Adrenergic β2-Receptors Mediates Visceral Hypersensitivity Induced by Heterotypic Intermittent Stress in Rats

Chronic visceral pain in patients with irritable bowel syndrome (IBS) has been difficult to treat effectively partially because its pathophysiology is not fully understood. Recent studies show that norepinephrine (NE) plays an important role in the development of visceral hypersensitivity. In this study, we designed to investigate the role of adrenergic signaling in visceral hypersensitivity induced by heterotypical intermittent stress (HIS). Abdominal withdrawal reflex scores (AWRs) used as visceral sensitivity were determined by measuring the visceromoter responses to colorectal distension. Colon-specific dorsal root ganglia neurons (DRGs) were labeled by injection of DiI into the colon wall and were acutely dissociated for whole-cell patch-clamp recordings. Blood plasma level of NE was measured using radioimmunoassay kits. The expression of β2-adrenoceptors was measured by western blotting. We showed that HIS-induced visceral hypersensitivity was attenuated by systemic administration of a β-adrenoceptor antagonist propranolol, in a dose-dependent manner, but not by a α-adrenoceptor antagonist phentolamine. Using specific β–adrenoceptor antagonists, HIS-induced visceral hypersensitivity was alleviated by β2 adrenoceptor antagonist but not by β1- or β3-adrenoceptor antagonist. Administration of a selective β2-adrenoceptor antagonist also normalized hyperexcitability of colon-innervating DRG neurons of HIS rats. Furthermore, administration of β-adrenoceptor antagonist suppressed sustained potassium current density (I K) without any alteration of fast-inactivating potassium current density (I A). Conversely, administration of NE enhanced the neuronal excitability and produced visceral hypersensitivity in healthy control rats, and blocked by β2-adrenoceptor antagonists. In addition, HIS significantly enhanced the NE concentration in the blood plasma but did not change the expression of β2-adrenoceptor in DRGs and the muscularis externa of the colon. The present study might provide a potential molecular target for therapy of visceral hypersensitivity in patents with IBS.


Introduction
Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is defined by recurrent symptoms of abdominal pain or discomfort associated with alterations in bowel habits [1]. Up to date, its cardinal feature, chronic visceral pain, has been difficult to treat effectively. Much progress has been made during last several decades in understanding the development of visceral hypersensitivity, but its pathophysiology is not fully understood. Clinical studies demonstrate that chronic stress plays an important role in the pathophysiology of IBS [2][3][4]. Alterations in adrenergic signaling have been implicated in the development of visceral hypersensitivity [5][6][7]. It is reported that chronic stress may induce abnormal expressions of brain G proteins, colonic alpha (2A)adrenoceptors, and norepinephrine (NE) reuptake transporter (NET), which may be responsible for the abnormalities of abdominal sensation in IBS [8]. Heterotypic chronic stress can increase sympathetic nervous system activity and induces the release of NE [5]. Once released, NE binds to its receptors. The receptors for NE are a class of G protein-coupled receptors, including a and b subtypes. a receptors have a 1 and a 2 subtypes while b receptors have b 1 , b 2 and b 3 subtypes. b 1 and b 2 adrenergic receptors involve in the adrenergic activation of electrogenic K + secretion in guinea pig distal colonic epithelium [9], which may play a role in colonic transit. b 2 adrenergic receptors located on primary afferent nociceptors are reported to produce a hyperalgesic state in rats [10][11][12]. b 3 adrenergic receptors, mainly expressed in brown and white adipose tissue, can regulate energy metabolism and thermogenesis [13]. Previous study showed that the blockade of a 1 /a 2 -and b 1 /b 2 -adernoreceptors before the daily application of chronic stress prevented the induction of visceral hypersensitivity in male Wistar rats [6]. However, which subtype of the adrenergic receptors involves in the induction of visceral hypersensitivity following chronic stress remains unclear.
Visceral hypersensitivity has merged as a key hypothesis or biological hallmark in explaining the painful symptoms in IBS [14][15][16]. Both the central sensitization and peripheral sensitization have been implicated in the development of visceral hypersensitivity [17,18]. The peripheral sensitization involves an enhanced excitability of primary afferent nociceptors, which are the information superhighway from the gut to the spinal cord and convey peripheral stimuli into action potentials that propagate to the central nervous system [17]. Sensitization of primary sensory afferents is maintained by a number of membrane receptors and ion channels. Voltage-gated potassium (Kv) channels play a fundamental role in controlling neuronal excitability. Suppression of K currents contributes to peripheral sensitization [19]. The transient A-type current (I A ) and the sustained delayed rectifier current (I K ) are two primary types of Kv currents that are important in modulating spike frequency and activation thresholds. The decrease in I A currents took part in enhanced excitability of pancreatic DRG neurons observed in chronic pancreatitis [20]. The decrease in I K took part in colonic DRGs of neonatal maternal separation rats [21] and contributed to hyperexcitability of colonic DRG neurons in mouse. Taken together, the changes in Kv channel function in DRG neurons are disease/organ specific. However, the role of Kv channels in HIS-induced visceral hypersensitivity remains unknown.
In this study, we investigated the contribution of different subtypes of adrenergic receptors to visceral hypersensitivity and to changes in the membrane properties and excitability of colon innervating DRG neurons in a rat model of visceral hypersensitivity induced by heterotypical intermittent stress (HIS). Furthermore, we specifically examined changes in Kv channel conductance in these cells. We tested the hypothesis that enhanced release of NE in response to HIS acts through activation of b 2adrenoreceptor to suppress I K and sensitize the colon-specific DRG neurons, thus induces visceral hypersensitivity to CRD. The present study suggests that adrenergic receptors are potential treatment targets for visceral pain in patents with IBS.

Animals and heterotypic intermittent stress protocol
Adult male Sprague-Dawley (SD) rats, weighing 250620 g, were obtained from the Animal Research Center of Soochow University. Rats were housed under controlled conditions (07:00,19:00 lighting, temperature: 2462uC) with free access to a standard laboratory diet and fresh water. Care and handling of these rats were approved by the Institutional Animal Care and Use Committee of the Soochow University and were in accordance with the guidelines of the International Association for the Study of Pain (IASP). Visceral hypersensitivity was induced by heterotypic intermittent stress (HIS), as described previously [6,22]. In brief, rats were subjected to an HIS protocol composed of 3 randomly arranged stressors: 45 minutes of cold restraint stress at 4uC, 60 minutes of water avoidance stress, or 20 minutes of forced swimming stress for 9 consecutive days.

Measurement of visceromoter response to graded colorectal distention
Visceral hypersensitivity was measured by grading the response of rats to colorectal distention (CRD) as described previously [23][24][25]. In brief, rats were lightly sedated with diethyl ether while a flexible balloon (6 cm) made of a surgical glove finger attached to a tygon tubing was inserted 8 cm into the descending colon and rectum via the anus and held in place by taping the tubing to the tail. SD rats were placed in small lucite cubicles and allowed to adapt for 30 mins. CRD was performed by rapidly inflating the balloon to a constant pressure measured using a sphygmomanometer connected to a pressure transducer. The balloon was inflated to various pressures: 20, 40, 60 and 80 mmHg, for a 20 sec stimulation period followed by a 2 mins rest. Behavioral responses to CRD were measured by visual observation of the abdominal withdrawal reflex (AWR) by a blinded observer and the assignments of an AWR score were as follows [23][24][25]: 0 = Normal behavior without response; 1 = Brief head movement at the onset of the stimulus followed by immobility; 2 = Contraction of abdominal muscles; 3 = Lifting of the abdomen off the platform; 4 = Body arching and lifting of pelvic structures. In addition, colonic distension threshold, the minimal pressure to induce abdominal muscle contraction, was used to measure the time course of HIS and drug effects.

Cell labeling
Colon-innervating DRG neurons were labeled by injection of 1, 19-dioleyl-3, 3, 39, 3-tetramethylindocarbocyanine methanesulfonate (DiI, Invitrogen) into the colon wall as described previously [25]. Briefly, 6-week-old rats were anaesthetized with ketamine (80 mg/kg, i. p.) plus xylazine (5,10 mg/kg, i. p.). The abdomen was opened by midline laparotomy and the colon was exposed. DiI was injected in ,1 ml volume (25 mg in 0.5 ml methanol) at 10 sites on the exposed colon extending from the level of the bladder to ,6 cm in an oral direction. To prevent possible contamination of adjacent organs with the dye, the needle was left in place for ,1 min and the injection site was washed with normal saline (NS) after each injection. The colon was gently swabbed prior to closing of the abdomen. Rats were returned to their housing and given free access to drinking water and standard food pellets. Ten days later, these rats were sacrificed for whole-cell patch clamp recordings.

Drug application
For behavioral, whole-cell patch clamp and protein experiments, phentolamine (Phen), propranolol (Prop), butoxamine (Buto), atenolol (Aten), SR-59230A (SR) or normal saline (NS) was intraperitoneally (i.p.) administrated 30 minutes before each stressor starting from the 1st day to the 9th day during the HIS protocol. Thus, antagonists or NS were injected for 9 consecutive days. AWR scores were tested 6 hours after termination of the last stressor. Animals were euthanized 6 hours after the last stressor and the colon innervating DRG neurons were harvested for whole-cell patch clamping. In some experiments, butoxamin was intraperitoneally (i.p.) administrated 30 min before NE injection intrathecally. For in vitro patch clamp recording study, NE at the concentration of 10 mM was used to incubate the acutely isolated DRG neurons for 3 minutes.

Western blotting
DRGs (T 13 -L 2 ) and the muscularis externa of the distal colon from HIS-treated rats or age-mateched controls were dissected out and lyzed in radioimmunoprecipitation assay buffer containing 1% NP-40, 0.5%Na deoxycholate, 0.1% SDS, PMSF (10 ml/ml), and aprotinin (30 ml/ml; Sigma). The lysates were then microfuged at 15,000 rpm for 30 minutes at 4uC. The concentration of protein in homogenate was determined using a BCA reagent (Beyotime, CHN). Twenty micrograms (20 mg) of proteins for b 2adernoceptor studies were loaded onto a 10% Tris-HCl SDS-PAGE gel (Bio-Rad, Hercules, CA). After electrophoresis, the proteins were electrotransferred onto polyvinyldifluoride membranes (Millipore) at 200 mA for 2 hours at 4uC. The membranes were incubated in 25 ml of blocking buffer (1XTBS with 5% w/v fat-free dry milk) for 2 hours at room temperature. The membranes were then incubated with the primary antibodies for 2 hours at room temperature. Primary antibodies used were mouse anti-b 2 -adrenoreceptor (1:100; abcam, USA) and mouse anti-actin (1:1000; Chemicon, Temecula, CA) or anti-GAPDH (1:1000; Goodhere, China). After incubation, the membranes were washed with TBST (1XTBS and 5% Tween 20) three times for 15 minutes each and incubated with anti-mouse HRP-conjugated secondary antibody (1:4000; Chemicon) for 2 hours at room temperature. The membrane was washed with TBST three times for 15 minutes each. The immunoreactive proteins were determined by enhanced chemiluminescence (ECL kit; Amersham Biosciences, Arlington Heights, IL). Bands were visualized by exposure of membranes onto an x-ray film. For quantification of b 2 -adernoceptor protein levels, photographs were digitalized and analyzed using a scanner (Bio-Rad imaging system Bio-Rad GelDoc XRS+). b-actin or GAPDH was used as an internal control. All samples were normalized to b-actin or GAPDH.

Measurement of norepinephrine (NE) in blood plasma
Blood samples were collected from the trunk in tubes containing 2.5% sodium citrate and 0.45% citric acid at the time of animal euthanasia by decapitation. Samples from HIS and age-matched control rats were spun in a refrigerated centrifuge; plasma was quickly aliquoted and stored at 280uC for assays. Plasma level of NE was measured using the radioimmunoassay kits from Abnova.

Data analysis
All data obtained are expressed as mean6SEM in the present study. Statistical analysis were performed using commercial software OriginPro 8 (OriginLab, US). Normality was checked before analyses. Significance was determined using paired sample t-test, paired sample sign test, Mann-Whitney test, Tukey post hoc test following Kruskal-Wallis ANOVA or one-way ANOVA, Dunn's post hoc test following Friedman ANOVA, two sample ttest, as appropriate. The level of significance was set at p,0.05.

HIS-induced visceral hypersensitivity was attenuated by b-adrenoceptor antagonist propranolol
In agreement with our previous report [6,22], AWR scores were significantly increased at distention pressures of 20, 40, 60, 80 mmHg at 6 hours after HIS and kept high at pressures of 20, 40 and 60 mmHg at 24 hours after HIS (

Butoxamine treatment reversed hyperexcitability of colon DRG neurons
To determine the effect of butoxamine on excitability of colon innervating DRG neurons (including T13, L1 and L2 DRGs), we measured passive and active membrane properties of these neurons from control (CON), HIS, NS-or Buto-treated HIS rats. Figure 3A shows a DiI labeled neuron (Arrow) and a non-DiI labeled neuron (Star). Whole-cell patch clamping was employed to record DiI-labeled neurons. Rheobase, action potential (AP) threshold, and pattern of firing in response to depolarizing current stimulations were determined. Rheobase is the minimal injected current to evoke an AP. The respective average rheobase of colon innervating DRG neurons from HIS-treated animals were much lower than those from the corresponding controls (  To further compare numbers of APs of colon DRG neurons after HIS and drug treatment, we also used 1-second ramp current stimulation ranging from 0 to 100 pA, 300 pA or 500 pA ( Figure 4). HIS remarkably increased the number of APs in response to a 100 pA, 300 pA and 500 pA ramp current stimulation when compared with controls ( Fig. 4A and B). The number of firings in responding to 100 pA ramp stimulation was 0.2460.12 in control group and was 4.2960.82 in HIS group ( Fig. 4A and B, n = 21 for each group, *p,0.05 vs. CON, Tukey post hoc test following Kruskal-Wallis ANOVA). The number of firings in response to 300 pA ramp stimulation was 6.3361.06 in control group and was 11.2961.15 in HIS group ( Fig. 4A and B, n = 21 for each group, *p,0.05 vs. CON, Tukey post hoc test following Kruskal-Wallis ANOVA). The number of firings in responding to 500 pA ramp current stimulation was 11.7161.42 in control rats and 20.0061.54 in HIS group ( Fig. 4A and B, n = 21 for each group, *p,0.05 vs. CON, Tukey post hoc test following Kruskal-Wallis ANOVA). In addition, the time to first spike (TTFS) in response to a 100 pA, 300 pA and 500 pA current injection was analyzed in the present study. HIS significantly decreased TTFS (Fig. 4C, 100   Buto on spike generation of colon specific DRG neurons of HIS rats.

Propranolol treatment increased sustained potassium current density
Since changes in activation thresholds and spike frequency suggest that there is an alteration in voltage-gated potassium (K V ) channels, we next performed patch-clamp recordings to examine K V currents under voltage-clamp conditions. As described previously [20], Na + in the normal external solution was replaced with equimolar choline. Ca 2+ concentration was reduced to 30 mM. A depolarization step from 250 to +90 mV in 10-mV increments with duration of 400 ms activated all K V channels (I Total ; Fig. 5A and B). The peak current-voltage (I-V) curves are shown in Figure 5B. However, HIS treatment greatly decreased peak current density in colon DRG neurons (Fig. 5A, *p,0.05, compared with CON, Tukey post hoc test following one-way ANOVA). The mean peak current density of I Total was 388.456 42.50 pA/pF (n = 8) from CON rats and 265.41628.06 pA/pF (n = 8) from HIS rats. Because there were two main types of K V currents (I A and I K ) described in nociceptive DRG neurons [20,26,27], we then isolated these two K V currents by manipulating the holding membrane potential. A depolarization step 250 to +90 mV with 400 ms in duration in 10-mV increments activated most of the sustained K V channels ( Fig. 5C and D) but not A-type K V channels. Subtraction of I K from I Total yielded I A (Fig. 5E and F). In the present study, the mean peak current density of I K was 221.15627.29 pA/pF (n = 8) from control rats and 104.7668.26 pA/pF from HIS rats (n = 8). I K density was markedly reduced following HIS (Fig. 5C, **p,0.01, compared with CON, Tukey post hoc test following one-way ANOVA). However, the I A density was not significantly changed (Fig. 5E, p. 0.05, compared with CON, Tukey post hoc test following one-way ANOVA). The mean peak current density of I A was 167.306 20.38 pA/pF (n = 8) from control rats and 160.65625.66 pA/pF from HIS rats (n = 8). These data suggest an involvement of I K in the development of visceral hypersensitivity induced by HIS in rats.
In an effort to investigate the contribution of b-adrenoceptor mediated signaling, propranolol was used in this experiment. Administration of propranolol (i.p.) significantly enhanced the I Total (Fig. 5 A and B) and I K density (Fig. 5 C and D) without any significant alteration in I A density (Fig. 5 E and F)  with NS-treated rats. The mean peak current density of I Total was 241.72616.94 pA/pF (n = 8) from NS rats and 394.126 23.57 pA/pF (n = 8) from Prop-treated rats. The mean peak current density of I K was 93.7969.19 pA/pF (n = 8) from NS rats and 206.18619.42 pA/pF from Prop-treated rats (n = 8). The mean peak current density of I A was 147.93613.44 pA/pF (n = 8) from NS rats and 187.94621.60 pA/pF from Prop-treated rats (n = 8). These data suggest an involvement of b-adrenoceptor mediated signaling in the suppression of I K induced by HIS in rats.

Administration of NE enhanced neuronal excitability of colon-specific DRG neurons
To further confirm the role of adrenergic signaling, we examined whether NE can increase neuronal excitability of colon innervating DRG neurons of healthy control rats. NE was freshly prepared at the concentration of 10 mM and added into the recording chamber for 3 minutes. Incubation of colon DRG neurons with NE significantly depolarized the resting membrane potential (Fig. 6A  NE incubation when compared with Pre-NE group ( Fig. 7A and B, for 100 pA, Pre: 0.7560.31, Post: 5.8361.42, n = 6, **p,0.01, paired sample t-test; for 300 pA, Pre: 6.0061.17, Post: 13.1762.56, n = 6, *p,0.05, paired sample sign test; for 500 pA, Pre: 16.7561.82, Post: 25.0062.85, n = 6, *p,0.05, paired sample t-test). NE treatment also significantly decreased the TTFP in response to a 100 pA (Fig. 7C, paired sample t-test), 300 pA (Fig. 7D, paired sample t-test) and 500 pA (Fig. 7E, paired sample t-test) ramp current injection. These data suggest that NE treatment enhanced the excitability of colon innervating DRG neurons of healthy control rats.

Intrathecal injection of NE produced visceral hypersensitivity
If NE generated endogenously contributes to the initiation and maintenance of visceral hypersensitivity in HIS rats, then application of exogenous NE to healthy rats should mimic the effects of HIS. In the present study, NE was intrathecally applied to healthy rats and behavioral responses were assessed. NE administration produced a significant increase in AWR scores at pressures of 20, 40, 60 and 80 mmHg 30 minutes after the injection (Fig. 8A, n = 10 for each group; *p,0.05 vs. NS group, Mann-Whitney test) and a significant decrease in distention threshold (Fig. 8B, n = 10 for each group, *p,0.05 vs. NS group, Mann-Whitney test). The AWRs and distention threshold returned to normal level 60 min after the injection (Fig. 8C and D, n = 10 for each group). Butoxamine administration blocked the NE effect ( Fig. 8E and F, n = 5 for each group; *p,0.05 vs. NE group, Mann-Whitney test). These data indicate that NE produced acute hyperalgesic effect in healthy rats, which was blocked by a b 2 -adrenoceptor antagonist.

HIS resulted in an elevation of NE concentration without alteration in expression of b 2 -adrenoceptors
We next measured the NE concentration in the blood plasma after HIS. The plasma concentration of NE significantly increased 6 hours after termination of the last stressor and return to base level 1 week after the last stressor (Fig. 9A, n = 4 for control and 6 hour group, n = 3 for 1 week group; **p,0.01 vs. CON, Tukey post hoc test following Kruskal-Wallis ANOVA). To determine whether expression of b 2 -adrenoceptor increases in T 13 -L 2 DRGs and in the muscularis externa of the distal colon after HIS, western Figure 5. Administration of propranolol increased voltage-gated potassium current. Currents were measured at different holding potentials. For recording total voltage-gated potassium current (I Total ), the membrane potential was held at 2100 mV and voltage steps were from 2 50 to +90 mV with10-mV increments and 400 ms duration. For recording the sustained K V current (I K ), the membrane potential was held at 250 mV and the voltage steps were the same as above. Currents generated by these two protocols were subtracted to produce I A . Bar graphs showed the mean peak I Total (A), I K (C), and I A (E) densities from control (CON), HIS, NS-and Prop-treated HIS (Prop) rats. The current density (pA/pF) was calculated by dividing the current amplitude by cell membrane capacitance. (A) HIS produced a significant reduction in I Total while Prop treatment caused a significant increase in I Total (*p,0.05, compared with CON, two sample t-Test; ##p,0.01, compared with NS). (B) HIS led to a significant decrease in I K while administration of Prop resulted in a marked potentiation in I K density (*p,0.05, compared with CON, two sample t-Test; ##p,0.01, blotting assay was performed. Anti-b 2 -adrenoceptor antibody labeled a 55 kDa molecular weight protein. Expression of b 2adrenoceptors in the DRGs 6 hours after end of the last stressor was not significantly changed when compared with controls ( Fig. 9B, n = 4 for each group, two sample t-test). In addition, expression of b 2 -adrenoceptors in the muscularis externa of the distal colon 6 hours after end of the last stressor was not significantly changed when compared with controls (Fig. 9C, n = 4 for each group, two sample t-test).

Discussion
In the present study, HIS induced visceral hypersensitivity to CRD lasted for 24 hours and returned to baseline by 48 hours in male SD rats after termination of the last stressor, which is consistent with our previous study [22]. By contrast, Winston et al. reported that HIS induced visceral hypersensitivity to CRD that lasted for at least 8 hours, and returned to baseline by 24 hours in male Wistar rats [6], indicating the mechanisms by which chronic stress leading to colonic dysfunction differs by animal species. Winston et al. reported that blockade of adrenergic signaling with a 1 /a 2 -and b 1 /b 2 -adrenoceptors before the daily application of stress prevented the induction of visceral hypersensitivity, but they didn't identify which type of adrenoceptors mainly mediates the prolonged induction of hypersensitivity in their model [6]. Our data provided additional evidence to confirm the idea that b 2adrenoceptor plays an important role in chronic stress-induced visceral hypersensitivity. This conclusion is based on the following observations. Firstly, we showed that HIS-induced visceral hypersensitivity was attenuated by non-selective b-adrenoceptor antagonist propranolol but not by non-selective a-adrenoceptor antagonist phentolamine (Fig. 1). Secondly, butoxamine, a selective b 2 -adrenoreceptor antagonist, significantly reversed visceral hyperalgesia in HIS-treated rats, in a dose-dependent manner (Fig. 2) while the selective b 1 -and b 3 -adrenoceptor antagonist did not produce any effect on visceral hyperalgesia in HIS-treated rats. Finally, nonselective antagonist of b 2 -adrenoceptor didn't produce any obvious effect in healthy control rats (data not shown), suggesting that butoxamine-induced effect was not a non-specific analgesic effect. This also suggested that the role of b 2 -adrenoceptor in signaling colonic distension may not be as important in healthy as in the sensitized state.
In addition to antagonism on pain behaviors, butoxamine treatment remarkably reduced excitability of colon innervating DRG neurons (Figs. 3 and 4). In agreement with our previous study [6], HIS significantly enhanced the excitability of colon specific DRG neurons, as was evidenced by our findings that HIS markedly reduced the rheobase and increased number of APs evoked by 2 and 3 times current stimulation, and by various amounts of ramp current stimulation. After butoxamine treatment, the rheobase was greatly increased and the number of APs evoked by 2 and 3 times current stimulation, and by 100, 300 and 500 pA ramp current stimulation was significantly decreased. Additionally, the time to first peak was also reversed in butoxamine-treated HIS rats. This raised a possibility that functions of voltage-gated Na + and/or K + channels may be modified following butoxamine treatment because these channels are major determinant factors for rheobase and repetitive firings. The present study showed that application of b-adrenoceptor antagonist significantly reversed the suppression of I K density caused by HIS in T 13 -L 2 DRGs (Fig. 5). The reduction in I K density induced by HIS may be a underlying mechanism for the enhanced excitability of DRG neurons, while the increase in I K density by application of b-adrenoceptor antagonist may well contribute to normalized excitability of DRG neurons. In addition to the effect of I K , the contribution of I A was also considered since I A has been reported to be one of the major players in control of neuronal excitability [26,28]. However, I A was not altered by HIS and propranolol in the present study. It is more likely that HIS plays a specific effect on I K . In addition to the suppression of I K , the present study cannot exclude the role of Na V channels since an increase in INa V also contributed to the increased excitability of T 13 -L 2 DRGs by HIS [22]. We have previously reported that the expression of cystathionine beta-synthetase (CBS) was upregulated by HIS [22] and that hydrogen sulfide (H 2 S), the product of CBS, suppresses I K of trigeminal neurons [29]. Further studies are warranted to determine the relationship between H 2 S pathway and adrenergic signaling and their roles in the modulation of I K in HIS model.
Another important finding is that HIS elevated the NE concentration in blood plasma without alteration of the expression of b 2 -adrenoreceptor. NE is one the most prominent mediators of stress response [30]. We measured the plasma concentration of NE to determine whether NE is a candidate to induce visceral hypersensitivity. In a line with previous report [5], the blood plasma level of NE remarkably increased by about 2-fold 6 hours after end of the last stressor. To further confirm the role of NE in the development of visceral hypersensitivity, additional experiments were performed. Firstly, i.t. injection of NE produced visceral hypersensitivity of healthy rats, which mimics the effect of HIS on visceral sensitivity. Secondly, incubation of colon innervating DRG neurons in vitro with NE greatly enhanced the excitability (Fig. 6). This conclusion was supported by our observations that NE significantly decreased the rheobase, increased the number of APs evoked by 2 and 3 times current stimulation, and by various ramp current stimulation. Together, these data suggest that NE mimics the effect of HIS on neuronal excitability. Of note is that the mechanism by which NE and HIS enhanced neuronal excitability and visceral hypersensitivity might differ to some degree. The acute application of NE is unlikely to increase channel expression. HIS, however, significantly enhanced expression of Nav 1.7 and Nav 1.8 of T 13 -L 2 DRGs [22]. NE elevated in the blood plasma and the colon wall could increase the expression of nerve growth factor in the colon wall, thus sensitizing primary afferents [6]. Nevertheless, our findings indicate a crucial role for NE signaling in the development of visceral hypersensitivity and in the hyperexcitability of colon-specific DRG neurons. The mechanism underlying the elevation of NE level is not clear. Several studies suggest that the hypothalamic-pituitary-adrenal  axis and the sympathetic system originating in the locus coeruleus are responsible for the elevated NE level [6,31]. It is also possible that NE reuptake transporter (NET) might play a role since NET was reported to be downregulated by chronic-acute combined stress [8]. Although the detailed mechanisms for an increase in NE concentration have yet to be investigated, our findings indicate that enhanced NE concentration might be the major contributor to visceral hypersensitivity following HIS.
Since only b 2 -adrenergic receptor antagonist attenuates the visceral hypersensitivity and reverses the neuronal hyperexcitability, we therefore assayed expression of b 2 -adrenergic receptors in rat DRGs and the colon after HIS. b 2 -adrenoceptors have been reported to exist widely both in central and in peripheral nervous system [32][33][34][35][36][37][38]. Since butoxamine could not cross blood brain barrier [39], the present study suggested an important role of peripheral b 2 -adrenoceptor activation in the development of HISinduced visceral hypersensitivity. In an effort to detect expression of b 2 -adrenoceptors, we showed that expression of b 2 -adrenoceptors was not altered in colon DRGs. It is possible that the expression of b 2 -adrenoceptors was not altered in DRGs, where the cell bodies locate, because b 2 -adrenoceptors after expression are transported to the peripheral terminals of DRGs. Therefore, we detected the expression of b 2 -adrenoceptors in the muscularis externa of the distal colon. We further confirm that the expression of b 2 -adrenoceptors in the muscularis externa of the colon was not altered. It is expected that function of b 2 -adrenoceptors would be enhanced in colon DRGs. Further experiments are warranted to investigate whether the function of b 2 -adrenergic receptors is enhanced after HIS.
In summary, these data provide evidence that NE elevated in blood plasma activates b 2 -adrenoreceptor to enhance the excitability of colon-specific DRG neurons, thus producing visceral hypersensitivity; Blockade of b 2 -adrenoreceptor reverses hyperexcitability and attenuates visceral hypersensitivity to colorectal distension after HIS. These findings suggest that b 2 -adrenoreceptor may be a potential target for novel agents for the treatment of visceral pain in patients with IBS and related disorders.

Author Contributions
Conceived and designed the experiments: GYX. Performed the experiments: CZ YYR YYZ ZJ. Analyzed the data: HZ CYH YX. Contributed reagents/materials/analysis tools: GYX. Wrote the paper: GYX YX HZ CYH.