Arun Chaudhury

Physiology of Normal Esophageal Motility

The esophagus consists of 2 different parts. In humans, the cervical esophagus is composed of striated muscles and the thoracic esophagus is composed of phasic smooth muscles. The striated muscle esophagus is innervated by the lower motor neurons and peristalsis in this segment is due to sequential activation of the motor neurons in the nucleus ambiguus. Both primary and secondary peristaltic contractions are centrally mediated. The smooth muscle of esophagus is phasic in nature and is innervated by intramural inhibitory (nitric oxide releasing) and excitatory (acetylcholine releasing) neurons that receive inputs from separate sets of preganglionic neurons located in the dorsal motor nucleus of vagus. The primary peristalsis in this segment involves both central and peripheral mechanisms. The primary peristalsis consists of inhibition (called deglutitive inhibition) followed by excitation. The secondary peristalsis is entirely due to peripheral mechanisms and also involves inhibition followed by excitation. The lower esophageal sphincter (LES) is characterized by tonic muscle that is different from the muscle of the esophageal body. The LES, like the esophageal body smooth muscle, is also innervated by the inhibitory and excitatory neurons. The LES maintains tonic closure because of its myogenic property. The LES tone is modulated by the inhibitory and the excitatory nerves. Inhibitory nerves mediate LES relaxation and the excitatory nerves mediate reflex contraction or rebound contraction of the LES. Clinical disorders of esophageal motility can be classified on the basis of disorders of the inhibitory and excitatory innervations and the smooth muscles.

Mounting evidence against the role of ICC in neurotransmission to smooth muscle in the gut

How nerves transmit their signals to regulate activity of smooth muscle is of fundamental importance to autonomic and enteric physiology, clinical medicine, and therapeutics. A traditional view of neurotransmission to smooth muscles has been that motor nerve varicosities release neurotransmitters that act on receptors on smooth muscles to cause their contraction or relaxation via electromechanical and pharmacomechanical signaling pathways in the smooth muscle. In recent years, an old hypothesis that certain interstitial cells of Cajal (ICC) may transduce neural signals to smooth muscle cells has been resurrected. This later hypothesis is based on indirect evidence of closer proximity and presence of synapses between the nerve varicosities and ICC, gap junctions between ICC and smooth muscles, and presence of receptors and signaling pathways for the neurotransmitters and ICC. This indirect evidence is at best circumstantial. The direct evidence is based on the reports of loss of neurotransmission in mutant animals lacking ICC due to c-Kit receptor deficiency. However, a critical analysis of the recent data show that animals lacking ICC have normal cholinergic and purinergic neurotransmission and tachykinergic neurotransmission is actually increased. The status of nitrergic neurotransmission in c-Kit deficient animals has been controversial. However, reports suggest that 1) nitrergic neurotransmission in the internal anal sphincter does not require ICC and 2) the in vivo phenotype of ICC deficiency does not resemble nNOS deficiency. 3) The most recent report, in this issue of the Journal, concludes that impaired nitrergic neurotransmission may be due to smooth muscle defects associated with c-Kit receptor deficiency.

Role of myosin Va in purinergic vesicular neurotransmission in the gut

We examined the hypothesis that myosin Va, by transporting purinergic vesicles to the varicosity membrane for exocytosis, plays a key role in purinergic vesicular neurotransmission. Studies were performed in wild-type (WT) and myosin Va-deficient dilute, brown, nonagouti (DBA) mice. Intracellular microelectrode recordings were made in mouse antral muscle strips. Purinergic inhibitory junction potential (pIJP) was recorded under nonadrenergic noncholinergic conditions after masking the nitrergic junction potentials. DBA mice showed reduced pIJP but normal hyperpolarizing response to P2Y1 receptor agonist MRS-2365. To investigate the mechanism of reduced purinergic transmission in DBA mice, studies were performed in isolated varicosities obtained from homogenates of whole gut tissues by ultracentrifugation and sucrose cushion purification. Purinergic varicosities were identified in tissue sections and in isolated varicosities by immunostaining for the vesicular ATP transporter, the solute carrier protein SLC17A9. The varicosities were similar in WT and DBA mice. Myosin Va was markedly reduced in DBA varicosities compared with the WT varicosities. Proximity ligation assay showed that myosin Va was closely associated with SLC17A9. Vesicular exoendocytosis was examined by FM1–43 staining of varicosities, which showed that exoendocytosis after KCl stimulation was impaired in DBA varicosities compared with WT varicosities. These studies show that SLC17A9 identifies ATP-containing purinergic varicosities. Myosin Va associates with SLC17A9-stained vesicles and possibly transports them to varicosity membrane for exocytosis. In myosin Va-deficient mice, purinergic inhibitory neurotransmission is impaired.

Pathogenesis of Achalasia: Lessons From Mutant Mice

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Role of PSD95 in membrane association and catalytic activity of nNOSα in nitrergic varicosities in mice gut

We have recently shown that membrane association of neuronal nitric oxide synthase-alpha (nNOSalpha) is critical in the regulation of synthesis of NO during nitrergic neurotransmission. The purpose of this study was to examine the role of the synapse-associated proteins (SAPs) in membrane association of nNOSalpha. Varicosities (swellings on terminal axons) were isolated from mice gastrointestinal tract and examined for nNOSalpha, postsynaptic density protein 95 (PSD95), and membrane interactions by coimmunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were present in the cytosol. nNOSalpha was associated with PSD95 but not SAP97. nNOSalpha-PSD95 complex was bound to the membrane via palmitoylation of PSD95. Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOSalpha and PSD95 from the varicosity membrane and abolishes NO production. These studies show that palmitoylation of PSD95 anchors nNOSalpha to the varicosity membrane and that it is obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a novel target for regulation of nitrergic neurotransmission.

Progress in understanding of inhibitory purinergic neuromuscular transmission in the gut.

Recent studies with genetic deletion of P2Y1 receptor (P2Y1−/−) have clinched its role in enteric purinergic inhibitory neurotransmission and suggested that β‐NAD may be the purinergic inhibitory neurotransmitter in the colon. In this issue of the Journal, Gil and colleagues extend their earlier observations to the cecum and gastric antrum, showing that P2Y1 receptor mediated purinergic inhibition may be a general phenomenon in the gut. However, the authors made an unexpected observation in contrast with their earlier findings in the colon that neither the selective P2Y1 receptor antagonist MRS2500, nor P2Y1 receptor deletion, blocked the hyperpolarizing action of β‐NAD in the cecum. These observations suggest that β‐NAD may be the purinergic inhibitory neurotransmitter in the colon, but not in the cecum. This group had previously reported that the selective P2Y1 receptor antagonist MRS 2179 suppressed the hyperpolarizing action of ATP or ADP. Further studies are now needed to determine whether the hyperpolarizing actions of ATP and ADP are suppressed by the more potent P2Y1 antagonist MRS2500, and in P2Y1−/− mutants to test the intriguing possibility that different purines serve as purinergic inhibitory neurotransmitters in the colon and cecum and perhaps in different parts of the gut. Studies in P2Y1−/− mice will resolve other issues in purinergic neurotransmission including cellular localization of the β‐NAD or ATP‐activated P2Y1 receptors on either smooth muscle cells or PDGFRα+ fibroblast‐like cells, relationship of purinergic to nitrergic neurotransmission and understanding the physiological and clinical importance of purinergic transmission in gastrointestinal motility and its disorders.

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

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.

Myosin Va Plays a Role in Nitrergic Smooth Muscle Relaxation in Gastric Fundus and Corpora Cavernosa of Penis

The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction.

Evidence for Dual Pathway for Nitrergic Neuromuscular Transmission in Doubt: Evidence Favors Lack of Role of ICC

Dear Sir: The evidence provided by Groneberg et al calls to question the central conclusions of “joint mediation of relaxant effect of enteric NO by ICCs and SMCs.” Rather, the novel evidence supports the alternate explanation of lack of role of interstitial cells of Cajal (ICC) in transducing nitrergic signal to smooth muscles after electrical field stimulation (EFS). Normal nitrergic relaxation was demonstrated in ICC–GCKO, but relaxation was impaired to nitric oxide (NO) donor DEA-NO in SM-GCKO fundic muscular strips precontracted with 10 mmol/L acetylcholine (ACh). This is direct evidentiary for the lack of role of ICC during nitrergic neuromuscular transmission. These data are in concordance with paradigm-shifting recent evidence of intact slow inhibitory junction potential, the electrophysiologic hallmark of evoked nitrergic neurotransmission, recorded after ICC depletion by tamoxifen treatment of c-KitCreERT2/þ;LSL-R26DTA/þmice.2 When a 100-fold lower dose of 0.1 mmol/L ACh was used to precontract the strips, there was only slight outward deviation of the relaxation inhibitory curve in ICC-GCKO. Based on this observation of percentage contractions/ relaxations (absolute values are not mentioned), an argument is presented of a possible parallel role of ICC in nitrergic neurotransmission, justifying the use of this lower agonist dose as appropriate for generating tetany of the strips. The authors forward the rationale that the lower dose “more closely mimics endogenous levels of evoked ACh release.” Few studies have addressed ACh release in gut neuromuscular strips under basal conditions or EFS. Neurotransmitter release is a non–steady-state phenomenon. Suboptimal dose of ACh makes most of the present evidence equivocal. Reliable in vitro and in vivo comparisons of neurotransmission cannot be made. Relaxation inhibition curves depend on agonist doses, making them unreliable to analyze inhibitory neurotransmission. EC99 of ACh or U46619 should have been calculated to induce optimal tetany of the strips before quantifying relaxation and, even if the mechanical recordings were challenging, log–log scale should have been used, which are known to generate linear responses with only few data points at a much lower concentration than EC50. The basal tone was increased in SM/ICC-GCKO. It is unclear how normalization was performed. sGC agonist BAY41-2722, which modifies b1 to effect haem on the b2 subunit, produced residual relaxation in SM-GCKO tissues, raising the possibility of incomplete sGCb1 deletion. Nearly 50% residual relaxation observed at 4 Hz EFS in SM-GCKO may be suggestive of incomplete knockdown of sGC, rather than intermediation by a third cell like ICC. Double knockout SM/1CC-GCKO showed 50% relaxation to externally applied DEA-NO and this responsewas abolished in the presence of sGC inhibitor ODQ, indicating residual sGC enzyme after genomic deletion. Critical missing information is the efficiency of recombinations. Germline-transmitted SMMHC-Cre, which was used to delete sGC in smoothmuscles, is known to only marginally transfect in stomach. Control data for gene targeting efficiency, like quantitative Southern for deleted sGC DNA, qRT-PCR for Cre mRNA, and Western blot analysis for sGC specifically in smooth muscles and double knockouts are lacking. The median values of whole gut transit times of all groups nearly overlap, again raising doubts about efficiency of genomic deletion. SM/ICC-GCKO showed scant (w10%) relaxation after EFS at 4 Hz. Relaxing responses to higher frequencies of stimulations were not examined in the current study. Earlier studies have utilized EFS frequencies up to 40 Hz to examine enteric nitrergic neurotransmission. The authors hypothesize that “relaxing responses on EFS with frequencies up to 4Hz are based on release of NO.” 1 What is intriguing is that slope of the relaxation curve in double knockouts remains unchanged after EFS even in the presence of BAY41-2722. This later curve should have shown a downward shift. Lack of this shift may have resulted from inadequate dose of agonist so that the muscle strip may not have acquired Emax at all, and so, failed to show any relaxation. This may also have resulted from inadequate stimulation frequencies. The discrepancy of why DEA-NO but not BAY41-2722 relaxes SM/ICC-GCKO merits resolution in future studies. The diffusion constant of NO is very high (3300 m/s). Actions of NO are long range and mainly determined by the cell’s preprogrammed characteristic response, rather than the widely held notion in the field of enteric neurotransmission of proximity to NO source (nerve varicosity). Although several aspects of experimental approaches of this current study may be critiqued, this study utilizing novel, cell-specific genomic deletion provides unique evidence of the lack of role of ICCs in nitrergic mechanical relaxation.

Tail Tale: nNOSdel1203-1434 Predicts Global Defects in Esophagogastrointestinal Transit

Dear Editor: Shteyer et al reports correlative evidence of a truncating mutation of nNOSa in 2 probands (siblings) of achalasia. This excellent study leaves scope for additional discussion. Achalasia results from prejunctional defects of evoked nitric oxide (NO) synthesis or postjunctional failure of signal transduction. In the present study, the affected children exhibits a nonsense mutation in which amino acids beyond 1202 of nNOSC-terminal is deleted. 1 The authors expressed the truncated enzyme (nNOSdel1203-1434) in vitro and demonstrated absent NO production. Protein gel separation analysis of the mutant nNOS shows an additional 250 kDa dimeric band, apart from the reported 135 kDa monomer. Thus, dimeric nNOS may not always be surrogate for active nNOS, contrary to what has been posited. Whether the ex vivo changes occur in patients is unknown as peripheral cultured fibroblasts revealed barely detectable nNOS transcripts. Peripheral biomarker identification that may provide surrogate estimates of neurotransmitter contents of biopsy inaccessible tissues like the gut may address these issues. nNOSdel1203-1434 is similar in molecular mass to nNOSb (135 kDa), which lacks the PDZN-terminal and thus unable to be membrane localized for active neurotransmission. nNOSdel1203-1434 may be membrane localized due to intact N-terminal region, but still represents inactive nNOS. These aspects should be considered when examining nNOS proteome. The motility problems were not limited to esophageal achalasia. One child had colonic perforation, which likely resulted from a distal intestinal obstruction. The co-existent autism of both these children merits to be emphasized. Specific genetic variants of autism are reported to result in defective enteric NO neurotransmission. Whether such additional defects were present in these children may be an important area of investigation. Interestingly, like the cyclical vomiting phenotype of Phelan–McDermid syndrome, both these children initially presented with refractory vomiting. Manometry showed a long segment of contraction in esophageal body proximal to and including LES, providing indirect evidence of functional esophageal nitrergic denervation. Repeated vomiting probably resulted from restricted gastric emptying (with achalasia also contributing), suggesting that nNOSdel1203-1434 also involved NO-neurotransmission of the pylorus. This underscores that the molecular defects in common pathways may affect both the proximal and distal gut and emphasizes the need for obtaining careful history to logically approach the possible underlying problem. Certain achalasic conditions do remain region-restricted and the basis for why the features of impaired neurotransmission is not manifested throughout the entire gastrointestinal tract remains largely unknown. Despite being an orphan disease, achalasia and other motility disorders calls for investigation into enteric neurotransmission. The authors are commended for pursuing the molecular medicine of the complex pathophysiology arising from founder mutation due to consanguinity. One child, though presented with initial failure to thrive, has now survived for 6 years, probably due to residual inhibitory musculomotor neurotransmission, at least in the post-gastric regions. Because in vivo tissues were not examined for pragmatic reasons, we do not know whether there was some residual nNOS function or alternatively upregulated purinergic neurotransmission. The resected bowel specimen during ileostomy construction, if available, may be examined for nNOS distribution in the nerve terminals. The kinetics of hydride transfer during NO synthesis requires electron flow from NADPH through FAD to FMN from nNOSreductase to nNOSoxidase. The rate limiting step of electron transfer involves shift from FMN to the oxidase domain. This regionwas unaffected in the currentmutation. Additionally, tail region in vitro mutants with deleted C-terminal and NADPH binding domains retain potential to synthesize NO in vitro. Surprisingly, the reported genetic mutation synthesizes a mutant form with similar deletions and additional partial deletion of FAD binding region (normal nNOS990-1038 and 1171-1231), but this mutant lacks complete capacity for in vitro NO production. However, nNOSdel12031434 demonstrated detectable NADPH oxidation, suggesting misdirection of electron transfer likely to superoxide synthesis. Neither sildenafil nor Heller’s myotomy provided sustained relief. Exploratory and empirical pharmacotherapy to sustain the faradaic flow using nNOSoxidase stimulant tetrahydrobiopterin (BH4) or simply saturating the enzyme with excess calmodulin to produce thermodynamically feasible electron transfer for enhancing NO synthesis may be considered. The pairing of these enzymatic augmentations with stimulus-evoked neurotransmission event is the grand challenge in designing bioelectronic medicine. Shteyer et al’s study emphasizes the need for examining molecular pathology in motility disorders, as impairment of nitrergic neurotransmission is not necessarily coupled with changes in nNOS expression. This report reiterates the need to investigate gastrointestinal motility disorders from the perspective of precision medicine.