Inhibitory motor neurons of the esophageal myenteric plexus are mechanosensitive

Abstract

Mittal RK. Inhibitory motor neurons of the esophageal myenteric plexus are mechanosensitive. Am J Physiol Cell Physiol 308: C405-C413, 2015. First published December 24, 2014; doi:10.1152/ajpcell.00159.2014.-Mechanosensitivity of enteric neurons has been reported in the small intestine and colon, but not in the esophagus. Our earlier in vivo studies show that mechanical stretch of the esophagus in the axial direction induces neurally mediated relaxation of the lower esophageal sphincter, possibly through mechanosensitive motor neurons. However, this novel notion that the motor neurons are mechanosensitive has not been examined in isolated esophageal myenteric motor neurons. The goal of our present study was to examine the mechanosensitivity of esophageal motor neurons in primary culture and elucidate the underlying molecular mechanisms. Immmunocytochemical analysis revealed that Ͼ95% cells were positive for the neuronal marker protein gene product 9.5 and that 66% of these cells costained with protein gene product 9.5 and neuronal nitric oxide (NO) synthase. Hypotonic solution induced an increase in the cytoplasm volume in all cells that was independent of extracellular Ca 2ϩ . Hypotonic solution and mechanical stretch induced cytoplasmic free Ca 2ϩ signaling in ϳ65% of neurons in the presence, but not absence, of extracellular Ca 2ϩ . Neurons grown on the elastic membrane responded to mechanical stretch by an increase in neuronal size and Ca 2ϩ signaling simultaneously. Hypotonic stretch-induced cytoplasmic free Ca 2ϩ signaling was not affected by extracellular Mg 2ϩ , 5-nitro-2-(3-phenylpropylamino)benzoic acid, and nifedipine but was attenuated by 2-aminoethoxydiphenyl borate, Gd 3ϩ , and Grammostola mechanotoxin 4, blockers of the stretch-activated ion channels. In ϳ57% of the neurons, hypotonic stretch also induced Ca 2ϩ -dependent cytoplasmic NO production, which was abolished by Grammostola mechanotoxin 4. These results prove that the esophageal inhibitory motor neurons possess a mechanosensitive property and also provide novel insights into the stretch-activated ion channel-Ca 2ϩ -NO signaling pathway in these neurons. neuronal nitric oxide synthase; nitric oxide production; cytoplasmic free calcium; stretch-activated cation channels THE ENTERIC NERVOUS SYSTEM regulates gastrointestinal (GI) functions, such as digestion, absorption, secretion, motility, and sensation (1, 14, 37). The enteric nervous system contains three types of neurons: 1) sensory neurons [intrinsic primary afferent neurons (IPANs)], 2) interneurons, and 3) motor neurons (1, 14). The latter are further subdivided into excitatory and inhibitory motor neurons on the basis of their regulatory functions (1, 15): excitatory motor neurons contain neurotransmitters, such as acetylcholine, substance P, and glutamate (14), and inhibitory motor neurons contain nitric oxide (NO), ATP, and vasoactive intestinal polypeptide (14, 37).Similar to neurons in the rest of the GI tract, neurons in the wall of esophagus also play an important role in the s...

Na+/Ca2+ exchanger 1 is a key mechanosensitive molecule of the esophageal myenteric neurons

“…Second, the mechanosensitivity of NCX1 may play a functional role in the EMN since the latter is subjected to mechanical distortion that could activate NCX1 during oesophageal motility. Third, in a study, 15 we found that mechanical stretch induced Ca 2+ -dependent NO production in the EMN, which is consistent with other reports that show flow-dependent changes in the NCX activity modulates endothelial NO production. 52 Based on the fact that operation of the Ca 2+ entry mode of NCX1 raises [Ca 2+ ] cyt and that nNOS is a Ca 2+ -dependent enzyme, NCX1 would produce NO production in the EMN.…”

“…12 More recently, we successfully cultured the EMN from the rat oesophagus. 14 Using the EMN grown in primary culture as cell model, we found that the oesophageal inhibitory motor neurons possessed mechanosensitive properties, 15 that is, they responded to osmotic and mechanical stretch, which was similar to the findings of Mazzuli and Schemann in the small intestine and colon. 7,16 However, so far there is almost no information on the nature of molecules responsible for the mechanosensitivity of enteric neurons.…”

“…44 We previously reported that the EMN are mechanosensitive and excluded the possibility of VGCC, TRPV1, TRPC and volume-sensitive Cl − channels as the molecular candidates for the mechanosensitivity of EMN. 15 The mechanosensitive property of NCX1 was first identified in the human myocardium and in the rat cardiac myocytes, 47,48 and confirmed in the CHO cells transfected with NCX1. 48 Indeed, we found that NCX1 is a possible molecular candidate for the mechanosensitivity of EMN for several reasons: (a) it is expressed in these neurons and regulates their Ca 2+ signalling; (b) the neuronal mechanosensitivity in humans and rats was tested using both hypotonic solution and mechanical stretch of the individual neurons; (c) NCX inhibitor blocked the stretch activated LES relaxation in vivo rat model; and (d) the mechanosensitivity of NCX1 was identified in the EMN and CHO cells transfected with NCX1 by combination of pharmacological inhibition and genetic knockdown.…”

“…After the EMNs were cultured for 2 weeks in a humidified tissue culture incubator, immmunocytochemical studies were performed and it was found that over 95% of these cells were positively stained for two generally accepted neuronal markers, that is, PGP 9.5 and Milli-Mark Fluoro. 15,16 A sample of fresh human oesophagus was procured from the National Disease Research Interchange (8 Penn Center,15th Floor. 1628 John F. Kennedy Blvd.…”

Mechanosensitive Piezo Channels in the Gastrointestinal Tract

“…Several studies have reported distinct mechanosensitive responses in enteric neurons to tensile stress (stretch), compressive stress (volume injection, glass probe, or von Frey hair), shear stress, and cell swelling (hypoosmotic solutions) (Dong, Jiang, Dong, & Mittal, 2014; Hibberd, Zagorodnyuk, Spencer, & Brookes, 2012; Kugler et al, 2015; Kunze et al, 1999; Kunze, Clerc, Furness, & Gola, 2000; Kunze et al, 1998; Mayer & Wood, 1975; Mazzuoli & Schemann, 2009, 2012; Spencer & Smith, 2004). While these studies suggest roles in mechanosen-sitivity sensing and control of muscle activity, as well as a servo-feedback loop (Mazzuoli-Weber & Schemann, 2015), the identities of the mechano-sensitive ion channels responsible for mechanotransduction are largely unknown.…”