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Muscarinic acetylcholine receptors (mAchRs) are guanosine nucleotide-binding protein (G protein) coupled receptors that crosstalk with receptor tyrosine kinases (RTKs) to signal mitogenic pathways. In particular, mAchRs are known to couple with RTKs for several growth factors to activate the mammalian target of rapamycin (mTOR)/Akt pathway, a regulator of protein synthesis. The RTK for the vascular endothelial growth factor (VEGF), VEGFR2, can signal protein synthesis but whether it cooperates with mAchRs to mediate mTOR activation has not been demonstrated. Using serum starved SK-N-SH neuroblastoma cells, we show that the muscarinic receptor agonists carbachol and pilocarpine enhance the activation of mTOR substrates p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6) in a VEGFR2 dependent manner. Treatments with carbachol increased VEGFR2 phosphorylation, suggesting that mAchRs stimulate VEGFR2 transactivation to enhance mTOR signaling. Inhibitor studies revealed that phosphoinositide 3 kinase resides upstream from S6K, S6 and Akt phosphorylation while protein kinase C (PKC) functions in an opposing fashion by positively regulating S6K and S6 phosphorylation and suppressing Akt activation. Treatments with the phosphatase inhibitors sodium orthovanadate and okadaic acid increase S6, Akt and to a lesser extent S6K phosphorylation, indicating that tyrosine and serine/threonine dephosphorylation also regulates their activity. However, okadaic acid elicited a far greater increase in phosphorylation, implicating phosphatase 2A as a critical determinant of their function. Finally, pilocarpine but not carbachol induced a time and dose dependent cell death that was associated with caspase activation and oxidative stress but independent of S6K and S6 activation through VEGFR2. Accordingly, our findings suggest that mAchRs crosstalk with VEGFR2 to enhance mTOR activity but signal divergent effects on survival through alternate mechanisms.

Duchenne muscular dystrophy (DMD) is a progressive primary myodegenerative disease caused by a genetic deficiency of the 427-kDa cytoskeletal protein dystrophin. Despite its single-gene etiology, DMD's complex pathogenesis remains poorly understood, complicating the extrapolation from results of preclinical studies in genetic homologs to the design of informative clinical trials. Here we describe novel phenotypic assays which when applied to the mouse resemble recently used primary end points for DMD clinical trials. By coupling force transduction, high-precision motion tracking, and respiratory measurements, we have achieved a suite of integrative physiological tests that provide novel insights regarding normal and pathological responses to muscular exertion. A common feature of these physiological assays is the precise tracking and analysis of volitional movement, thereby optimizing the relevance to clinical tests. Unexpectedly, the measurable biological distinction between dystrophic and control mice at early time points in the disease process is better resolved with these tests than with the majority of previously used, labor-intensive studies of individual muscle function performed ex vivo. For example, the dramatic loss of volitional movement following a novel, standardized grip test distinguishes control mice from mice by a 17.4-fold difference of the means (3.5 ± 2.2 vs. 60.9 ± 12.1 units of activity, respectively; effect size 1.99). The findings have both mechanistic and translational implications of potential significance to the fields of basic myology and neuromuscular therapeutics. This study uses novel phenotypic assays which when applied to the mouse resemble recently used primary end points for DMD clinical trials. A measurable distinction between dystrophic and control mice was seen at early time points in vivo compared with invasive muscle studies performed ex vivo. These assays shed light on normal and pathological responses to muscular exertion and have significant mechanistic and translational implications for the fields of basic myology and neuromuscular therapeutics.