Abstract: 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.
Abstract: Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.
Abstract: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle.
Abstract: The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 +/- 1.0 microm; N = 213), the diameter of ectopic fibers (59.1 microm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.
Abstract: cyclic AMP (cAMP)-mediated pathway regulates several aspects of muscle and neuromuscular synapse physiology such as anabolism, neurotransmitter release and expression and function of crucial synaptic proteins like acetylcholinesterase (AChE) and nicotinic receptors, not to mention muscle contraction itself. The synthesis of this second messenger is mediated by stimulatory G protein coupled receptors (GsPCR), which activate adenylyl cyclase (AC), the enzyme that converts ATP in cAMP. Until now, nine different membrane-bound AC isoforms (AC1–AC9) are known, each one of them with different regulatory and enzymatic properties. Previous studies from our group showed that long-term stimulation (24 h) with the neuropeptide CGRP, a GsPCR agonist, or forskolin (FSK), a direct activator of isoforms AC1–AC8, attenuates AChE expression and cAMP formation induced by a subsequent stimulus, suggesting an adaptative mechanism of the muscle fiber in response to superstimulation (da Costa, Lapa and Godinho 2001). Besides, AC activation leads to extracellular accumulation of cAMP through probenecide-sensitive transporters (Godinho and Costa-Jr, 2003). Outside the cell, the cyclic nucleotide is converted into adenosine through the extracellular cAMP–adenosine pathway, suggesting an extracellular autocrine action of this second messenger (Chiavegatti 2005; Chiavegatti et al. 2008). Thus, the objective of this work was to: a) evaluate the expression pattern of AC isoforms in fast (extensor digitorum longus, EDL) and slow (soleus) muscles or cultured rat skeletal muscle; b) determine the mechanisms responsible for the diminished cAMP synthesis after long-term stimulation; and c) to analyze the role of the extracellular cAMP–adenosine pathway on the intracellular cyclic nucleotide signaling. RT-PCR analysis revealed transcripts for isoforms AC2–AC9 in EDL, soleus, primary culture and L6 cells. Confirming previous results, long-term stimulation (24 h) of AC with FSK reduced the cAMP generation by subsequent treatment with FSK, isoproterenol (ISO) or CGRP. This effect was explained by the down-regulation of AC detected by 3H-FSK binding assay. In addition, treatment with FSK for 24 h reduced the affinity of the enzyme for its substrate ATP, while long-term stimulation with CGRP or ISO increased the affinity for the substrate and reduced Vmax, indicating that direct- or G protein-mediated continuous activation has different effects on AC regulation. Long-term stimulation with FSK also reduced the mRNA for AC5, AC6 and AC9, while transcripts for AC2 and AC4 were increased, demonstrating a differential regulation of AC isoforms under superstimulation. On the other hand, the basal activity of AC was increased in membranes from cultures stimulated for 24 h with ISO or FSK. The sensitization of AC was confirmed by the increased cAMP generation in membranes from cells treated for 12 h with ISO or FSK and allowed to recover for 24 h. Denervation also increased AC activity, increasing Vmax and Km on soleus muscles, but not in EDL. At the same time, denervation reduced the amount of mRNA for AC2 but not for AC9 in soleus, while the expression of these two isoforms remained unchanged in EDL and diaphragm muscles, suggesting that the effect of denervation on AC depend on trophic properties of each muscle. Finally, we demonstrated that the extracellular cAMP¡Vadenosine pathway acts in an autocrine fashion on adenosine receptors coupled to GƒÑs, providing a positive feedback response to intracellular cAMP signalling on the skeletal muscle fibers. These results indicate that cAMP signaling on skeletal muscle may be regulated by multiple mechanisms, including genomic and intra- or extracellular non-genomic pathways that may be targets for new pharmacological interventions.
Notes: Part of the results were published at the paper Skeletal muscle expresses the extracellular cyclic AMP-adenosine pathway. Br J Pharmacol (2008) 153: 6. 1331-1340 Mar
