Sensitive Afferents Research Articles

A Peripheral Adrenoceptor-mediated Sympathetic Mechanism Can Transform Stress-induced Analgesia into Hyperalgesia

Stress has paradoxical effects on pain, causing stress-induced analgesia but also exacerbating pain via poorly understood mechanisms. Adrenergic neurotransmission is integral in pathways that regulate the response to both pain and stress. Hyperalgesia is often associated with enhanced adrenergic sensitivity of primary afferents, but sympathetic nervous system outflow has not been demonstrated to exacerbate pain perception after stress. Rats or C57/BL6 wild-type mice treated with α-2 receptor antagonists or α-2A receptor knockout mice were exposed to ultrasonic noise stress or footshock stress and subsequently tested for hotplate paw withdrawal latencies. The sensory sensitivity of α-2A knockout mice to electrical and chemical stimuli was tested neurophysiologically and behaviorally. The effects of sympatholytic treatments were investigated. Noise and footshock stressors induced thermal hyperalgesia in rats pretreated systemically with α-2 antagonists. Wild-type mice pretreated with α-2 antagonists and α-2A knockout mice also exhibited thermal hyperalgesia induced by noise stress. Local spinal or intraplantar injection of an α-2 antagonist counteracted stress-induced analgesia without causing hyperalgesia. The α-2A knockout mice had decreased thresholds for peripheral sensitization with sulprostone and for windup of the dorsal horn neuronal response to repetitive electrical stimuli. Stress-induced hyperalgesia was abolished and the sensitization was attenuated by sympathectomy or systemic administration of an α-1-adrenergic antagonist. Sympathetic postganglionic nerves can enhance pain sensation via a peripheral α-1-adrenoceptor mechanism when sympathetic outflow is disinhibited. The net effect of stress on pain sensation reflects a balance between descending spinal inhibition and sympathetic outflow that can shift toward pain facilitation when central and peripheral α-2-adrenoceptor inhibitory mechanisms are attenuated.

Researching bladder afferents—determining the effects of β3‐adrenergic receptor agonists and botulinum toxin type‐A

A substantial portion of the current research on lower urinary tract dysfunction is focused on afferent mechanisms. The main goals are to define and modulate the signaling pathways by which afferent information is generated, enhanced and conveyed to the central nervous system. Alterations in bladder afferent mechanisms are a potential source of voiding dysfunction and an emerging source for drug targets. Established drug therapies such as muscarinic receptor antagonists, and two emerging therapies, β(3) -adrenergic receptor agonists and botulinum toxin type-A, may act partly through afferent mechanisms. This review focuses on these two new principles and new and established methods for determining their sites of action. It also provides brief information on the innervation of the bladder, afferent receptors and transmitters and how these may communicate with the urothelium, interstitial cells and detrusor smooth muscle to regulate micturition. Peripheral and central mechanisms of afferent sensitization and myogenic mechanisms that lead to detrusor overactivity, overactive bladder symptoms and urgency sensations are also covered. This work is the result from 'Think Tank' presentations, and the lengthy discussions that followed, at the 2010 International Consultation on Incontinence Research Society meeting in Bristol, UK.

Neonatal cystitis-induced colonic hypersensitivity in adult rats: a model of viscero-visceral convergence

The objective of this study was to determine if neonatal cystitis alters colonic sensitivity later in life and to investigate the role of peripheral mechanisms. Neonatal rats received intravesical zymosan, normal saline, or anesthesia only for three consecutive days [(postnatal (PN) days 14-16)]. The estrous cycle phase was determined prior to recording the visceromotor response (VMR) to colorectal distension (CRD) in adult rats. Eosinophils and mast cells were examined from colon and bladder tissues. CRD- or urinary bladder distension (UBD)-sensitive pelvic nerve afferents (PNAs) were identified and their responses to distension were examined. The relative expression of N-methyl-d-aspartic acid (NMDA)-NR1 subunit in the lumbo-sacral (L6-S1) spinal cord was examined using Western blot. The VMR to CRD (≥10mmHg) in the neonatal zymosan group was significantly higher than control in both the diestrus, estrus phase and in all phases combined. There was no difference in the total number of eosinophils, mast cells or number of degranulated mast cells between groups. The spontaneous firing of UBD, but not CRD-sensitive PNAs from the zymosan-treated rats was significantly higher than the saline-treated control. However, the mechanosensitive properties of PNAs to CRD or UBD were no different between groups (P>0.05). The expression of spinal NR1 subunit was significantly higher in zymosan-treated rats compared with saline-treated rats (P<0.05). Neonatal cystitis results in colonic hypersensitivity in adult rats without changing tissue histology or the mechanosensitive properties of CRD-sensitive PNAs. Neonatal cystitis does result in overexpression of spinal NR1 subunit in adult rats.

Mesenteric Afferent Nerve Sensitivity in the Small Intestine During Mechanical Ileus in Mice

Mesenteric Afferent Nerve Sensitivity in the Small Intestine During Mechanical Ileus in Mice

Role of ATP in the Mechanisms Underlying Intestinal Afferent Sensitivity to Hydrogen Sulphide

the 3rd CRD, 24 h after the last WAS (day 5). The VMR to CRD after WAS was expressed as percentage of the respective baseline values at different pressures of CRD. Data were analyzed using two-way ANOVA followed by a multiple comparison Bonferroni post-hoc test. Results: Immediately after the 1h exposure to WAS, on day 4, non-injected male and female rats exhibited visceral analgesia at 40 mmHg (p<0.05). Saline or naloxone injected sc before each WAS enhanced the visceral analgesia in both sexes in comparison to noninjected rats (analgesic response at 40 and 60 mmHg). On day 5, 24h after the last session of stress, the VMR was back to baseline values in all non-injected and injected male and female rats, except in females injected with naloxone which exhibited an analgesia at 60 mmHg (74.8±11.9 vs 100.0±0.0 %, p<0.05). Conclusions:When monitored non-invasively, intermittent repeated exposure to a psychological stress induces an immediate naloxoneindependent visceral analgesia in male and female rats. Repeated sc injections potentiate the immediate analgesic response suggesting that the concomitant somatic noxious stimuli may exert heterotopic analgesic effects. The sex-dependent delayed restoration of the VMR to baseline values by naloxone indicates an opioid-dependent component in females' stressinduced visceral response. Supported by NIH P50 DK-64539, The Kosciuszko Foundation (AM) and Wonkwang University (YSK) *ML, AM equal contribution

TRP Channels-Mediated Intestinal Afferent Sensitivity to Hydrogen Sulphide

the 3rd CRD, 24 h after the last WAS (day 5). The VMR to CRD after WAS was expressed as percentage of the respective baseline values at different pressures of CRD. Data were analyzed using two-way ANOVA followed by a multiple comparison Bonferroni post-hoc test. Results: Immediately after the 1h exposure to WAS, on day 4, non-injected male and female rats exhibited visceral analgesia at 40 mmHg (p<0.05). Saline or naloxone injected sc before each WAS enhanced the visceral analgesia in both sexes in comparison to noninjected rats (analgesic response at 40 and 60 mmHg). On day 5, 24h after the last session of stress, the VMR was back to baseline values in all non-injected and injected male and female rats, except in females injected with naloxone which exhibited an analgesia at 60 mmHg (74.8±11.9 vs 100.0±0.0 %, p<0.05). Conclusions:When monitored non-invasively, intermittent repeated exposure to a psychological stress induces an immediate naloxoneindependent visceral analgesia in male and female rats. Repeated sc injections potentiate the immediate analgesic response suggesting that the concomitant somatic noxious stimuli may exert heterotopic analgesic effects. The sex-dependent delayed restoration of the VMR to baseline values by naloxone indicates an opioid-dependent component in females' stressinduced visceral response. Supported by NIH P50 DK-64539, The Kosciuszko Foundation (AM) and Wonkwang University (YSK) *ML, AM equal contribution

Degeneration of capsaicin sensitive sensory nerves enhances myocardial injury in acute myocardial infarction in rats

BackgroundEvidence indicated an involvement of afferent nerves in the pathology of acute myocardial infarction. This study was undertaken to clarify the role and mechanisms by which the sensory afferent degeneration exacerbates the myocardial injury in acute myocardial infarction in rats. MethodsThe myocardial injury was assessed by analysis of 1) the differences in the infarct size, myocyte apoptosis, the caspase activity in the myocardium and cardiac troponin I in serum between the denervated and non-denervated rats; 2) the differences in the size of infarctiom with and without antagonisms of endogenous neurokinin 1 receptor or calcitonin gene related peptide receptor in acute myocardial infarction. ResultsDegeneration of the afferent nerves resulted in marked increase in the pain threshold and decrease in substance P and calcitonin gene related peptide in dorsal root ganglia, spinal dorsal horn and myocardium. Increases of the infarction size (39%±4% vs. 26%±4%,), troponin-I (28.4±8.89ng/ml, vs. 14.6±9.75ng/ml), apoptosis of myocytes (by 1.8±0.2 folds) and caspase-3 activity (1.6±0.3 vs. 1.05±0.18) were observed in the denervated animals at 6h of myocardial infarction, compared with the non-denervated rats. Antagonisms of the endogenous neurokinin 1 receptor or calcitonin gene related peptide receptor caused increase of the size of infarction in the animals. ConclusionDegeneration of capsaicin sensitive afferent nerves enhances the myocardial injury of acute myocardial infarction, possibly due to reduction of endogenous calcitonin gene related peptide and substance P.

Obesity‐induced hypertension is associated with blunted renal sympathoinhibitory responses to gastric peptides

The gastric peptides leptin and cholecystokinin (CCK) induce vagally-mediated splanchnic sympathoinhibition, and a medium high fat diet (MHFD) attenuates this response. Our aim was to evaluate renal sympathetic nerve discharge (RSND) responses to these peptides to determine whether blunted sympathoinhibition is linked to hypertension in obesity. Male Sprague-Dawley rats were fed either a low fat diet (LFD, control; n=8) or a MHFD (n=24) for 13 weeks; the latter were grouped as obesity prone (OP; n=8) or obesity resistant (OR; n=8) based on their weight gain falling into the upper or lower tertile, respectively. RSND and arterial pressure (AP) were recorded in isoflurane-anesthetised animals. Blood was collected for determination of plasma leptin and fat pads were excised postmortem. OP rats had higher baseline AP, plasma leptin and adiposity than control/OR rats (P < 0.05 for all). Renal sympathoinhibitory responses to CCK (0.5–8 μg/kg) administered close to the celiac artery were attenuated in OP compared to control/OR animals (P < 0.05). Close arterial leptin (15 μg/kg) induced renal sympathoinhibition in controls, and this was abolished in OP animals (P < 0.01). Diet-induced obesity may be associated with altered sensitivity of vagal afferents to gastrointestinal hormones. This may lead to increased vascular resistance in the splanchnic and renal beds, possibly contributing to hypertension in obesity.

Alteration in Skeletal Muscle Afferents in Rats with Chronic Heart Failure

An exaggerated exercise pressor reflex (EPR) contributes to exercise intolerance and excessive sympatho-excitation in the chronic heart failure (CHF) state. However, the components of this reflex that are responsible for the exaggerated EPR in CHF remain unknown. To determine whether muscle afferent function is altered in CHF, we recorded the discharge of group III and IV afferents in response to static contraction, passive stretch and hindlimb intra-arterial injection of capsaicin in sham and CHF rats. We also investigated the roles of purinergic 2X receptor (P2X) and the transient receptor potential vanilloid 1 (VR1) in mediating the altered sensitivity of muscle afferents. Compared with sham rats, CHF rats exhibited greater responses of group III afferents to contraction and stretch whereas the responses of group IV afferents to contraction and capsaicin were blunted. Hindlimb intra-arterial infusion of pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a P2X antagonist, attenuated the responses of group III afferents to contraction and stretch in CHF rats to a greater extent than in sham rats. Western blot data showed that P2X3 receptors were significantly upregulated in doral root ganglion (DRG) of CHF rats whereas VR1 receptors were significantly downregulated. Immunohistochemical evidence showed that immunostaining of the P2X3 receptors was more intense in both IB4-positive (C-fibre) and NF200-positive (A-fibre) neurons in DRG of CHF rats whereas the immunostaining of the VR1 receptors was decreased in IB4-positive neurons. These data suggest that group III afferents are sensitized whereas group IV afferents are desensitized in CHF, which is related to the dysfunction of P2X and VR1 receptors.

Exercise with angina and cramp?

Exercise with angina and cramp?

Local Activation of Cannabinoid CB1 Receptors in the Urinary Bladder Reduces the Inflammation-Induced Sensitization of Bladder Afferents

BackgroundSystemic administration of cannabinoid agonists is known to reduce pain induced by bladder inflammation and to modulate cystometric parameters in vivo. We have previously reported that intravesical administration of a cannabinoid agonist reduces the electrical activity of bladder afferents under normal conditions. However, the effects of local activation of bladder cannabinoid receptors on afferent activity during inflammation are unknown. This study was aimed to assess the effects of intravesical administration of a cannabinoid agonist on the discharges of afferent fibers in inflamed bladders ex vivo. We also characterized the expression of CB1 receptors in the bladder and their localization and co-expression with TRPV1, a marker of nociceptive afferents.ResultsCompared to untreated animals, afferent fiber activity in inflamed bladders was increased for intravesical pressures between 10 and 40 mmHg. Local treatment with a non selective cannabinoid agonist (AZ12646915) significantly reduced the afferent activity at intravesical pressures above 20 mmHg. This effect was blocked by AM251 but not by AM630 (selective for CB1 and CB2 respectively). Finally, CB1 was co-expressed with TRPV1 in control and inflamed bladders.ConclusionThese results demonstrate that sensitization of bladder afferents induced by inflammation is partly suppressed by intravesical activation of cannabinoid receptors, an effect that appears to be mediated by CB1 receptors. Also, TRPV1 positive fibers were found to co-express CB1, supporting the hypothesis of a direct action of the cannabinoid agonist on nociceptive afferents. Taken together, these results indicate a peripheral modulation by the cannabinoid system of bladder hypersensitivity during inflammation.

Transient Inflammation-Induced Ongoing Pain is Driven by TRPV1 Sensitive Afferents

BackgroundTissue injury elicits both hypersensitivity to evoked stimuli and ongoing, stimulus-independent pain. We previously demonstrated that pain relief elicits reward in nerve-injured rats. This approach was used to evaluate the temporal and mechanistic features of inflammation-induced ongoing pain.ResultsIntraplantar Complete Freund's Adjuvant (CFA) produced thermal hyperalgesia and guarding behavior that was reliably observed within 24 hrs and maintained, albeit diminished, 4 days post-administration. Spinal clonidine produced robust conditioned place preference (CPP) in CFA treated rats 1 day, but not 4 days following CFA administration. However, spinal clonidine blocked CFA-induced thermal hyperalgesia at both post-CFA days 1 and 4, indicating different time-courses of ongoing and evoked pain. Peripheral nerve block by lidocaine administration into the popliteal fossa 1 day following intraplantar CFA produced a robust preference for the lidocaine paired chamber, indicating that injury-induced ongoing pain is driven by afferent fibers innervating the site of injury. Pretreatment with resiniferatoxin (RTX), an ultrapotent capsaicin analogue known to produce long-lasting desensitization of TRPV1 positive afferents, fully blocked CFA-induced thermal hypersensitivity and abolished the CPP elicited by administration of popliteal fossa lidocaine 24 hrs post-CFA. In addition, RTX pretreatment blocked guarding behavior observed 1 day following intraplantar CFA. In contrast, administration of the selective TRPV1 receptor antagonist, AMG9810, at a dose that reversed CFA-induced thermal hyperalgesia failed to reduce CFA-induced ongoing pain or guarding behavior.ConclusionsThese data demonstrate that inflammation induces both ongoing pain and evoked hypersensitivity that can be differentiated on the basis of time course. Ongoing pain (a) is transient, (b) driven by peripheral input resulting from the injury, (c) dependent on TRPV1 positive fibers and (d) not blocked by TRPV1 receptor antagonism. Mechanisms underlying excitation of these afferent fibers in the early post-injury period will offer insights for development of novel pain relieving strategies in the early post-traumatic period.

Anti-inflammatory effects of limb ischaemic preconditioning are mediated by sensory nerve activation in rats

We have shown that ischaemic preconditioning ameliorates both the local periosteal and the systemic leukocyte activation evoked by limb ischaemia-reperfusion. We hypothesized that the activation of chemosensitive afferent nerves by transient ischaemia contributes to the protective mechanisms of ischaemic preconditioning via a calcitonin gene-related peptide (CGRP)-dependent mechanism. In Sprague-Dawley rats, 60-min complete limb ischaemia was followed by 180min of reperfusion. In further experiments, the CGRP analogue hCGRP (0.3μgkg(-1)) or ischaemic preconditioning (2 × 10-min ischaemia/10-min reperfusion) was applied prior to the ischaemia-reperfusion insult. Ischaemic preconditioning was performed in three subgroups in which animals received the CGRP receptor antagonist CGRP(8-37) (30μgkg(-1)h(-1)), the chemosensitive afferent nerve inactivator resiniferatoxin (3 × 15μgkg(-1), sc), or vehicle. The effects of CGRP(8-37) and resiniferatoxin on ischaemia-reperfusion without ischaemic preconditioning were also evaluated. In the tibial periosteum of rats, intravital fluorescence microscopy and immunohistochemistry revealed significant attenuations of ischaemia-reperfusion-induced post-ischaemic leukocyte-endothelial interactions (rolling and adherence in the postcapillary venules) and tissue intracellular adhesion molecule expression following ischaemic preconditioning or hCGRP administration. Administration of CGRP(8-37) or pretreatment of animals with resiniferatoxin reversed the anti-inflammatory effects of limb ischaemic preconditioning, but failed to affect the microcirculatory consequences of ischaemia-reperfusion without ischaemic preconditioning. The results suggest that activation of the chemo- (capsaicin-) sensitive afferent nerves is involved in the mechanisms of microcirculatory anti-inflammatory protection provided by limb ischaemic preconditioning. Controlled activation of chemosensitive C-fibres or the CGRP receptors by the induction of ischaemic preconditioning or other means may furnish therapeutic benefit by ameliorating the periosteal microcirculatory consequences of tourniquet ischaemia.

Alteration in skeletal muscle afferents in rats with chronic heart failure

An exaggerated exercise pressor reflex (EPR) contributes to exercise intolerance and excessive sympatho-excitation in the chronic heart failure (CHF) state. However, the components of this reflex that are responsible for the exaggerated EPR in CHF remain unknown. To determine whether muscle afferent function is altered in CHF, we recorded the discharge of group III and IV afferents in response to static contraction, passive stretch and hindlimb intra-arterial injection of capsaicin in sham and CHF rats. We also investigated the roles of purinergic 2X receptor (P2X) and the transient receptor potential vanilloid 1 (VR1) in mediating the altered sensitivity of muscle afferents. Compared with sham rats, CHF rats exhibited greater responses of group III afferents to contraction and stretch whereas the responses of group IV afferents to contraction and capsaicin were blunted. Hindlimb intra-arterial infusion of pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS), a P2X antagonist, attenuated the responses of group III afferents to contraction and stretch in CHF rats to a greater extent than in sham rats. Western blot data showed that P2X3 receptors were significantly upregulated in doral root ganglion (DRG) of CHF rats whereas VR1 receptors were significantly downregulated. Immunohistochemical evidence showed that immunostaining of the P2X3 receptors was more intense in both IB4-positive (C-fibre) and NF200-positive (A-fibre) neurons in DRG of CHF rats whereas the immunostaining of the VR1 receptors was decreased in IB4-positive neurons. These data suggest that group III afferents are sensitized whereas group IV afferents are desensitized in CHF, which is related to the dysfunction of P2X and VR1 receptors.

Perception of gastro-oesophageal reflux

Gastro-oesophageal reflux disease (GORD) is common and often associated with unpleasant symptoms requiring utilisation of health care resource. While in the majority of patients symptom resolution occurs with acid suppressant therapy, in a proportion this treatment is ineffective in resolving symptoms. This is particularly the case in patients with non-erosive reflux disease (NERD) and functional heartburn (FH). It is increasingly being recognised that the presence of acid in the oesophagus can cause dilated intercellular spaces (DIS) which increases the exposure of the sub-epithelial nerves to the acid. Experimental studies in both animals and humans suggest that a variety of receptors on afferent nerves can be sensitised upon exposure to acid so that there is increased afferent input to the spinal cord dorsal horn neurons which leads to a reduction in threshold of these neurons together with an increase in their receptive field. This increased sensitivity of primary afferent nerves is described as peripheral sensitisation, whereas the consequent increase in sensitivity of the spinal dorsal horn neurons is described as central sensitisation. Once these mechanisms have been established they can cause a long term increase in sensitivity of tissues to previously innocuous stimuli. Furthermore, psychological stress has been shown to increase DIS and may therefore facilitate peripheral sensitisation. Currently peripheral and central sensitisations are considered to be important mechanisms of oesophageal pain hypersensitivity and occurrence of symptoms to even physiological amounts of acid. In these patients treatments aimed at reducing neuronal sensitivity may be effective in the management.

Expression of E‐series prostaglandin (EP) receptors and urodynamic effects of an EP4 receptor antagonist on cyclophosphamide‐induced overactive bladder in rats

To investigate the expression of four subtypes of E-series prostaglandin (EP(1) -EP(4) ) receptors and the urodynamic effects of an EP(4) receptor antagonist (AH23848) in cyclophosphamide (CYP)-induced overactive bladder (OAB) in rats, as intravesical prostaglandin E(2) (PGE(2) ) induces OAB via activation of EP receptors and sensitization of afferent nerves. Experimental and control rats were injected with CYP (200 mg/kg, intraperitoneally) or saline, respectively. Continuous cystometrograms (CMGs) were performed 48 h after CYP or saline injection under urethane anaesthesia. AH23848 was given intravenously at doses of 0.01 and 0.1 mg/kg. The bladder was then harvested for histology. Some bladders were harvested for analysis of EP receptors expression by Western blotting without a CMG study. CMG variables (baseline pressure; intercontraction interval [ICI], pressure threshold [PT], contraction amplitude) and histological changes were measured. CYP-induced up-regulation of EP(4) receptor (100% increase) accompanied by detrusor overactivity (ICI 70.5% decrease; PT, 67.7% increase). However, CYP down-regulated EP(1) receptor expression (51.9% decrease), but had no significant effects on the EP(2) and EP(3) receptors. AH23848 significantly extended the ICI in CYP-treated rats but it had no effects on other urodynamic variables or in control rats. Modulation of EP receptors plays a role in CYP-induced OAB. Antagonists to the EP(4) receptor may be a new target for treatment of patients with OAB.

Muscle metaboreflex activation by static exercise dilates pupil in humans

We examined a hypothesis that static exercise and activation of sympathetic activation by metabolically sensitive skeletal muscle afferents (metaboreflex) influence the sympathetic nervous activity modulating pupil diameter. Nine subjects performed 2 min isometric handgrip exercise at 30% maximal voluntary contraction, which was followed by either 2 min of postexercise muscle ischemia (PEMI) in the forearm or no PEMI (control trial). The pupil diameter and mean blood pressure (MAP) increased significantly from rest during exercise in PEMI and control trials (5 ± 1 and 7 ± 1% in diameter; 13 ± 2 and 12 ± 2 mmHg in MAP, respectively). These increases in the diameter and MAP were maintained during PEMI (7 ± 2% and 9 ± 2 mmHg) but not during the recovery period in the control trial (3 ± 2% and 1 ± 2 mmHg). These results demonstrate that static handgrip exercise increases the pupil diameter, and this increase is partly due to the activation of metaboreflex in humans.

ERK1/2 signaling pathway in mast cell activation-induced sensitization of esophageal nodose C-fiber neurons

Sensitization of esophageal nociceptive afferents by inflammatory mediators plays an important role in esophageal inflammatory nociception. Our previous studies demonstrated that esophageal mast cell activation increases the excitability of esophageal nodose C-fibers. But the intracellular mechanism of this sensitization process is still less clear. We hypothesize that extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway plays an important role in mast cell activation-induced sensitization of esophageal nodose C-fiber neurons. Mast cell activation and in vivo esophageal distension-induced phosphorylations of ERK1/2 were studied by immuno-staining and Western blot in esophageal nodose neurons. Extracellular recordings were performed from nodose neurons using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Nerve excitabilities were compared by action potentials evoked by esophageal distensions before and after mast cell activations with/without pretreatment of mitogen-activated protein kinases (MAPK)/ERK kinase inhibitor U0126. The expressions of phospho-ERK1/2 (p-ERK1/2) in the same nodose ganglia were then studied by Western blot. Mast cell activation enhances in vivo esophageal distension-induced phosphorylation of ERK1/2 in nodose neurons. This can be prevented by pretreatment with mast cell stabilizer cromolyn. In ex vivo esophageal-vagal preparations, both mast cell activation and proteinase-activated receptor 2 (PAR2)-activating peptide perfusion increases esophageal distension-induced mechano-excitability of esophageal nodose C-fibers and phosphorylation of ERK1/2 in nodose neurons. Pretreatment with MAPK/ERK kinase inhibitor U0126 prevents these potentiation effects. Collectively, our data demonstrated that mast cell activation enhances esophageal distension-induced mechano-excitability and phosphorylation of ERK1/2 in esophageal nodose C-fiber neurons. This reveals a new intracellular pathway of esophageal peripheral sensitization and inflammatory nociception.

Increased sensitivity of group III and group IV afferents from incised muscle in vitro

Understanding deep muscle pain is of increasing importance for evaluating clinical pathologic pain states. Previously, a central role of deep muscle tissue in the development of ongoing pain behavior after incision was demonstrated. The underlying mechanisms, however, remain unclear. Using a new in vitro plantar flexor digitorum brevis (FDB) muscle–nerve preparation, we investigated properties of mechanosensitive group III and IV afferents innervating incised and unincised muscle, and explored potential mediators of afferent excitation after incision. Afferents of uninjured muscle had a low incidence (14.3%) of ongoing activity. A high proportion (65.8%) of afferents responded to heat and a minority, 20.8%, were activated by pH 6.0 lactic acid. Incision increased the prevalence of afferents with ongoing activity to 54.7%. A greater proportion of group III and IV afferents responded to pH 6.0 lactic acid after incision compared to control (55.4% vs. 20.8%). Sensitization of afferents to heat and mechanical stimulation was prominent in group IV afferents after incision; both heat (38.0 vs. 40.5°C in control) and mechanical response threshold (median: 5.0 vs. 22.0mN in control) were decreased. The finding hat incision increased ongoing activity of muscle afferents is consistent with our previous in vivo studies and supports the idea that deep muscle tissue has a prominent role in the genesis of ongoing pain after incision. The enhanced chemosensitivity of muscle afferents to lactic acid after incision suggests an increased response to an ischemic mediator that may contribute to pain and hyperalgesia caused by surgery in deep tissues.

A new function for ATP: activating cardiac sympathetic afferents during myocardial ischemia

Myocardial ischemia activates cardiac sympathetic afferents leading to chest pain and reflex cardiovascular responses. Brief myocardial ischemia leads to ATP release in the interstitial space. Furthermore, exogenous ATP and α,β-methylene ATP (α,β-meATP), a P2X receptor agonist, stimulate cutaneous group III and IV sensory nerve fibers. The present study tested the hypothesis that endogenous ATP excites cardiac afferents during ischemia through activation of P2 receptors. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T(2)-T(5)) in anesthetized cats. Single fields of 45 afferents (conduction velocities = 0.25-4.92 m/s) were identified in the left ventricle with a stimulating electrode. Five minutes of myocardial ischemia stimulated 39 of 45 cardiac afferents (8 Aδ, 37 C fibers). Epicardial application of ATP (1-4 μmol) stimulated six ischemically sensitive cardiac afferents in a dose-dependent manner. Additionally, epicardial ATP (2 μmol), ADP (2 μmol), a P2Y agonist, and α,β-meATP (0.5 μmol) significantly activated eight other ischemically sensitive afferents. Third, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, a P2 receptor antagonist, abolished the responses of six afferents to epicardial ATP (2 μmol) and attenuated the ischemia-related increase in activity of seven other afferents by 37%. In the absence of P2 receptor blockade, cardiac afferents responded consistently to repeated application of ATP (n = 6) and to recurrent myocardial ischemia (n = 6). Finally, six ischemia-insensitive cardiac spinal afferents did not respond to epicardial ATP (2-4 μmol), although these afferents did respond to epicardial bradykinin. Taken together, these data indicate that, during ischemia, endogenously released ATP activates ischemia-sensitive, but not ischemia-insensitive, cardiac spinal afferents through stimulation of P2 receptors likely located on the cardiac sensory neurites.