Multiple Opioid Receptors in Endotoxic Shock

Home , Naloxazone

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. cies. These data indicate that the au-selective antagonist 3- Our results clearly establish that centrally or peripherally FNA significantly affected the antinociceptive actions of the administered 8 antagonist ICI reversed endotoxic shock hy- a manner i.v. 15- ,4 agonist morphine; by contrast, the 8-selective antagonist potension in dose-related (effective doses, ICI was without effect upon morphine (,) antinociception. 60 mg/kg; see refs. 25 and 26) via sites within the brain (Fig. These differing nociceptive effects provide one index of the 3). By contrast, the irreversible At antagonist P-FNA was relative selectivities of ,B-FNA and ICI for ,u and 8 receptors, without therapeutic effect in endotoxic shock hypotension at and additional studies were conducted to further pedigree a dose that antagonized morphine analgesia (Fig. 1). Thus, these antagonists (19). Using flurothyl seizure thresholds, within the constraints of present knowledge of multiple endocrine responses, and cardiorespiratory function as end opioid receptor pharmacology, our use of these selective re- points, selectively different responses to physiological or ceptor antagonists has established that endotoxic hypoten- pharmacological opioid challenge were observed when com- sion is mediated by endogenous opioids acting upon 8 opioid bined with ,B-FNA and ICI treatment (19). Other reports also receptors. establish the receptor selectivities of these compounds in vi- These findings are consistent with other observations that tro and in vivo (12-16, 19, 20). indirectly suggested an importance of 8 receptors in shock Data in Fig. 2 Top demonstrate that i.c.v. pretreatment hypotension. For example, higher doses of naloxone are re- quired to reverse shock than to antagonize morphine-mediated (A) analgesia (1-9), indicating a possible action of nalox- 35- one upon 8 receptors. Third ventricular injection of the 8 cm agonist [D-Ala2,D-Leu5]enkephalin produced a shock-like 0 hypotension, whereas similar injection of the ,u agonist mor- x 30- phine failed to do so (27). Additionally, Curtis and Lefer

C.).E have recently concluded that opioid receptors other than K are involved in the pathophysiology of hemorrhagic shock @) 25- (28). Cf) Prior studies have established that naloxone and other 0 nonselective opioid antagonists rapidly reverse the hemody- X) 20- Cu namic, metabolic, autonomic, and neurologic effects of cir- a) culatory shock and spinal trauma (4-8, 17, 18, 29-31). How- ever, relatively nonselective opioid antagonists such as nal- 15 oxone may be expected to have the adverse effect of intensi- fying traumatic pain (32-34). Since, at the doses used in the 2 4 8 16 32 present studies, ICI reversed shock without affecting mor- Morphine sulfate, mg/kg phine-induced analgesia, the further development of selective 8 antagonists may provide an opportunity to reverse cir- FIG. 1. Antinociceptive effects of i.v. morphine sulfate are com- culatory shock without affecting the simultaneous use of pared among rats treated i.c.v. with 13FNA (o--o), ICI (A-.-A), or opioid agonists for the treatment of traumatic pain. saline (e-e) (see text for treatment details). Dose-response data We were were obtained from integrated area responses under dose-time surprised to find that pretreatment with p-FNA curves for individual rats. Lines were obtained by best-fit regres- significantly attenuated the therapeutic effects of subse- sion; vertical bars are SEM. Only the &3-FNA-pretreated rats experi- quent ICI injections in endotoxemic rats. In recent studies enced a significant antagonism of morphine antinociception, as indi- we have shown that naloxazone, an opioid antagonist that cated by a 3-fold rightward shift of the dose-response data. irreversibly blocks high-affinity (A.l-site) binding, failed to Downloaded by guest on September 23, 2021 2900 Medical Sciences: D'Amato and Holaday Proc. NatL Acad ScL USA 81 (1984)

110 -

100 - 12-

90 - II -

80- 10-

70- 9-

60- 8- 50 7 X 40- 125 10 IS 30 45 60 75 90 105 120 Saline pIFm Time after endotoxin, min n = 8 n -ru8= 8 FIG. 2. In rats subjected to endotoxic shock hypotension, arte- 100i rial pressure changes (mm Hg) are compared between saline controls 95 and experimental animals treated 90 with the A antagonist f3-FNA (Top; *-*, 9.8 ,ug of /3-FNA 85 m 12- i.c.v.; o--o, saline i.c.v.), the 8 80 antagonist ICI (Middle; *-*, 60 x 11 mg of ICI per kg i.v.; o-o, saline 0) 75 0) T* i.v.), and the two combined (Bot- -I.05 70- _ 10- tom; o-o, 1-FNA/ICI; o--o, f- E _FNA/saline). Because of the E 9- E 65 E complexity of these experiments, la 60 each of these three groups was 8- run on separate days, with control 55. di and experimental animals simulta- t l' oID . 50. i.v. neously evaluated. In all three injection 0co;O graphs, saline-treated control ani- 45J 10' I ,II mals demonstrate the typical bi- t 125 10 15 30 45 60 75 90 l05 120 Before Saline ICI phasic response to endotoxin in- Time after treatment, min n = 12 n = 10 jection. The irreversible ,u antago- endotoxin nist /3-FNA was injected i.c.v. 18 o00o hr prior to endotoxin (Top and I Bottom), whereas the short-last- 95- ing 8 antagonist ICI was acutely administered i.v. after the onset 90 of endotoxic hypotension (Middle 85- 12- and Bottom). Comparisons be- 80- tween experimental and control I' II- groups were performed by inte- 75- grating areas under response- 10- 70- time data for individual rats, and averages of area scores were com- 65- 9- pared among groups using Stu- dent's t comparison. Asterisk in- 60 8 i dicates significant effect of ICI in 55 reversing the hypotension pro- t 71 duced by endotoxemia (Middle; P 50 I.v. injection < 0.01). Although without effect 45 by itself (Top), pretreatment with I 125 10 15 30 45 60 75 90 105 120 p-FNA/saline p-FNA/ICI 3FNA attenuated the pressor ef- Before Time after treatment, min n = 8 = 8 fect of ICI (Bottom). Vertical bars endotoxin are SEM.

alter endotoxic shock hypotension by itself, yet it also signif- elevation of flurothyl seizure thresholds produced by the 8 icantly attenuated the usual pressor response to naloxone in agonist [D-Ala2,D-Leu5]enkephalin, yet prevented reversal rats subjected to endotoxic shock hypotension (35). Taken of this effect by the 8 antagonist ICI or high doses of nalox- together, these findings indicate that prior occupancy of A one (36). receptor sites by "irreversible" antagonists prevents actions Since our data indicate that irreversible A& antagonists se- of other opioid antagonists that are presumably acting at 8 lectively inhibit the subsequent actions of antagonists at the binding sites. Since the intensity of endotoxic shock hypo- 8 receptor, we suggest that ,u and 8 receptor sites may be tension per se was unaltered by irreversible Au receptor an- part ofthe same macromolecular complex that produce func- tagonists, the binding of endogenously released 8 agonists in tional interactions through noncompetitive systems, possi- endotoxemia (which are presumably responsible for the bly involving allosteric coupling. These observations are in opioid component of shock hypotension) appears to be rela- general accordance with molecular interactions or intercon- tively unaffected by prior occupancy of the A site with irre- versions among opioid receptors as proposed by several in- versible antagonists. This observation has recently been vestigators (21-24). Our results are also consistent with the confirmed by using another pharmacological end point. It observations of Villarreal and co-workers (37, 38), who have was demonstrated that f3-FNA failed to completely block the reported that certain opioid antagonists that have little direct Downloaded by guest on September 23, 2021 Medical Sciences: D'Arnato and Holaday Proc. NatL Acad. Sci. USA 81 (1984) 2901

110-

100 - m, 12- 0 90 - x II- I cm o 80- " 10 T E - 0 E E - 70- E 9-

60- E 8 0 0 50- cn 7 i.c.v. c0 I injection 40- < O.i 1 025 10 15 30 45 60 75 90 105 120 Saline IC0 Before Time after treatment, min n=7 n=8 endotoxin

FIG. 3. Changes in arterial pressure following injection of the 8 antagonist ICI (e*-, 200 /.g) or saline (o--o) i.c.v. in endotoxemic, hypotensive rats. Asterisk indicates significant response to ICI injection when compared to saline control data. Thus, doses of this 8 antagonist that are too low to be effective upon peripheral injection are effective when injected i.c.v., indicating a central site of action. Within the first 5 min, the pattern of response to centrally injected 8 antagonist is more gradual and sustained than following iv. injection (Fig. 2 Middle), where a biphasic response is seen.

actions of their own can block the actions of other antago- 15. Ward, S. J. & Holaday, J. W. (1982) Abstr. Soc. Neurosci. 8, nists such as naloxone. 388. The collective findings of the present studies provide evi- 16. Holaday, J. W. & Ward, S. J. (1982) Abstr. Soc. Neurosci. 8, dence that endogenous opioid systems exert pathophysiolog- 389. 17. Holaday, J. W. & Faden, A. I. (1978) Nature (London) 275, ical effects in endotoxic shock by acting at 8 opioid receptors 450-451. within the central nervous system. Since the analgesic ef- 18. Holaday, J. W. & Faden, A. I. (1980) Brain Res. 189, 295-299. fects of opioids are predominantly mediated by receptors, 19. Holaday, J. W. & Tortella, F. C. (1984) in Recent Progress in the use of selective 8 opioid antagonists affords the opportu- Opioid Research, Central and Peripheral Endorphins, eds. nity to reverse circulatory shock without affecting pain per- Genazzani, A. R. & Muller, E. E. (Raven, New York), in ception or the concomitant use of u opioid analgesics. Addi- press. tionally, the simultaneous use of these unique opioid antago- 20. Krulich, L. & Koenig, J. I. (1984) in Recent Progress in Opioid nists in endotoxic shock has provided evidence supporting Research, The Opioid Modulation of the Endocrine Function, M. functional interactions among multiple opioid receptor sub- eds. Giusti, G., Martini, L., Pepeu, G. C. & Serio, (Raven, New York), in press. as well. types that may pertain to other biological systems 21. Lee, H. M. & Smith, A. P. (1980) Life Sci. 26, 1459-1464. We thank Charles Glatt, Lauren Black, Lydia Robles, Julie Ken- 22. Bowen, W. D., Gentleman, S., Herkenham, M. & Pert, C. B. ner, and Annabelle Trees for their expert assistance. The 3-FNA (1981) Proc. Natl. Acad. Sci. USA 78, 4818-4822. was a generous gift of Drs. A. Takemori and P. Portoghese (Univer- 23. Vaught, J. L., Rothman, R. B. & Westfall, T. C. (1982) Life sity of Minnesota), and the ICI was supplied by Drs. M. Turnbull Sci. 30, 1443. and J. Shaw (ICI Pharmaceuticals, Macclesfield, England). 24. Li, C. H. (1982) Cell 31, 504-505. 25. Holaday, J. W., D'Amato, R. J. & Glatt, C. (1983) Fed. Proc. 1. Krieger, D. T. (1982) Dis.-Mon. 28, 1-53. Fed. Am. Soc. Exp. Biol. 42, 498 (abstr.). 2. Henry, J. L. (1982) Neurosci. Biobehav. Rev. 6, 229-245. 26. Holaday, J. W. & D'Amato, R. J. (1983) Life Sci. 33, 703-706. 3. Holaday, J. W. & Loh, H. H. (1981) in Hormonal Proteins and 27. Holaday, J. W. (1982) Peptides 3, 1023-1029. Peptides, ed. Li, C. H. (Academic, New York), Vol. 10, pp. 28. Curtis, M. T. & Lefer, A. I. (1983) Circ. Shock 10, 131-145. 202-290. 29. Tiengo, M. (1980) Lancet i, 690. 4. Holaday, J. W. (1983) Annu. Rev. Pharmacol. Toxicol. 23, 30. Peters, W. P., Johnson, M. W., Friedman, P. A. & Mitch, 541-594. W. E. (1981) Lancet i, 529-532. 5. Gurll, N. J., Reynolds, D. G., Vargish, T. & Lechner, R. 31. Gurll, N. J. (1983) in Advances in Shock Research, eds. Rei- (1982) J. Pharmacol. Exp. Ther. 202, 625-628. chard, S., Reynolds, D. & Adams, H. (Liss, New York), Vol. 6. Curtis, M. T. & Lefer, A. M. (1982) Eur. J. Pharmacol. 78, 10, pp. 63-71. 307-313. 32. Belenky, G. L., Ruvio, B. A. & Holaday, J. W. (1981) Abstr. 7. Chance, E., Todd, M. H. & Waterfall, J. F. (1981) Br. J. Phar- Soc. Neurosci. 7, 798. macol. 300, 123P (abstr.). 33. Holaday, J. W., D'Amato, R. J. & Faden, A. I. (1981) Science 8. Amir, S. (1982) Eur. J. Pharmacol. 80, 161-162. 213, 216-218. 9. Terenius, L. (1978) Annu. Rev. Pharmacol. Toxicol. 18, 189- 34. Faden, A. I., Jacobs, T. P. & Holaday, J. W. (1981) N. Engl. 204. J. Med. 305, 1063-1067. 10. Iwamoto, E. T. & Martin, W. R. (1981) Med. Res. Rev. 1, 411- 440. 35. Holaday, J. W., Pasternak, G. W., D'Amato, R. J., Ruvio, 11. Zukin, R. S. & Zukin, S. R. (1981) Life Sci. 29, 2681-2690. B. A. & Faden, A. I. (1983) Eur. J. Pharmacol. 89, 293-296. 12. Shaw, J. S., Miller, L., Turnbull, M. J., Gormley, J. J. & Mor- 36. Tortella, F. C. & Holaday, J. W. (1984) Proc. West. Pharma- ley, J. S. (1982) Life Sci. 31, 1259-1262. col. Soc., in press. 13. Takemori, A. E., Larson, D. L. & Portoghese, P. S. (1981) 31. Villarreal, J. E., Herrera, J. E., Salazar, L. A. & Moreno, F. Eur. J. Pharmacol. 70, 445-451. (1982) Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 1303 (abstr.). 14. Ward, S. J., Portoghese, P. S. & Takemori, A. E. (1982) J. 38. Herrera, J. E., Salazar, L. A. & Villarreal, J. E. (1982) Fed. Pharmacol. Exp. Ther. 220, 494. Proc. Fed. Am. Soc. Exp. Biol. 41, 1314 (abstr.). Downloaded by guest on September 23, 2021