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Transmembrane signaling of M1 muscarinic receptors in the rat brain and cell cultures
Vol.
52,
No.s
5 & 6, 1993
Abstracts
565
31 F U N C T I O N A L ACTIVITIES OF THE NOVEL C H O L I N O M I M E T I C S RU 35926 (CI 979) AND RU 35963 WITH RESPECT TO MUSCARINIC RECEPTORS. P. Hunt, C. Bardelay, A. Jouanen, D. Massardier, A-M. Tremblet and D. Van de Velde. Centre de Recherches Roussel Uclaf, 93230 Romainville, France RU 35926 and RU 35963 are potent cholinomimetic compounds, structurally related to arecoline. Their agonist profiles with respect to different muscarinic receptor subtypes in the rat have been evaluated in binding and functional models in vitro. The ratios of the low affinity (K1) and high affinity (Kh) components of agonist competition curves were determined for M1, M2 and M3 receptors using antagonist ligands. With respect to M1 and M3, both RU 35963 and RU 35926 show low K1/Kh ratios, indicative of partial agonists. At the M2 receptor the K1/Kh ratio for RU 35963 is nearer to that of a full agonist, while that of RU 35926 is significantly lower. Phosphatidyl-inositol (PI) hydrolysis (M1 / M3) was measured in both hippocampal slices and in primary cultures of cerebellar neurons. In the former system, RU 35963 like carbachol, strongly stimulates the formation of inositol phosphates (ECs0 values = 0.4 and 1.9 mM, respectively) while RU 35926 (10-2M), only produces - 3 0 % of the maximal effect of carbachol. In the latter system where only the M3 receptor is involved, both compounds show partial PI responses. Presynaptically (probably M2), RU 35963 causes a complete and dose dependent inhibition of acetylcholine release from electrically stimulated hippocampal slices (EC50 = 1.9 mM), while RU 35926 partially inhibits (-60%). At the M2 receptor in cardiac membrane homogenates however, both compounds (EC50 = 0.88 and 3.5 I.tM respectively), like carbachol (ECs0 = 6.1 ~tM), are efficient inhibitors of adenylate cyclase. These results confirm the muscarinic agonist properties of both RU 35926 and RU 35963 but also show that their efficacy varies according to the tissue preparation and the receptor subtype involved. Functionally, RU 35926 more often shows partial agonist character while RU 35963 tends to behave more as a full agonist.
32 T R A N S M E M B R A N E SIGNALING OF MI MUSCARINIC R E C E P T O R S IN THE RAT BRAIN AND CELL CULTURES.
Z_.=Pittel, A. Fisher, Z. Vogel # and E. Heldman. Israel Inst. Biolog. Res., Ness-Ziona, #The Weizmann Inst., Rehovot, ISRAEL. Full muscarinic agonists increase phosphoinositide (PI) hydrolysis and cAMP levels in mammalian cells transfected with ml muscarinic receptors (mAChRs). However, it is still not clear whether the artificial combination of the transfected receptors and the endogenous G-proteins in such cells reflects the situation in neuronal tissues. The rat brain contains a mixture of ml-m4 AChR subtypes, and their respective signal transduction mechanisms in their native environment. In this study, we used AF102B, a relatively selective M1 agonist (Fisher et al., JPET 257: 392, 1991), in order to elucidate the nature of signal transductions by the M1 AChRs in rat brain. Synaptosomes, slices and homogenates from rat forebrain (rich in ml and m3 AChRs) were used to determine PI hydrolysis and adenylyl cyclase (AC) activity. AFI02B (1 /~M-10 mM) neither inhibited AC activity, nor did it potentiate PI hydrolysis. Yet, coincubation of AF102B and l mM carbachol (CCh) inhibited CCh-induced PI hydrolysis with an IC50=75 #M without affecting CCh-induced inhibition of AC. Preincubation (30 rain) of synaptosomes, slices or membranes with AFI02B (100/~M-10 mM), followed by extensive washing, also inhibited CCh (0.1 )~[1 mM)-induced PI hydrolysis, without any effect on mAChR binding sites as determined by H]QNB binding. In Chinese hamster ovary cells transfected with human ml AChR preincubation (120 min) with AF102B (1 #M-10 mM) potentiated (1-100/~M) or inhibited (1-10 mM) CCh-induced PI hydrolysis. Notably, AFI02B acts as a partial agonist on ml AChRs in these cells, stimulating PI hydrolysis yet, unlike CCh, lacking an effect on cAMP levels in the same cell preparation (Fisher et al., Biorg. Med. Chem. Lett., in press). We suggested that AF102B is selective at the level of both ml AChR and distincts signal transduction pathway. The present results further support the notion that an agonist like AFI02B can modulate indirectly the interaction of mAChRs with discrete signal transduction processes.
Supported by Snow Brand, Japan.
52,
No.s
5 & 6, 1993
Abstracts
565
31 F U N C T I O N A L ACTIVITIES OF THE NOVEL C H O L I N O M I M E T I C S RU 35926 (CI 979) AND RU 35963 WITH RESPECT TO MUSCARINIC RECEPTORS. P. Hunt, C. Bardelay, A. Jouanen, D. Massardier, A-M. Tremblet and D. Van de Velde. Centre de Recherches Roussel Uclaf, 93230 Romainville, France RU 35926 and RU 35963 are potent cholinomimetic compounds, structurally related to arecoline. Their agonist profiles with respect to different muscarinic receptor subtypes in the rat have been evaluated in binding and functional models in vitro. The ratios of the low affinity (K1) and high affinity (Kh) components of agonist competition curves were determined for M1, M2 and M3 receptors using antagonist ligands. With respect to M1 and M3, both RU 35963 and RU 35926 show low K1/Kh ratios, indicative of partial agonists. At the M2 receptor the K1/Kh ratio for RU 35963 is nearer to that of a full agonist, while that of RU 35926 is significantly lower. Phosphatidyl-inositol (PI) hydrolysis (M1 / M3) was measured in both hippocampal slices and in primary cultures of cerebellar neurons. In the former system, RU 35963 like carbachol, strongly stimulates the formation of inositol phosphates (ECs0 values = 0.4 and 1.9 mM, respectively) while RU 35926 (10-2M), only produces - 3 0 % of the maximal effect of carbachol. In the latter system where only the M3 receptor is involved, both compounds show partial PI responses. Presynaptically (probably M2), RU 35963 causes a complete and dose dependent inhibition of acetylcholine release from electrically stimulated hippocampal slices (EC50 = 1.9 mM), while RU 35926 partially inhibits (-60%). At the M2 receptor in cardiac membrane homogenates however, both compounds (EC50 = 0.88 and 3.5 I.tM respectively), like carbachol (ECs0 = 6.1 ~tM), are efficient inhibitors of adenylate cyclase. These results confirm the muscarinic agonist properties of both RU 35926 and RU 35963 but also show that their efficacy varies according to the tissue preparation and the receptor subtype involved. Functionally, RU 35926 more often shows partial agonist character while RU 35963 tends to behave more as a full agonist.
32 T R A N S M E M B R A N E SIGNALING OF MI MUSCARINIC R E C E P T O R S IN THE RAT BRAIN AND CELL CULTURES.
Z_.=Pittel, A. Fisher, Z. Vogel # and E. Heldman. Israel Inst. Biolog. Res., Ness-Ziona, #The Weizmann Inst., Rehovot, ISRAEL. Full muscarinic agonists increase phosphoinositide (PI) hydrolysis and cAMP levels in mammalian cells transfected with ml muscarinic receptors (mAChRs). However, it is still not clear whether the artificial combination of the transfected receptors and the endogenous G-proteins in such cells reflects the situation in neuronal tissues. The rat brain contains a mixture of ml-m4 AChR subtypes, and their respective signal transduction mechanisms in their native environment. In this study, we used AF102B, a relatively selective M1 agonist (Fisher et al., JPET 257: 392, 1991), in order to elucidate the nature of signal transductions by the M1 AChRs in rat brain. Synaptosomes, slices and homogenates from rat forebrain (rich in ml and m3 AChRs) were used to determine PI hydrolysis and adenylyl cyclase (AC) activity. AFI02B (1 /~M-10 mM) neither inhibited AC activity, nor did it potentiate PI hydrolysis. Yet, coincubation of AF102B and l mM carbachol (CCh) inhibited CCh-induced PI hydrolysis with an IC50=75 #M without affecting CCh-induced inhibition of AC. Preincubation (30 rain) of synaptosomes, slices or membranes with AFI02B (100/~M-10 mM), followed by extensive washing, also inhibited CCh (0.1 )~[1 mM)-induced PI hydrolysis, without any effect on mAChR binding sites as determined by H]QNB binding. In Chinese hamster ovary cells transfected with human ml AChR preincubation (120 min) with AF102B (1 #M-10 mM) potentiated (1-100/~M) or inhibited (1-10 mM) CCh-induced PI hydrolysis. Notably, AFI02B acts as a partial agonist on ml AChRs in these cells, stimulating PI hydrolysis yet, unlike CCh, lacking an effect on cAMP levels in the same cell preparation (Fisher et al., Biorg. Med. Chem. Lett., in press). We suggested that AF102B is selective at the level of both ml AChR and distincts signal transduction pathway. The present results further support the notion that an agonist like AFI02B can modulate indirectly the interaction of mAChRs with discrete signal transduction processes.
Supported by Snow Brand, Japan.