,345 or FK 888. 1. Petitet, F., SatTroy,M., Totrens, Y., Lavielle, S., Chassaing, G., Loeuillet, D., Glowinski, J. and J. C. Beaujouan (1992). Possible existence of a new tachykinin receptor subtype in the guinea-pig ileum. Peptides, 13,383-398.
Cl9 Evidence for the Presence of a Septidesensitive Tachykinin Receptor in the Circular Muscle of the Guinea-pig Ileum
C20 Effect of SP and Selective NKl Receptor Agonist on Inoritol Phosphates and Cytosolic Calcium Levels in Cultured Human Fibroblasts
R. Pa&chin& S. Meini, S. Giuliani and C. A. Maggi Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3 5013 1, Florence, Italy We have investigated the ability of the tachykinin NK-1 receptor antagonists (*)CP 96,345, GR 82,334 and FK 888 to antagonize contractions produced by the NK-1 receptor agonists septide and [Sa?]substance P (SP) sulfone in the circular muscle of the guinea-pig ileum (GPI) 231
M. Ziche, A. Parenti, L. Morbidelli, S. Atnerini, F. Ledda, E. Baldi* and C. A. Maggit Departments of Pharmacology and *Clinical Physiopathology, University of Florence, Viale Morgagni 65, Firenze, Italy and; ~Pharmacology Department, Menarini S.r.1. Firenze, Italy We previously reported that SP sustains proliferation of cultured human fibroblasts (HF) and that this effect is
232 mediated by the interaction with NKl receptors. In this study we have investigated the intracellular mechanisms involved in this receptor-mediated activity. Subconfluent HF labelled with [3H]-myoinositol were exposed to SP and to the selective NKl receptor agonist [Sar9]-SP sulphone. IP3 levels were measured at 3, 6, 10 s following exposure to the peptides and IPl at 15,30 and 60 min. IP3 levels were increased at 3 s after exposure to both peptides and peaked at 6 s (130% and 122% over basal for SP and [S&l-SP sulphone respectively). IPl levels peaked at 15 min in presence of both peptides declining toward basal levels at 60 min. The effect of SP and of the NKl receptor agonist were also investigated on cytosoiic calcium. I-IF were grown on plastic coverslips and loaded with the fluorescent dye FURA-2. Cytosolic calcium was calculated on the base of fluorescence emission recorded with a spettrofluorimetric apparatus. Both peptides almost doubled intracellular calcium levels (243 and 223 r&l for SP and the NKl receptor agonist respectively; 125.2 nM basal). We conclude that binding of SP to its NKl receptor on human skin fibroblasts generates inositol 1,4,5trisphosphate which releases Ca++ ions into the cytosol.
C21 Differences in the Pharmacological Binding Profile of Rat and Human NK2 Tachykinin Receptors V. Fardin, A. Jolly, F. Bellot*, A. Crespo* and F. Vaissade Rhi$e-Poulenc Rorer S.A., Departments of Biology and Biotechnology*, Centre de Recherche de VitryAlfortville, 94403 Vitry-sur-Seine, Cedex, France The binding of *Z510do-hystidylNKA (lz51-NKA) was studied in rat duodenum and urinary bladder and compared to that observed in HSKR-2 cells, a mouse 3T3 fibroblast cell line which expresses the human NK2 receptor cloned from jejunum. 12’1~NKAbinds with high affinity to a single population of sites in all tissues with similar Kd values. The total number of sites was more than double in urinary bladder than in duodenum and HSKR-2 membrane preparations. The binding (at 100 pM ‘*9+JKA) had the same pharmacological characteristics as that expected for NKz sites in all tissues: high inhibitory potency of NKA, (Lys5, MeLe&‘, NorLeu’O)NKA.+.loand SR 48968 and little or no inhibition of NKI [(Pro9) SP, septide, RP 67580 and CP96,345] or m (senktide) selective compounds. However, the rank order of potency for the two NKz peptide antagonists, L-659,877 and MEN-10,207, clearly differed between the two species, being in rat tissues: L-659,877 > MEN-lo,207 and in HSKR-2: MEN-lo,207 > L-659,877. These data reveal differences between rat and human NI& receptors, but not between rat duodenum and urinary bladder and emphasize the importance of developing a human model to search for NKz antagonists with potent activity in humans.
NEUROPEPTIDES
We are grateful to Dr. R. Kris and Dr. J. Schlessinger for the gift of HSKR-2.
C22 A Novel Peptidic Tachykinin Antagonist Which is Potent at NK3 Receptors J. M. Stables, S. Arkinstall*, I. J. M. Beresford, P. Sealet, P. Ward? and R. M. Hagan Glaxo Group Research, Ware, UK, *Glaxo Institute of Molecular Biology, Geneva and tMedicina1 Chemistry, Glaxo Group Research, Greenford, UK While selective and potent NKI and NKz antagonists have been reported, no potent NK, antagonists have been developed. Here we report on a novel, potent NI& antagonist, GR138676, a conformationally constrained analogue of neurokinin B.
In the m receptor-containing rat portal vein preparation, GR138676 caused a concentration-related (3 nM-1 pM) antagonism of se&tide-induced contractions (PKB = 8.24 f 0.09, n = 15, slope = 1.3 (l&.6)). GR138676 (10-200 &I) antagonised se&tide-evoked release of preloaded arachidonic acid from Chinese hamster ovary cells transfected with a human m receptor gene’@& = 8.27 f 0.04, n = 15, slope = 1.2 (1.0-1.3)). However, GR138676 also antagonised the increase in intracellular calcium evoked by the selective NKI agonist, GR73632, in U373MG cells, @I(8 = 8.26 f 0.04, n = 10, slope = 1.1 (0.9-I .3)). It had little activity at NKz receptors, inhibiting binding of the NK, antagonist ligand [3H]-GR100679 to rat colon membranes with a pKi of 5.8. GR138676 has high affinity for the NKLl receptor. Although it is equiactive at NK receptors, will be a usell tool for characterisation of N& receptors in vitro and investigation of behaviours induced by the selective N& agonist senktide in vivo. 1. Buell, G., Schulz, M. F., Arkinstall, S., Maury, K., Missotten, N., Adami, N., Talabot, F. and Kawashima, E. (1992). Molecular characterisation, expression and localisation of human neurokinin-3 receptor. FEBS Lett. 299: 90-95.
P105 Neurokinin Receptor Antagonists D. Regoli* and R. Couturef *Department of Pharmacology, Medical School, Universiti de Sherbrooke, Sherbrooke (Quebec) Canada, JlH 5N4 and TDepartment of Physiology, Medical School, Universitk de Mont&al, Mont&al (Qukbec) Canada, H3C 3J7