Discovery of non-zwitterionic aryl sulfonamides as Nav1.7 inhibitors with efficacy in preclinical behavioral models and translational measures of nociceptive neuron activation.

Since zwitterionic benzenesulfonamide Nav1.7 inhibitors suffer from poor membrane permeability, we sought to eliminate this characteristic by replacing the basic moiety with non-basic bicyclic acetals and monocyclic ethers. These efforts led to the discovery of the non-zwitterionic aryl sulfonamide 49 as a selective Nav1.7 inhibitor with improved membrane permeability. Despite its moderate cellular activity, 49 exhibited robust efficacy in mouse models of neuropathic and inflammatory pain and modulated translational electromyogram measures associated with activation of nociceptive neurons.

Lower esophageal sphincter is achalasic in nNOS(-/-) and hypotensive in W/W(v) mutant mice.

BACKGROUND AND AIMS: It has been proposed that nitrergic nerves mediate lower esophageal sphincter (LES) relaxation with intramuscular interstitial cells of Cajal (ICC-IM) as an intermediary. Dysfunction of the nitrergic pathway has been shown to cause LES hypertension and impaired relaxation in achalasia. We determined whether mice with neuronal nitric oxide synthase gene disruption (nNOS(-/-)) and W/W(v) mice lacking ICC-IM have achalasia-like LES dysfunction. METHODS: Intraluminal manometry using a customized micro-sized catheter assembly was performed in anesthetized mice. Basal LES pressure and swallow- and vagal-evoked LES relaxations were quantified in wild-type, Nomega-nitro-L-arginine methyl ester HCl salt (L-NAME)-treated, nNOS(-/-), and W/W(v) mice. RESULTS: Wild-type mouse LES maintained a basal pressure (24 +/- 3 mm Hg; N = 8) and relaxed normally to swallow (87% +/- 3%; N = 8) and vagal stimulation (91% +/- 4% mm Hg; N = 6). Pretreatment with L-NAME (100 mg/kg, intravenously) attenuated LES relaxation to both stimuli (P < 0.05). The LES in nNOS(-/-) was significantly hypertensive (36 +/- 5 mm Hg; N = 10; P < 0.05) with a markedly impaired relaxation (P < 0.05). In contrast, W/W(v) mouse LES was significantly hypotensive (11 +/- 2 mm Hg; N = 6; P < 0.05) with normal relaxation that was blocked by L-NAME. CONCLUSIONS: nNOS(-/-) mice have LES hypertension with impaired relaxation resembling achalasia. In contrast, W/W(v) mice have hypotensive LES with unimpaired relaxation, suggesting that ICC-IM do not play a role in nitrergic neurotransmission.

Functional anatomy and physiology of the upper esophageal sphincter.

Upper esophageal sphincter (UES) refers to the high-pressure zone located in between the pharynx and the cervical esophagus. The physiological role of this sphincter is to protect against reflux of food into the airways as well as prevent entry of air into the digestive tract. UES is a musculocartilaginous structure with its anterior wall being formed by the full extent of the posterior surface of the cricoid cartilage and arytenoid and interarytenoid muscles in the upper part. Posteriorly and laterally the cricopharyngeus (CP) muscle is a definitive component of the UES. CP has many unique characteristics: it is tonically active, has a high degree of elasticity, does not develop maximal tension at basal length, and is composed of a mixture of slow- and fast-twitch fibers, with the former predominating. These features enable the cricopharyngeus to maintain a resting tone and yet be able to stretch open by distracting forces, such as a swallowed bolus and hyoid and laryngeal excursion. CP, however, constitutes only the lower one third of the entire high-pressure zone. The thyropharyngeus (TP) muscle accounts for the remaining upper two thirds of the UES. The UES pressure is not entirely the result of myogenic activity, as a component of the pressure is the result of passive elasticity of the tissues. The opening of the UES involves relaxation of CP and TP muscles and forward movement of the larynx by the contraction of hyoid muscles. The UES function is controlled by a variety of reflexes that involve afferent inputs to the motorneurons innervating the sphincter. These physiological reflexes elicit either contraction or opening of the UES. Inability of the sphincter to open leads to difficulty in swallowing. Opening of the sphincter without associated CP relaxation leads to the clinical syndrome of cricopharyngeal bar.

Excitation of dorsal motor vagal neurons evokes non-nicotinic receptor-mediated gastric relaxation.

Vagal stimulation results in both gastric motor excitatory and non-adrenergic non-cholinergic (NANC) inhibitory responses. The NANC pathway involves preganglionic cholinergic neurons, which act through nicotinic receptors to ultimately evoke gastric smooth muscle relaxation via release of nitric oxide (NO) and other neurotransmitters. Within the dorsal motor nucleus of the vagus (DMN), some preganglionic neurons also contain NO synthase. The NO synthase-containing neurons innervate the gastric fundus where adaptive relaxation occurs. This study tests the hypothesis that chemical stimulation of vagal motor neurons in animals, in which nicotinic receptors are blocked, evokes an NO-dependent gastric relaxation. A cell body excitant, N-methyl-D-aspartate (NMDA, 0.03-3 nmol), was microinjected into the DMN in anesthetized rats while recording intragastric pressure (IgP). The first group received NMDA before and after administration of a ganglionic blocker, hexamethonium bromide (15 mg/kg, i.v.) and atropine (1.0 mg/kg). Significant dose-dependent increases in IgP and gastric motility occurred before hexamethonium after the 0.3 and 3 nmol doses of NMDA. After hexamethonium, 0.3 and 3 nmol NMDA evoked significant decreases in IgP. A second group of rats was hexamethonium-pretreated and received NMDA microinjection into the DMN before and after an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (10 mg/kg, i.v.). The NMDA-evoked decrease in IgP was completely abolished by the NO synthase inhibitor. These data support the novel idea that NO synthase-containing preganglionic neurons mediate gastric relaxation that is independent of nicotinic receptors.

Vagally-regulated gastric motor activity: evidence for kainate and NMDA receptor mediation.

Blockade of GABA(A) receptors in the dorsal vagal complex produces marked gastric motor excitation. This effect is abolished by a prior microinjection of a non-selective excitatory amino acid receptor antagonist. Here we present functional evidence for kainate and NMDA receptor-mediated gastric excitation in the dorsal vagal complex. Microinjections into the dorsal vagal complex were performed in alpha-chloralose-anesthetized rats using multi-barrelled glass micropipettes while recording intragastric pressure and motility. Kainic acid (30 and 100 pmol in 30 nl) and NMDA (100 and 300 pmol) produced dose-related increases in intragastric pressure and motility. The gastric responses to kainate (30 pmol) and NMDA were selectively abolished by prior microinjection 6,7-dinitroquinoxaline-2,3-dione (600 pmol, 60 nl) and DL-2-amino-5-phosphanopentanoic acid (2 nmol), respectively. Atropine (1 mg/kg, i.v.) pretreatment blocked kainate-, NMDA- and L-glutamate-induced gastric excitation. Thus, both kainate- and NMDA-receptors in the dorsal vagal complex can independently cause vagally-mediated gastric motor excitation.

Role of GABAA receptors in rat hindbrain nuclei controlling gastric motor function.

It has been shown in cats that gastric motor control by the dorsal vagal complex and nucleus ambiguus is under a tonic GABAergic influence. Since much more work has been performed in rats to define vago-vagal reflexes controlling gastrointestinal function, an understanding of the potential inhibition by candidate neurotransmitters such as GABA (gamma aminobutyric acid) in the rat dorsal vagal complex (DVC) is essential to assess. Multiple-barrelled micropipettes were used to apply to the dorsal vagal complex the GABAA antagonist, bicuculline methiodide (0.1-1 nmol), and a GABAA agonist, muscimol (10 nmol) prior to micro-injection of the GABAA antagonist. Micro-injections of bicuculline (353 pmol and 1 nmol), which were localized primarily in the dorsal motor nucleus of the vagus, produced significant increases in intragastric pressure and pyloric motility. These responses were abolished by vagotomy and by a prior micro-injection of muscimol. To determine whether GABAergic blockade in the dorsal vagal complex results in gastric motor excitation through excitatory amino acid receptors, kynurenic acid (5 nmol), a kainate/NMDA (N-methyl D-aspartic acid) receptor antagonist, was micro-injected prior to bicuculline. This abolished the increase in gastric motor function normally evoked by bicuculline. In the other two important hindbrain nuclei controlling gastric function, the nucleus raphe obscurus and nucleus ambiguus, bicuculline (353 pmol) significantly increased intragastric pressure via vagally mediated pathways. These data demonstrate that all three rat hindbrain nuclei known to influence gastric function via the vagus nerve are under tonic GABAergic control. In addition, in the dorsal vagal complex, relief from GABAergic inhibition results in increases in gastric motor function through kainate/NMDA receptor-mediated excitation.

Intracisternal antisense oligonucleotides to TRH receptor abolish TRH-evoked gastric motor excitation.

Thyrotropin-releasing hormone (TRH) from the nucleus raphe obscurus (nROb) innervates the dorsal vagal complex (DVC) and activates gastric motor function. Assessment of the importance of TRH has been hampered by the lack of TRH receptor antagonists. To overcome this, rats were given intracisternal antisense oligonucleotides against the first 18 bases of TRH receptor mRNA, mismatch oligonucleotides, or saline. Rats were anesthetized, and L-glutamate (15 nmol), TRH (1 and 10 pmol), and saline were microinjected into the DVC and nROb while gastric motor function was monitored. Intracisternal TRH mRNA antisense oligonucleotides abolished the gastric excitatory affects of microinjection of TRH, but not L-glutamate, into the DVC, and the response to TRH recovered after 2 wk of no antisense treatment. Chemical stimulation of the nROb increased intragastric pressure in saline- and mismatch- but not antisense-treated animals. These studies demonstrate that intracisternal TRH receptor antisense oligonucleotides produce a selective and reversible "knockdown" of responsiveness to exogenous TRH in the DVC, as well as to excitation of an endogenous TRH pathway controlling gastric function. It also provides a new tool for assessment of TRH pathways in hindbrain control of gastric function.

2-Butoxyethanol autoprotection is due to resiliance of newly formed erythrocytes to hemolysis.

Pretreatment with a low dose of a toxic chemical protecting the animals from a subsequently administered lethal dose of the same chemical is called autoprotection. Autoprotection by model hepatotoxicants has been recently shown to be due to augmentation of cell division and tissue repair as well as an inherent resiliance of newly divided cells. The present studies were designed to investigate if an autoprotection model could be established in an extrahepatic tissue. The second objective was to test the hypothesis that inherent resiliance of newly divided cells is a major contributing mechanism for autoprotection. Female Sprague-Dawley rats (200-250 g) received a single administration of a moderately toxic but nonlethal dose (500 mg/kg, p.o.) 7 days prior to the administration of an LD90 dose (1500 mg/kg, p.o.) of the same compound. All rats receiving the initial protective dose are able to survive the lethal dose of butoxyethanol, in contrast to the death of those receiving the lethal dose alone. Following the administration of butoxyethanol, the hematocrit decreased from the normal 45% to 18% and by day 7, recovered to normal levels. Following the lethal challenge, hematocrit decreased to 13% in the naive rats, while decreasing only to 27% in rats receiving the protective dose, permitting animal survival. Administration of pyrazole to inhibit metabolism of butoxyethanol to butoxyacetic acid abolished autoprotection.(ABSTRACT TRUNCATED AT 250 WORDS)