ABSTRACT
Seven-transmembrane receptors mediate diverse skeletal muscle responses for a wide variety of stimuli, via activation of heterotrimeric G-proteins. Herein we evaluate the expression and activation of rat diaphragm or cultured skeletal muscle G-proteins using [(35)S]GTPγS. Total membrane Gα subunit content was 4-7 times higher in rat primary cultured myotubes and L6 cell line than in diaphragm (32.6±1.2fmol/mg protein) and 7-27% of them were in the active conformational state. Immunoprecipitation assay showed equal expression of diaphragm Gαs, Gαq and Gαi/o. Addition of GDP allowed the measurement of G-protein activation by different GPCR, including adrenoceptor, adenosine, melatonin and muscarinic receptors. Diaphragm denervation resulted in a marked increase in both total and active state G-protein levels. Together, the results show that [(35)S]GTPγS binding assay is a sensitive and valuable method to evaluate GPCR activity in skeletal muscle cells, which is of particular interest for pharmacological analysis of drugs with potential use in the management of respiratory muscle failure.
Subject(s)
Diaphragm/enzymology , Heterotrimeric GTP-Binding Proteins/physiology , Animals , Cells, Cultured , Diaphragm/drug effects , Diaphragm/innervation , Guanosine Diphosphate/pharmacology , Male , Muscarinic Agonists/pharmacology , Oxotremorine/pharmacology , Phrenic Nerve/drug effects , Phrenic Nerve/physiology , Rats , Rats, Wistar , Receptors, Adrenergic/drug effects , Receptors, Adrenergic/physiology , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/physiology , Receptors, Melatonin/drug effects , Receptors, Melatonin/physiology , Receptors, Muscarinic/drug effects , Receptors, Muscarinic/physiology , Receptors, Purinergic P1/drug effects , Receptors, Purinergic P1/physiologyABSTRACT
The role of acetylcholinesterase (AChE) in the termination of the cholinergic response through acetylcholine (ACh) hydrolysis and the involvement of plasma butyrylcholinesterase (BuChE), mainly of hepatic origin, in the metabolism of xenobiotics with ester bonds is well known. Besides, BuChE has a crucial role in ACh hydrolysis, especially when selective anticholinesterases inhibit AChE. Herein, we analyzed the gender-related differences and the circadian changes of rat plasma cholinesterases. Plasma and liver cholinesterase activities were evaluated in control or 2-30-day castrated adult male and female rats. Plasma and liver AChE activities did not differ between genders and were not influenced by sex hormone deprivation. BuChE plasma activity was 7 times greater in female, reflecting gender differences in liver enzyme expression. Castration increased liver and plasma BuChE activity in male, while reduced it in female, abolishing gender differences in enzyme activity. Interestingly, female AChE and BuChE plasma activities varied throughout the day, reaching values 27% and 42% lower, respectively, between 2 p.m. and 6 p.m. when compared to the morning peaks at 8 a.m. Castration attenuated daily female BuChE oscillation. On the other hand, male plasma enzymes remained constant throughout the day. In summary, our results show that liver and plasma BuChE, but not AChE, expression is influenced by sex hormones, leading to high levels of blood BuChE in females. The fluctuation of female plasma BuChE during the day should be taken into account to adjust the bioavailability and the therapeutic effects of cholinesterase inhibitors used in cholinergic-based conditions such Alzheimer's disease.
