- Email: [email protected]
Activity of selective muscarinic agonists at human M1 receptors expressed at varying levels in A9 L cells
Vol. 64, Nos. 6/7, 1999
571
Abstracts
34 Ml MUSCARINIC RECEPTORS POTENTIATE CRH-STIMULATED CYCLASE ACTIVITY IN MEMBRANES OF RAT FRONTAL CORTEX. M.C. Olianas and P. Onali, Section on Biochemical Pharmacology, University of Cagliari, 09124 Cagliari, Italy.
Department
ADENYLYL
of Neuroscience,
Stimulation of cortical Mi muscarinic receptors has been identified as a preferential target for drugs effective in the treatment of cognitive deficits. Moreover, it has been proposed that potentiation of central corticotropin releasing hormone (CRH) transmission may be beneficial in the treatment of Alzheimer’s disease (Behan et al., Nature, 378, 284-287, 1995). Here we report that in membranes of rat frontal cortex acetylcholine (ACh) and other cholinergic agonists potentiate the stimulation of adenylyl cyclase activity elicited by CRH. Oxotremorine-M, carbachol and methacoline were as effective as ACh, whereas oxotremorine and arecoline were much less effective. The facilitating effect of ACh was potently blocked by the Mi antagonists Rtrihexyphenidyl, telenzepine and pirenzepine, whereas the Mz-M4 antagonists himbacine, methoctramine, AQ-RA 741 and AF-DX 116 were less potent. The Mi potentiation was pertussis toxin-insensitive and not affected by the phospholipase C inhibitor U-73122, the protein kinase inhibitor staurosporine and by arachidonic acid metabolism inhibitors. Conversely, the Mi potentiation was reduced by the Gq antagonist GPant-2A and by membrane treatment with the GDP-bound form of the a subunit of transducin. Addition of exogenous G protein l3r subunits mimicked the muscarinic potentiation. The data indicate that Mi and CRH receptors are colocalized on cellular structures of rat frontal cortex and that Mi stimulation positively regulates CRH receptor activity through /3r subunits of Gq.
35 ACTIVITY OF SELECTIVE MUSCARINIC AGONISTS EXPRESSED AT VARYING LEVELS IN A9 L CELLS
AT HUMAN
M, RECEPTORS
Y. Cao, D.D. Wise, H. Zhang, X.-P. Huang, and W.S. Messer, Jr., The University Toledo, OH, U.S.A.
of Toledo,
Selective Mr muscarinic agonists might be useful in the treatment of Alzheimer’s disease. Numerous compounds have been synthesized and characterized for muscarinic agonist activity, and a few have entered into clinical studies. Relatively few studies have compared the activity of these compounds at MI receptors under the same conditions. Here we compare the activity of three Mi agonists at human MI receptors expressed in A9 L cells. 3-(3-Hexyloxy-1,2,5-thiadiazol4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline), (4R,S)-Z-l-azabicyclo[2.2.1]heptan-3one-0-[3-(3’-methoxyphenyl)-2-propynylloxime [(+)-PD1518321 and CDD-0102 were synthesized according .to published procedures. M 1receptors were stably expressed in A9 L cells at low and high levels. Agonist activity was assessed by examining [3H]-inositol phosphate (IP) production. Each agonist displayed robust activity at Mi receptors expressed at high levels. Xanomeline was the most potent (EC 50 = 9.4 nM) and efficacious [59 % of carbachol (CCh)], followed by CDD-0102 (3.8 pM; 53 % of CCh) and (k)-PD151832 (6.6 pM; 42 % of CCh). At lower expression levels, xanomeline also was the most potent (24 nM; 41 % of CCh), followed by CDD-0102 (4.8 pM; 42 % of CCh) and (+PD151832 (5.0 pM; 58 % of CCh). In each assay, IP levels dropped dramatically at higher concentrations of xanomeline and (?)-PD 15 1832. In contrast to the stimulation of IP levels, this effect of xanomeline was not blocked by I-hyoscyamine. The data indicated that xanomeline is the most potent of the ligands tested, yet its potency and activity depended upon the level of receptor expression. Receptor expression level is an important determinant of agonist activity. The work was supported by NS 01493, NS 31173 and NS 35127.
571
Abstracts
34 Ml MUSCARINIC RECEPTORS POTENTIATE CRH-STIMULATED CYCLASE ACTIVITY IN MEMBRANES OF RAT FRONTAL CORTEX. M.C. Olianas and P. Onali, Section on Biochemical Pharmacology, University of Cagliari, 09124 Cagliari, Italy.
Department
ADENYLYL
of Neuroscience,
Stimulation of cortical Mi muscarinic receptors has been identified as a preferential target for drugs effective in the treatment of cognitive deficits. Moreover, it has been proposed that potentiation of central corticotropin releasing hormone (CRH) transmission may be beneficial in the treatment of Alzheimer’s disease (Behan et al., Nature, 378, 284-287, 1995). Here we report that in membranes of rat frontal cortex acetylcholine (ACh) and other cholinergic agonists potentiate the stimulation of adenylyl cyclase activity elicited by CRH. Oxotremorine-M, carbachol and methacoline were as effective as ACh, whereas oxotremorine and arecoline were much less effective. The facilitating effect of ACh was potently blocked by the Mi antagonists Rtrihexyphenidyl, telenzepine and pirenzepine, whereas the Mz-M4 antagonists himbacine, methoctramine, AQ-RA 741 and AF-DX 116 were less potent. The Mi potentiation was pertussis toxin-insensitive and not affected by the phospholipase C inhibitor U-73122, the protein kinase inhibitor staurosporine and by arachidonic acid metabolism inhibitors. Conversely, the Mi potentiation was reduced by the Gq antagonist GPant-2A and by membrane treatment with the GDP-bound form of the a subunit of transducin. Addition of exogenous G protein l3r subunits mimicked the muscarinic potentiation. The data indicate that Mi and CRH receptors are colocalized on cellular structures of rat frontal cortex and that Mi stimulation positively regulates CRH receptor activity through /3r subunits of Gq.
35 ACTIVITY OF SELECTIVE MUSCARINIC AGONISTS EXPRESSED AT VARYING LEVELS IN A9 L CELLS
AT HUMAN
M, RECEPTORS
Y. Cao, D.D. Wise, H. Zhang, X.-P. Huang, and W.S. Messer, Jr., The University Toledo, OH, U.S.A.
of Toledo,
Selective Mr muscarinic agonists might be useful in the treatment of Alzheimer’s disease. Numerous compounds have been synthesized and characterized for muscarinic agonist activity, and a few have entered into clinical studies. Relatively few studies have compared the activity of these compounds at MI receptors under the same conditions. Here we compare the activity of three Mi agonists at human MI receptors expressed in A9 L cells. 3-(3-Hexyloxy-1,2,5-thiadiazol4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline), (4R,S)-Z-l-azabicyclo[2.2.1]heptan-3one-0-[3-(3’-methoxyphenyl)-2-propynylloxime [(+)-PD1518321 and CDD-0102 were synthesized according .to published procedures. M 1receptors were stably expressed in A9 L cells at low and high levels. Agonist activity was assessed by examining [3H]-inositol phosphate (IP) production. Each agonist displayed robust activity at Mi receptors expressed at high levels. Xanomeline was the most potent (EC 50 = 9.4 nM) and efficacious [59 % of carbachol (CCh)], followed by CDD-0102 (3.8 pM; 53 % of CCh) and (k)-PD151832 (6.6 pM; 42 % of CCh). At lower expression levels, xanomeline also was the most potent (24 nM; 41 % of CCh), followed by CDD-0102 (4.8 pM; 42 % of CCh) and (+PD151832 (5.0 pM; 58 % of CCh). In each assay, IP levels dropped dramatically at higher concentrations of xanomeline and (?)-PD 15 1832. In contrast to the stimulation of IP levels, this effect of xanomeline was not blocked by I-hyoscyamine. The data indicated that xanomeline is the most potent of the ligands tested, yet its potency and activity depended upon the level of receptor expression. Receptor expression level is an important determinant of agonist activity. The work was supported by NS 01493, NS 31173 and NS 35127.