Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2898-2901, May 1984 Medical Sciences Multiple opioid receptors in endotoxic shock: Evidence for 6 involvement and ,u-6 interactions in vivo (endogenous opiates/receptor allosterism/cardiovascular function/opiate antagonists/rats) ROBERT D'AMATO AND JOHN W. HOLADAY* Neuropharmacology Branch, Department of Medical Neurosciences, Division of Neuropsychiatry, Walter Reed Army Institute of Research, Washington, DC 20012 Communicated by Choh Hao Li, January 30, 1984 ABSTRACT The use of selective 8 and it opioid antago- macology is less well established and selective antagonists nists has provided evidence that 8 opioid receptors within the are as yet unavailable. brain mediate the endogenous opioid component of endotoxic In this report, we demonstrate that the 8 antagonist ICI, at shock hypotension. The selectivity of these 6 and ,u antagonists doses shown to be without effect upon analgesia (15, 16), was demonstrated by their differing effects upon morphine an- significantly reversed endotoxic hypotension following cen- algesia and endotoxic hypotension. The !t antagonist (-funal- tral or peripheral injection. By contrast, the A antagonist P- trexamine, at doses that antagonized morphine analgesia, FNA failed to alter the pattern of hypotension produced by failed to alter shock, whereas the 6 antagonist M 154,129: endotoxemia at a dose that significantly antagonized mor- [N,N-bisallyl-Tyr-Gly-Gly-I.(CH2S)-Phe-Leu-OH] (ICI) re- phine-induced analgesia (15, 16). Unexpectedly, pretreat- versed shock at doses that failed to block morphine analgesia. ment with ,B-FNA antagonized the ability of ICI to reverse Therefore, selective 8 antagonists may have therapeutic value endotoxic hypotension. These collective observations indi- in reversing circulatory shock without altering the analgesic cate that 8 (not ,u) opioid receptors mediate endotoxic shock actions of endogenous or exogenous opioids. Additional data hypotension. Additionally, the failure of the 8 antagonist ICI revealed that prior occupancy of ,u binding sites by irrevers- to improve arterial pressure following pretreatment with the ible opioid antagonists may allosterically attenuate the actions irreversible A antagonist /-FNA provides in vivo evidence of of antagonists with selectivity for 8 binding sites. For endoge- functional interactions between ,u and 8 binding sites. nous opioid systems, this observation provides an opportunity to link in vivo physiological responses with receptor-level bio- MATERIALS AND METHODS chemical interactions. Following anesthesia with intraperitoneal pentobarbital (20 Several physiological and pathophysiological actions of en- mg/kg) and intramuscular ketamine (60 mg/kg), male dogenous opioid systems have been defined through the use Sprague-Dawley rats (Zivic-Miller, Pittsburgh; 250-300 g) of opioid antagonists (1-3). For example, it has been estab- were surgically implanted with PE50 catheters in the ventral lished that many hemodynamic, autonomic, and metabolic tail artery and external jugular vein as described (17). In ad- effects of circulatory shock following endotoxemia, sepsis, dition, an intracranial guide tube was implanted to facilitate hemorrhage, spinal transection, burn, anaphylaxis, and vari- intracerebroventricular (i.c.v.) injections into the right later- ous other causes are promptly reversed by the opioid antago- al ventricle (18). One day later, arterial catheters from these nist naloxone as well as several other antagonists that have conscious rats were connected to microtransducers (Narco the Bio-systems, RP 1500i), and cardiovascular data were re- simultaneous actions at more than one of opioid receptor corded and analyzed with a Narcotrace 80 physiograph sys- subtypes (4-8). Evidence has also accrued to indicate that tem. Rats were randomly preassigned to drug treatment endogenous opioid systems play a critical analgesic role in groups, and all animals were then injected intravenously responses to painful stimuli (1-3, 9). Because of the duality (i.v.) with Escherichia coli lipopolysaccharide endotoxin (15 of action of endogenous opioids upon cardiovascular and an- mg/kg, Difco, lot no. 654109). Mean arterial pressure (MAP) algesic systems, administration of naloxone for the treat- was monitored; ==20% of the rats failed to demonstrate a pre- ment of circulatory shock may enhance pain by antagonizing cipitous 25-30 mm of Hg fall in MAP within the first 10 min, endogenous or exogenous opioid analgesia. If shock and an- and they were excluded. Rats were injected i.c.v. with Ev- algesia are mediated by different types of opioid receptors, ans blue dye immediately following acute cardiovascular the use of opioid antagonists with receptor selectivity could measurements; complete ventricular perfusion was con- allow for a dissociation of these effects. firmed at necropsy. At least five different opioid receptor subtypes have been For 3FNA studies, rats were treated i.c.v. with either ,- proposed, including morphine-preferring (,) and enkephalin- FNA [20 nM (or 9.8 jig) in 20 ,ul over 20 sec] or an equal preferring (8) receptors (10, 11). As of yet, the precise phys- volume of saline 18 hr prior to endotoxin challenge. This pre- iological functions of distinct multiple opioid receptor sub- treatment time and dose were chosen since ,3-FNA has been types are poorly understood, largely due to an absence of shown to have acute K-agonist properties that diminish with- selective antagonists. The recent development of M 154,129: in hours, whereas the irreversible ,u-antagonist properties of [N,N-bisallyl-Tyr-Gly-Gly-T-(CH2S)-Phe-Leu-OHI (ICI) ,B3FNA (as demonstrated by its actions in blocking morphine (12), a reversible antagonist with selectivity for 8 opioid re- analgesia) persist at least 24 hr (13-16). Because of the scar- ceptors, and -funaltrexamine (,B-FNA), an irreversible ,u city of ,B-FNA, the efficacy of i.v. injections could not be antagonist (13, 14), provided a unique opportunity to evalu- evaluated. ate the relative importance of 8 and u opioid receptors in the Unlike the effects of ICI treatment were exam- endogenous opioid component ofendotoxic shock. Although 3FNA, other opioid receptors may be involved in shock, their phar- Abbreviations: ICI, M 154,129: [N,N-bisallyl-Tyr-Gly-Gly-T- (CH2S)-Phe-Leu-OH]; 3-FNA, ,B-funaltrexamine; i.c.v., intracere- The publication costs of this article were defrayed in part by page charge broventricular(ly); iv., intravenous(ly); MAP, mean arterial pres- payment. This article must therefore be hereby marked "advertisement" sure. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2898 Downloaded by guest on September 23, 2021 Medical Sciences: D'Arnato and Holaday Proc. NatL. Acad. Sci. USA 81 (1984) 2899 ined after the onset of endotoxic hypotension due to its short with f3-FNA failed to alter the typical pattern of arterial pres- duration of action. After MAP fell by 25 mm of Hg, the cen- sure changes produced by endotoxemia. By contrast, injec- tral actions of i.c.v. ICI (200 nM or 200 ,g) or equivolume tion of ICI at an i.v. dose of60 mg/kg resulted in a significant saline (20 41 over 20 sec) were compared to the peripheral and sustained increase in MAP when compared to saline- effects of ICI (15-60 mg/kg) or saline administered i.v. (1.0 treated control rats (Fig. 2 Middle; P < 0.001). As seen in ml/kg). Fig. 3, the i.c.v. injection of ICI also produced a significant To establish their receptor selectivity, 3-FNA and ICI and sustained increase in MAP (P < 0.01), indicating a cen- were injected i.c.v. and hot-plate escape latencies were eval- tral site of action. Note that the i.v. administration of ICI uated following i.v. challenge with varying doses of the A- produced an initial spike in MAP (Fig. 2 Middle and Bottom) selective agonist morphine sulfate (15, 16). The hot-plate that was not observed upon i.c.v. injection (Fig. 3), possibly was maintained at 520C, and maximal cutoff latencies were representing a peripheral action of ICI in this initial pressor 60 sec. Morphine sulfate was administered i.v. (in a volume response. These doses and routes of antagonist injection of 1.0 ml/kg) in rats pretreated with ICI or /-FNA according were without significant effect upon MAP in normotensive, to the same protocol as with endotoxin administration. nonshocked animals, although a slight increase in MAP (-=7 Time-response data for individual rats were integrated and mm of Hg; P = not significant) was observed following i.c.v. averaged according to morphine dose and treatment groups. injection of ICI (data not shown). These data indicate that In conducting the research described in this report, we ad- ICI predominantly acts to reverse shock hypotension and is hered to the "Guide for the Care and Use of Laboratory Ani- not acting as a pressor substance per se. mals" as promulgated by the Committee on Care and Use of Since others have indicated that interactions among opioid Laboratory Animals of the Institute of Laboratory Animal receptor subtypes may occur in various biological systems Resources, National Research Council. (21-24), we were interested in evaluating the influence of pri- or ,u receptor antagonism upon the cardiovascular responses RESULTS AND DISCUSSION produced by the 8 antagonist following the onset of endotox- Neither f3-FNA nor ICI by itself resulted in alterations in ic hypotension. As shown in Fig. 2 Bottom, pretreatment baseline hot-plate escape latencies. Furthermore, the data in with f3-FNA significantly attenuated the protracted pressor Fig. 1 indicate that ICI did not affect the antinociceptive effects of this dose of ICI (compare Fig. 2 Middle and Bot- responses to morphine challenge, whereas f3-FNA pretreat- tom). Thus, although without hemodynamic effects by itself ment resulted in a significant 3-fold antagonism of mor- in endotoxemic rats, f3-FNA significantly attenuated the phine's antinociceptive effects upon hot-plate escape laten- usual pressor response produced by ICI.