Vicente Martinez

Effects of pregabalin on visceral pain responses and colonic compliance in rats

Pregabalin, which binds to the α2‐δ subunit of voltage‐gated calcium channels, increased the threshold for pain during colorectal distension (CRD) in irritable bowel syndrome (IBS) patients. We tested the effects of oral pregabalin on the visceral pain‐related viscerosomatic and autonomic cardiovascular responses to CRD and colonic compliance in rats.

Involvement of metabotropic glutamate 5 receptor in visceral pain

&NA; Metabotropic glutamate 5 receptor (mGluR5) antagonists are effective in animal models of inflammatory and neuropathic pain. The involvement of mGluR5 in visceral pain pathways from the gastrointestinal tract is as yet unknown. We evaluated effects of mGluR5 antagonists on the colorectal distension (CRD)‐evoked visceromotor (VMR) and cardiovascular responses in conscious rats, and on mechanosensory responses of mouse colorectal afferents in vitro. Sprague–Dawley rats were subjected to repeated, isobaric CRD (12 × 80 mmHg, for 30 s with 5 min intervals). The VMR and cardiovascular responses to CRD were monitored. The mGluR5 antagonists MPEP (1–10 μmol/kg, i.v.) and MTEP (1–3 μmol/kg, i.v.) reduced the VMR to CRD dose‐dependently with maximal inhibition of 52 ± 8% (p < 0.01) and 25 ± 11% (p < 0.05), respectively, without affecting colonic compliance. MPEP (10 μmol/kg, i.v.) reduced CRD‐evoked increases in blood pressure and heart rate by 33 ± 9% (p < 0.01) and 35 ± 8% (p < 0.05), respectively. Single afferent recordings were made from mouse pelvic and splanchnic nerves of colorectal mechanoreceptors. Circumferential stretch (0–5 g force) elicited slowly‐adapting excitation of action potentials in pelvic distension‐sensitive afferents. This response was reduced 55–78% by 10 μM MTEP (p < 0.05). Colonic probing (2 g von Frey hair) activated serosal splanchnic afferents; their responses were reduced 50% by 10 μM MTEP (p < 0.01). We conclude that mGluR5 antagonists inhibit CRD‐evoked VMR and cardiovascular changes in conscious rats, through an effect, at least in part, at peripheral afferent endings. Thus, mGluR5 participates in mediating mechanically evoked visceral nociception in the gastrointestinal tract.

The GABAB receptor agonist, baclofen, and the positive allosteric modulator, CGP7930, inhibit visceral pain-related responses to colorectal distension in rats

Activation of GABA(B) receptors by the selective agonist baclofen produces anti-nociceptive effects in animal models of somatic pain. The aim of the present study was to evaluate the effect of baclofen and the GABA(B) receptor positive allosteric modulator CGP7930 on pseudo-affective responses to colorectal distension in rats. Female Sprague-Dawley rats were subjected to repeated, noxious colorectal distension (CRD) (12 distensions at 80 mmHg, for 30 s with 5 min intervals). The visceromotor response (VMR) and cardiovascular responses (mean arterial blood pressure (ABP) and heart rate (HR)) to CRD were monitored in conscious, telemetrized animals. Baclofen (0.3-3 micromol/kg, i.v.) reduced the VMR to CRD dose-dependently, reaching a 61% maximal inhibition (p < 0.001). The highest doses of baclofen attenuated CRD-evoked increases in ABP by 17% (p > 0.05) and reduced the change in HR by 48% (p < 0.01). CGP7930 (3-30 micromol/kg, i.v.) reduced the VMR to CRD in a dose-dependent fashion with a maximal inhibition of 31% (p < 0.05). The highest dose of CGP7930 also attenuated the increase in ABP by 18% (p > 0.05) and inhibited the increase in HR by 24% (p < 0.05) associated with CRD. Neither baclofen nor CGP7930 affected colorectal compliance. The results suggest that activation of GABA(B) receptors produces anti-nociceptive effects in a rat model of mechanically induced visceral pain. While CGP7930 was less efficacious than baclofen overall, positive allosteric modulation of GABA(B) receptors may represent a valid approach in the treatment of visceral pain conditions, with the possibility of an improved safety profile compared to full agonism.

Involvement of the transient receptor potential vanilloid 1 (TRPV1) in the development of acute visceral hyperalgesia during colorectal distension in rats.

Transient receptor potential vanilloid 1 (TRPV1) channels have been implicated in pain mechanisms and, particularly, in the development of hyperalgesia. We used selective TRPV1 antagonists (NGV-1, SB-750364 and JYL 1421) to assess the role of TRPV1 channels in repetitive noxious colorectal distension (CRD)-induced visceral pain responses in rats. Isobaric CRD (80 mmHg) induced a viscerosomatic response, indicative of visceral pain associated to the distension procedure. Repetition (12 consecutive distensions) of the CRD resulted in an increase in the response over time (119+/-23% increase at distension 12, P<0.05 vs response during the 1st distension) indicative of acute mechanical sensitization. NGV-1 (0.1, 0.3, 1 or 3 micromol/kg, i.v.) prevented in a dose-related manner the development of sensitization, without inducing hypoalgesic responses. SB-750364 (30 micromol/kg, i.v.) had a transitory effect, partially reducing the sensitization response, while JYL 1421 (4.7 micromol/kg, i.v.) was without effect. In the same conditions, the cannabinoid receptor 1 (CB(1)) agonist, WIN55,212-2 (0.1 micromol/kg) reduced pain responses leading to a hypoalgesic state. At 3 micromol/kg, NGV-1, did not affect the pressure-volume relationship during CRD, indicating that TRPV1 channels do not modulate colonic compliance. These observations suggest that TRPV1 channels are involved in the development of acute mechanical colonic hyperalgesia during repetitive noxious CRD in rats. Antagonism of TRPV1 channels might result in antihyperalgesic effects without hypoalgesic activity and might be beneficial in the treatment of visceral pain disorders, such as irritable bowel syndrome. These observations warrant the clinical assessment of TRPV1 antagonists for the treatment of visceral pain.

Characterisation of colonic accommodation in Wistar Kyoto rats with impaired gastric accommodation

Defective colonic and gastric accommodations have been related to altered viscerosensitivity in irritable bowel syndrome and to functional dyspepsia, respectively. We assessed colonic accommodation in rats with impaired gastric accommodation to determine if altered accommodation can be regarded as a widespread pathophysiological alteration within the gastrointestinal (GI) tract. Colonic accommodation during colorectal distension (CRD) was assessed in Wistar Kyoto rats (WKY), an animal model of impaired gastric accommodation, and in Sprague–Dawley (SD) and Wistar rats, considered normal. CRD (10–80xa0mmHg)-induced visceral pain responses were also evaluated in the same strains of rats. During gastric distension, WKY rats had lower intra-gastric volume (0.96u2009±u20090.22xa0ml) than SD (1.85u2009±u20090.19xa0ml, Pu2009<u20090.05) or Wistar rats (2.80u2009±u20090.26xa0ml, Pu2009<u20090.05), indicating impaired gastric accommodation. In the same animals, pressure–volume curves were constructed during CRD as a measure of colonic accommodation. During short-lasting (1xa0min) phasic CRD (2–20xa0mmHg), the pressure–volume curve in WKY rats was displaced to the right compared with SD or Wistar rats, indicative of reduced colonic accommodation (maximal volume: SD, 1.22u2009±u20090.05xa0ml; Wistar, 1.07u2009±u20090.04xa0ml; WKY, 0.87u2009±u20090.07xa0ml; Pu2009<u20090.01). Pre-treatment with atropine normalised the pressure–volume responses in WKY rats. No differences among strains were observed during the 2-min phasic or ramp-tonic CRD. Visceral pain responses during CRD (10–80xa0mmHg) were, overall, similar in the three strains, although WKY rats showed lower thresholds for pain (28.0u2009±u20094.9xa0mmHg) than SD (42.3u2009±u20096.6xa0mmHg, Pu2009=u20090.072) or Wistar rats (48.3u2009±u20096.0xa0mmHg, Pu2009<u20090.05). WKY rats, although having impaired gastric accommodation, have the ability to fully accommodate the colon to increasing pressures. In WKY rats, impaired accommodation of the smooth muscle might not be a widespread phenomenon along the GI tract but rather a local disturbance.

Role of somatostatin receptors on gastric acid secretion in wild-type and somatostatin receptor type 2 knockout mice

Somatostatin, probably acting through somatostatin type 2 receptors (SSTR2), is the main inhibitor of gastric acid secretion. We characterized gastric acid secretion in SSTR2 knockout mice, and used preferential somatostatin receptor agonists to assess the relative role of SSTR1, 2, 3, 4, and 5 on gastric acid secretion. Basal gastric acid secretion and the secretory response to a meal were similar in conscious wild-type and knockout mice. However, under urethane anesthesia, which releases endogenous somatostatin, SSTR2 knockout mice had a basal secretion 11–15-fold higher than wild-type animals (μmol/10 min:1.40±0.09 vs. 0.10±0.01, p<0.05). Gastrin immunoneutralization or H2 receptors blockade (cimetidine), but not cholinergic blockade (atropine), reduced the high basal secretion in SSTR2 knockout mice. In SSTR2 knockout mice, gastrin and histamine stimulated acid secretion with similar efficacy, while in wild-type mice histamine was more effective than gastrin. SSTR2 knockout mice showed also a hypersecretory response to pylorus ligation compared with wild-type animals. In wild-type mice, somatostatin-14, SMS 201-995, and the SSTR2-preferential agonist, DC 32–87, inhibited gastrin-stimulated acid secretion with an order of potency SMS 201–995>DC 32–87>somatostatin-14. Preferential agonists for the SSTR1, 3, 4, and 5 were devoid of any effect. None of the compounds tested affected the high basal secretion observed under urethane anesthesia in SSTR2 knockout mice. These results show that gastric antisecretory effects of peripheral somatostatin are mediated solely through SSTR2. In the absence of functional SSTR2 other somatostatin receptors do not compensate for the lack somatostatin-SSTR2-mediated inhibition. Basal acid secretion and the response to a meal are normal in conscious SSTR2 knockout mice, suggesting the presence of somatostatin-independent mechanisms that compensate for the lack of somatostatin-SSTR2-mediated inhibitory responses.

Role of TRPV1 in high-threshold rat colonic splanchnic afferents is revealed by inflammation

The vanilloid-1 receptor TRPV1 is known to play a role in extrinsic gastrointestinal afferent function. We investigated the role of TRPV1 in mechanosensitivity in afferents from normal and inflamed tissue. Colonic mechanosensitivity was determined in an in vitro rat colon preparation by recording from attached splanchnic nerves. Recordings were made from serosal/mesenteric afferents responding only at high thresholds to graded mechanical stimulation with von Frey probes. Colonic inflammation was induced by adding 5% dextran sulphate sodium (DSS) to the drinking water for 5 days, and was confirmed by histopathology. The selective TRPV1 antagonist, SB-750364 (10(-8) to 10(-6)M), was tested on mechanosensory stimulus response functions of afferents from normal and inflamed preparations (N=7 each). Mechanosensory responses had thresholds of 1-2g, and maximal responses were observed at 12 g. The stimulus response function was not affected by DSS-induced colitis. SB-750364 had no effect on stimulus response functions in normal preparations, but reduced (up to 60%) in a concentration-dependent manner those in inflammation (2-way ANOVA, p<0.05). Moreover, in inflamed tissue, spontaneous afferent activity showed a dose-dependent trend toward reduction with SB-750364. We conclude that mechanosensitivity of high-threshold serosal colonic splanchnic afferents to graded stimuli is unaffected during DSS colitis. However, there is a positive influence of TRPV1 in mechanosensitivity in inflammation, suggesting up-regulation of excitatory TRPV1-mediated mechanisms.

Oral clonidine inhibits visceral pain-related viscerosomatic and cardiovascular responses to colorectal distension in rats.

The alpha(2)-adrenoceptor agonist, clonidine, modulates colorectal sensorimotor functions in humans and, given intrathecally, has analgesic effects in the colorectal distension (CRD) model in rats. We tested the effects of systemic clonidine on the visceral pain-related viscerosomatic and autonomic cardiovascular responses to CRD and colonic compliance in rats using clinically relevant CRD protocols. The activity of the abdominal musculature (viscerosomatic response), monitored by electromyography and intracolonic manometry, and changes in arterial blood pressure and heart rate, monitored by telemetry, were assessed simultaneously in conscious rats during CRD. Pressure-volume relationships during CRD served as a measure of colonic compliance. Clonidine (50-200 nmol/kg, p.o.) dose-dependently inhibited the viscerosomatic response to phasic, noxious CRD (12 distension at 80 mm Hg). At 200 nmol/kg clonidine also attenuated the increase in blood pressure (70+/-7% inhibition, P<0.05) and heart rate (67+/-16% inhibition, P<0.05) associated to noxious CRD. Similar effects were observed after i.v. administration. Likewise, clonidine (200 nmol/kg, p.o.) reduced the response to ascending phasic CRD (10-80 mm Hg) and significantly increased the threshold pressure for pain-related responses. Clonidine (50 or 150 nmol/kg, i.p.) did not affect the pressure-volume relationship during phasic CRD (2-20 mm Hg). These results show that systemic clonidine, at doses devoid of visible side effects, has analgesic effects in the CRD model of visceral pain in rats without affecting colonic compliance. These observations confirm the analgesic activity of systemic clonidine on visceral pain, support the translational value of the rat CRD model to humans and show that manometry is more sensitive than electromyography detecting pain-related responses.

Acute colonic ischaemia in rats results in long-term structural changes without alterations of colonic sensitivity

Colonic ischaemia and mast cells have been involved in the pathophysiology of the functional gastrointestinal disorder irritable bowel syndrome, although the cause–effect relationships remain unknown. We assessed long‐term histopathological and functional changes associated to an acute ischaemic episode (1u2003h) of the colon, followed by 8‐week recovery, in rats. Functional colonic alterations [sensitivity during colorectal distension (CRD), compliance and propulsive motility] were assessed regularly during the recovery. Colonic histopathology (presence of inflammation, morphometric alterations and variations in neuronal density in the enteric nervous system) 8‐week postischaemia was assessed. Following ischaemia, none of the functional parameters tested (motility, sensitivity and compliance) were affected. At necropsy, the colon presented an overall normal appearance with an increase in weight of the ischaemic area (mg/cm: 99u2003±u20036; Pu2003<u20030.05 vs. control: 81u2003±u20034 or sham ischaemia: 81u2003±u20033). Histopathological evaluations revealed the presence of a local infiltrate of mast cells in the area of ischaemia (nb of mast cells: 142u2003±u200350; Pu2003<u20030.05 vs. control, 31u2003±u200314 or sham ischaemia: 40u2003±u200316), without other significant alterations. Animals subjected to colonic ischaemia and treated 8u2003weeks later with the mast cell degranulator, compound 48/80, showed no changes in CRD‐related pain responses. These studies show that acute colonic ischaemia is associated with the presence of a long‐term local infiltration of mast cells, located within the serosa and muscle layers, despite the absence of functional changes, including colonic sensitivity. Considering the important pathophysiological functions of mast cells, the observed mast cell infiltration may be involved in ischaemia‐induced functional changes yet to be characterized.

Role of tachykinin NK1 and NK2 receptors in colonic sensitivity and stress-induced defecation in gerbils

The pharmacology of tachykinin NK receptors varies greatly among species. The aim of the present study was to assess the role of NK(1) and NK(2) receptors in mediating colorectal distension-evoked nociception and psychological stress-induced defecation in gerbils, a species with human-like NK receptor pharmacology. The effects of the selective NK(1) and NK(2) receptor antagonists, aprepitant and saredutant, on acute (1 h) restraint stress-evoked defecation and plasma adenocorticotropin (ACTH) levels in gerbils were assessed. The effects of antagonists alone or in combination on colorectal distension-evoked visceral pain in conscious gerbils were evaluated using the visceromotor response as a surrogate marker of pain. Restraint stress increased fecal pellet output 2-3-fold and plasma ACTH levels 9-fold. Aprepitant inhibited the defecatory and endocrine responses to stress by 50%, while saredutant completely normalized the same parameters. Visceral pain responses during colorectal distension were attenuated by both compounds, but aprepitant (19+/-6% inhibition, P<0.01) was slightly more effective than saredutant (10+/-9% inhibition, P<0.05). A combination of both compounds resulted in an additive effect (30+/-10% inhibition, P<0.01). The results demonstrate that NK(1) and NK(2) receptors are involved in stress-related colonic motor alterations and visceral pain responses in gerbils and that combined antagonism provides enhanced inhibition of visceral pain responses. This suggests that for therapeutic use in for instance functional gastrointestinal disorders, dual NK(1)/NK(2) receptor antagonists may provide better clinical outcome than selective compounds.