Adenosine A1 receptor agonist induces visceral antinociception via 5-HT1A, 5-HT2A, dopamine D1 or cannabinoid CB1 receptors, and the opioid system in the central nervous system

We have recently demonstrated that N(6)-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, acts centrally to induce a visceral antinociception. Since serotonin (5-HT), cannabinoid (CB), dopamine or opioid signaling in the central nervous system is involved in the regulation of visceral sensation, we made a hypothesis that the signaling may play a role in the CPA-induced visceral antinociception. Visceral sensation was evaluated by colonic distension-induced abdominal withdrawal reflex (AWR) in conscious rats. Subcutaneously administered CPA significantly increased the threshold of colonic distension-induced AWR. Intracisternal injection of either 5-HT1A or 5-HT2A receptor antagonist blocked the CPA-induced visceral antinociception while 5-HT1B antagonist did not block the CPA-induced visceral antinociception. Subcutaneous injection of dopamine D1 receptor antagonist, CB1 receptor antagonist or naloxone significantly blocked the CPA-induced visceral antinociception while neither subcutaneous injection of dopamine D2 receptor antagonist nor CB2 receptor antagonist blocked the CPA-induced anti-pain action. These results suggest that 5-HT1A, 5-HT2A, dopamine D1, CB1 receptors and the opioid system in the CNS may specifically mediate the CPA-induced visceral antinociception. These findings may help in understanding the physiological relevance of central adenosine with special reference to the pathophysiology of altered visceral sensation especially in irritable bowel syndrome.


Introduction
Adenosine is a ubiquitous endogenous neuromodulator or neurotransmitter, which plays an important role in pain modulation [1]. It is known that spinal or systemic administration of adenosine and its analogs leads to antinociception in response to somatic pain in various animal models [1][2][3][4][5][6][7][8]. Although the effects of adenosine on antinociceptive responses have been widely studied, little is known whether it is also involved in the antinociceptive action related to visceral pain sensation. We have recently demonstrated that subcutaneous or intracisternal injection of N(6)-cyclopentyladenosine (CPA), an adenosine A1 receptor (A1R) agonist, induced an antinociceptive action against colonic distension in a dose-dependent manner in rats, where antinociception was observed when much smaller doses of CPA were administered centrally rather than peripherally, suggesting that adenosine acts in the central nervous system (CNS) to produce an antinociceptive action against colonic distension [9]. The antinociceptive effect of intracisternal CPA was blocked completely by the subcutaneous administration of a specific A1R antagonist, which indicates that the centrally injected CPA-induced antinociception was indeed mediated by A1Rs.
Increasing findings have suggested that visceral sensation is regulated by some molecules other than adenosine in the CNS. For instance, we have showed that dopamine, serotonin (5-HT), cannabinoid (CB) or opioid signaling in the CNS could also induce visceral antinociception in rats [10][11][12][13]. The present study was performed to further clarify whether dopamine, 5-HT, CB or opioid signaling may be involved in the mechanisms of the visceral antinociception by CPA.

Animals
Male Sprague-Dawley rats (Charles River Laboratory, Atsugi, Japan) weighing about 200 g were housed under controlled light/dark conditions (lights on: 07:00-19:00), and the room temperature was regulated at 23°C-25°C. Rats were allowed free access to standard rat chow (solid rat chow; Oriental Yeast Co., Tokyo, Japan) and tap water. All of the experiments were performed using conscious animals, which had been deprived of food for 24 h but with free access to water until the initiation of the experiments.

Implantation of electrodes and placement of colorectal balloon
The electrodes used to obtain electrophysiological measurements of abdominal muscle contractions were implanted acutely on the day of the experiment, as described previously [14,15]. Briefly, a skin incision measuring 5 mm was created while the rats were under isoflurane anesthesia. The electrodes (Teflon-coated stainless steel, 0.05 mm diameter; MT Giken, Tokyo, Japan) were inserted approximately 2 mm into the left side of the external oblique musculature through the incision and fixed to the incised skin with cyanoacrylate instant adhesive (Aron Alpha, Toagosei, Tokyo, Japan). The electrode leads were externalized through this closed incision and threaded through a urethane tube. Immediately after implanting the electrodes, a distension balloon was inserted intraanally where the distal end was positioned 2 cm proximal to the anus. A 6-Fr (2 mm external diameter) disposable silicon balloon-urethral catheter for pediatric use (JU-SB0601; Terumo, Tokyo, Japan) was employed. The maximal inflation volume of the balloon was 1.5 ml, and the length of the maximally inflated balloon was 1.2 cm. The balloon was secured in place by taping the catheter to the tail.

Detection of visceral sensitivity
The abdominal withdrawal reflex (AWR) test was performed as described previously to detect the pain threshold, which was defined as the intensity of colorectal distension that elicited AWR [16]. Briefly, colonic distension was performed as described previously [14,15], that is, an ascending method for limited phasic distension was applied by inflating the balloon manually with water using a syringe until the AWR was detected by the EMG. After completing the surgery for electrode implantation and balloon placement, the sedated rats were placed in Bollman cages where they were allowed to recover and adjust for 20 min before testing. Next, the electrode leads were connected to a custom-made EMG amplifier. The EMG signals were amplified, filtered (3,000 Hz) and digitized using a PowerLab system and recorded using computer software (LabChart 7). The pain threshold was assessed two times (2-min interval), and the mean of the threshold was calculated as the results for the animals. In the majority of animals, the pain threshold in the first test was consistent with that in the second test.

Experimental procedures
Initially, to clarify whether the 5-HT signaling is involved in the CPA-induced antinociceptive action against colonic distension, we examined the effect of intracisternal injection of either WAY100635, 5-HT 1A receptor antagonist, isamoltane, 5-HT 1B receptor antagonist or ketanserin, 5-HT 2A receptor antagonist at a dose of 1 μg/10 μl on the subcutaneously administered CPA (0.2 mg/rat)-induced visceral antinociception. Intracisternal injection was performed under brief isoflurane anesthesia using a 10-μl Hamilton microsyringe after the rats were mounted in a stereotaxic apparatus (David Kopf Instruments, Tijunga, CA, USA), as reported previously [17]. Next, to clarify whether the dopamine, cannabinoid or opioid signaling is involved in the CPAinduced antinociceptive action against colonic distension, we examined the effect of the subcutaneous injection of SCH23390 (0.2 mg/rat), sulpiride (40 mg/rat), naloxone hydrochloride (0.5 mg/rat), AM251 (0.5 mg/rat) or AM630 (0.5 mg/rat) on the CPA-induced antinociceptive action against colonic distension. We selected the doses of WAY100635, isamoltane or ketanserin [12,[18][19][20], and AM251, AM630, SCH23390, sulpiride or naloxone hydrochloride according to previous studies [10,11,13,15]. In the previous studies, we have clearly demonstrated that the same dose of WAY100635, isamoltane, ketanserin, AM251, AM630, SCH23390, or sulpiride alone failed to change the AWR threshold in the same experimental setting [10][11][12]. Following subcutaneous and/or intracisternal injection, the rats were implanted with electrodes, and the balloons were inserted, after which the rats were moved into Ballman cages to evaluate the AWR threshold volume as described above. In rats that received subcutaneous and intracisternal injections, the procedures on each rat were performed within 5 min.

Statistical analysis
The data were expressed as means ± standard error (SE). The data were compared with the one-way analysis of variance followed by Dunnett's multiple comparisons test. P < 0.05 was considered statistically significant.

Results
We examined the effect of intracisternal injection of 5-HT receptor antagonists on the subcutaneously administered CPA-induced visceral antinociception. As shown in Fig. 1, subcutaneously administered CPA at a dose of 0.2 mg significantly increased the AWR threshold volume. Either intracisternal WAY 100635, 5-HT 1A receptor antagonist or ketanserin, 5-HT 2A receptor antagonist significantly blocked the CPA-induced visceral antinociception while intracisternal isamoltane, 5-HT 1B receptor antagonist failed to change the AWR threshold volume (Fig. 1), suggesting that 5-HT 1A and 5-HT 2A receptor signaling may specifically mediate the CPA-induced visceral antinociception.
To identify whether CB signaling may be involved in the CPA-induced visceral antinociception, we examined the effect of subcutaneous injection of either AM251, CB 1 receptor antagonist or AM630, CB 2 receptor antagonist on the subcutaneously administered CPA-induced visceral antinociception. As shown in Fig. 2, AM251 but not AM630 potently blocked the CPA-induced visceral antinociception, suggesting that CB 1 receptor signaling may be implicated in the CPA-induced visceral antinociception while CB 2 receptor could not mediate the CPAinduced visceral antinociception.
The effect of subcutaneous injection of either SCH23390, dopamine D 1 receptor antagonist, or sulpiride, dopamine D 2 receptor antagonist on the subcutaneously administered CPA-induced visceral antinociception was examined to identify whether dopamine signaling may be involved in the CPA-induced visceral antinociception, we examined SCH23390 but not sulpiride potently blocked the CPA-induced visceral antinociception (Fig. 3), suggesting that dopamine D 1 receptor signaling may be implicated in the CPA-induced visceral antinociception while dopamine D 2 receptor signaling could not mediate the CPA-induced visceral antinociception.
To clarify whether the opioid system is involved in the CPA-induced antinociceptive action during colonic distension, the effect of naloxone was evaluated. As shown in Fig. 4, naloxone by itself did not change the threshold of colonic distension-induced AWR as shown in our recent   publication [15]. The antinociceptive effects of subcutaneous CPA was significantly reversed by subcutaneously injected naloxone, indicating that the opioid system is involved in the CPA-induced visceral antinociception.

Discussion
We have recently showed that intracisternal injection of ketanserin, a 5-HT 2A atagonist, but not WAY-100635, 5-HT 1A receptor antagonist or isamoltane, 5-HT 1B receptor antagonist blocked the levodopa-induced visceral antinociception [12], suggesting that 5-HT 2A receptors in the CNS, especially the spinal cord specifically plays a role in the visceral antinociception by levodopa. The present findings also showed that 5-HT 2A receptor antagonist potently blocked the CPA-induced visceral antinociception. These results indicate that 5-HT 2A receptors in the CNS are involved in the visceral antinonociception by not only levodopa but also activation of A1R signaling. With regard to the 5-HT 1A receptors in the CNS, Barriere et al. [21] have demonstrated that intrathecal injection of 5-HT 1A receptor antagonist, WAY-100635, prevented the antinociceptive action of 4-aminophenol, suggesting that 5-HT 1A receptors in the spinal cord plays a role in the pain control. The 5-HT 1A receptor antagonist significantly attenuated the CPA-induced visceral antinociception as shown in the present study. We would therefore speculate that not only 5-HT 2A but 5-HT 1A receptors in the spinal cord may be implicated in the CPA-induced visceral antinociception. A couple of papers have demonstrated a tight relationship between 5-HT 2A or 5-HT 1A receptor, and adenosine. For instance, the involvement of 5-HT 1A receptors in the antidepressant-like effect of adenosine was reported in the mouse forced swimming test [22]. Stutzmann et al. [23] have demonstrated adenosine preferentially suppresses 5-HT 2A receptor-enhanced excitatory postsynaptic currents in layer V neurons of the rat medial prefrontal cortex. These findings may support the functional relation between adenosine and 5-HT 1A and 5-HT 2A receptors in the CPA-induced visceral antinociception.
As shown in this study, CPA-induced visceral antinociception was blocked by dopamine D 1 receptor antagonist but not dopamine D 2 receptor antagonist, suggesting that dopamine D 1 receptors may be involved in the A1R-mediated visceral antinociception. We have recently showed that either central injection of A1R agonist or dopamine D 1 receptor agonist increased the AWR threshold volume against colonic distension in rats, suggesting that adenosinergic or dopamine D 1 receptor signaling is capable of inducing visceral antinociceptive action. With regard to the relationship between adenosine and dopamine D 1 receptor signaling, adenosine-dopamine interaction in the pathophysiology of CNS disorders has been established [24]. Yabuuchi et al. [25] have demonstrated a role of A1Rs in the modulation of dopamine D 1 receptor signaling in the neostriatum. According to Hobson et al. [26], A1R and dopamine D 1 receptor regulate cocaine-seeking behavior in mice. Thus, there is a functional relationship between A1Rs and dopamine D 1 receptors in the CNS. Based on the findings, the present results might support an existence of functional relationship between A1R and dopamine D 1 receptors to regulate visceral sensation.
The CB system plays a role in the regulation of visceral pain sensation. We have very recently demonstrated that intraperitoneal injection of either CB 1/2 receptor agonist, WIN 55212 or O-Arachidonoyl ethanolamine increased the threshold volume of colonic distension-induced AWR in rats, suggesting CB could induce visceral antinociception [10]. Pretreatment with either the CB 1 or CB 2 receptor antagonist potently blocked the centrally injected orexin-A-induced antinociceptive action against colonic distension [10], suggesting that endogenous CB may be involved in the central orexin-induced antinociceptive action through the CB 1 or CB 2 receptors. Thus, the CB system plays a role in the regulation of visceral sensation through the specific receptors, CB 1 or CB 2. We have examined the roles of CB receptors in the CPA-induced visceral antinociception and showed that CB 1 but not CB 2 receptor antagonist significantly blocked the CPA-induced visceral antinociception, suggesting that CB 1 receptors are involved in the visceral antinociceptive action by CPA. Interaction between A1R and CB 1 receptors has been reported in in vivo studies, where an A1Rmediated enhancement of cannabinoid CB 1 receptor-induced impairment of short-term spatial memory and motor incoordination were observed [27,28]. In addition, Serpa et al. [29] have shown the combined inhibitory effect of the A1R and cannabinoid CB1 receptors on cAMP accumulation in the hippocampus, suggesting a functional interaction of adenosine and CB1 receptors in the CNS. These findings may support the functional relation between adenosine and CB1 receptors in the CNS to evoke the CPA-induced visceral antinociception. In order to assess the possible involvement of the opioid system in the CPA-induced antinociceptive effect during colonic distension, the opioid antagonist, naloxone hydrochloride was administered. The antinociceptive action during colonic distension of CPA was reversed by subcutaneous injection of naloxone hydrochloride, indicating that the CPA-induced visceral antinociceptive action is mediated by the opioid system. A couple of reports have shown the close relation between adenosine and opioid pathways. Demirkapu et al. [30] have demonstrated that administration of the A1R agonist significantly decreased opioid-withdrawal behaviors. Liao et al. [31] have shown electroacupuncture attenuates induction of inflammatory pain by regulating opioid and adenosine pathways in mice. Such tight relationship between adenosine and opioid pathways might play a role in the CPAinduced visceral antinociception.
Whether adenosine A1 signaling is involved in related receptors directly or indirectly to induce visceral antinociception is not clarified by the present limited pharmacological experimental data. Although the present study could not identify the interactive sites of action of molecules between adenosine A1 signaling and receptors such as 5-HT, dopamine, cannabinoid and opioid, we might be allowed to speculate a possible mechanism by which adenosine A1 signaling induces visceral antinociception (Fig. 5). Earlier investigators have demonstrated that spinal 5-HT 1A [32,33] or 5-HT 2A receptors [34,35] play an antinociceptive role. We would therefore suggest that 5-HT 1A or 5-HT 2A receptors in the spinal cord may be involved in the visceral antinociception by adenosinergic signaling. Immunohistochemical [36] and electrophysiological [37] studies suggested that adenosine A 1 receptors localize in the dopamine-containing neurons in the ventral tegmental area (VTA). Since dopamine D1 receptor in the VTA is involved in the descending pain control system [38], dopamine D1 receptor in the VTA may mediate the visceral antinociception by adenosine A1 receptor activation. A well-characterised descending pathway originates within the periaqueductal grey (PAG) and projects to the spinal dorsal horn via the rostral ventromedial medulla (RVM) [39]. Activation of this descending system, either from within the PAG, RVM or higher centers, elicits analgesia by inhibiting ascending nociceptive transmission at the spinal cord level. Opioids and cannabinoid agonists influences PAG and RVM neurons via presynaptic μ-opioid and CB 1 receptors, respectively [40][41][42][43]. Considering the findings, we would suggest that opioid and CB1 receptor in the PAG-RVM-spinal pathway may play a role in regulation of visceral sensation by adenosinergic signaling. A possibility of interaction of receptors such as relation between 5-HT receptors and dopamine D1 receptor to induce antinociception could not be excluded. Further studies should be needed to clarify the issues.
Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal tract [44,45]. Brain-gut interaction plays an important role in the pathophysiology of IBS. Visceral hypersensitivity to mechanical distension of the gut is related to symptom severity and it is considered to play a role in the pathophysiology of IBS [33]. However, a therapeutic strategy to improve visceral hypersensitivity in IBS has not been established. In other words, we are able to provide a novel therapeutic option if we could clarify mechanisms in the CNS of visceral hypersensitivity and control it. Based on the present study, we would therefore suggest that activation of adenosine A 1 signaling through 5- HT 2A, 5-HT 1A , dopamine D 1 or CB 1 receptors, or opioid signaling in the CNS should be considered to be a novel therapeutic target for IBS patients. These findings help us understand the pharmacological property of CPA and the pathophysiology of visceral hypersensitivity especially in IBS.

Conflicts of interest
We have no conflicts of interest to declare.