Neuronal nicotinic AChRs (nAChRs) are implicated in the pathogenesis of diverse neurological disorders and in the regulation of small-cell lung carcinoma growth. Twelve subunits have been identified ...in vertebrates, and mutations of one are recognized in a rare form of human epilepsy. Mice with genetically manipulated neuronal nAChR subunits exhibit behavioral or autonomic phenotypes. Here, we report the first model of an acquired neuronal nAChR disorder and evidence for its pertinence to paraneoplastic neurological autoimmunity. Rabbits immunized once with recombinant alpha 3 subunit (residues 1-205) develop profound gastrointestinal hypomotility, dilated pupils with impaired light response, and grossly distended bladders. As in patients with idiopathic and paraneoplastic autoimmune autonomic neuropathy, the severity parallels serum levels of ganglionic nAChR autoantibody. Failure of neurotransmission through abdominal sympathetic ganglia, with retention of neuronal viability, confirms that the disorder is a postsynaptic channelopathy. In addition, we found ganglionic nAChR protein in small-cell carcinoma lines, identifying this cancer as a potential initiator of ganglionic nAChR autoimmunity. The data support our hypothesis that immune responses driven by distinct neuronal nAChR subtypes expressed in small-cell carcinomas account for several lung cancer-related paraneoplastic disorders affecting cholinergic systems, including autoimmune autonomic neuropathy, seizures, dementia, and movement disorders.
Background
Otilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are ...well‐characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function.
Methods
Adult Sprague–Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB‐treated and age‐matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology.
Key Results
Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti‐Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential.
Conclusions & Inferences
Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.
Chronic administration of otilonium bromide (OB) provides relief from irritable bowel syndrome (IBS) in humans; yet the underlying mechanisms remain to be fully elucidated. In studies on rat colon, chronic OB administered at a dose relevant to human pharmacotherapy altered neurokinin1 receptor, substance P and neuronal nitric oxide synthase expression and neuromuscular transmission but had no effect on interstitial cells of Cajal and the electrical slow wave. A novel mechanism for the effects of chronic OB administration involving the myenteric plexus is revealed that, together with the established inhibitory effects of OB acting directly on smooth muscle cells, modulates colonic function.
Intestinofugal afferent neurones (IFANs) are a unique subset of myenteric ganglion neurones that regulate normal gastrointestinal function. The IFANs relaying mechanosensory information to ...sympathetic neurones of the prevertebral ganglion (PVG) function as volume detectors. It is possible that mechanosensory information arriving in the PVG via axon collaterals of visceral spinal afferent nerves can be modulated entirely within the PVG itself.
Hydrogen sulfide (H2S) plays important roles in the enteric system in the wall of the gastrointestinal tract. There have been no studies on whether H2S is endogenously generated in peripheral ...sympathetic ganglia and, if so, its effect on synaptic transmission. In this study, we examined the effect of H2S on cholinergic excitatory fast synaptic transmission in the mouse superior mesenteric ganglion (SMG). Our study revealed that NaHS and endogenously generated H2S selectively potentiated cholinergic fast EPSPs (F-EPSPs) evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. The H2S-producing enzyme cystathionine-γ-lyase (CSE) was expressed in both neurons and glial cells. The CSE blocker PAG (dl-propargylglycine) significantly reduced the amplitude of F-EPSPs evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. Inhibiting the breakdown of endogenously generated H2S with stigmatellin potentiated the amplitude of F-EPSPs evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. Splanchnic F-EPSPs but not colonic F-EPSPs were reduced in CSE knock-out (KO) mice. Functional studies showed that NaHS enhanced the inhibitory effect of splanchnic nerve stimulation on colonic motility. Colonic motility in CSE-KO mice was significantly higher than colonic motility in wild-type mice. We conclude that endogenously generated H2S acted selectively on presynaptic terminals of splanchnic nerves to modulate fast cholinergic synaptic input and that this effect of H2S modulates CNS control of gastrointestinal motility. Our results show for the first time that the facilitatory effect of endogenous H2S in the mouse SMG is pathway specific.
Interstitial cells of Cajal (ICC) generate the electrical slow wave required for normal gastrointestinal motility. The ionic conductances expressed in human intestinal ICC are unknown. The aim of ...this study was to determine expression of a Na+ current in human intestinal ICC and to determine the effects of the Na+ current on the slow wave. Visually identified, freshly dissociated, single ICC were verified by the presence of c-kit mRNA by using single-cell RT-PCR. Standard whole cell currents were recorded from patch-clamped ICC held at -100 mV between pulse protocols. A Na+ current was identified in human intestinal ICC. The current activated at -55 mV and peaked at -30 mV. Extracellular N-methyl-d-glucamine abolished and QX-314 (500 microM) blocked the Na+ current, but nifedipine and Ni2+ did not. The Na+ current was activated by shear stress. Single-cell RT-PCR detected mRNA for the Na+ alpha-subunit SCN5A in single human intestinal ICC. Lidocaine (200 microm) and QX-314 (500 microM) decreased slow wave frequency, and stretch increased slow wave frequency. A mechanosensitive Na+ channel current is present in human intestinal ICC and appears to play a role in the control of intestinal motor function.
Autoimmune autonomic neuropathy (AAN) is an acquired, often severe, form of dysautonomia. Many patients with AAN have serum antibodies specific for the neuronal ganglionic nicotinic acetylcholine ...receptor (AChR). Rabbits immunized with a fusion protein corresponding to the N-terminal extracellular domain of the ganglionic AChR alpha3 subunit produce ganglionic AChR antibodies and develop signs of experimental AAN (EAAN) that recapitulate the cardinal autonomic features of AAN in man. We now demonstrate that EAAN is an antibody-mediated disorder by documenting sympathetic, parasympathetic, and enteric autonomic dysfunction in mice injected with rabbit IgG containing ganglionic AChR antibodies. Recipient mice develop transient gastrointestinal dysmotility, urinary retention, dilated pupils, reduced heart rate variability, and impaired catecholamine response to stress. The autonomic signs are associated with a reversible failure of nicotinic cholinergic synaptic transmission in superior mesenteric ganglia. Mice injected with IgG from two patients with AAN (of three tested) demonstrated a milder phenotype with evidence of urinary retention and gastrointestinal dysmotility. The demonstration that ganglionic AChR-specific IgG causes impaired autonomic synaptic transmission and autonomic failure in mice implicates an antibody-mediated pathogenesis for AAN. The antibody effect is potentially reversible, justifying early use of immunomodulatory therapy directed at lowering IgG levels and abrogating IgG production in patients with AAN.
The objective of this study was to determine the distribution of enteric nerves and interstitial cells of Cajal (ICC) in the normal human appendix and in type 1 diabetes. Appendixes were collected ...from patients with type 1 diabetes and from non‐diabetic controls. Volumes of nerves and ICC were determined using 3‐D reconstruction and neuronal nitric oxide synthase (nNOS) expressing neurons were counted. Enteric ganglia were found in the myenteric plexus region and within the longitudinal muscle. ICC were found throughout the muscle layers. In diabetes, c‐Kit positive ICC volumes were significantly reduced as were nNOS expressing neurons. In conclusion, we describe the distribution of ICC and enteric nerves in health and in diabetes. The data also suggest that the human appendix, a readily available source of human tissue, may be useful model for the study of motility disorders.
Background & Aims: Gastrointestinal smooth muscle strips devoid of enteric nerve cells can contract in response to stretch, suggesting that mechanosensitivity and mechanotransduction can occur at the ...level of the smooth muscle cell. The aim of this study was to determine whether stretch-activated calcium channels are present in gastrointestinal smooth muscle cells.
Methods: Whole-cell and single-channel calcium currents were measured from human jejunal circular smooth muscle cells in response to increased intracellular pressure, bath perfusion, and membrane stretch.
Results: At 10 mm Hg positive pressure, peak calcium current increased from −36 ± 10 pA to −53 ± 13 pA. Bath perfusion at 10 mL/min increased calcium current from −97.7 ± 14 pA to −122 ± 16 pA. Single-channel open probability increased in response to negative pipette pressure. All increases were blocked by nifedipine.
Conclusions: A stretch-activated, nifedipine-sensitive calcium channel is present in human jejunal circular smooth muscle cells. The channel is activated by both an increase in intracellular pressure and by external shear forces. The presence of a stretch-activated calcium channel in gastrointestinal smooth muscle cells may allow the smooth muscle cells to act directly as mechanotransducers and to participate in the regulation of smooth muscle tone and intestinal motility.
GASTROENTEROLOGY 1999;117:900-905
Tetrodotoxin‐resistant Na+currents are expressed in a variety of muscle cells including human jejunal circular smooth muscle (HJCSM) cells. The aim of this study was to determine the molecular ...identity of the pore‐forming α‐subunit of the HJCSM Na+channel. Degenerate primers identified a cDNA fragment of 1.5 kb with 99% nucleotide homology with human cardiac SCN5A. The identified clone was also amplified from single smooth muscle cells by reverse transcriptase–polymerase chain reaction (RT–PCR). Northern blot analysis showed expression of full‐length SCN5A. Laser capture microdissection was used to obtain highly purified populations of HJCSM cells. RT–PCR on the harvested cells showed that SCN5A was present in circular but not in longitudinal muscle. A similar result was obtained using a pan‐Na+channel antibody. The full‐length sequence for SCN5A was obtained by combining standard polymerase chain reaction with 5′ and 3′ rapid amplification of cDNA end techniques. The intestinal SCN5A was nearly identical to the cardiac SCN5A. The data indicate that SCN5A is more widely distributed than previously thought and encodes the pore‐forming α‐subunit of the tetrodotoxin‐resistant Na+current in HJCSM cells.
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