Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOSα to enteric varicosity membrane

Abstract

Nitrergic neurotransmission at the smooth muscle neuromuscular junctions requires nitric oxide (NO) release that is dependent on the transport and docking of neuronal NO synthase (nNOS) α to the membrane of nerve terminals. However, the mechanism of translocation of nNOSα in actin-rich varicosities is unknown. We report here that the processive motor protein myosin Va is necessary for nitrergic neurotransmission. In wild-type mice, nNOSα-stained enteric varicosities colocalized with myosin Va and its tail constituent light chain 8 (LC8). In situ proximity ligation assay showed close association among nNOSα, myosin Va, and LC8. nNOSα was associated with varicosity membrane. Varicosities showed nitric oxide production upon stimulation with KCl. Intracellular microelectrode studies showed nitrergic IJP and smooth muscle hyperpolarizing responses to NO donor diethylenetriamine-NO (DNO). In contrast, enteric varicosities from myosin Va-deficient DBA (for dilute, brown, non-agouti) mice showed near absence of myosin Va but normal nNOSα and LC8. Membrane-bound nNOSα was not detectable, and the varicosities showed reduced NO production. Intracellular recordings in DBA mice showed reduced nitrergic IJPs but normal hyperpolarizing response to DNO. The nitrergic slow IJP was 9.1 ± 0.7 mV in the wild-type controls and 3.4 ± 0.3 mV in the DBA mice (P < 0.0001). Deficiency of myosin Va resulted in loss of nitrergic neuromuscular neurotransmission despite normal presence of nNOSα in the varicosities. These studies reveal the critical importance of myosin Va in nitrergic neurotransmission by facilitating transport of nNOSα to the varicosity membrane.

Colligative Property of ATP: Implications for Enteric Purinergic Neuromuscular Neurotransmission

“…However, the prolonged release of nitric oxide prevents the restoration of membrane potential to baseline and aims to maintain the hyperpolarization. This is manifested as the slow inhibitory junction potential (sIJP), unambiguously recorded by several investigators across decades (Figure 1; Bennett et al, 1966; Atanasova et al, 1972; Smith et al, 1990; Hirst et al, 2004; Allego et al, 2008; Chaudhury et al, 2011, 2012; Chaudhury, 2016a). Following the paradigm-shifting demonstration of ATP as a neurotransmitter using gut tissues (Burnstock et al, 1970), there was a gap of several decades in which the VNUT could not be identified within the synaptic vesicles.…”

“…Evoked enteric inhibitory neuromuscular neurotransmission involves the sequential release of purines (most importantly, ATP) and the gas nitric oxide (NO), synthesized by neuronal nitric oxide synthase (nNOS) at the membranes of nerve terminals (Chaudhury et al, 2011, 2012; Chaudhury, 2014, 2015a, 2016a,b). While ATP is stored in the vesicles of the nerve terminals, NO is synthesized de novo (Chaudhury, 2016a).…”

Furthering the debate on the role of interstitial cells of Cajal in enteric inhibitory neuromuscular neurotransmission

“…There is no obvious direct evidence to this hypothesis. I have reported the role of prejunctional cytoskeletal force-generating proteins like myosin Va in the tandem neural release of the vesicular and nonvesicular neurotransmitters (ATP and NO, respectively) (4,7,8), which may explain the coordinative response of fast and slow IJPs on a scale of a few seconds.…”

2D DIGE Does Not Reveal all: A Scotopic Report Suggests Differential Expression of a Single “Calponin Family Member” Protein for Tetany of Sphincters!

“…The cutting-edge technologies of advanced proteomics shall help form rationale approaches to disorders currently labeled as “functional,” including dyspepsia, irritable bowel syndrome (IBS), unexplained constipation, and intestinal pseudo-obstruction (37). It should be noted that not all gastrointestinal motility disorders manifest changes in protein expression and may involve changes in the protein function or post-translational modification(s).…”