RS09 increased antibody production in a vaccine setting suggests that this LPS peptide mimic can function as an adjuvant and thus serve as a novel candidate to be considered as a new class of TLR4 agonist adjuvants. Acute exposure of skeletal muscles to low concentrations of acetylcholine stimulates Na,K-ATPase electrogenic activity through a regulatory interaction between the muscle nicotinic acetylcholine receptor and the Na,K-ATPase. The nAChR specifically co- immunoprecipitates with both a1 and a2 isoforms of the Na,K-ATPase a-subunit and phospholemman, a muscle-specific auxiliary subunit of Na,K-ATPase which modulates NSC 632839 enzyme activity. This suggests that these proteins assemble in a macromolecular complex capable of functional interactions. Acute stimulation of enzyme activity by ACh produces membrane hyperpolarization. Because the hyperpolarization occurs in the voltage range where Na + channel slow inactivation is steeply voltage-dependent, it increases membrane excitability by shifting Na + channels from the inactive to available conformation. Skeletal muscles are normally exposed to nanomolar concentrations of ACh for some time following nerve excitation, after the larger bolus of ACh has been hydrolyzed by acetylcholinesterase. MDL-29951 Therefore, when a quiescent muscle is stimulated by nerve input, this ACh-induced hyperpolarization primes the muscle to respond to an increased level of nerve activity. The hyperpolarizing effect of acute exposure to ACh is specific for the Na,K-ATPase a2 isoform and is most likely mediated by a desensitized state of the nAChR. Acute nanomolar concentrations of nicotine, an exogenous non-hydrolyzable nAChR agonist, also stimulate the Na,K-ATPase a2 isoform by this mechanism. This finding suggested that chronic in vivo exposure to nicotine, which reaches hundreds of nM up to mM levels during tobacco use, might produce long-term effects on the Na,K-ATPase and membrane potentials in skeletal muscle. This question has not been investigated previously. It is commonly thought that muscle nAChRs are largely spared the effects of nicotine use because the affinity of the muscle nAChR for nicotine is significantly lower than that of brain nAChRs. However, chronic agonist exposure promotes nAChR desensitization, and the desensitized state of the nAChR is the favored conformation which interacts with the Na,K-ATPase. In addition, the stable association of PLM with the Na,K-ATPase isoforms suggested that this regulatory interaction may modulate enzyme activity through a kinase-dependent phosphorylation of PLM. In cardiac and smooth muscle cells, phosphorylation of PLM at Ser63 and Ser68 by PKC stimulates Na,K-ATPase a2 activity by relieving an inhibitory interaction of PLM with the enzyme. The role of PLM in regulating Na,K-ATPase activity in skeletal muscle is not known and the significance of its association with the nAChR- Na,K-ATPase complex has not been previously investigated.