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.

• 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

The effect of lobeline on rat α4β2 nicotinic receptors expressed in COS cells was studied using the patch-clamp technique. Currents were recorded in whole-cell mode 2-4 days after cell transfection by plasmids coding the α4β2 combination of receptor subunits. In cells sensitive to acetylcholine, the application of lobeline evoked minor responses (up to 2% of maximal acetylcholine response). When acetylcholine was applied to the background of an already running application of lobeline, acetylcholine responses were inhibited in a concentration- and time dependent manner. However, when lobeline was applied simultaneously with acetylcholine without any prepulse or during an already running application of acetylcholine, the acetylcholine responses were potentiated up to 300-600% of that of the control. The site of lobeline action overlaps with the cholinergic site, as was proven by the partially protective effect of (+)-tubocurarine. Thus, lobeline can apparently desensitize receptors when applied alone (inhibition) whereas its binding to a second agonist site with the first one already occupied by acetylcholine leads to channel opening (potentiation).

• MeSH
• acetylcholin farmakologie   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• krysa rodu rattus MeSH
• lobelin farmakologie   MeSH
• neurony metabolismus   MeSH
• nikotinové receptory metabolismus   MeSH
• nikotinoví agonisté farmakologie   MeSH
• nikotinoví antagonisté farmakologie   MeSH
• synergismus léků MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH