Capsaicin-sensitive Sensory Neurons Research Articles

Epac activation sensitizes rat sensory neurons through activation of Ras.

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization.

Regulatory mechanism of the gastric hyperemic response following barrier disruption: roles of cyclooxygenase-1, the prostaglandin E2/EP1 receptor and sensory neurons.

We herein reviewed the mechanism underlying the gastric hyperemic response following barrier disruption, with a focus on cyclooxygenase (COX) isozymes, prostaglandin (PG) E2, and capsaicin-sensitive afferent neurons. Mucosal damage was induced by exposing the stomach to 20 mM taurocholate (TC) with 50 mM HCl. The TC treatment disrupted surface epithelial cells, and then increased acid back-diffusion and mucosal blood flow (GMBF) in the stomachs of rats or wild-type mice. This hyperemic response in the rat stomach was inhibited by indomethacin without affecting acid back-diffusion, which resulted in the aggravation of lesions. The effect of indomethacin was mimicked by loxoprofen and the selective COX-1 inhibitor, SC-560, but not by the selective COX-2 inhibitor, celecoxib. The GMBF responses induced by TC were similarly observed in the stomachs of wild-type mice and EP3 receptor knockout mice, but not in mice lacking the EP1 receptor or pretreated with an EP1 antagonist. The increase in the GMBF response associated with acid back-diffusion after the TC treatment was also inhibited by the chemical ablation of capsaicin-sensitive afferent neurons, but not capsazepine, a TRPV1 antagonist. Thus, endogenous PGE2 produced by COX-1 plays a role in the gastric hyperemic response following barrier disruption of the stomach by interacting with capsaicin-sensitive afferent neurons, mainly through EP1 receptors, and facilitating the GMBF response to acid back-diffusion. These findings have also contributed to a deeper understanding of mucosal defensive mechanisms following barrier disruption and the development of new strategies for the treatment of gastrointestinal diseases.

Role of hydrogen sulfide, a gasotransmitter, in colonic pain and inflammation

Hydrogen sulfide (H2S), the third known gaseous transmitter following nitric oxide and carbon monoxide, is generated by multiple enzymes including cystathionine-γ-lyase (CSE) in vivo. We previously demonstrated that H2S activates Cav3.2 T-type Ca(2+) channels expressed on sensory neurons, leading to hyperalgesia and facilitation of inflammation. Here, we describe the role of H2S in processing of colonic pain and inflammation. Intracolonic (i.col.) administration of NaHS, an H2S donor, to mice evoked colonic pain-like nociceptive behavior and referred hyperalgesia accompanied by phosphorylation of ERK in the superficial layers of spinal dorsal horn, a marker for excitation of nociceptive neurons. The pronociceptive effect of NaHS was abolished by inhibitors or knockdown of Cav3.2 and by an inhibitor of TRPA1, another target molecule of H2S. In rats with colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS), on the other hand, repeated i.col. administration of NaHS prevented colonic ulcer and inflammatory symptoms, which were inhibited by ablation of capsaicin-sensitive sensory neurons or T-type Ca(2+) channel inhibitor. NaHS, given i.col., caused phosphorylation of ERK in the spinal dorsal horn of rats with TNBS-induced colitis, but not of naïve rats. In TNBS-treated rats, Cav3.2 was upregulated in the dorsal root ganglia, while CSE was downregulated in the colon. Taken together, these findings suggest that inhibitors of the CSE/H2S/Cav3.2 or TRPA1 pathways might be useful for the treatment of colonic pain diseases such as irritable bowel syndrome, while H2S donors or Cav3.2 activators might be useful for the treatment of inflammatory bowel disease including Crohn's disease.

H2S-induced HCO3− secretion in the rat stomach – Involvement of nitric oxide, prostaglandins, and capsaicin-sensitive sensory neurons

Hydrogen sulfide (H2S) is known to be an important gaseous mediator that affects various functions under physiological and pathological conditions. We examined the effects of NaHS, a H2S donor, on HCO3− secretion in rat stomachs and investigated the mechanism involved in this response. Under urethane anesthesia, rat stomachs were mounted on an ex vivo chamber and perfused with saline. Acid secretion had been inhibited by omeprazole. The secretion of HCO3− was measured at pH 7.0 using a pH-stat method and by the addition of 10 mM HCl. NaHS (0.5–10 mM) was perfused in the stomach for 5 min. Indomethacin or L-NAME was administered s.c. before NaHS treatment, while glibenclamide (a KATP channel blocker), ONO-8711 (an EP1 antagonist), or propargylglycine (a cystathionine γ-lyase inhibitor) was given i.p. before. The mucosal perfusion of NaHS dose-dependently increased the secretion of HCO3−, and this effect was significantly attenuated by indomethacin, L-NAME, and sensory deafferentation, but not by glibenclamide or ONO-8711. The luminal output of nitric oxide, but not the mucosal production of prostaglandin E2, was increased by the perfusion of NaHS. Mucosal acidification stimulated HCO3− secretion, and this response was inhibited by sensory deafferentation, indomethacin, L-NAME, and ONO-8711, but not by propargylglycine. These results suggested that H2S increased HCO3− secretion in the stomach, and this effect was mediated by capsaicin-sensitive afferent neurons and dependent on nitric oxide and prostaglandins, but not ATP-sensitive K+ channels. Further study is needed to define the role of endogenous H2S in the mechanism underlying acid-induced gastric HCO3− secretion.

Differential regulatory role of pituitary adenylate cyclase-activating polypeptide in the serum-transfer arthritis model.

ObjectivePituitary adenylate cyclase–activating polypeptide (PACAP) expressed in capsaicin-sensitive sensory neurons and immune cells has divergent functions in inflammatory and pain processes. This study was undertaken to investigate the involvement of PACAP in a mouse model of rheumatoid arthritis.MethodsArthritis was induced in PACAP−/− and wild-type (PACAP+/+) mice by K/BxN serum transfer. General features of the disease were investigated by semiquantitative scoring, plethysmometry, and histopathologic analysis. Mechano- and thermonociceptive thresholds and motor functions were also evaluated. Metabolic activity was assessed by positron emission tomography. Bone morphology was measured by in vivo micro–computed tomography, myeloperoxidase activity and superoxide production by bioluminescence imaging with luminol and lucigenin, respectively, and vascular permeability by fluorescent indocyanine green dye study.ResultsPACAP+/+ mice developed notable joint swelling, reduced grasping ability, and mechanical (but not thermal) hyperalgesia after K/BxN serum transfer. In PACAP−/− mice clinical scores and edema were significantly reduced, and mechanical hyperalgesia and motor impairment were absent, throughout the 2-week period of observation. Metabolic activity and superoxide production increased in the tibiotarsal joints of wild-type mice but were significantly lower in PACAP−/− animals. Myeloperoxidase activity in the ankle joints of PACAP−/− mice was significantly reduced in the early phase of arthritis, but increased in the late phase. Synovial hyperplasia was also significantly increased, and progressive bone spur formation was observed in PACAP-deficient mice only.ConclusionIn PACAP-deficient mice with serum-transfer arthritis, joint swelling, vascular leakage, hyperalgesia, and early inflammatory cell accumulation are reduced; in the later phase of the disease, immune cell function and bone neoformation are increased. Elucidation of the underlying pathways of PACAP activity may open promising new avenues for development of therapy in inflammatory arthritis.

Role of capsaicin-sensitive peripheral sensory neurons in anorexic responses to intravenous infusions of cholecystokinin, peptide YY-(3-36), and glucagon-like peptide-1 in rats.

Cholecystokinin (CCK)-induced suppression of feeding is mediated by vagal sensory neurons that are destroyed by the neurotoxin capsaicin (CAP). Here we determined whether CAP-sensitive neurons mediate anorexic responses to intravenous infusions of gut hormones peptide YY-(3-36) [PYY-(3-36)] and glucagon-like peptide-1 (GLP-1). Rats received three intraperitoneal injections of CAP or vehicle (VEH) in 24 h. After recovery, non-food-deprived rats received at dark onset a 3-h intravenous infusion of CCK-8 (5, 17 pmol·kg⁻¹·min⁻¹), PYY-(3-36) (5, 17, 50 pmol·kg⁻¹·min⁻¹), or GLP-1 (17, 50 pmol·kg⁻¹·min⁻¹). CCK-8 was much less effective in reducing food intake in CAP vs. VEH rats. CCK-8 at 5 and 17 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 39 and 71% in VEH rats and 7 and 18% in CAP rats. In contrast, PYY-(3-36) and GLP-1 were similarly effective in reducing food intake in VEH and CAP rats. PYY-(3-36) at 5, 17, and 50 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 15, 33, and 70% in VEH rats and 13, 30, and 33% in CAP rats. GLP-1 at 17 and 50 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 48 and 60% in VEH rats and 30 and 52% in CAP rats. These results suggest that anorexic responses to PYY-(3-36) and GLP-1 are not primarily mediated by the CAP-sensitive peripheral sensory neurons (presumably vagal) that mediate CCK-8-induced anorexia.

S6-4 Stimulation of duodenal HCO3− secretion by H2S in rats: Regulatory mechanism and importance in mucosal defense

We examined the effect of H2S on duodenal HCO 3 - secretion in rats and investigated the mechanism involved in this response. Animals were fasted overnight and anesthetized with urethane. A duodenal loop was perfused with saline, and HCO 3 - secretion was measured at pH 7.0 using a pH stat-method. The loop was perfused at a rate of 0.2 mL/min with NaHS (H2S donor) for 5 min or 10 mM HCl for 10 min. Indomethacin or l -NAME was given s.c. 30 min or 3 h, respectively, before NaHS or acidification, while glibenclamide (KATP channel blocker) or propargylglycine (cystathionine-g-lyase inhibitor) was given i.p. 30 min before. Mucosal perfusion with NaHS dose-dependently increased the HCO 3 - secretion, and this effect was significantly attenuated by indomethacin and l -NAME as well as sensory deafferentation, but not by glibenclamide. Mucosal PGE2 and NO production were both increased by NaHS perfusion. Mucosal acidification stimulated HCO 3 - secretion concomitant with increase in PGE2 and NO production, and these responses were mitigated by propargylglycine. The duodenal damage induced by acid (100 mM HCl for 4 h) was aggravated by pretreatment with propargylglycine. These results suggest that H2S increases HCO 3 - secretion in the duodenum, and this action is partly mediated by PG and NO as well as by capsaicin-sensitive afferent neurons. It is assumed that endogenous H2S is involved in the regulatory mechanism of acid-induced HCO 3 - secretion and mucosal protection in the duodenum.

N-type calcium current, Cav2.2, is enhanced in small-diameter sensory neurons isolated from Nf1+/− mice

Major aspects of neuronal function are regulated by Ca2+ including neurotransmitter release, excitability, developmental plasticity, and gene expression. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/−) exhibited both greater excitability and evoked release of neuropeptides compared to wildtype mice. Furthermore, augmented voltage-dependent sodium currents but not potassium currents contribute to the enhanced excitability. To determine the mechanisms giving rise to the enhanced release of substance P and calcitonin gene-related peptide in the Nf1+/− sensory neurons, the potential differences in the total voltage-dependent calcium current (ICa) as well as the contributions of individual Ca2+ channel subtypes were assessed. Whole-cell patch-clamp recordings from small-diameter capsaicin-sensitive sensory neurons demonstrated that the average peak ICa densities were not different between the two genotypes. However, by using selective blockers of channel subtypes, the current density of N-type (Cav2.2) ICa was significantly larger in Nf1+/− neurons compared to wildtype neurons. In contrast, there were no significant differences in L-, P/Q- and R-type currents between the two genotypes. Quantitative real-time polymerase chain reaction measurements made from the isolated but intact dorsal root ganglia indicated that N-type (Cav2.2) and P/Q-type (Cav2.1) Ca2+ channels exhibited the highest mRNA expression levels although there were no significant differences in the levels of mRNA expression between the genotypes. These results suggest that the augmented N-type (Cav2.2) ICa observed in the Nf1+/− sensory neurons does not result from genomic differences but may reflect post-translational or some other non-genomic modifications. Thus, our results demonstrate that sensory neurons from Nf1+/− mice, exhibit increased N-type ICa and likely account for the increased release of substance P and calcitonin gene-related peptide that occurs in Nf1+/− sensory neurons.

OP24 Characterization of TRPA1-mediated and TRPA1-independent microcirculatory responses to hydrogen sulfide in mice

Capsaicin-sensitive sensory neurons express several ion channels of the TRP family, such as transient receptor potential ankyrin 1 (TRPA1) receptors. TRPA1 can be activated by several chemical stimuli, such as allylisothiocyanate (AITC) or cinnamaldehyde resulting in the release of inflammatory neuropeptides, e.g. calcitonin gene-related peptide (CGRP) [1] . Recently, the gaseous mediator hydrogen sulfide (H2S) was suggested to have effect on capsaicin-sensitive sensory neurons acting via TRPA1 receptors. Microcirculatory changes in the mouse ear and hind paw have been investigated in this study. Balb/c, C57BL/6, TRPA1 KO and TRPV1 KO mice (20–30 g) were treated with sodium hydrogen sulfide (NaHS) or AITC. Ipsilateral ears of the animals were treated with 2% AITC or 5% NaHS. Contralateral ears received respective vehicles. Balb/c mice received selective TRPA1 receptor antagonist HC-030031 (i.p., 30 or 100 mg/kg). NaHS (0.5 μmol/10 μL Tyrode) was injected s.c. into hind paws of C57BL/6 and TRPA1 KO mice. Skin blood flow was detected by laser Doppler imaging. AITC treatment of mouse ears led to 29.8 ± 2.8% increase in blood flow. NaHS elevated cutaneous blood flow of the mouse ears by 61 ± 4.5%. Effect of NaHS on microcirculation was ameliorated by HC-030031. Blood flow of TRPA1 KO mice showed significantly smaller increase in response to NaHS compared to the wild-type counterparts. Vasodilatation in TRPV1 KO animals did not differ from control. CGRP8−37 peptide antagonist of CGRP only partly inhibited the response. Intraplantar administration of NaHS elevated blood flow of murine hind paws by 34.1 ± 2.8%. The plantar microcirculatory response to NaHS was unaffected by genetic lack of TRPA1 receptor or resiniferatoxin pretreatment. We conclude that NaHS-induced vasodilatation in the mouse ear is partly mediated by TRPA1 receptors, but is independent of sensory nerves in the skin of the hind paw.

Hyperexcitability of Bladder Afferent Neurons Associated with Reduction of Kv1.4 α-Subunit in Rats with Spinal Cord Injury

To clarify the functional and molecular mechanisms inducing hyperexcitability of C-fiber bladder afferent pathways after spinal cord injury we examined changes in the electrophysiological properties of bladder afferent neurons, focusing especially on voltage-gated K channels. Freshly dissociated L6-S1 dorsal root ganglion neurons were prepared from female spinal intact and spinal transected (T9-T10 transection) Sprague Dawley® rats. Whole cell patch clamp recordings were performed on individual bladder afferent neurons. Kv1.2 and Kv1.4 α-subunit expression levels were also evaluated by immunohistochemical and real-time polymerase chain reaction methods. Capsaicin sensitive bladder afferent neurons from spinal transected rats showed increased cell excitability, as evidenced by lower spike activation thresholds and a tonic firing pattern. The peak density of transient A-type K+ currents in capsaicin sensitive bladder afferent neurons from spinal transected rats was significantly less than that from spinal intact rats. Also, the KA current inactivation curve was displaced to more hyperpolarized levels after spinal transection. The protein and mRNA expression of Kv1.4 α-subunits, which can form transient A-type K+ channels, was decreased in bladder afferent neurons after spinal transection. Results indicate that the excitability of capsaicin sensitive C-fiber bladder afferent neurons is increased in association with reductions in transient A-type K+ current density and Kv1.4 α-subunit expression in injured rats. Thus, the Kv1.4 α-subunit could be a molecular target for treating overactive bladder due to neurogenic detrusor overactivity.

Capsaicin-sensitive vagal afferent neurons contribute to the detection of pathogenic bacterial colonization in the gut

Vagal activation can reduce inflammation and disease activity in various animal models of intestinal inflammation via the cholinergic anti-inflammatory pathway. In the current model of this pathway, activation of descending vagal efferents is dependent on a signal initiated by stimulation of vagal afferents. However, little is known about how vagal afferents are activated, especially in the context of subclinical or clinical pathogenic bacterial infection. To address this question, we first determined if selective lesions of capsaicin-sensitive vagal afferents altered c-Fos expression in the nucleus of the solitary tract (nTS) after mice were inoculated with either Campylobacter jejuni or Salmonella typhimurium. Our results demonstrate that the activation of nTS neurons by intraluminal pathogenic bacteria is dependent on intact, capsaicin sensitive vagal afferents. We next determined if inflammatory mediators could cause the observed increase in c-Fos expression in the nTS by a direct action on vagal afferents. This was tested by the use of single-cell calcium measurements in cultured vagal afferent neurons. We found that tumor necrosis factor alpha (TNFα) and lipopolysaccharide (LPS) directly activate cultured vagal afferent neurons and that almost all TNFα and LPS responsive neurons were sensitive to capsaicin. We conclude that activation of the afferent arm of the parasympathetic neuroimmune reflex by pathogenic bacteria in the gut is dependent on capsaicin sensitive vagal afferent neurons and that the release of inflammatory mediators into intestinal tissue can be directly sensed by these neurons.

Characteristics of capsaicin-sensitive afferent neurons of spinal nerve ganglia

Morphological peculiarities of neurons that had TRPV1, SP, CGRP, and NF200 markers in thoracic spinal ganglia nerves were studied in 3-month-old rats subjected to chemical deafferentation produced by capsaicin. The obtained results have shown that from 6.5 to 41.3% of ganglion neurons in the control group of animals contain, as a rule, one of the above-mentioned markers. The heterogeneity of nociceptive neurons observed in a control group of rats was also preserved in the capsaicin-treated animals. In both groups, the spinal ganglion TRPV1 neurons were predominant, whereas populations of SP, CGRP, and NF200 neurons formed smaller groups. In each population, sensitivity to capsaicin was shown in the largest neurons, regardless of marker; this sensitivity was pronounced to the greatest degree in the group of TRPV1 neurons.

Role of transient receptor potential vanilloid 1 in inflammation and autoimmune diseases.

Transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel, is a receptor activated by high temperatures and chemical agonists such as the vanilloids and protons. Because of these properties, TRPV1 has emerged as a polymodal nocisensor of nociceptive afferent neurons. TRPV1 is thought to be a central transducer of hyperalgesia and a prime target for controlling pain pharmacologically because it is a point where many proalgesic pathways converge and it is upregulated and sensitized by inflammation and injury. However, whether TRPV1 agonists promote or inhibit inflammation remains unclear. We recently demonstrated that SA13353 (1-[2-(1-adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea), a novel TRPV1 agonist, inhibits tumor necrosis factor-a production by the activation of capsaicin-sensitive afferent neurons and reduces the severity of symptoms in kidney injury, lung inflammation, arthritis, and encephalomyelitis. These results suggest that TRPV1 agonists may act as anti-inflammatories in certain inflammatory and autoimmune conditions in vivo. Given the potential deleterious effects of inhibiting the population of channels with a protective function, caution should be taken in the use of potent TRPV1 antagonists as a general strategy to treat inflammation. Further studies are required to clarify the role of TRPV1 and neuropeptides, which are released because of TRPV1 activation in inflammation and autoimmune diseases.

Exacerbation of Nonsteroidal Anti-Inflammatory Drug-Induced Small Intestinal Lesions by Antisecretory Drugs in Rats: The Role of Intestinal Motility

Antisecretory drugs such as histamine H2-receptor antagonists (H2-RAs) and proton pump inhibitors (PPIs) are commonly used for the treatment of gastric and duodenal ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs). However, the effects of these drugs on NSAID-induced small intestinal ulcers are not fully understood. The effects of H2-RAs and PPIs on NSAID-induced gastrointestinal lesions and small intestinal motility were examined in rats. Male Wistar rats (180-220 g) were used. Indomethacin (10 mg/kg) was administered orally in fasted or fed rats, and gastrointestinal lesions were examined 24 h after indomethacin administration. Intestinal motility was measured by using a balloon method under urethane anesthesia. Indomethacin produced multiple lesions in the gastric corpus in fasted rats and in the small intestine in fed rats: 1) H2-RAs (cimetidine, ranitidine, and famotidine) and PPIs (omeprazole, lansoprazole, and rabeprazole) markedly inhibited the formation of gastric lesions. 2) The drugs, except for lansoprazole, increased intestinal lesions. 3) H2-RAs augmented the increase in intestinal motility caused by indomethacin, and the effects of H2-RAs on motility and intestinal lesions were markedly inhibited by atropine. 4) Lansoprazole inhibited the formation of intestinal lesions, and the effect was prevented by both pharmacological ablation of capsaicin-sensitive sensory neurons and pretreatment with N-nitro-l-arginine methyl ester, a selective inhibitor of nitric-oxide synthesis. The results suggest that: 1) inhibition of acid secretion by antisecretory drugs may exacerbate NSAID-induced intestinal lesions, 2) H2-RAs further aggravate lesions by increasing intestinal motility via the activation of cholinergic pathways, and 3) lansoprazole protects the intestinal mucosa against NSAID-related ulcerative stimuli.

The role of transient receptor potential ankyrin 1 (TRPA1) receptor activation in hydrogen-sulphide-induced CGRP-release and vasodilation

Activation of transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) channels on capsaicin-sensitive sensory neurons causes release of inflammatory neuropeptides, including calcitonin gene-related peptide (CGRP). We investigated whether the hydrogen sulphide (H2S)-evoked CGRP release from sensory neurons of isolated rat tracheae and H2S-induced increases in the microcirculation of the mouse ear were mediated by TRPA1 receptor activation.Allylisothiocyanate (AITC) or the H2S donor sodium hydrogen sulphide (NaHS) were used as stimuli and CGRP release of the rat tracheae was measured by radioimmunoassay. AITC or NaHS were applied to the ears of Balb/c, C57BL/6, TRPA1 and TRPV1 receptor gene knockout mice and blood flow was detected by laser Doppler imaging.Both AITC and NaHS increased CGRP release from isolated rat tracheae, and both responses were inhibited by the TRPA1 antagonist, HC-030031, but was not affected by the TRPV1 receptor blocker, BCTC. Application of AITC or NaHS increased the cutaneous blood flow in the mouse ears. Similarly to the effect of AITC, the vasodilatory response to NaHS was reduced by HC-030031 or in TRPA1 deleted mice. In contrast, genetic deletion of TRPV1 did not affect the increase in the ear blood flow evoked by AITC or NaHS.We conclude that H2S activates TRPA1 receptors causing CGRP release from sensory nerves of rat tracheae, as well as inducing cutaneous vasodilatation in the mouse ear. TRPV1 receptors were not involved in these processes. Our results highlight that TRPA1 receptor activation should be considered as a potential mechanism of vasoactive effects of H2S.

Comparison of Lafutidine and Rabeprazole in 7‐day Second‐line Amoxicillin‐ and Metronidazole‐Containing Triple Therapy for Helicobacter pylori: A Pilot Study

Lafutidine is an H2-receptor antagonist with gastroprotective action through capsaicin-sensitive afferent neurons and relatively inexpensive compare to proton-pump inhibitors (PPIs). A 7-day course of PPIs-amoxicillin-metronidazole is recommended as standard second-line Helicobacter pylori therapy and is covered by national health insurance in Japan. The aim of this study was to determine the efficacy and safety of second-line eradication using the H2-receptor antagonist lafutidine as a substitute for a PPI. Fifty-two patients who failed in first-line eradication using PPI-amoxicillin-clarithromycin were randomly assigned to a 7-day course of rabeprazole at 10 mg b.i.d., amoxicillin at 750 mg b.i.d., and metronidazole at 250 mg b.i.d. (RPZ-AM) or a 7-day course of lafutidine at 10 mg t.i.d., amoxicillin at 750 mg b.i.d., and metronidazole at 250 mg b.i.d. (LFT-AM) as second-line therapy. Eradication was assessed by the (13) C urea breath test. A drug susceptibility test was performed before the second-line therapy. Prior to second-line H. pylori eradication, the rate of resistance to clarithromycin was 86.5% and the rate of resistance to metronidazole was 3.8%. The eradication rates for both LFT-AM and RPZ-AM groups were 96% (95%CI = 88.6-100%). There were no severe adverse events in either group. Lafutidine plus metronidazole-amoxicillin as second-line therapy provided a high eradication rate and safe treatment similar to a PPI-based regimen. Lafutidine-based eradication therapy is therefore considered to be a promising alternative and is also expected to reduce health care costs in H. pylori eradication.

Oxytocin Inhibits the Membrane Depolarization-Induced Increase in Intracellular Calcium in Capsaicin Sensitive Sensory Neurons

Lumbar intrathecal injection of oxytocin produces antinociception in rats and analgesia in humans. Classically, oxytocin receptors couple to stimulatory G proteins, increase inositol-3-phosphate production, and result in neuronal excitation. Most work to date has focused on a spinal site of oxytocin to excite γ-aminobutyric acid interneurons to produce analgesia. Here we ask whether oxytocin might also affect primary sensory afferents by modulating high voltage-gated calcium channels, such as it does in the brain. Dorsal root ganglion cells from adult rats were acutely dissociated and cultured, and changes in intracellular calcium determined by fluorescent microscopy using an indicator dye. The effects of oxytocin alone and in the presence of transient depolarization from increased extracellular KCl concentration were determined, and the pharmacology of these effects were studied. Cells from injured dorsal root ganglion cells after spinal nerve ligation were also studied. Oxytocin produced a concentration-dependent inhibition of the increase in intracellular calcium from membrane depolarization, an effect blocked more efficiently by oxytocin- than vasopressin-receptor selective antagonists. Oxytocin-induced inhibition was present in cells responding to capsaicin, and when internal stores of calcium were depleted with thapsigargin. Oxytocin produced similar inhibition in cells from animals with spinal nerve ligation. These data suggest that oxytocin produces antinociception after intrathecal delivery in part by reducing excitatory neurotransmitter release from the central terminals of nociceptors.

Use and limitations of three TRPV-1 receptor antagonists on smooth muscles of animals and man: A vote for BCTC

Specificity of the effect is a crucial factor in using antagonists for detecting the physiological/pathophysiological roles of receptors. Here we examined the capsaicin receptor antagonist effects of three commercially-available substances, capsazepine, iodo-resiniferatoxin (I-RTX) and BCTC, on isolated smooth muscle preparations, including the human intestine. Care was taken to observe possible non-specific effects, to find out safe and effective concentrations. Capsazepine appeared to have a low margin of safety. I-RTX (up to 1μM) specifically inhibited capsaicin-induced contractions in the guinea-pig ileum and urinary bladder. I-RTX showed agonist activity on the rat urinary bladder. BCTC (1μM) abolished the contractile effects of capsaicin (1 or 2μM) on all preparations tested (guinea-pig ileum, bladder, trachea, as well as rat and mouse bladder), and on the guinea-pig renal pelvis, where it failed to influence capsaicin-sensitive, sensory neuron-mediated positive inotropy in response to field stimulation. On human intestinal preparations BCTC prevented the relaxant effect of capsaicin. It is concluded that of the three antagonists tested BCTC seems the safest one for inhibiting TRPV-1 receptors. The effect of capsazepine may be complicated by non-specific inhibition of smooth muscle contractility and that of I-RTX by agonist activity. The “local efferent” function of capsaicin-sensitive sensory neurons is not influenced by BCTC, as shown by the results obtained in the renal pelvis. In conclusion, of the TRPV-1 receptor antagonists studied, BCTC (1μM) seems the most reliable in isolated organ experiments. This substance is also effective in the human intestine.

Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKMζ

Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK1/2 (PD-98059), PLC (U-73122, neomycin), or conventional/novel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKMζ, PKCζ, and PKCλ/ι) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKCζ. Both Western blots and quantitative real-time PCR established that PKMζ, but neither full-length PKCζ nor PKCλ/ι, was significantly reduced after siRNA exposure. Treatment with these labeled siRNA prevented the NGF-induced enhancement of excitability. Furthermore, consistent with the high degree of catalytic homology for aPKCs, internal perfusion with active recombinant PKCζ or PKCι augmented excitability, recapitulating the sensitization produced by NGF. Internal perfusion with recombinant PKCζ suppressed the total potassium current and enhanced the tetrodotoxin-resistant sodium current. Pretreatment with the myristoylated pseudosubstrate inhibitor blocked the increased excitability produced by ceramide or internal perfusion with recombinant PKCζ. These results demonstrate that NGF leads to the activation of PKMζ that ultimately enhances the capacity of small-diameter capsaicin-sensitive sensory neurons to fire APs through a PI3K-dependent signaling cascade.

Glucocorticoids are gastroprotective under physiologic conditions

Stress may contribute to the development and progression of gastrointestinal disorders. Activation of the hypothalamic-pituitary-adrenocortical (HPA) axis is one of the main characteristics of stress. For several decades it was generally accepted that glucocorticoids released during stress are ulcerogenic hormones. We designed some experimental studies in rats to clarify the validity of this widely held view. To achieve this goal, we examined the effect of glucocorticoid deficiency followed by corticosterone replacement or the glucocorticoid receptor antagonist, RU-38486, on stress-induced gastric erosion and the parameters of gastric function in rats. The data obtained shows that the reduction in the stress-induced corticosterone release, or its actions, aggravates stress-caused gastric erosion. It is suggested that an acute increase in corticosterone during stress protects the stomach against stress-induced injury. According to our results, various ulcerogenic stimuli, similar to stress, induce an increase in corticosterone that helps the gastric mucosa to resist against a harmful action of ulcerogenic stimuli. Glucocorticoids exhibit their gastroprotective effect by both maintaining local defensive factors and inhibiting pathogenic elements. Furthermore, the contribution of glucocorticoids to gastroprotection is tightly related to their contribution to general body homeostasis. Glucocorticoids provide gastroprotective actions in co-operation with prosta-glandins, nitric oxide and capsaicin-sensitive sensory neurons. The results obtained do not support the traditional paradigm and suggest that glucocorticoids released during acute activation of the HPA axis are naturally occurring gastroprotective factors. In this article, we review our recent publications on the role of glucocorticoids in gastroprotection.