We have found earlier that changes in membrane cholesterol content have distinct impact on signaling via the M1, M2, or M3 receptors expressed in CHO cells (CHO-M1 through CHO-M3). Now we investigated whether gradual changes in membrane cholesterol exerts differential effects on coupling of the M1 and M3 muscarinic receptors to preferential signaling pathways through Gq/11 and non-preferential Gs G-proteins signaling. Changes in membrane cholesterol resulted in only marginal alterations of antagonist and agonist affinity of the M1 and M3 receptors, and did not influence precoupling of either subtype. Changes in membrane cholesterol did not influence parameters of carbachol-stimulated GTP-γ(35)S binding in CHO-M1 membranes while reduction as well as augmentation of membrane cholesterol lowered the efficacy but increased the potency of carbachol in CHO-M3 membranes. Gradual increase or decrease in membrane cholesterol concentration dependently attenuated agonist-induced inositolphosphates release while only cholesterol depletion increased basal values in both cell lines. Similarly, membrane cholesterol manipulation modified basal and agonist-stimulated cAMP synthesis via Gs in the same way in both cell lines. These results demonstrate that changes in membrane cholesterol concentration differentially impact preferential and non-preferential M1 and M3 receptor signaling. They point to the activated G-protein/effector protein interaction as the main site of action in alterations of M1 receptor-mediated stimulation of second messenger pathways. On the other hand, modifications in agonist-stimulated GTP-γ(35)S binding in CHO-M3 membranes indicate that in this case changes in ligand-activated receptor/G-protein interaction may also play a role.

• Klíčová slova
• Agonist binding, Cholesterol, G-Proteins, Muscarinic receptors, Signal transduction, cAMP synthesis,
• MeSH
• CHO buňky MeSH
• cholesterol metabolismus   MeSH
• Cricetulus MeSH
• karbachol farmakologie   MeSH
• lidé MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptor muskarinový M1 účinky léků   metabolismus   MeSH
• receptor muskarinový M3 účinky léků   metabolismus   MeSH
• signální transdukce MeSH
• systémy druhého messengeru fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• karbachol MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M1 MeSH
• receptor muskarinový M3 MeSH

We investigated the influence of membrane cholesterol content on preferential and non-preferential signaling through the M(2) muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M(2) receptors for [(3)H]-N-methylscopolamine ([(3)H]-NMS) binding and increased B(max) in intact cells and membranes. Membranes displayed two-affinity agonist binding sites for carbachol and cholesterol depletion doubled the fraction of high-affinity binding sites. In intact cells it also reduced the rate of agonist-induced receptor internalization and changed the profile of agonist binding from a single site to two affinity states. Cholesterol enrichment by 137% had no effects on carbachol E(max) of cAMP synthesis inhibition and on cAMP synthesis stimulation and inositolphosphates (IP) accumulation at higher agonist concentrations (non-preferred pathways). On the other hand, cholesterol depletion significantly increased E(max) of cAMP synthesis inhibition or stimulation without change in potency, and decreased E(max) of IP accumulation. Noteworthy, modifications of membrane cholesterol had no effect on membrane permeability, oxidative activity, protein content, or relative expression of G(s), G(i/o), and G(q/11) alpha subunits. These results demonstrate distinct changes of M(2) receptor signaling through both preferential and non-preferential G-proteins consequent to membrane cholesterol depletion that occur at the level of receptor/G-protein/effector protein interactions in the cell membrane. The significant decrease of IP accumulation by cholesterol depletion was also observed in cells expressing M(3) receptors and by both cholesterol depletion and enrichment in cells expressing M(1) receptors indicating relevance of reduced G(q/11) signaling for the pathogenesis of Alzheimer's disease.

• MeSH
• acetylcholin analogy a deriváty   MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• beta-cyklodextriny farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• CHO buňky MeSH
• cholesterol metabolismus   MeSH
• Cricetulus MeSH
• karbachol analogy a deriváty   metabolismus   farmakologie   MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin metabolismus   farmakologie   MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptor muskarinový M2 metabolismus   MeSH
• systémy druhého messengeru * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acetylcholin MeSH
• antagonisté muskarinových receptorů MeSH
• beta-cyklodextriny MeSH
• cholesterol MeSH
• karbachol MeSH
• methyl-beta-cyclodextrin MeSH Prohlížeč
• N-methylskopolamin MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M2 MeSH

It is generally accepted that the crucial events in the pathogeny of Alzheimer's disease (AD) are the increased accumulation of amyloidogenic peptides derived from amyloid precursor protein and the harmful actions of these peptides on neurons, which bring about neurodegeneration. The enhanced beta-amyloid accumulation is known to be caused by mutations of specific genes in patients who suffer from the familial (hereditary) form of AD but who represent just a minor group within the total population of AD patients. The reasons for beta-amyloid accumulation are not known in the much larger group of patients with the sporadic form of the disease. A biochemical feature common to either form of the disease is the preferential atrophy and degeneration of cholinergic neurons, which is probably responsible for much of the cognitive decline characteristic of the disease. We present an overview of recent investigations on the interactions between beta-amyloid and cholinergic neurons.

• MeSH
• amyloidní beta-protein metabolismus   MeSH
• amyloidový prekurzorový protein beta metabolismus   MeSH
• cholinergní vlákna fyziologie   MeSH
• fenotyp MeSH
• glukosa metabolismus   MeSH
• lidé MeSH
• neurony fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• amyloidní beta-protein MeSH
• amyloidový prekurzorový protein beta MeSH
• glukosa MeSH

Recent advances in neuroscience and molecular neurochemistry have substantially increased the knowledge of the neuropathobiology of senile dementia and Alzheimer's disease. On the basis of various hypotheses concerning degenerative processes in aging brains, new therapeutic strategies have been developed, including nootropic drugs with different mechanisms of action and heterogenous chemical structures. Mutual relationships exist between neuroscientific research and nootropic drug development. To date, such areas of research and drug development have involved deficits of brain neurotransmission (cholinergic, monoaminergic, peptidergic), free radical-induced damage, disturbances of calcium homeostasis and excitatory amino acid function, and deposition of amyloid protein.

• MeSH
• demence farmakoterapie   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• psychotropní léky farmakologie   MeSH
• senioři MeSH
• stárnutí fyziologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• psychotropní léky MeSH