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Respiratory Physiology & Neurobiology 191 (2014) 95– 105

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

j ourna l ho me pa ge: www.elsev ier .com/ loca te / resphys io l

ole of central and peripheral opiate receptors in the effects ofentanyl on analgesia, ventilation and arterial blood-gas chemistry inonscious rats

raser Hendersona, Walter J. Maya, Ryan B. Gruberb, Joseph F. Discalab, Veljko Puskovicb,lex P. Younga, Santhosh M. Babyb, Stephen J. Lewisc,∗

Pediatric Respiratory Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908, USADivision of Biology, Galleon Pharmaceuticals, Horsham, PA 19044, USADepartment of Pediatrics, Case Western Reserve University, Cleveland, OH 44106-4984, USA

r t i c l e i n f o

rticle history:ccepted 18 November 2013

eywords:entanylaloxone methiodide

a b s t r a c t

This study determined the effects of the peripherally restricted �-opiate receptor (�-OR) antagonist,naloxone methiodide (NLXmi) on fentanyl (25 �g/kg, i.v.)-induced changes in (1) analgesia, (2) arterialblood gas chemistry (ABG) and alveolar-arterial gradient (A-a gradient), and (3) ventilatory parameters, inconscious rats. The fentanyl-induced increase in analgesia was minimally affected by a 1.5 mg/kg of NLXmibut was attenuated by a 5.0 mg/kg dose. Fentanyl decreased arterial blood pH, pO2 and sO2 and increased

entilationrterial blood gasesnalgesia, Rats

pCO2 and A-a gradient. These responses were markedly diminished in NLXmi (1.5 mg/kg)-pretreated rats.Fentanyl caused ventilatory depression (e.g., decreases in tidal volume and peak inspiratory flow). Pre-treatment with NLXmi (1.5 mg/kg, i.v.) antagonized the fentanyl decrease in tidal volume but minimallyaffected the other responses. These findings suggest that (1) the analgesia and ventilatory depressioncaused by fentanyl involve peripheral �-ORs and (2) NLXmi prevents the fentanyl effects on ABG byblocking the negative actions of the opioid on tidal volume and A-a gradient.

. Introduction

The peripheral administration of opioids such as morphine elic-ts analgesia in humans (DeHaven-Hudkins and Dolle, 2004; Steinnd Lang, 2009) and rodents (Reichert et al., 2001; Lewanowitschnd Irvine, 2002; Lewanowitsch et al., 2006; Stein et al., 2009) viactivation of opiate receptors (ORs) in the central nervous systemCNS) and on peripheral nociceptive afferents. Analgesic doses ofpioids are associated with a significant incidence of ventilatoryepression in humans and rodents via activation of ORs within theNS and also key peripheral structures such as the carotid bodiesnd neuromuscular components of the chest-wall and diaphragmDahan et al., 2010). In addition, opiates increase pulmonary vas-ular resistance suggesting decreased perfusion of alveoli (Schurigt al., 1978; Hakim et al., 1992).

Fentanyl is a high-potency opiate that is widely prescribed toreat acute and chronic pain (Nelson and Schwaner, 2009; Johnston,010). Abuse or misuse lead to significant consequences, including

∗ Corresponding author at: Department of Pediatrics, Case Western Reserve Uni-ersity, 0900 Euclid Avenue, Cleveland, OH 44106-4984, USA. Tel.: +1 216 368 3482;ax: +1 216 368 4223.

E-mail address: sjl78@case.edu (S.J. Lewis).

569-9048/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.resp.2013.11.005

© 2013 Elsevier B.V. All rights reserved.

death via depression of ventilation (Nelson and Schwaner, 2009).The mechanisms responsible for the analgesic and ventilatorydepressant effects of fentanyl and analogs have been extensivelyinvestigated (Dahan et al., 1998; Sarton et al., 1999; Stein et al.,2009). Although fentanyl is thought of as a selective �-OR agonist(Trescot et al., 2008; Hajiha et al., 2009) and has high affinity for�-ORs (Raynor et al., 1994; Huang et al., 2001), it also activates�- and �-ORs with affinities/intrinsic activities of biological signifi-cance (Yeadon and Kitchen, 1990; Zhu et al., 1996; Butelman et al.,2002; Gharagozlou et al., 2006). For example, whereas fentanyl haslow affinity for �-ORs it has a remarkably high efficacy at these ORs(Gharagozlou et al., 2006).

The relative contributions of central and peripheral ORs in theanalgesic and ventilatory effects of fentanyl have received littleattention. One approach to evaluating these contributions is tocompare the effects of centrally-penetrant and -impenetrant ORantagonists on the fentanyl-induced responses. Naloxone (NLX) isan OR antagonist that readily enters the CNS (DeHaven-Hudkinsand Dolle, 2004; Stein et al., 2009). NLX is an effective antago-nist of �-, �- and �-ORs although it has approximately twice the

affinity for �-ORs than for �-ORs and ≈15 times greater affin-ity for �-ORs than �-ORs (see Lewanowitsch and Irvine, 2003).In contrast, it appears that naloxone methiodide (NLXmi) doesnot cross the blood–brain barrier in rodents (Lewanowitsch and

9 iology

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6 F. Henderson et al. / Respiratory Phys

rvine, 2002; Lewanowitsch et al., 2006; Inglis et al., 2008; Het al., 2009). NLXmi has lower affinities for �-, �- and �-ORs thanLX (Lewanowitsch and Irvine, 2003). For example: (1) NLXmi has20 times lower affinity for �-ORs in rat brain membranes thanLX (Bianchetti et al., 1983; Valentino et al., 1983), (2) NLXmiad ≈10 times lower affinity for �-, �- and �-ORs in guinea pigrain homogenates than NLX (Magnan et al., 1982), and (3) bind-

ng affinities for NLX versus NLXmi in mouse brain homogenatesas 15:1 for �-, 6:1 for �- and 330:1 for �-ORs (Lewanowitsch and

rvine, 2003). Evidence that the analgesic and ventilatory depres-ant effects of morphine, methadone and heroin in mice wereeversed by NLXmi (Lewanowitsch and Irvine, 2002; Lewanowitscht al., 2006) provides evidence that peripheral ORs are involved inhe effects of these opioids.

Since the relative contributions of central and peripheral ORsn the analgesic and ventilatory effects of fentanyl are not known,he aims of this study were to use conscious rats to determine1) the effects of NLXmi (1.5 mg/kg, i.v.) on fentanyl-inducedhanges in analgesia status, arterial blood-gas chemistry (ABG) andlveolar-arterial (A-a) gradient, an index of ventilation–perfusion

Stein et al., 1995; Story, 1996) and (2) the effects of 1.5 mg/kgoses of NLXmi or NLX on fentanyl-induced changes in ventilatoryarameters. These studies were designed to discern which of theentilatory responses and/or changes in A-a gradient were respon-ible for the fentanyl-induced changes in ABG chemistry, and theole of peripheral ORs in these responses.

. Methods

.1. Rats and surgeries

All studies were carried out in accordance with the Nationalnstitutes of Health Guide for the Care and Use of Laboratorynimals (NIH Publication No. 80-23) revised in 1996. The pro-

ocols were approved by the Animal Care and Use Committeef the University of Virginia. Adult male Sprague-Dawley ratsere purchased from Harlan (Madison, WI) with jugular vein

atheters and/or femoral artery catheters (surgeries performednder ketamine–xylazine). The rats were allowed 5 days to recoverrom surgery and transport and were used for the blood-gas andnalgesia studies. Other adult male Sprague-Dawley rats were pur-hased from Harlan and venous catheters were implanted under% isoflurane anesthesia. These rats were given 4 days to recoverrom surgery and were used for the plethysmography and bodyemperature (TB) studies.

.2. Protocols for blood gas measurements and determination ofrterial-alveolar gradient

Study 1: Arterial blood samples (200 �L) were taken beforend 5 and 12 min after injection of vehicle (saline, i.v.; n = 6 rats;19 ± 3 g) or NLXmi (1.5 mg/kg, i.v.; n = 6 rats; 323 ± 4 g) and 1,

and 9 min after injection of fentanyl (25 �g/kg, i.v.). Study 2:rterial blood samples (200 �L) were taken before and 5, 10 and5 min after injection of vehicle (n = 6 rats; 312 ± 2 g) or NLXmi1.5 mg/kg, i.v.; n = 6 rats; 310 ± 3 g) and 5, 10 and 15 min afternjection of fentanyl (50 �g/kg, i.v.). The pH, pCO2, pO2 and sO2f arterial blood samples (100 �L) were measured by a Radiome-er blood-gas machine (ABL800 FLEX). The A-a gradient measureshe difference between alveolar and arterial blood concentrationsf O2 (Stein et al., 1995; Story, 1996). A decrease in PaO2, with-

ut a change in A-a gradient is caused by hypoventilation whereas

decrease in PaO2 with an increase in A-a gradient indicatesentilation–perfusion mismatch or shunting (Stein et al., 1995).-a gradient = PAO2 − PaO2, where PAO2 is the partial pressure of

& Neurobiology 191 (2014) 95– 105

alveolar O2 and PaO2 is pO2 in arterial blood. PAO2 = [(FiO2 ×(Patm − PH2O) − (PaCO2/respiratory quotient)], where FiO2 is thefraction of O2 in inspired air; Patm is atmospheric pressure, PH2Ois the partial pressure of water in inspired air; PaCO2 is pCO2in arterial blood; and respiratory quotient (RQ) is the ratio ofCO2 eliminated/O2 consumed. We took FiO2 of room-air to be21% = 0.21, Patm to be 760 mmHg, and PH2O to be 47 mmHg (seeStein et al., 1995; Story, 1996). We took the RQ value of our adultmale Sprague-Dawley rats to be 0.9 (Stengel et al., 2010; Chapmanet al., 2012).

2.3. Antinociception protocols

Antinociception was determined by the radiant heat tail-flick(TF) assay (Lewis et al., 1991). The intensity of the light was adjustedso that baseline TF latencies were ≈3 s. A cutoff time of 12 s wasset to minimize damage to the tail. Rats were injected with vehi-cle (saline, i.v.; n = 4 rats; 300 ± 1 g) or NLXmi (1.5 mg/kg, i.v.; n = 4;290 ± 2 g) and after 15 min the rats received fentanyl (25 �g/kg,i.v.). Other rats received vehicle (saline; n = 5 rats; 315 ± 7 g) orNLXmi (5 mg/kg, n = 5 rats; 317 ± 5 g) and after 15 min, an injectionof fentanyl (25 �g/kg, i.v.). The rats were tested for antinocicep-tion at 15, 45, 60, 90 and 120 min post-fentanyl. Data are presentedas TF latencies (s) and as “maximum possible effect” (%MPE)using the formula, %MPE = [(post-injection TF latency − baseline TFlatency)/(12 − baseline TF latency)] × 100.

2.4. Body temperature (TB) protocols

Changes in TB can significantly impact the magnitude ofrecorded flow-related variables in plethysmography chambers(Mortola and Frappell, 1998). Although our plethysmographychambers are not equipped to continuously monitor TB, it remainsimperative to record TB to better understand the influences ofNLXmi and NLX on fentanyl-induced changes in ventilation andRQ. Adult male Sprague-Dawley rats of approximately 300 g wereplaced in separate open plastic boxes and allowed 60–90 min toacclimatize. TB was recorded as described previously (Kregel et al.,1997). In brief, a thermistor probe was inserted 5–6 cm into therectum to allow regular recording of TB. A 2–3 in. length of theprobe cable, which was connected to a telethermometer (YellowSprings Instruments, South Burlington, Vermont), was taped tothe tail. TB was recorded every 5 min during the acclimatizationperiod to establish baseline values. One group of rats (n = 6) receivedan injection of vehicle (saline, i.v.) whereas other groups (n = 6rats per group) received either NLX (1.5 mg/kg, i.v.) or NLXmi (1.5or 5.0 mg/kg, i.v.). After 15 min, the four groups of rats receivedfentanyl (25 �g/kg, i.v.). TB was recorded 5, 10 and 15 min afterinjection of vehicle or the OR antagonists, and 5, 10, 15 and 20 minafter injection of fentanyl. These and all rats used in the otherdescribed studies were not fasted prior to use in the experiments.

2.5. Ventilatory protocols

Ventilatory parameters were continuously recorded in ratsusing a whole body plethysmography system (PLY 3223; BuxcoInc., Wilmington, NC, USA), as described previously (Kanbar et al.,2010; Young et al., 2013). The parameters were (1) frequencyof breathing (fR), (2) tidal volume (VT), (3) minute ventilation(V = fR × VT), (4) inspiratory time (TI), (5) expiratory time (TE), (6)end inspiratory pause (EIP), time between end of inspiration andstart of expiration, (7) peak inspiratory flow (PIF), and (8) peak

expiratory flow (PEF). Provided software constantly corrected dig-itized values for changes in chamber temperature and humidityand a rejection algorithm was included in the breath-by-breathanalysis to exclude nasal breathing. The rats were placed in the

F. Henderson et al. / Respiratory Physiology & Neurobiology 191 (2014) 95– 105 97

Fig. 1. Effects of fentanyl (25 �g/kg, i.v.) on tail-flick latencies in rats pretreated with (a) vehicle (n = 4) or naloxone methiodide (NLXmi; 1.5 mg/kg, i.v., n = 4) (upper-leftp pper-p st-dru

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anel) or (b) vehicle (n = 5) or naloxone methiodide (NLXmi; 5.0 mg/kg, i.v., n = 5) (uanels). All data are presented as mean ± SEM. *P < 0.05, significant change from po

lethysmography chambers and allowed 45–60 min to acclimatize.ne group of rats received a bolus injection of vehicle (saline, i.v.)hereas another group received an injection of NLX (1.5 mg/kg,

.v.). After 15 min, both groups of rats received an injection of fen-anyl (25 �g/kg, i.v.) and parameters recorded for a further 20 min.n another study, one group of rats received an injection of vehiclesaline, i.v.) and another received an injection of NLXmi (1.5 mg/kg,.v.). After 15 min, both groups of rats received an injection of fen-anyl (25 �g/kg, i.v.) and parameters recorded for a further 20 min.he body weights of the four groups of rats (n = 6 rats per group)ere 328 ± 3, 331 ± 3, 334 ± 3 and 329 ± 5 g, respectively (P > 0.05,

or all between-group comparisons). Due to the closeness of theseody weights, ventilatory data are presented without body weightorrections.

.6. Statistics

All data are presented as mean ± SEM and were analyzedy one-way or two-way analysis of variance followed by Stu-ent’s modified t test with Bonferroni corrections for multiple

able 1ffects of fentanyl on arterial blood gas values and Arterial-alveolar (A-a) gradients in rat

Parameter Group Pre Post-treatment (min)

T5 T10

pH Vehicle 7.45 ± 0.01 7.46 ± 0.01 7.45 ± 0.02

NLXmi 7.46 ± 0.02 7.43 ± 0.04 7.45 ± 0.02

pCO2 Vehicle 35.9 ± 0.5 36.3 ± 0.5 36.1 ± 0.5

NLXmi 36.2 ± 0.6 36.5 ± 0.5 36.4 ± 0.4

pO2 Vehicle 94.3 ± 1.2 92.6 ± 1.1 94.2 ± 1.5

NLXmi 93.0 ± 0.5 92.2 ± 1.6 92.8 ± 0.8

sO2 Vehicle 99.2 ± 1.0 99.5 ± 1.3 98.3 ± 0.7

NLXmi 98.7 ± 0.8 99.7 ± 0.9 99.2 ± 0.7

A-agradient

Vehicle 15.5 ± 2.6 16.8 ± 2.5 15.4 ± 3.2

NLXmi 16.5 ± 1.5 17.0 ± 1.1 16.5 ± 1.9

ata are shown as mean ± SEM. There were 6 rats in each group. The dose of fentanyl werms T5, T10 and T15 refer to values taken 5, 10 and 15 min after administration of vehicdministration of fentanyl. *P < 0.05, significant response. †P < 0.05, NLXmi versus vehicle.

right panel). The data are also expressed as maximal possible effect (%MPE) (lowerg value. †P < 0.05, NLXmi versus vehicle.

comparisons between means using the error mean square termsfrom the analyses of variance (Wallenstein et al., 1980). A value ofP < 0.05 was taken to denote statistical significance.

3. Results

3.1. Tail-flick latencies

Fentanyl (25 �g/kg) elicited a robust antinociception of at least2 h in duration in vehicle-treated (VEH) rats and smaller but not sta-tistically different responses in NLXmi (1.5 mg/kg)-treated (NLXmi)rats (Fig. 1). Pretreatment with a higher dose of NLXmi (5 mg/kg)markedly attenuated but did not abolish the antinociceptive effectsof the 25 �g/kg dose of fentanyl (Fig. 1).

3.2. Arterial blood gas chemistry

The effects of fentanyl (25 �g/kg) on ABG chemistry and A-agradients in VEH or NLXmi (1.5 mg/kg) rats are summarized inFig. 2. The terms F1, F5 and F9 on the x-axis refer to the values

s pretreated with vehicle (saline) or naloxone methiodide.

Post-fentanyl (min)

T15 F5 F10 F15

7.46 ± 0.02 7.15 ± 0.02* 7.21 ± 0.02* 7.33 ± 0.01*7.45 ± 0.02 7.35 ± 0.01*,† 7.38 ± 0.02† 7.44 ± 0.01†

35.8 ± 0.7 61.2 ± 2.5* 49.6 ± 1.8* 43.3 ± 1.4*36.1 ± 0.5 38.0 ± 0.8† 36.0 ± 0.4† 35.5 ± 0.7†

94.1 ± 1.2 52.2 ± 1.2* 59.7 ± 1.8* 76.1 ± 1.0*92.4 ± 1.6 88.7 ± 2.2† 93.3 ± 1.0† 95.6 ± 1.1†

99.0 ± 1.0 55.8 ± 1.4* 70.8 ± 2.9* 92.5 ± 2.299.3 ± 0.9 90.2 ± 1.7*,† 94.3 ± 2.1† 98.8 ± 1.815.9 ± 2.8 29.5 ± 4.3* 34.9 ± 4.4* 25.5 ± 2.6*17.2 ± 1.5 18.8 ± 3.3† 16.4 ± 2.2† 14.7 ± 1.6†

as 50 �g/kg, i.v. The dose of naloxone methiodide (NLXmi) was 1.5 mg/kg, i.v. Thele or NLXmi. The terms F5, F10 and F20 refer to values taken 5, 10 and 20 min after

98 F. Henderson et al. / Respiratory Physiology & Neurobiology 191 (2014) 95– 105

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35

45

55

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Pos t-Drug (min)

F1 F5 F9

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100 110

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Pos t-Drug (min)

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Table 2Effects of treatments on body temperatures.

Group Bodyweight (g)

Body temperature (◦C)

Pre Post Change (◦C)

Vehicle 332 ± 3 37.8 ± 0.1 37.8 ± 0.1 +0.02 ± 0.09NLX – 1.5 mg/kg 330 ± 4 37.9 ± 0.1 37.8 ± 0.1 −0.05 ± 0.08NLXmi – 1.5 mg/kg 328 ± 3 37.7 ± 0.1 37.8 ± 0.1 +0.04 ± 0.10NLXmi – 1.5 mg/kg 334 ± 2 37.8 ± 0.1 37.8 ± 0.1 +0.03 ± 0.06

Data are presented as mean ± SEM. NLX, naloxone. NLXmi, naloxone methiodide.There were 6 rats in each group. There were no between-group differences in bodyweights or body temperatures (P > 0.05, for all comparisons). None of the treatmentsaffected baseline body temperatures (P > 0.05, for all comparisons).

0

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ig. 2. Effects of fentanyl (25 �g/kg, i.v.) on arterial blood pH, pO2, pCO2 and sO2 vaaloxone methiodide (NLXmi; 1.5 mg/kg, i.v.). The terms F1, F5 and F9 refer to vaean ± SEM. There were 6 rats in each group. *P < 0.05, significant change from pos

hat were recorded 1, 5 and 9 min after the injection of fentanyl.either VEH nor NLXmi affected ABG chemistry or A-a gradients.

n VEH rats, fentanyl decreased pH, pO2 and sO2 values whereas itlevated pCO2 and A-a gradient values. These fentanyl responsesere virtually absent in NLXmi rats. The effects of a higher dose

f fentanyl (50 �g/kg) on ABG and A-a gradient in VEH or NLXmi1.5 mg/kg) rats are summarized in Table 1. This higher dose ofentanyl elicited (1) a decrease in pH at 5, 10 and 15 min, (2) anncrease in pCO2 at 5, 10 and 15 min, (3) a decrease in pO2 at 5,0 and 15 min, (4) a decrease in sO2 at 5 and 10 but not 15 min,nd (5) an increase in A-a gradient at 5, 10 and 15 min. The fen-anyl responses were markedly attenuated but not abolished byLXmi.

.3. Effects of vehicle, NLX and NLXmi on fentanyl inducedhanges in TB

Resting TB was similar in the four groups of rats before drugnjection (Table 2). The injection of vehicle, NLX (5.0 mg/kg, i.v.) orLXmi (1.5 or 5 mg/kg, i.v.) did not affect TB as recorded at +15 min

Table 2). The changes in TB elicited by fentanyl (25 �g/kg) in theseats are summarized in Fig. 3. Fentanyl elicited relatively minor

hanges in TB in vehicle-treated rats over the 20 min recordingeriod. Pretreatment with the 1.5 or 5 mg/kg doses of NLXmi didot affect these increases in TB whereas pretreatment with NLXbolished the hyperthermic responses.

Fig. 3. Effects of fentanyl (25 �g/kg, i.v.) on body temperatures (arithmetic change)in conscious rats pretreated with vehicle (saline), naloxone methiodide (NLXmi;1.5 or 5.0 mg/kg, i.v.) or naloxone (NLX, 1.5 mg/kg, i.v.). The data are presented asmean ± SEM. There were 6 rats in each group. *P < 0.05, significant change frompost-drug value. †P < 0.05, NLXmi or NLX versus vehicle.

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.4. Effects of vehicle, NLX and NLXmi on baseline ventilatoryarameters

Resting parameters before injection of VEH, NLX or NLXmi areummarized in Table 3. There were no between-group differencesn any of these parameters. As seen in Figs. 4–6, the injection of VEHlicited transient changes in some ventilatory parameters (i.e., fR,˙ , TI, TE, PIF and PEF) but not others (VT or EIP). These responsesesolved before the injection of fentanyl (25 �g/kg). The injectionsf NLX or NLXmi elicited responses that were similar to the vehicleesponses (Figs. 4–6).

.5. Effects of NLX or NLXMI on fentanyl-induced changes inentilatory parameters

.5.1. fR, VT and VAs seen in Fig. 4, fentanyl (25 �g/kg) elicited an increase in fR of

bout 5 min in duration (F1–F5) in VEH rats, which was followed by more sustained decrease (F6–F20). The increases in fR were notffected by NLX whereas they were reversed to a decrease in NLXmiats. The fentanyl-induced decreases in fR were absent in NLX ratsut were not affected by NLXmi. Fentanyl decreased VT for about

min in VEH rats (F1–F5) with minor F6–F20 changes (Fig. 4). Theecreases in VT were abolished by NLX and markedly attenuatedy NLXmi. The minor F6–F20 changes in VT were not affected byLX or NLXmi. Fentanyl decreased VT for about 15 min in VEH rats

Fig. 4). The F1–F5 decreases in V were converted to increases byLX whereas the F6–F20 responses were abolished. None of the

entanyl changes in V were affected by NLXmi. Response areas (RA,umulative arithmetic change from pre) for the F1–F5 (Table 4) and6–F20 (Table 5) time periods confirm that (1) the fentanyl-inducedncreases in fR were attenuated by NLXmi but not NLX whereas theentanyl decreases in fR were attenuated by NLX but not NLXmi,2) the fentanyl-induced decreases in VT (F1–F6) were eliminatedy NLX and markedly attenuated by NLXmi whereas the F6–F20esponses were unaffected by NLX or NLXmi, and (3) the fentanyl-nduced decreases in V in NLX rats were reversed to increases at1–F6 and were abolished at F6–F20, and that NLXmi was withoutffect.

.5.2. TI, TE and EIPFentanyl (25 �g/kg) elicited sustained increases in TI in VEH rats

Fig. 5). The F1–F5 increases in TI were reversed to decreases byLX but were augmented by NLXmi. The F6–F20 increases in TIere abolished by NLX but were unaffected by NLXmi. Fentanyl

licited minor changes in TE in VEH or NLX rats (Fig. 5). The F1–F5nd F6–20 TE values in NLXmi rats were higher than in VEH rats.hese responses were absent in NLX rats. Fentanyl increased EIPor at least 20 min in VEH rats (Fig. 5). The increases in EIP at1–F10 were similar whereas the F11–F20 values were smallern NLXmi rats. RAs for the F1–F5 (Table 4) and F6–F20 (Table 5)eriods confirm that (1) the fentanyl F1–F5 increases in TI wereeversed to decreases by NLX but were augmented by NLXmi, andhat the F6–F20 increases in TI were blocked by NLX only, (2) fen-anyl elicited minimal effects on TE in VEH, NLX and NLXmi rats,nd (3) the fentanyl increases in EIP were eliminated by NLX andhat the latter time-points were attenuated by NLXmi.

.5.3. PIF and PEFFentanyl decreased PIF for at least 20 min in VEH rats (Fig. 6). The

1–F5 decreases in PIF were converted to increases in the presencef NLX whereas F6–F20 responses were abolished. These fentanyl

esponses were not affected by NLXmi. Fentanyl decreased in PEFor about 5 min in VEH rats (Fig. 6). The F1–F5 decreases in PEFere converted to increases by NLX but were unaffected by NLXmi.

he F6–F20 values were similar in VEH or NLX rats whereas these

& Neurobiology 191 (2014) 95– 105 99

values tended to be lower in NLXmi rats. RAs for the F1–F5 (Table 4)and F6–F20 (Table 5) periods confirm that (1) the fentanyl-induceddecreases in PIF were reversed to increases at F1–F6 and were abol-ished at F6–F20 in NLX rats, and that NLXmi was without effectand (2) the fentanyl decreases in PEF were reversed to increases atF1–F5 in NLX rats and that NLXmi was without effect.

4. Discussion

The present study provides compelling evidence that the ven-tilatory depressant and analgesic effects of fentanyl involve theactivation of peripheral �-ORs that were sensitive to blockadewith NLXmi. On the basis of our findings with NLXmi, it wasapparent that the key mechanisms responsible for the deleteri-ous effects of fentanyl on ABG chemistry were mainly dependenton peripheral �-OR-mediated reductions in VT and mismatch inventilation–perfusion.

4.1. NLXmi and fentanyl-induced changes in antinociception

A major finding of this study was that although the analgesicaction of fentanyl was minimally affected by 1.5 mg/kg of NLXmi,it was markedly affected by a 5 mg/kg dose of this peripherallyrestricted OR antagonist. These findings support evidence thatperipheral ORs participate in the analgesic actions of other opi-ates (Stein et al., 2009). In contrast to the analgesia, the lower doseof NLXmi virtually eliminated the effects of fentanyl on ABG chem-istry and A-a gradient (see below). As such, NLXmi may have greateraccess to and/or greater affinity/intrinsic activity at OR sub-typesmediating the ventilatory depressant effects of fentanyl than forthose mediating the analgesic actions of this opioid. This scenariocould arise if the relative roles of �-, �-and �-ORs in the venti-latory depressant effects of 25 �g/kg fentanyl differ from thosemediating the analgesia. Indeed, �1-, �2- and �-ORs play differ-ent roles in morphine-induced (Ling et al., 1985; Su et al., 1998)and sufentanil-induced (Verborgh and Meert, 1999a,b; Verborghet al., 1997; Latasch and Freye, 2002) analgesia and respiratorydepression. Based on the known pharmacology of NLXmi, it couldbe argued that the effects of the 1.5 mg/kg dose of NLXmi are duemainly to blockade of �-ORs, lesser blockade of �-ORs and minimalblockade of �-ORs. As such, if the ventilatory depressant effects offentanyl (especially those mediating the changes in ABG and A-a gradient) are mediated primarily via �-ORs and �-ORs whereasthe analgesic actions are primarily via �-ORs and �-ORs, then the1.5 mg/kg dose of NLXmi would be expected to have relativelygreater effects on the ventilatory depressant effects than the anal-gesic effects of fentanyl.

4.2. Effects of NLX/NLXmi on fentanyl-induced changes in TB

The observation that the injections of NLX and NLXmi had min-imal effects on resting TB is consistent with evidence that blockadeof peripheral and central �-ORs has minimal effects on TB in rats(Geller et al., 1983; Colman and Miller, 2002; Cao and Morrison,2005). The administration of fentanyl elicits a gradual hyperther-mia in rats with maximal responses (0.5–0.7 ◦C) occurring between45 and 60 min after administration (Geller et al., 1983; Colmanand Miller, 2002; Cao and Morrison, 2005; Savic Vujovic et al.,2013). The findings that the 25 �g/kg dose of fentanyl elicited minorresponses during the 20 min post-injection period is consistentwith the above findings. Moreover, our finding that this minorhyperthermia was blocked by NLX but not NLXmi (and therefore

likely to be of central origin) is also consistent with previous find-ings (Geller et al., 1983; Colman and Miller, 2002; Cao and Morrison,2005). It is important to note that the minor changes in TB observedin this study (about 0.25 ◦C) are likely to have a minimal impact

100 F. Henderson et al. / Respiratory Physiology & Neurobiology 191 (2014) 95– 105

Table 3Baseline ventilatory values in study groups prior to injection of vehicle, naloxone or naloxone methiodide.

Parameter Naloxone study Naloxone methiodide study

Vehicle NLX Vehicle NLXmi

Frequency (breaths/min) 124 ± 13 112 ± 10 122 ± 17 94 ± 11Tidal volume (ml) 2.11 ± 0.19 2.29 ± 0.14 2.29 ± 0.13 2.45 ± 0.12Minute ventilation (ml/min) 246 ± 23 252 ± 27 274 ± 31 235 ± 34Inspiratory time (s) 0.17 ± 0.01 0.18 ± 0.01 0.17 ± 0.01 0.21 ± 0.03Expiratory time (s) 0.37 ± 0.04 0.40 ± 0.04 0.37 ± 0.05 0.46 ± 0.05End inspiratory pause (ms) 6.9 ± 0.4 7.6 ± 0.5 8.7 ± 0.7 8.6 ± 0.5Peak inspiratory flow (ml/s) 19.1 ± 1.7 19.9 ± 1.5 21.7 ± 1.9 19.7 ± 3.1Peak expiratory flow (ml/s) 14.0 ± 1.0 13.0 ± 0.8 13.4 ± 1.2 11.8 ± 1.4

Data are shown as mean ± SEM; NLX, naloxone; NLXmi, naloxone methiodide. There were 6 rats in each group. There were no significant between-group differences for anyparameter (P > 0.05, for all comparisons).

Table 4Cumulative responses recorded 1–5 min after injection of fentanyl in vehicle-, naloxone- or naloxone-methiodide-treated rats.

Parameter NLX study NLXmi study

Vehicle NLX Vehicle NLXmi

Frequency (breaths/min) × min 88 ± 18* 196 ± 19*,† 200 ± 39* −164 ± 18*,†

Tidal volume (ml) × min −2.53 ± 0.43* 0.26 ± 0.14† −4.27 ± 0.52* −1.49 ± 0.30*,†

Minute ventilation (ml/min) × min −454 ± 58* 441 ± 36*,† −604 ± 73* −471 ± 66*Inspiratory time (s) × min 0.30 ± 0.08 −0.27 ± 0.03*,† 0.26 ± 0.06* 0.68 ± 0.09*,†

Expiratory time (s) × min −0.00 ± 0.06 −0.12 ± 0.06 −0.19 ± 0.08 0.20 ± 0.13End inspiratory pause (ms) × min 19 ± 3* −8 ± 3† 13 ± 1 16 ± 2Peak inspiratory flow (ml/s) × min −50 ± 5* 56 ± 9*,† −53 ± 6* −38 ± 5*Peak expiratory flow (ml/s) × min −23 ± 8 68 ± 10*,† −19 ± 7 −16 ± 8

Data are presented as mean ± SEM; NLX, naloxone; NLXmi, naloxone methiodide. There were 6 rats in each group. *P < 0.05, significant response area. †P < 0.05, NLX or NLXmiversus relevant vehicle.

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VEH or NLX

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Fenta nyl

0.5

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Fentanyl

VEH or NLX

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Post -Fentanyl (min)

Fentanyl

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Fentanyl

Fig. 4. Effects of fentanyl (25 �g/kg, i.v.) on frequency of breathing (top panels), tidal volume (middle panels) and minute volume (bottom panels) in rats pretreated witheither vehicle or naloxone (NLX; 1.5 mg/kg, i.v.) (left-hand panels) or vehicle or naloxone methiodide (NLXmi; 1.5 mg/kg, i.v.) (right-hand panels). The stippled horizontalline denotes average resting values immediately before injection of fentanyl. The data are presented as mean ± SEM. There were 6 rats in each group.

F. Henderson et al. / Respiratory Physiology & Neurobiology 191 (2014) 95– 105 101

0.1

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NL Xmi (1 .5 mg/kg, i.v.)

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VEH or NLX mi

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Fentany l

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NLXmi (1.5 mg/kg, i.v.)

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Fentany l

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NLX (1.5 mg/kg, i .v.)

Pre (min ) Post-Dr ug (min)

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Post-Fenta nyl (min)

Fentanyl

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sec)

VEH NLX (1.5 mg/kg, i.v.

Pre (min) Post-Drug (min)

VEH or NLX

Post-Fenta nyl (min)

Fentanyl

Fig. 5. Effects of fentanyl (25 �g/kg, i.v.) on inspiratory time (top panels), expiratory time (middle panels) and end inspiratory pause (bottom panels) in rats pretreated witheither vehicle or naloxone (NLX; 1.5 mg/kg, i.v.) (left-hand panels) or vehicle or naloxone methiodide (NLXmi; 1.5 mg/kg, i.v.) (right-hand panels). The stippled horizontalline denotes average resting values immediately before injection of fentanyl. The data are presented as mean ± SEM. There were 6 rats in each group.

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NLXmi (1.5 mg /kg, i.v.)

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Post-Fent any l (min)

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NLX (1.5 mg/kg, i.v.)

Pre (min ) Post -Drug (min)

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Post-Fent any l (min)

Fenta nyl

Fig. 6. Effects of fentanyl (25 �g/kg, i.v.) on peak inspiratory flow (top panels) and peak expiratory flow (bottom panels) in rats pretreated with either vehicle or naloxone(NLX; 1.5 mg/kg, i.v.) (left-hand panels) or vehicle or naloxone methiodide (NLXmi; 1.5 mg/kg, i.v.) (right-hand panels). The stippled horizontal line denotes average restingvalues immediately before injection of fentanyl. The data are presented as mean ± SEM. There were 6 rats in each group.

102 F. Henderson et al. / Respiratory Physiology & Neurobiology 191 (2014) 95– 105

Table 5Cumulative responses recorded 6–20 min after injection of fentanyl in vehicle-, naloxone- or naloxone-methiodide-treated rats.

Parameter Naloxone study Naloxone methiodide study

Vehicle NLX Vehicle NLXmi

Frequency (breaths/min) × min −582 ± 69* 246 ± 38*,† −435 ± 51* −502 ± 60*Tidal volume (ml) × min 2.14 ± 0.56 −0.13 ± 0.18 4.06 ± 0.52* 5.04 ± 0.58*Minute ventilation (ml/min) × min −679 ± 73* 455 ± 67*,† −444 ± 53* −337 ± 46*Inspiratory time (s) × min 2.27 ± 0.36* −0.25 ± 0.08† 2.25 ± 0.37* 1.80 ± 0.29*Expiratory time (s) × min 0.13 ± 0.06 −0.16 ± 0.08 −0.15 ± 0.06 −0.20 ± 0.07End inspiratory pause (ms) × min 121 ± 17* −9 ± 6 109 ± 15* 66 ± 9*,†

Peak inspiratory flow (ml/s) × min −141 ± 13* 20 ± 3*,† −91 ± 9* −78 ± 9*,†

D ere wv

(aFe

4a

roifadaaoi5AmieOs2co1

tti

TSr

N0

Peak expiratory flow (ml/s) × min 24 ± 3*

ata are presented as mean ± SEM. NLX, naloxone. NLXmi. Naloxone methiodide. Thersus relevant vehicle.

less than 2%) effect on the flow-dependent variables (e.g., VT, PIF,nd PEF) recorded in the plethysmography chambers (Mortola andrappell, 1998). Moreover, these minor changes in TB would not bexpected to have major effects on RQ (van Klinken et al., 2013).

.3. Effects of fentanyl on ventilatory parameters, ABG chemistrynd A-a gradient

The 25 �g/kg dose of fentanyl elicited (1) a decrease in V viaeductions in fR and VT, (2) increases in TI and EIP with no effectn TE, and (3) decreases in PIF and PEF (see Table 6). The decreasesn VT and PEF were relatively transient whereas the decreases inR and therefore V were more sustained. The increases in Ti, EIPnd PIF lasted for at least 20 min. As such, it is evident that thisose of fentanyl diminished V by a combination of reduced ratend efficiency of breathing. The finding that fentanyl markedlyffected TI and PIF whereas it elicited relatively minor effectsn TE and PEF suggests that fentanyl primarily affected activenspiration rather than passive expiration. In addition, the 25 and0 �g/kg doses of fentanyl produced changes in ABG chemistry and-a gradient that were consistent with hypoventilation and mis-atch of ventilation–perfusion. Specifically, fentanyl decreased pH,

ncreased pCO2, decreased pO2 and sO2, and increased A-a gradi-nt. The above effects of fentanyl are likely to involve activation ofRs on neurons in ventilatory control regions of the brainstem and

pinal cord (Laferriere et al., 1999; Wang et al., 2002; Haji et al.,003; Lonergan et al., 2003a,b) and peripheral tissues such as thearotid bodies, pulmonary arteries and neuromuscular componentsf the chest-wall and diaphragm (Schurig et al., 1978; Shook et al.,990; Hakim et al., 1992; Haji et al., 2000; Dahan et al., 2010).

The novel finding that fentanyl increased A-a gradient suggestshat it increased pulmonary vascular resistance and/or exacerbatedhe hypoxic pulmonary vasoconstriction due to the fentanyl-nduced reduction in ventilation and the concomitant decreases in

able 6ummary of the effects of naloxone and naloxone methiodide on fentanyl-inducedesponses.

Parameter Fentanyl responses Effects ofantagonists

Direction Duration (min) NLX NLXmi

Frequency ↑*↓ 1–5, 6–20 +++ 0Tidal volume ↓ 5 +++ ++Minute ventilation ↓ 20 +++ 0Inspiratory time ↑ 20 +++ 0Expiratory time ≈0 n.a. n.a. n.a.End inspiratory pause ↑ >20 +++ +Peak inspiratory flow ↓ 20 +++ 0Peak expiratory flow ↓ 4 +++ 0

LX, naloxone; NLXmi, naloxone methiodide; +++ to + complete to partial blockade;, no effect; n.a., not applicable.

1 ± 6* 28 ± 4* 18 ± 4*

ere 6 rats in each group. P < 0.05, significant response area. †P < 0.05, NLX or NLXmi

arterial blood pO2. These findings are consistent with evidence thatother opiates increase pulmonary vascular resistance in humans(e.g., Popio et al., 1978; Mitaka et al., 1985) and animals (Schuriget al., 1978; Gentil et al., 1989; Hakim et al., 1992). ORs exist onalveolar walls, smooth muscle of the trachea and bronchi, and onautonomic nerve terminals and vagal afferents in the lungs (Cabotet al., 1996; Zebraski et al., 2000; Groneberg and Fischer, 2001). Assuch, the increase in A-a gradient elicited by fentanyl is also likely toinvolve increases in upper and/or lower airways resistance therebydiminishing O2 delivery to alveoli. Indeed, fentanyl increases air-way resistance in rats (Willette et al., 1982, 1983, 1987; Bennettet al., 1997) and in rabbits (Taguchi et al., 1986). In humans, fen-tanyl and sufentanil increase overall airway resistance (Cohendyet al., 1992; Ruiz Neto and Auler Júnior, 1992) via tracheal constric-tion (Yasuda et al., 1978) and bronchoconstriction (Ruiz Neto andAuler Júnior, 1992). Moreover, fentanyl increases the tendency forairway obstruction at glottic and supraglottic levels and producesrigidity of chest wall and abdominal musculature (Niedhart et al.,1989; Bennett et al., 1997; Bowdle, 1998).

4.4. Effects of NLX/NLXmi on fentanyl changes in ventilation, ABGchemistry, and A-a gradient

The finding that NLX and NLXmi did not elicit ventilatoryresponses or changes in ABG chemistry or A-a gradients that weredifferent to those of VEH, is consistent with evidence that blockadeof endogenous �1,2-OR-dependent pathways does not have majoreffects on these parameters (Trescot et al., 2008; Stein and Lang,2009; Stein et al., 2009; Dahan et al., 2010). As expected, pretreat-ment with NLX blocked the effects of fentanyl on ABG chemistryand A-a gradient. NLX also blocked some of the ventilatory depres-sant effects of fentanyl (i.e., the decreases in fR and increases in EIP)whereas it actually reversed (i.e., decrease converted to an increase)many of the depressant effects of fentanyl (i.e., uncovered a facili-tatory response) including V , Ti, PIF and PEF. Since NLXmi did notuncover facilitatory response to fentanyl, the ability of NLX to elicitsuch responses to fentanyl suggests that blockade of central �-ORsallows �- and �-ORs, ORL1, and/or non-OR-dependent mechanismsto facilitate ventilation (see below).

A key conclusion from the NLXmi studies is that the majormechanisms responsible for fentanyl-induced disturbances in ABGchemistry are the peripheral OR-mediated reductions in VT and themismatch in ventilation–perfusion (as denoted by the increase inA-a gradient). More specifically, our study demonstrated that theability of the low dose of NLXmi (1.5 mg/kg) to attenuate the effectsof fentanyl on ABG chemistry was correlated with attenuation of thenegative effects of fentanyl on (1) A-a gradient, and (2) VT specifi-

cally, since NLXmi did not blunt the effects of fentanyl on the otherventilatory parameters except for a minor effect on EIP (Table 6).This raises the possibility that the primary mechanisms by whichfentanyl disturbs ABG chemistry is via activation of peripheral

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F. Henderson et al. / Respiratory Phys

Rs on structures regulating VT including the carotid bodies andeuromuscular elements of the chest wall and diaphragm, andRs within the lungs and pulmonary vasculature (decreased bloodow to alveoli and an increase in upper/lower airways resistance).he findings that NLXmi did not uncover ventilatory excitatoryesponses to fentanyl as effectively as NLX gives confidence toscribing the effects of fentanyl to central and peripheral ORs andurther supports evidence that NLXmi is peripherally restricted inhe rat.

It should be noted that the accurate estimation of A-a gra-ient would normally require a detailed analysis of the changes

n RQ. This quotient can be affected by numerous factors (e.g.,hanges in TB, hypoxia as elicited by opioids) that directly or indi-ectly affect metabolic rate and carbohydrate and lipid metabolismsee Mendoza et al., 2013). A switch to carbohydrate metabolismould increase RQ whereas a switch to lipid metabolism wouldecrease RQ (see Mendoza et al., 2013; van Klinken et al., 2013).he minor changes in BT observed in this study would minimallyffect RQ. In addition, the majority of relevant studies provide com-elling evidence that fentanyl in similar or higher doses used inhe present study have minimal direct effects on carbohydratend lipid metabolism, which is a major determinant of RQ (vanlinken et al., 2013). For example, (1) fentanyl (50 and 100 �g/kg,

.v.) or sufentanil (10–200 �g/kg, i.v.) had minimal effect on cere-ral metabolism/energy status in dogs (Milde et al., 1989, 1990),2) high-dose fentanyl (100 �g/kg, i.v. loading dose followed byn infusion of 200 �g/kg/h, i.v.), did not alter glucose, lactater high-energy metabolite levels in the brains of rats (Keykhaht al., 1988), (3) intra-coronary fentanyl produced minor effectsn left-ventricular mechanoenergetics in dog hearts over a wideange of blood concentration (Kohno et al., 1997), and (4) fen-anyl was devoid of major effects on myocardial metabolism insolated rat hearts (Blaise et al., 1990). Although several stud-es have provided evidence that morphine can either enhance orecrease lipid metabolism (see Mendoza et al., 2013). There is littleirect evidence as to the effects of fentanyl on lipid metabolism.owever, Reneman and Van der Vusse (1982) reported that fen-

anyl (25 �g/kg, i.v.) decreased fatty acid metabolism slightly inhe ischemic myocardium of open-chest dogs. A decrease in fat

etabolism, would shift RQ to a value greater than 0.9, which wouldncrease the calculated A-a gradient (van Klinken et al., 2013). Asuch, keeping to an RQ of 0.9 in the calculations of A-a gradi-nt, would underestimate the negative effects of morphine on gasxchange in the lungs.

. Summary

This study provided evidence that the fentanyl-induced changesn ABG chemistry are closely related to the temporal decreases inT and the increases in A-a gradient. The finding that the abilityf NLXmi to block the effects of fentanyl on ABG chemistry wastrongly correlated with its ability to diminish the fentanyl-inducedecrease in VT and the increase in A-a gradient, provides novelvidence that fentanyl exerts detrimental ventilatory responsesia activation of peripheral �1,2-ORs. The importance of periph-ral �1,2-ORs in the ventilatory depressant effects of fentanyl inur rats is consistent with evidence that NLXmi attenuates thenhibitory effects of morphine in mice (Lewanowitsch and Irvine,002; Lewanowitsch et al., 2006). It is tempting to assume thathe ability of NLXmi to blunt some of the ventilatory effects ofentanyl (i.e., depression of VT and increase in EIP) is due to block-

de of �-ORs in peripheral structures such as the carotid bodies,ulmonary circulation and the chest and diaphragm. However, anxtensive search of the literature could not confirm that direct evi-ence (e.g., analyses of brain levels after peripheral administration)

& Neurobiology 191 (2014) 95– 105 103

exists as to whether NLXmi actually does enter the brain. In addi-tion, NLXmi has at least 10 times lower affinity for opioid receptorsthan NLX (e.g., Lewanowitsch and Irvine, 2003) and we have notfound any studies that compare the effects of, for example, a1.5 mg/kg a dose of intravenous NLX with a 10 times higher dose ofNLXmi to provide more definitive evidence as to whether NLXmiis centrally-active. Moreover, the ventilatory effects of systemi-cally injected opiates may involve ORs in brain structures devoidof a blood–brain barrier, sites that would be readily accessible toNLXmi. These circumventricular organs of the brain, which includethe area postrema (Johnson and Gross, 1993), express �-, �-and �-ORs (Pert et al., 1976; Atweh and Kuhar, 1977; Guan et al., 1999),and direct injections of OR agonists into these structures elicitphysiological responses (Hattori et al., 1991; Bhandari et al., 1992;Mosqueda-Garcia et al., 1993).

Although fentanyl inhibits the carotid chemoreceptor reflex inconscious dogs (Mayer et al., 1989), it is not known whether thisinvolves activation of �1,2-ORs in the carotid bodies or whetherfentanyl depresses the responses of carotid body chemoafferentsupon exposure to hypoxia and/or hypercapnia. The relative contri-bution of central and peripheral ORs in the analgesic and ventilatorydepressant effects of opiates in humans is still open to question.However, peripherally restricted �-OR antagonists (e.g., methyl-naltrexone) attenuate opiate-induced constipation and prurituswhile preserving (the presumably) centrally mediated analgesia(Moss and Rosow, 2008). Of interest is that these antagonists alsoattenuate opiate-induced nausea, vomiting and urinary retention,which are traditionally thought to be due to central actions of opi-ates (Moss and Rosow, 2008).

Conflicts of interest

None.

Acknowledgement

This work was supported by grants from Galleon Pharmaceut-icals.

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