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doi: 10.1152/japplphysiol.01282.200498:2056-2063, 2005. First published 13 January 2005;J Appl Physiol

Melissa M. Crisostomo, Peng Li, Stephanie C. Tjen-A-Looi and John C. Longhurstratsinhibition of cardiovascular reflex excitatory response inNociceptin in rVLM mediates electroacupuncture

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Nociceptin in rVLM mediates electroacupuncture inhibition of cardiovascular

reflex excitatory response in rats

Melissa M. Crisostomo, Peng Li, Stephanie C. Tjen-A-Looi, and John C. Longhurst

Department of Medicine, Susan Samueli Center for Integrative Medicine,

College of Medicine, University of California, Irvine, CA 92697-4075

Submitted 15 November 2004; accepted in final form 12 January 2005

Crisostomo, Melissa M., Peng Li, Stephanie C. Tjen-A-Looi,and John C. Longhurst. Nociceptin in rVLM mediates electroacu-puncture inhibition of cardiovascular reflex excitatory response inrats. J Appl Physiol 98: 20562063, 2005. First published January 13,2005; doi:10.1152/japplphysiol.01282.2004.Electroacupuncture(EA) at Neiguan-Jianshi acupoints through an opioid mechanisminhibits the cardiovascular pressor response induced by mechanicalstimulation of the stomach. Because nociceptin also may regulatecardiovascular activity through its action in the brain stem, wehypothesized that this neuromodulator serves a role in the EA-relatedinhibitory effect. Blood pressure in ventilated male Sprague-Dawley

rats (400 600 g) anesthetized by ketamine and -chloralose wasmeasured during balloon inflation of the stomach. Gastric distensionwith 68 ml of air induced consistent pressor reflexes of 26 1mmHg that could be repeated every 10 min for 100 min. Whennociceptin (10 nM) was microinjected into the rostral ventrolateralmedulla (rVLM), the pressor response induced by gastric distensionwas inhibited by 68 6%. Thirty minutes of EA also decreased thereflex response by 75 11%; microinjection of saline into the rVLMdid not alter the inhibitory effect of EA. In contrast, microinjection ofa nociceptin receptor antagonist into the rVLM promptly reversed theEA response. Pretreatment with the opioid receptor antagonist nalox-one did not influence the EA-like inhibitory effect of nociceptin on thedistension-induced pressor reflex (22 1 to 8 2 mmHg). Further-more, a -opioid receptor agonist microinjected into the rVLM aftermicroinjection of a nociceptin receptor antagonist during EApromptly reversed the nociceptin receptor antagonist-related inhibi-tion of the EA effect. Thus, in addition to the classical opioid system,nociceptin, through opioid receptor-like-1 receptor stimulation in therVLM, participates in the modulatory influence of EA on reflex-induced increases in blood pressure.

opioids; gastric distension; somatic afferent

RECENTLY, A NOVEL OPIATE-LIKE heptadecapeptide was identifiedin rat brain extracts (41, 46). The peptide, called nociceptin(41) or Orphanin FQ (46), has a high amino acid sequencehomology to classical endogenous opioid peptides (i.e., endor-phins, enkephalins, endomorphins, and dynorphins) and espe-

cially to dynorphin A. Furthermore, amino acid sequencing ofthe receptor believed to be specific for nociceptin, or opioidreceptor-like-1 (ORL-1), was found to be 4750% identical tothat of the coding sequences of -, -, and -opioid receptors(10, 42, 46).

Nociceptin and ORL-1 receptors are reported to be abun-dantly distributed in neurons throughout the central nervoussystem (CNS), including the rostral ventrolateral medulla(rVLM), sensory trigeminal complex, raphe nuclei, locus cer-

uleus, periacqueductal gray, amygdala, and hypothalamic andseptal regions (1, 29, 30, 42, 44, 49). These locations suggestthat the nociceptinergic system may play a role in modulationof physiological activities, such as regulation of pain andautonomic outflow, much like the classical endogenous opioidsystem.

For centuries, acupuncture has been used by Eastern culturesto treat cardiovascular diseases such as hypertension, hypoten-sion, arrhythmias, and conditions associated with cardiac orlimb ischemia. In recent years, Western society has demon-

strated a heightened interest in this treatment modality. In1993, Eisenberg et al. (22) reported that one in three individ-uals obtained acupuncture or another alternative therapy. Aconsensus conference was convened in 1997 by the NationalInstitutes of Health to address the efficacy and biologicaleffects of acupuncture (42a). This conference concluded thatacupuncture was useful as a complementary or alternativetreatment to conventional therapy for pain, nausea, and vom-iting and possibly for cardiovascular disease such as myocar-dial ischemia, arrhythmias, and hypertension. Additionally, theconference reported that acupuncture was being widely prac-ticed by thousands of health practitioners for the managementof pain and other health conditions. Although the medicalcommunity is beginning to recognize the value of acupunctureas a viable form of therapy, there is still much to be learnedabout its mechanisms of action.

Studies have begun to elucidate the mechanisms of acupunc-ture with regard to its therapeutic effects in cardiovasculardisorders (4, 6, 7, 15, 24, 31, 34, 38, 47, 53). For example, ithas been suggested that the influence of electroacupuncture(EA) on the cardiovascular system is mediated, at least par-tially, by the release of endogenous opioid peptides, which actas neurotransmitters that modulate the activity of premotorsympathoexcitatory cells in the brain stem medulla (15, 34, 53,54). Our laboratory previously reported that EA modulates thereflex cardiovascular response induced by chemical and elec-trical stimulation of visceral afferent nerves in cats (15, 33, 34,

53, 54). In these studies, we noted that EA improves ischemicmyocardial dysfunction (15, 34) by reducing demand-inducedischemia (33, 34). These responses are mediated through inhi-bition of autonomic reflex responses (34) specifically throughthe influence of EA on cardiovascular premotor sympatheticneuronal discharge in the rVLM (53, 54). In these studies, wedemonstrated that the cardiovascular effects of EA were me-diated by endogenous opioids and that an important site ofaction was the rVLM.

Address for reprint requests and other correspondence: J. C. Longhurst,Medical Science 1, C240, College of Medicine, Univ. of California, Irvine, CA92697-4075 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Appl Physiol 98: 20562063, 2005.First published January 13, 2005; doi:10.1152/japplphysiol.01282.2004.

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Nociceptin appears to regulate cardiovascular activity byinfluencing the peripheral nervous system (2, 13, 14) and CNS(13, 14, 1719). Like classical opioids, nociceptin inhibitscardiovascular activity (18), decreases cardiac output and totalperipheral resistance (13), and inhibits neuronal activity in therVLM (19). It is possible that nociceptin in the CNS alsocontributes to the effects of EA. Nociceptin could function

through the opioid system or in parallel with opioids to evokeinhibitory cardiovascular effects of EA. At present, there is noevidence to implicate the endogenous nociceptinergic systemin the rVLM in the acupuncture-induced inhibition of thecardiovascular sympathoexcitatory reflexes.

In the present study, we hypothesized that, like the opioidsystem, nociceptin in the rVLM modulates cardiovascularreflex responses during visceral afferent stimulation. We fur-ther hypothesized that nociceptin works independently fromthe classical opioid system. To investigate these hypotheses, ina model of gastric distension-induced reflex cardiovascularexcitatory responses, we utilized percutaneous EA and block-ade and stimulation of the classical opioid system in the rVLMbefore and after pharmacological manipulation of the nocicep-tin system. A preliminary report of this work has been pre-sented (20).

METHODS

Surgical Preparations

Experimental preparations and protocols were reviewed and ap-proved by the Institutional Animal Care and Use Committee of theUniversity of California, Irvine, CA. The study conformed to theAmerican Physiological Societys Guiding Principles for ResearchInvolving Animals and Human Beings (American PhysiologicalSociety, 2002). Studies were performed on adult Sprague-Dawleymale rats (400600 g). After an 18-h overnight fast, anesthesia wasinduced with ketamine (100 mg/kg im) and was followed by -chlor-

alose (5 mg/kg iv). Additional doses of -chloralose were given asnecessary to maintain an adequate level of anesthesia, as determinedby the lack of response to noxious toe pinch, a respiratory pattern thatfollowed the respirator, as well as a stable blood pressure and heartrate (HR). The right or left jugular vein was cannulated for adminis-tration of fluids. The trachea was intubated, and the animals wereartificially ventilated with a respirator (model 661, Harvard Appara-tus). The right or left carotid artery was cannulated and attached to apressure transducer (P23XL, Ohmeda) to monitor arterial blood pres-sure. HR was derived from the pulsatile blood pressure signal. Arterialblood gases and pH were measured periodically with a blood-gasanalyzer (ABL5, Radiometer America) and were kept within normalphysiological limits (PCO2 3040 Torr, PO2 100 Torr) by adjustingventilatory rate or volume and enriching the inspired O2 supply.Arterial pH was maintained between 7.35 and 7.43 by infusion of a

solution of 8% sodium bicarbonate. Body temperature was keptbetween 36 and 38C with a heating pad.

A 2-cm-diameter (unstressed dimension) latex balloon (Traub) wasattached to a polyurethane tube (3-mm diameter) that was insertedinto the stomach through the mouth and esophagus (31). The balloonwas manually palpated during insertion as it passed through theesophagus into the stomach to verify positioning of the balloon insidethe stomach. A syringe was attached to the cannula to inflate anddeflate the balloon with air, while a manometer through a T-connec-tion was used to monitor balloon pressure. Transmural pressure wasdetermined by measuring the pressure required to inflate the balloonwith the various volumes of air before it was inserted into thestomach. The balloon then was inflated inside the stomach, andpressure was recorded.

Experimental Procedures

Transmural pressure was calculated as the difference in pressure ofthe balloon inside and outside the stomach at corresponding volumes.Volumes ranging between 6 and 8 ml of air were injected into thestomach of each animal, which generated transmural pressures of11 1 mmHg. Injection volumes were selected to achieve transmuralpressures 20 mmHg, a value that has been reported by others to be

below the pain threshold (4) in rats (52). These distension pressuresfall within the range that a rat normally experiences during ingestionof food and fluids in a single meal (21).

Animals were placed in a stereotaxic head frame, and their headswere positioned such that the floor of the fourth ventricle washorizontal. A partial occipital craniotomy was performed to exposethe dorsal medulla. A three-barrel pipette (A-M Systems, Everette,WA; total outer diameter of the multibarrel tip, 4560 m) wasinserted unilaterally (side chosen randomly) into the medulla at anangle of 90 relative to the dorsal surface of the medulla, 1.5 mmlateral from the midline and 1.8 mm rostral to the obex, and wasadvanced 3.2 mm from the dorsal surface toward the ventral surface.The pipette was positioned relative to the obex (interaural 4.3 mm)using landmarks, as depicted in the atlas of Paxinos and Watson (45).These coordinates provide access to a region in the rVLM that has

been found by others to contain premotor sympathoexcitatory cells(25). An injection cannula and microsyringe (Hamilton) fixed to oneof the barrels of a pipette were used to administer a small dose ofDL-homocysteic acid (DLH, 34 nM, 100 nl) to identify cardiovas-cular pressor sites at the beginning of each experiment. Each siteinitially was confirmed by observing a small increase in blood pres-sure (510 mmHg) without damaging the neuron (39). In controlexperiments, one of the micropipette barrels was filled with saline,whereas the remaining two barrels were filled with either DLH or dye.

At the end of each experiment following administration of druginto the medulla, the injection site was marked with an injection of100 nl of Chicago Sky Blue dye (5% in 0.5 M sodium acetate).Thereafter, rats were euthanized with intravenous saturated KCl underdeep anesthesia. The stomach then was exposed to verify the locationof the balloon. Only animals in which the balloon was observed to be

fully within the stomach were used for data analysis. The brain stemwas removed and fixed in 4% paraformaldehyde and 20% sucrose forat least 4 days. Frozen 60-m coronal sections were cut with acryostat microtome (CM 1850, Leica) and then stained with 1%neutral red in acetate buffer with 2% acetic acid, pH 4.8. To confirmthe injection sites histologically, dye spots were identified accordingto the atlas of Paxinos and Watson (45). Diffusion distances for dyespots ranged between 80 and 100 m.

Chemicals

All experimental drugs were purchased from Sigma Aldrich (St.Louis, MO). Drugs were dissolved in normal saline at room temper-ature to an initial concentration of 1 mg/ml. The stock solutions werestored in a freezer and used within 2 wk, after which a fresh stocksolution was prepared. On the day of the experiment, appropriateserial dilutions were made to obtain desired concentrations. Concen-trations of the DLH and -opioid agonist {[D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin acetate salt (DAGO)} were 34 and 612 nM, respec-tively. The concentration of the final solution of nociceptin, naloxone,and nociceptin receptor antagonist was 10 nM for each 100-nl micro-injection into the rVLM.

Experimental Protocols

Following the surgical procedure, we allowed a 30- to 60-minperiod of stabilization before beginning the experimental protocol.Gastric distension was induced by inflating the balloon slowly within10 s and was held for an additional 10 s, and then the injected air wasslowly withdrawn from the balloon. We typically noted an increase in

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systemic arterial blood pressure within 510 s following inflation.After the maximal cardiovascular pressor response, air was withdrawnfrom the balloon. Ten-minute recovery intervals prevented attenuationof the cardiovascular responses (31).

Protocol 1A: responses to repeated gastric distension. In the timecontrol group, rats were subjected to 10 repeated periods of gastricdistension without EA. The total time over which the stomach wasrepeatedly stimulated lasted 100 min.

Protocol 1B: response to nociceptin antagonist in rVLM. Weexamined the effect of nociceptin antagonist on the reflex pressorresponses to gastric distension. Nociceptin was delivered unilaterallyinto the rVLM 5 min after the second gastric distension, followed bythree more stimulations. In addition to determining the medullaryinfluence of nociceptin in the gastric distension reflex in the absenceof EA, this protocol served as a volume control for injections in therVLM.

Protocol 2A: response to nociceptin in rVLM. After recording tworepeatable control responses to gastric distension, 0.1 l nociceptin(10 nM) was microinjected into the rVLM, in the absence of EA,followed by an 80-min period during which the reflex responses togastric distension were examined repeatedly. Thus the stomach wasinflated at 10-min intervals over a period of 100 min, two timesbefore, and eight times after microinjection of nociception.

Protocol 2B: influence of naloxone on nociceptin response. Similarto protocol 1, animals were instrumented to allow microinjection intothe rVLM. After two repeatable control responses to gastric distensionwere recorded, naloxone (10 nM, 0.1 l), which we have shownblocks opioid receptors (31), was microinjected unilaterally into therVLM. After 5 min, the response to gastric distension was recorded.Five minutes after gastric distension, nociceptin was microinjected atthe same site in the rVLM, followed by a 70-min period during whichthe reflex responses to repeated gastric distension were recorded. Thecontrol group in experiment 1B served as control for this protocol.

Protocol 3A: influence of EA on cardiovascular response to gastricdistension, microinjection of vehicle in rVLM. After recording tworepeatable pre-EA control responses to gastric distension, percutane-ous EA (12 mA, 2 Hz, 0.5-ms duration) was performed bilaterallywith 32-gauge stainless steel acupuncture needles (Suzhou Medical

Appliance) at the Neiguan-Jianshi acupoints (over median nerveabove the paw, pericardial meridian, P 56) for 30 min in rats (16,35). Needles were inserted perpendicularly to a depth of 13 mm.Correct positioning of the needles at the acupoint was confirmed byobserving slight repetitive paw twitches during stimulation, indicatingstimulation of motor fibers in the median nerve (15, 31, 33). Twenty-five minutes after beginning EA, a saline injection, which functionedas a control for the effects of microinjection, was administeredunilaterally into the rVLM. During EA and for 50 min after itstermination, responses to gastric distension were recorded. The 50-min period after EA was found to be adequate for recovery from theinhibitory effects of EA on the reflex pressor response. Responses togastric distension were induced at 10-min intervals for a total of 100min, including two before, three during, and five after EA.

Protocol 3B: effect of nociceptin receptor antagonists on EA

response. The ORL-1 receptor antagonists [Phe1-(CH2-NH)-Gly2]-nociceptin(113)-NH2 (Sigma), [N-Phe

1]-nociceptin(113)NH2 (Sigma)(10 nM, 0.1 l), or an equal volume of saline was microinjectedunilaterally into the rVLM. At the time of commencement of thestudy, only [Phe1-(CH2-NH)-Gly

2]-nociceptin(1-13)-NH2 wasavailable commercially. When the more potent antagonist [N-Phe1]-nociceptin(1-13)NH2 became available and its activity was verified tolack agonist effects (12), we incorporated [N-Phe1]-nociceptin(1-13)NH2 into this protocol. Because we observed similar responses toboth antagonists, we combined these data in this study. Two repro-ducible pre-EA control values were obtained followed by 30 min ofEA at the Neiguan-Jianshi acupoints. Twenty-five minutes after be-ginning EA, the nociceptin antagonist was administered unilaterallyinto the rVLM. Distension-response measurements were taken every

10 min for a total of 130 min, including two before, three during, andeight after EA.

Protocol 3C: effect of -opioid agonist on nociceptin antagonistresponse during EA. The -opioid agonist DAGO was diluted to aconcentration of 612 nM (51). Similar to protocols 1B, 2, 3A, and3B, animals were instrumented for microinjection into the rVLM.Two reproducible pre-EA control values were obtained followed by30 min of EA at the Neiguan-Jianshi acupoints. Twenty-five minutes

after the onset of EA, the nociceptin antagonist [N-Phe1]-nocicep-tin(1-13)NH2 was administered unilaterally into the rVLM. After 5min, the response to gastric distension was recorded. Five minuteslater, DAGO was microinjected into the same side of the rVLM,followed by repeated gastric distensions during a 70-min recoveryperiod. Thus the stomach was inflated at 10-min intervals over aperiod of 120 min, four times before and one time after microinjectionof the nociceptin antagonist, and seven times after microinjection ofDAGO.

Statistical Analysis

Data are presented as means SE. Change in mean arterialpressures of distension responses before, during, and after EA and afterdelivery of saline and experimental drugs were compared by one-way

repeated-measures analysis of variance and post hoc by the Student-Newman-Keuls test. All statistical analyses were performed with thesoftware package Sigma Stat (Jandel Scientific). The 0.05 probabilitylevel was used to determine statistically significant differences.

RESULTS

Responses to Repeated Gastric Distension

Ten repeated gastric distensions caused consistent pressorresponses over a 100-min period in six rats (Fig. 1A). In theabsence of EA, the pressor response was constant, averaging26 1 mmHg during the duration of the protocol.

Response to Nociceptin Antagonist in rVLM

Microinjection of a nociceptin antagonist did not alter thepressor responses in four rats (Fig. 1B), indicating that 100 nlof volume injection into the rVLM do not alter the reflexes, andthe reflex response is not mediated by nociceptin in the rVLM.

Gastric Distension Response to Nociceptin in rVLM

The gastric distension-induced pressor response of 19 3mmHg was reduced to 6 2 mmHg by nociceptin adminis-tered unilaterally into the rVLM in six rats, representing a 68%change. Original blood pressure tracings are displayed in Fig.2A. Inhibition lasted for 60 min before returning to 15 2mmHg, 80 min after microinjection of nociceptin (Fig. 2A).Injection of nociceptin did not alter HR or resting blood

pressure before gastric distension.Influence of Opioid Antagonism on Nociceptin Response

The magnitude of the pressor responses induced by gastricdistension was unaltered by naloxone microinjected unilater-ally into the rVLM. However, in the presence of opioidreceptor blockade, nociceptin in the rVLM promptly reducedthe pressor response by 64% to 8 2 mmHg (Fig. 2B) in sevenanimals. Inhibition lasted for 40 min before returning to 22 1 mmHg 60 min after nociceptin. Injection of nociceptin didnot change the HR responses or baseline blood pressure beforegastric distension. Resting arterial pressure and HR remainedconstant throughout this protocol.

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Effect of EA on Distension of the Stomach, Influence of

Saline Microinjected in rVLM

The inhibitory effect of low-frequency EA at P 56 wasevaluated in five animals (Fig. 3A). Although the baselineblood pressures showed some fluctuation, blood pressuresbefore the pressor responses were not significantly different.HR also was not significantly altered during the several hoursof the experiment. Before EA, the pressor responses weresimilar to the time control groups, averaging 22 3 and 20 4 mmHg, respectively. After 30 min of EA, the pressor reflexduring distension of the stomach was reduced to 5 4 mmHg(75%), a response that persisted for 50 min after termination ofEA. Microinjection of saline into the rVLM did not alter theinhibitory effect of EA.

Influence of Nociceptin Antagonist on EA Response

EA at P 56 inhibited the reflex response to gastric disten-sion from 19 2 mmHg before compared with 7 1 mmHgduring EA in seven rats. Blood pressure tracings of an indi-vidual animal demonstrate the inhibitory modulation by EA ofthe pressor reflex as well as the effect of a nocieptin antagonistduring EA (Fig. 3B). Unilateral microinjection of a nociceptinantagonist into the rVLM immediately following EA promptlyreversed the pressor response to 16 3 mmHg, a value notdifferent than the response during the pre-EA control period,but one which was significantly higher than the response to EAbefore administration of the nociceptin antagonist (Fig. 3B).

Resting blood pressure and HR remained constant throughoutthe protocol.

Response of Nociceptin Antagonism to -Opioid Agonist

During Effect of EA

EA inhibited the reflex response to gastric distension from

21 1 to 7 1 mmHg in this group of five rats. Unilateralinjection of a nociceptin antagonist into the rVLM promptlyreversed the pressor response to 18 1 mmHg (Fig. 3C).Subsequent administration of the -opioid agonist DAGO atthe same site in the rVLM neutralized the antagonists effects,noted by a prompt return of the reduced pressor response of7 1 mmHg. This inhibition lasted 20 min, increasing to 12 2 mmHg after 30 min. Injection of DAGO did not change theHR responses during gastric distension. Resting mean arterialpressure and HR were unchanged throughout this protocol.

Fig. 2. A: increase in blood pressure in response to gastric distension andmicroinjection of nociceptin. B: mean reflex blood pressure responses tonociceptin (10 nM, 0.1 l) microinjected unilaterally into rVLM in the absenceof electroacupuncture (EA) in 6 rats. C: effect of opioid antagonist onnociceptin response in rVLM. Naloxone (opioid antagonist, n 7) wasmicroinjected unilaterally into the rVLM 10 min before nociceptin. Baselineblood pressures before distension are indicated below each bar. Bars showincrease in MAP (SE) induced by distension of the stomach. a, b, and c inthe bars (B) correspond with raw data (A). *Significant difference aftermicroinjection of nociceptin, P 0.05.

Fig. 1. A: responses of mean arterial pressure (MAP) in 6 rats to repeateddistension of the stomach with air every 10 min. B: nociceptin antagonistmicroinjection in rostral ventrolateral medulla (rVLM) during repeated disten-sion of the stomach. Values are means SE. Bars represent pressor responsesfollowing gastric distension. Values below each bar indicate baseline bloodpressures before each distension. , Change.



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Location of Injection Sites

Examination of the rat brain slices verified that all injectionslocated within the rVLM had significantly influenced the reflexresponses to gastric distension, whereas five injections outsidethe rVLM (Fig. 4) did not alter the reflex cardiovascularresponses. All effective sites for each of the protocols wereconfined to an area 1.002.50 mm rostral to the obex (inter-aural 4.3 mm) and the hypoglossal rootlets, 2.503.3 mm tothe right or left of the midline, 0.11.7 mm from the ventralsurface, lateral to the nucleus inferior olive and pyramidal tract,and ventral and medial to the facial and retrofacial nuclei andincludes the medial and caudal parts of nucleus paragiganto-

cellularis (Fig. 4). These sites are located within the rVLM, asdefined previously by others (8, 25, 26, 28, 40).

DISCUSSION

The inhibitory effect of EA on the visceral cardiovascularsympathoexcitatory pressor reflexes has been well character-ized (15, 31, 34, 54). Several studies have observed differentialcardiovascular responses to gastric distension. Our laboratoryhas developed a rodent model that yields consistent pressorresponses (31). The present study has used this model to assessthe role of the novel opiate-like peptide nociceptin in the rVLMand its contribution to EAs inhibitory effects on the cardio-

Fig. 3. A: gastric distension responses before,during, and after 30 min of EA (12 mA, 2 Hz)at the Neiguan-Jianshi acupoints (pericardialmeridian, P 56) in 5 rats with control salineinjection. B: blood pressure responses (means SE) to unilateral microinjection of nociceptinreceptor antagonist (10 nM, 0.1 l) into rVLM25 min after onset of EA (12 mA, 2 Hz at P

56 acupoints) in 7 rats. a-e in the bars (Bb)correspond with raw data (Ba). C: stimulationof -opioid receptors following blockade ofnociceptin receptors during EA. Nociceptinantagonist was microinjected unilaterally intothe rVLM 10 min before microinjection of[D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin (DAGO)(-opioid agonist, 612 nM) in 5 rats 25 minafter onset of EA. Gastric distension was re-peated every 10 min. Values below each barrepresent baseline blood pressures before dis-tension. *Significant difference compared withpre-EA, P 0.05. **Significant differenceafter microinjection with DAGO, P 0.05.



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vascular system. Microinjection of either nociceptin receptorantagonist [N-Phe1]-nociceptin(113)NH2 or [Phe

1-(CH2-NH)-Gly2]-nociceptin(113)-NH2 into the rVLM markedly re-duced the EA-related response. As a surrogate for EA, injec-tion of nociceptin into this same region of the brain stem alsoinhibited the reflex pressor response caused by mechanicaldistension of the stomach. Pretreatment with the nonselectiveopioid receptor antagonist naloxone in the absence of EA didnot alter the gastric distension-induced pressor response, nor

did it alter the inhibitory effect of nociceptin in the rVLM onthe response to gastric distension. Lastly, blockade of theEA-induced response by a nociceptin receptor antagonist didnot prevent the inhibitory response to a specific -opioidreceptor agonist DAGO. Taken together, these results suggestthat nociceptin and its associated ORL-1 receptor in the rVLMcontribute to the inhibitory effect of EA on the reflex auto-nomic responses during mechanical stimulation of the stom-ach, in a manner that is independent of the classical opioidsystem.

We began this study using [Phe1-(CH2-NH)-Gly2]-noci-

ceptin(113)-NH2 as the antagonist in the nociceptin antago-nist protocols (protocols 1B and 3B), as this was the only

inhibitor that was available commercially. It later becameapparent that this inhibitor exhibited both antagonist and partialagonist behaviors (12). When the new antagonist [N-Phe1]-nociceptin(113)NH2 became available (12), we incorporatedthis inhibitor into protocols 1B and 3B. In 11 animals, both[Phe1-(CH2-NH)-Gly

2]-nociceptin(113)-NH2 (n 5) andthe newer antagonist [N-Phe1]-nociceptin(113)NH2 (n 6)

exhibited similar antagonistic activity when they were micro-injected locally into the rVLM during pressor reflexes and EA.Similarly, protocol 3C, utilizing only the newer antagonist,caused similar degrees of inhibition of the EA-related cardio-vascular response. Thus the two protocols reinforce eachother and suggest that at least part of the EA-inducedcardiovascular inhibitory influence in the rVLM is mediatedthrough nociceptin.

Previous investigations have been limited by the absence ofselective pharmacological antagonists to nociceptin. The factthat two structurally dissimilar selective nociceptin receptorantagonists, [N-Phe1]-nociceptin(113)NH2 and [Phe

1-(CH2-NH)-Gly2]-nociceptin(113)-NH2, produced similar inhibitionof the EA-related cardiovascular response strongly suggeststhat nociceptin plays an important role in the EA-cardiovascu-lar response. Furthermore, demonstration in the present inves-tigation that the endogenous ligand nociceptin caused inhibi-tory responses much like acupuncture supports the hypothesisthat this system inhibits sympathetic outflow to the cardiovas-cular system.

The inhibitory effect of EA on the cardiovascular systemlikely is the result of excitation of group III and possibly groupIV somatic afferent nerve fibers beneath the acupoint (33, 37).Stimulation of these afferents can activate inhibitory systems inthe brain, resulting in the release of endogenous neurotrans-mitters such as -amino--butyric acid, serotonin, and endog-enous opioids (32). These neurotransmitters, along with noci-

ceptin, seem to function as neuromodulators by inhibiting areasthat regulate sympathetic neuronal activity like the rostralventrolateral medulla, a key integrative center responsible, inpart, for maintaining blood pressure and cardiovascular func-tion (9, 50).

Several regions in the CNS involved in the sympathoinhibi-tory effect of EA have been reported, including the nucleusarcuatus in the hypothalamus, the ventral periaqueductal gray,and the nucleus raphe obscurus with a projection to the rVLM(32). Furthermore, Liu et al. (36) reported that c-fos expressionin the nucleus tractus solitarius during EA may involve thisnucleus in the effects of acupuncture. In situ hybridization andimmunocytochemistry have been used to localize nociceptinreceptors and the peptide in the sensory trigeminal complex,

raphe nuclei, locus coeruleus, periacqueductal gray, amygdala,and hypothalamic and septal regions (49), as well as abundantdistribution in the rVLM (2, 43). Location of the nociceptin-ergic system in several areas involved in central regulation ofcardiovascular function suggest that it may participate in theEA-cardiovascular responses not only in the rVLM but in otherregions as well.

Many reports describe the wide range of nociceptins car-diovascular and nociceptive effects, varying from inhibitory(17, 18) to analgesic (48) to excitatory (3) and hyperalgesic(11). Like the endogenous opioid peptides, the global action ofnociceptin is dependent on the distribution of its receptors, aswell as the nature of the cell that has been activated. At the

Fig. 4. Sections of the rat medulla illustrating the microinjection sites ofnociceptin ( ), naloxone nociceptin ( ), nociceptin antagonist (), noci-ceptin antagonist -opioid agonist DAGO (), saline (E), and injectionsoutside the rVLM (*). All injections were unilateral (side chosen randomly);although for ease of reading, all injection sites are superimposed on left.Sections are 1.001.34, 1.342.00, and 2.002.50 mm rostral to the obex[Paxinos and Watson (45)]. rVLM, rostral ventrolateral medulla; Py, pyramidal

tract; Amb, ambiguus nucleus; 7, facial nucleus; Sp5, spinal trigeminalnucleus; IO, inferior olive nucleus; MVe, medial vestibular nucleus; SuVe,superior vestibular nucleus; LVe, lateral vestibular nucleus; Sol, solitary tractnucleus; 4V, 4th ventricle.



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cellular level, nociceptins actions are generally inhibitory(27). However, Feuerstein and Fadden (23) demonstrated thatmicroinjection of an opioid peptide agonist into two hypotha-lamic nuclei located only 1 m apart led to opposite effects onarterial blood pressure in the rat. Such differences in cellularresponse could potentially account for the previous disparatereports characterizing nociceptins effects. More investigation

will be required to determine whether these mechanisms ac-count for nociceptins varied actions on the cardiovascular orpain systems. However, the present study provides strongevidence that nociceptin in the rVLM inhibits reflex-inducedsympathoexcitatory cardiovascular responses and serves as oneof the mechanisms of EA-related inhibition of excitatory pres-sor reflexes.

One potential limitation of this study is that the gastricdistension responses in the time control group were slightlylarger than the gastric distension responses observed in proto-cols 1B, 2A, 2B, 3A, 3B, and 3C. We suggest that this obser-vation likely was due to the less invasive surgery in animals inthe time control group compared with animals in the microin-

jection studies. However, it is important to note that, before EAor microinjection studies, control gastric distension responseswere consistent, and all of the interventions were reversible.Therefore, we do not believe that differences in the controlresponses vs. those in the microinjection studies affect ourconclusions.

In conclusion, these data provide the first documentation thatthe endogenous nociceptinergic system in the rVLM contrib-utes to the inhibitory actions of EA on the excitatory reflexeselicited by mechanical distension of the stomach. In thisregard, antagonism of nociceptins action in the rVLM duringEA reversed the inhibitory actions of EA on the pressor reflexduring visceral afferent stimulation. Additionally, in the ab-sence of EA, exogenous nociceptin microinjected into the

rVLM elicited EA-like attenuation of the reflex increase inblood pressure. Pretreatment of nociceptin with a nonselectiveopioid receptor antagonist, naloxone, did not alter the EA-likeinhibitory influence of nociceptin on the gastric distension-induced pressor reflex, suggesting that at least part of nocicep-tins actions are independent of the opioid system. Also,microinjection of a selective -opioid receptor agonist imme-diately after microinjection of a nociceptin receptor antagonistduring EA rapidly reversed the nociceptin receptor antagonist-related inhibition of the EA response, indicating that activationof the opioid system during EA can function independentlyfrom the nociceptinergic system. Taken together, these datasuggest that nociceptin, similar to but separate from the clas-

sical opioid system, functions as an important neuromodulatorof visceral reflex cardiovascular stimulation during EA-relatedsomatic afferent activation.

GRANTS

This project was supported in part by National Heart, Lung, and BloodInstitute Grants HL-72125 and HL-63313 and the Larry K. Dodge EndowedChair (J. C. Longhurst).

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