A recent report by Estévez-Herrera et al. (2016) suggested that ATP, the energy coin, also a neurotransmitter, controls the osmotic pressure of vesicular contents (Estévez-Herrera et al., 2016). Since the discovery and description of quantal transmission (Katz, 1971; Bennett and Kearns, 2000), membrane-delimited packeted structures, the vesicles, have been morphologically identified and correlated with secretion in several secretory tissues, including nerve terminals of central and peripheral neurons, chromaffin cells, platelets, and insulin-secreting beta cells of the pancreas (Goyal and Chaudhury, 2013; Südhof, 2013; Thorn et al., 2016). Though it is now known for some time that ATP is concentrated in the vesicles, the demonstration that ATP, by virtue of being negatively charged, and its ability to associate with positively charged molecules like amines, agglomerates vesicular particles, is a significant finding. Vesicles do not release their contents randomly within the cytosol, but rather are transported to the membranes for exocytosis (Jena, 2009). An important contributor to this site-specific release of vesicular contents may be the constant fine tuning and maintenance of osmotic pressure isotonic with the cytosol of the vesicle-containing structure. In the light of such plausible dynamic regulation of osmotic pressure of vesicles, the demonstration of the likely role of ATP in regulating this vesicular osmotic pressure acquires importance (Estévez-Herrera et al., 2016). In this perspective, we discuss the implications of these findings on enteric purinergic inhibitory musculomotor neurotransmission.

Osmotic property is a colligative property: it depends on the number of particles, as suggested by Raoult:

pV = nRT…standard gas equation

The process of release of neurotransmitters is highly coordinated, involving several 100 proteins with graded responses to intracellular calcium fluctuations (Goyal and Chaudhury, 2013; Südhof, 2013). In neurosecretory processes like stimulation-evoked neurotransmission, the pool of readily releasable vesicles empty contents after docking at the cell membrane of the active zones. Electron micrographs of nerve terminal varicosities always demonstrate intact membranes of vesicles within the cytosol of the terminal (Collman et al., 2015). This structural integrity of vesicles strongly suggests that the contents of the vesicles are isotonic with the matrix of the varicosities.

Earlier, an interesting study investigated the osmotic pressure of synaptic vesicles (Kopell and Westhead, 1982). This study revealed that the vesicles obtained from chromaffin cells of the adrenal gland, despite their high concentrations of various amines, and peptides, remained isotonic (Kopell and Westhead, 1982). This study hypothesized that the highly negatively charged ATP molecules, a major co-constituent of chromaffin cells beside the positively-charged amines, forms a polymeric complex within the vesicles (Kopell and Westhead, 1982).

The leading hypothesis by Estévez-Herrera et al. (2016) is that when ATP agglomerates the vesicular contents, there is a reduction in the number of free particles, leading to balance of pressures across the vesicular membrane. This physical property may be potentially a critical determinant of membrane integrity of vesicles from their biogenesis until the time they receive the necessary stimuli for exocytosis.

Estévez-Herrera et al. (2016) show that SLC17A9, the vesicular nucleotide transporter (VNUT; Sawada et al., 2008), performs a rate-limiting step to the transport of ATP within the large dense core (LDC) particles of the chromaffin cells. It has been specifically demonstrated earlier and well-known for some time that the drive for ATP entry is regulated by a proton motive force (Sawada et al., 2008). Thus, it is imperative that the colligative property has a direct relationship with the intravesicular acidity.

The biophysical characteristics of particle-based actions of ATP may have important implications for enteric neuromuscular transmission. 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). The released ATP during evoked neurotransmission hyperpolarizes the smooth muscle membrane (Chaudhury, 2016a). In a span of a few 100 ms, the membrane potential endeavors to swing back to its resting stage. 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. Quinacrine, the antimalarial drug, robustly stains ATP containing nerve terminals (Belai and Burnstock, 1994), but this never could provide insights into how ATP, a highly negatively charged molecule, could be shuttled across the cell membrane of the vesicles. Following the report by Sawada et al. (2008) of the molecular identity of VNUT as the solute carrier protein SLC17A9 (Sawada et al., 2008), a commercially available antibody was used to demonstrate the existence of SLC17A9 in the enteric musculomotor nerve terminals (Chaudhury et al., 2012), providing the preliminary critical evidence of fulfillment of the Sherringtonian criterion (Levine, 2007) for the existence of the transporter of a neurotransmitter.

ATP is widely distributed in enteric musculomotor nerve terminals. It is present in both VIP containing large dense core vesicles, as well as acetylcholine (Ach) containing small clear vesicles (Figure 1). While VIP plays a significant role in inhibitory neurotransmission and smooth muscle relaxation, Ach facilitates excitatory neurotransmission and smooth muscle contraction. Alternate relaxation and contractions of smooth muscles at the same location are the key factors that determine transit of luminal contents but very little is known regarding the release kinetics of VIP and Ach, and parallel release of ATP. Below, we discuss some of the possibilities that may happen to execute these complex release of excitatory and inhibitory neurotransmitters during enteric nerve-smooth muscle neurotransmission. We also discuss the potential role of colligative property of ATP in influencing these functions.

The biophysical experiments of estimating osmotic pressure of vesicles are challenging to perform, and more so in an in vivo context. Enteric synaptosomal preparations may be used to examine whether the mechanisms of ATP contributing to particle stability (Estévez-Herrera et al., 2016) is a general phenomenon seen across all vesicular structures, for example cholinergic containing small synaptic vesicles and VIP containing large dense core vesicles. Additionally, polymeric vesicular contents with (ATP-neurotransmitter)_n needs demonstration, likely by estimation of the polymeric masses or by surrogate measures of vesicular acidity. The specific gravity of the clear and dense core vesicles have been reported (Table 1). A relevant hypothesis that may be examined is whether the ATP contents are different between exclusive Ach containing vesicles vs. Ach-VIP containing vesicles. If so, the particle aggregating effects of ATP may differentially regulate release of Ach and VIP during excitatory and inhibitory neurotransmission, respectively.

Most esophagogastrointestinal motility disorders involve dysfunction of nitrergic biosynthesis and postjunctional smooth muscle responses (Chaudhury, 2015a,b, 2016b). Varied mechanisms of pathophysiology finally converge on the nitrergic pathways to cause diseases like achalasia, gastroparesis, pseudo-obstruction, megacolon, and constipation. There are virtually no disorders in which purinergic inhibitory neurotransmission has been found as the solitary basis of the gastrointestinal motility disorder. There are incipient suggestions that the purinergic fast IJP may be impaired, for example in the transitional zone in Hirschsprung's disease (Jiménez et al., 2015). It is possible that defective ATP production or vesicular shuttle may cause subtle defects in inhibitory neuro-smooth muscle neurotransmission. Mitochondrial ATP production is defective in diabetes (Bagkos et al., 2014). ATP gates SLC17A9 (Sawada et al., 2008). Thus, deficient ATP production may cause SLC17A9 channelopathy. SLC17A9 knockout mice do not show any frank gastrointestinal phenotypic abnormalities and have normal body weight (Dr. Richard Palmiter, personal communication). In SLC17A9 knockout mice, insulin vesicular exocytosis is accelerated (Sakamoto et al., 2014). This may result from deficient particle aggregating action of ATP. Whether such defects also occur in enteric vesicular release remains to be tested. We plan to undertake further studies of the enteric synaptosomal properties during neurotransmission to test the generalizability of colligative property of ATP and any effect of its deficiency on purinergic neuromuscular transmission.

AC conceptualized and drafted manuscript. VD important intellectual participation. WM important intellectual participation, overall supervision. All authors read and approved final version of manuscript.