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Effect of muscarinic stimulation on β-amyloid precursor protein processing in rat brain and primary cultures
572
Vol. 64, Nos. 617, 1999
Abstracts
36 MUSCARINIC RECEPTOR SUBTYPES AND AGONIST-MEDIATED IN CULTURED CELL LINES
SIGNAL TRANSDUCTION
B.V. Rama Sastry and Clifford C. Stephan, Vanderbilt Medical Center, Nashville, TN 37232-4125 U.S.A. There are at least five subtypes (Ml -M5) or more (M6, Mi, MS) of muscarinic receptors (l-3). Two signal transduction mechanisms, attenuation of hormone-stimulated c-AMP formation and phosphatidylinositol (PI) hydrolysis, are coupled to M receptors in NG-108-15 neuroblastoma x glioma (NC) and 131-lN1 human astrocytoma (AC) cells, respectively. Using selective antagonists, the receptors in NC and AC cells were characterized as M2 and M3 sub-types (1,2). In a search to find selective agonists, we investigated the effects of several muscarinic agonists on these transduction mechanisms (3). The potencies of full agonists for c-AMP attenuation has the order: carbachol > 5-methylfurmethide (SMFT) > dl-muscarine. All are full agonists and produced maximum inhibition of 73-76%. These three agonists maximally stimulated PI hydrolysis (100%) in AC cells. Their relative potencies have a different order 5-MFT > carbachol > dl-muscarine. Agonists which are considered to be selective for Ml (MCN A345) MS (5-methoxyfurmethide) and Mi (5-hydroxymethylfurmethide) exhibited low activity on CAMP attenuation in NC cells (maximum inhibition, < 42-46%), and PI hydrolysis in AC cells (maximum stimulation O-22%). These observations suggest that (a) non-selective full agonists mediate both effects, and (b) selective agonists at M 1, MS and Mi receptors do not mediate these effects. Selective M2 and M3 agonists have yet to be discovered to support the previous observations with antagonists that M2 receptors mediate CAMP attenuation while M3 receptors mediate PI hydrolysis. (Supported by the Smokeless Tobacco Research Council, Inc.). References: 1.) Stephan, C.C. and Sastry, B.V.R., Cell. Mol. Biol. 38(6):601-612, 1992. 2.) Stephan, C.C. and Sastry, B.V.R., Cell. Mol. Biol. 38(7):701-712, 1992. 3.)Sastry,B.V.R.andOchillo,R.F., LijbSci.,60(13/14), 1194, 1996.
37 EFFECT OF MUSCARINIC STIMULATION ON P-AMYLOID PROCESSING IN RAT BRAIN AND PRIMARY CULTURES
PRECURSOR
PROTEIN
Z. Pittel, A. Fisher, N. Eshhar, R. Haring and E. Heldman. Israel Institute for Biological Research P. 0. Box. 19, Ness-Ziona 74100, ISRAEL. Metabolism of l3-amyloid precursor protein (APP) can lead to the formation of amyloid g-protein (Ap), neurotoxic agent found in brains of Alzheimer’s patients. An inverse relationship is reported between the levels of Al3 and secreted APP (APPs). Activation of Mi and MS muscarinic acetylcholine receptors (mAChR) increases APPs secretion in cell lines expressing these mAChR and in cerebrocortical slices (Pittel et al., 1996). We examined the effect of muscarinic stimulation on APPs secretion in primary neuronal cultures and in rat brain in vivo. Primary cell cultures derived from the hippocampus and the cortex of 18 days old rat fetuses were used. Exposure of neuronal cultures for 1 h to the non selective muscarinic agonist, carbachol and to the functionally Mi selective muscarinic agonists, AF102B and AFl5O(S) (each at 100 @I) significantly increased APPs secretion by 2-6 fold over basal level. These effects were blocked by 10 @I pirenzepine, an Ml selective antagonist, suggesting the enhancing role of Mi mAChR in APPs secretion. Studies in viva indicate that more than one subtype of mAChR might be responsible for APP processing. Administrations of oxotremorine (1 mg/kg; ip) and the cholinesterase inhibitor, physostigmine (0.25 mg/kg; im) to rats reduced the levels of APPs in the soluble fraction of the hippocampi 1 h after the injections by 52 and 44%, respectively, compared to control rats. It seems that following muscarinic stimulation dierent mAChR subtypes may dominate the regulation of APP processing in the brain (e.g. M2 or M inhibitory mAChR) although other factors can be considered. The relative interplay between two or more mAChR subtypes (excitatory vs inhibitory) on APPs processing needs further investigation.
Vol. 64, Nos. 617, 1999
Abstracts
36 MUSCARINIC RECEPTOR SUBTYPES AND AGONIST-MEDIATED IN CULTURED CELL LINES
SIGNAL TRANSDUCTION
B.V. Rama Sastry and Clifford C. Stephan, Vanderbilt Medical Center, Nashville, TN 37232-4125 U.S.A. There are at least five subtypes (Ml -M5) or more (M6, Mi, MS) of muscarinic receptors (l-3). Two signal transduction mechanisms, attenuation of hormone-stimulated c-AMP formation and phosphatidylinositol (PI) hydrolysis, are coupled to M receptors in NG-108-15 neuroblastoma x glioma (NC) and 131-lN1 human astrocytoma (AC) cells, respectively. Using selective antagonists, the receptors in NC and AC cells were characterized as M2 and M3 sub-types (1,2). In a search to find selective agonists, we investigated the effects of several muscarinic agonists on these transduction mechanisms (3). The potencies of full agonists for c-AMP attenuation has the order: carbachol > 5-methylfurmethide (SMFT) > dl-muscarine. All are full agonists and produced maximum inhibition of 73-76%. These three agonists maximally stimulated PI hydrolysis (100%) in AC cells. Their relative potencies have a different order 5-MFT > carbachol > dl-muscarine. Agonists which are considered to be selective for Ml (MCN A345) MS (5-methoxyfurmethide) and Mi (5-hydroxymethylfurmethide) exhibited low activity on CAMP attenuation in NC cells (maximum inhibition, < 42-46%), and PI hydrolysis in AC cells (maximum stimulation O-22%). These observations suggest that (a) non-selective full agonists mediate both effects, and (b) selective agonists at M 1, MS and Mi receptors do not mediate these effects. Selective M2 and M3 agonists have yet to be discovered to support the previous observations with antagonists that M2 receptors mediate CAMP attenuation while M3 receptors mediate PI hydrolysis. (Supported by the Smokeless Tobacco Research Council, Inc.). References: 1.) Stephan, C.C. and Sastry, B.V.R., Cell. Mol. Biol. 38(6):601-612, 1992. 2.) Stephan, C.C. and Sastry, B.V.R., Cell. Mol. Biol. 38(7):701-712, 1992. 3.)Sastry,B.V.R.andOchillo,R.F., LijbSci.,60(13/14), 1194, 1996.
37 EFFECT OF MUSCARINIC STIMULATION ON P-AMYLOID PROCESSING IN RAT BRAIN AND PRIMARY CULTURES
PRECURSOR
PROTEIN
Z. Pittel, A. Fisher, N. Eshhar, R. Haring and E. Heldman. Israel Institute for Biological Research P. 0. Box. 19, Ness-Ziona 74100, ISRAEL. Metabolism of l3-amyloid precursor protein (APP) can lead to the formation of amyloid g-protein (Ap), neurotoxic agent found in brains of Alzheimer’s patients. An inverse relationship is reported between the levels of Al3 and secreted APP (APPs). Activation of Mi and MS muscarinic acetylcholine receptors (mAChR) increases APPs secretion in cell lines expressing these mAChR and in cerebrocortical slices (Pittel et al., 1996). We examined the effect of muscarinic stimulation on APPs secretion in primary neuronal cultures and in rat brain in vivo. Primary cell cultures derived from the hippocampus and the cortex of 18 days old rat fetuses were used. Exposure of neuronal cultures for 1 h to the non selective muscarinic agonist, carbachol and to the functionally Mi selective muscarinic agonists, AF102B and AFl5O(S) (each at 100 @I) significantly increased APPs secretion by 2-6 fold over basal level. These effects were blocked by 10 @I pirenzepine, an Ml selective antagonist, suggesting the enhancing role of Mi mAChR in APPs secretion. Studies in viva indicate that more than one subtype of mAChR might be responsible for APP processing. Administrations of oxotremorine (1 mg/kg; ip) and the cholinesterase inhibitor, physostigmine (0.25 mg/kg; im) to rats reduced the levels of APPs in the soluble fraction of the hippocampi 1 h after the injections by 52 and 44%, respectively, compared to control rats. It seems that following muscarinic stimulation dierent mAChR subtypes may dominate the regulation of APP processing in the brain (e.g. M2 or M inhibitory mAChR) although other factors can be considered. The relative interplay between two or more mAChR subtypes (excitatory vs inhibitory) on APPs processing needs further investigation.