Enkephalin (opiate) receptors in the intestine

328

T I P S - D e c e m b e r 1981

binding to isoproterenol whereas the remaining native receptors behave as do receptors from naive cells. An intriguing interpretation of these observations becomes apparent when it is recognized that continued exposure of ceils to agonists leads to loss of receptors from the cell. The mechanism qnderlying the loss of B A R is not known, but an interesting possibility is consistent with a variety of independent observatitms. First, it is known that down-regulation of plasma membrane receptors for polypeptide hormones involves an agonist-induced endoc) totic process '6. Dansyleadavarin, which blocks the coated pit-mediated endocytosis of polypcptide hormone receptors has been reported also to block the catecholamine-induced loss of B A R in BHK cells '7. Second, Chuang and Costa 's reported that "soluble" B A R could be detected in the cytosol of frog crythrocyte:; desensitized by exposure to cateeholamines. Our observation 's that up to 60% of the B A R of astrocytoma cells exposed to catecholamines co-migrate in sucrose gradients with enzyme markers for endoplasmic reticulum and golgi apparatus is consistent with the possibility that such uncoupled receptors reside on small endocytotic vesicles. O u r most recent studies have shown that the so-called lost B A R ( B A R , ) can be completely recovered even in the presence of cycloheximide at a concentration sufficient to block protein synthesis by 9 0 + % . This implies that

•

the

modification

,

,

subserving

•

growing body of evidence indicates the existence of cellular mechanisms fl~r modification of the n u m b e r of functional B A R as an adaptive rcsp~msc to fluctuating levels of catccholamil~esin vivo. The study of these processes in model systems may help to provide the details of what appears to be an elaborate set of regulatory processes that modulate catecholamine-mediated intercellular communiication. Reading list I Bloom, F. (1975)Rev, Physiol. Biochem. Phar. macol. 74, 1-1(13 2 Perkins,J, P.,Su, Y. F. md Harden, T, K. (1979) Drug Alcohol 1.Pep.4, 2 79-94 3 Duly.J. W. (! ~77) Cydw Nucleotides in the Nervous System, Plenum Press 4 Kalisker. A, Rutlcdge, C. O. and Perkins, J. P. (I)73)MOt. Pharmacol 9, 619-29 5 Dismukes, R. k. and Daly, J.W. (1975)Exp. Neurol. 49, ! 51J-Ir30 6 Wolfe, B.B., Harden, T K., Sporn, J. R. and M.dinoff, P. It. {It~78) J. Pharmacol. Exp. Therap. 207, 446-57 7 Terasaki, W.L.. Brtx~ker, G., deVellis, J., English, D., Hsu, C. Y. and Moylan,R. P. (1978) Adv. Cyclic Nucleotide Res. 9, 33-52 8 Lefkowitz,R. J, Weasels. M, R. and Stadel,J. M. (I980)Curr. Top. Ceil. Regul. Vol. 17, 205-230 9 Mukherjee, C. and Lefkowitz.R. J. (1977) 114o1. Pharmacol. ! 3, 291-303 I0 Shear, M.. lnsel, P.A., Melmon, K.L. and Coffino,P. (1976)1. Biol. Chem. 251,7572-7576 ! ! Su, Y.-F., Harden, T, K. and Perkins,J. P. (1980) J. Biol. Chem. 255.7410--7419 12 ~es~ls. M. R,, Mullikm,D. and Lefkowitz,R. J. ~!979)Mol. PharmacoL 16, 10-20

13 lyengar, R.. Bhat. M.K., Ri~r, M.E. and Birnbamner, L. (1'~81)J. Biol. Chem. 25(~. 48111--4815 14 Greene. D. A, and Clark. R. B. (1981)J. Biol. Chem. 25h, 2105-211114 15 Harden, 1". K., Cotton, C. U., Waldon. G. L.. Lunon.J. K. aml PerkinxJ. P. (I 9811)Sck.nor,211), 441-.443 16 Pastan. I. H. and Willingham,M. C. (iqSI IAnn. Rev. Physiol. 43, 23q-250 17 Reggiani,A.. Vernaleone,F. and Robi~m, G. A. (1980)Neurtt~ci. Ahstr. 6, 534 18 Chuang, D. M. and Costa, E. (1971))Proc. Natl Acad. Aci. U,S.A. 76, 31)24-31)28

John Perkins receivedhis Ph.D. in Pharmacology from Yale University in 1966. He then joined Dr Edwin Krebs at the Universityof Washington as a postdoctoral fellow. In 1068, he joined the factdo, of the Department of Pharmacology at the University of Colorado. Since 1977, he ha~ been Professor and Chairman of the Deparmzent of Pharmacology at the Uniz~ersi~. of North Carolina at Chapel Hill. The major l~;rtion ofhiz re,arch career has involved a study of hormonal regu. lation of the adenylate cyclasesystem.

Enkephali,,,opiate)-receptors in' the

the

p~

-

ence ofcvcloheximide begins after a 4 h lag

and proceeds to 100% recovery at a rate that is 35--40% that observed in the absence of cycloheximide. Thus, if an endocyto~ic mechanism is involved in the process of desensitization it apparently does not result in the rapid proteolysis of BAR: perhaps other reversible chemical modifications (glycosylation, phosphorylation?) are involved in the reversible loss of the capacity of B A R to bind to IHYP. It would be premature to conclude from such data that a process of endoeytosis and subsequent modification of receptors is involved in agonist-induced downregulation of BAR. However, a potentially fruitful direction for further study is indicated, Although some progress has been made in underst,mding the mechanisms of catecholamine-indueed desensitization in vitro, the physiological significance of the,~ processes is still not clear. Nonetheless, a ~' Elsevicr/Noflh-HollandBiomedicalPre~s1981 tlIt~.~- h147181t(l~l~- 011OO/102.75

intestine

Tetsuo Oka Department of Pharmacol,gy. School of Aledicine. l'okai Univcrxity. l~ehara 259--1 I, Japan.

The presence of enkephalins in the intestitle of various species' and the existence of enkephalin (opiate) receptors in the guinea-pig ileum are well-known 2. However, the significance of enkephalins in the intestine of animals other tikan the guinea pig has been uncertaiq, sine:: they 'rove scarcely been studied. Recently, the rabbit ileum was shown to contain a r,ew type of enkephalin receptor a. w h k n had significantly different characteristics from the well known opiate receptor in the guinea-pig ileum. Moreover, results from experiments perfi~n acd on the chronic spinal dog 4 and on isola ed preparations such as guinea-pig ileum s, ~qou~ vas defercns 5 and rat vas

deferens '~ strongly suggested the existence of multiple opioid peptide (opiate) receptors (i.e., putatiee #, K, or, ~5 a n d , receptors). Thus, it has been of interest and importance to characterize the eakephalin receptors in the intestine of various species. In order to do this, effects of the classical non-peptide narcotic analgesics and opioid peptides on electrically-induced contractions of the myenterie plexus-longitudinal muscle (MP-LM) preparation of the ileum of various species have been studied to determine the rank order of agonist activities and the Ke (equilibrium dissociation constant) value of narcotic antagonists against opioids in the assay tissues. Results

T I P S - D e c e m b e r 1981

have shown thal enkephalin receptors in the MP-I.M preparation of the ileum of the dog, rabbit, rat and mouse had significantly difR'rent characteristics from the wellknown opiate receptor in the guinea-pig ileum~.

Enkephalinreceptor in the rabbit Ileum

3 2,~

Mc Phe'.Mctl())=~d]-cnkephalin. mhich had been shox~,n to be relati~clx ~lectite mu-recepior agonisP, were less potent in inhibiting the c o n t r a c t i o n s o f the s t r i p t h a n

cnkephalins. in contrast to opioid peptides, nmrphinc at concentrations up to i II 4 NIdid not pn,duce 511% inhibition of the eleetricalbevoked contraclions. The other classical non-peptide narcotic analgesics such a,, methadone and pethidine at conccntralions ranging from il~ + to Ill ." M ,,htw+ed no significant depression of contraction,, of the strip. Thus. the MP-I+M preparation o[ the rabbit ileum scart~ly contains mureceptors.

Electrically-evoked contractions of the MP-LM strip of the rabbit ileum were completely inhibited either by letrodotoxin, atropine or opioid peptides. U..te magnitude of the inhibition was dose dependent (Fig. I ). In contrast. Sl~mtaneous contractions of the strip were only slightly depressed (usually less than 20%) by these agents (Fig. I). I n c o n t r a M to m u - r e c e p t o r agonists. Both [MeP]-enkephalin and [l.euS] - kappa-receptor agonists such as ketoc.xcenkephalin, which was shown to be pre.~nt lazocine' and bremazocine ~ had ~er~ in the intestineL significantly depres.~d the strong inhibitory, actions on the electriccovtractions of the strip. The inhibitoD ally-induced contractions of the strip. l~)tcncy of these two pentapeptides was Bremazocine had the greatest inhibitor~ essentially the same. The synthetic potcncy among opioids employed. The Ke enkephalin analogues, [D-Alaa,MeP] - value against ketocydazocine of naloxone enkephalin, [ D-AlaLLeuS]-enkephalin. was significantly higher than that of Mr [ D. AlaLMePl-enkephalinamide and 2266. which was reported to be a relatively [D-Ala=,D-Leu~l-enkephalin, were mor~. selective kappa-receptor antagonistL indila~tent in inhibiting the twitch-like ta)ntrac- cating that ketocx'clazocine inhibited the tions of the strip than naturally occurring contractions by interacting with kappaenkephalins. Both another endogenous receptors in the rabbit ileum. Ho+vevcr. the opioid peptide, /3-endorphin, which was Ke value either against IMePi-enkephalin not detected in the intestineL and another or against [D-AlaLMeP]-enkephalin of synthetic em, ephalin analogue. [D-Ala ". naloxone was not significantly different from that of Mr 22<+6. indicating that the+~ two pentapeptides inhibited the contractions by interacting mainly xqth receptors other than kappa-receptors. ] h e Kc value of naloxone either against [MePIenkephalin or against [D-Aia=,MeP] enkephalin obtained in the rabbit ileum was similar to that obtained in the mou~" vas deferens. However. enkephalin receptors in the rabbit ileum are likch t o be different from those in the mouse vas deR, rens since [D-AlaLD-LeuS]-enkepbalin, x~hich was reported to be a relatively specific L.d,..,.l, L---- + . d , = . t = . i ,.~.a_a..l delta-receptor agoni.q t°, had .,ignificantl.x higher inhibitory potency than [l)Ah.i=,MeP]-enkephalin in the mouse vas (.) (O-Ato~.Mel 6)- onkopholin 2 x tO"e M defcrens while the latter had significantly higher potency than the t'ormcr in the rabbit ileum. F g, I, The effect of [D-Ala=,MeP.].enkephalin on ez+.('tricully.evokedcontractions of the MP. I- M Sml) o.f tl e rabbit ih,unl, Iiy wcltching tilt" ,~l)ontallecm.~ ('+mtrcw. Enkephalinreceptors in the dog ileum U ms ,m an ink ,'riling pen o.wilh~gnll~h, eh,ctrical In contrast to the rabhit ileunl, I~+th ~1iml~hlti¢m was apidied manually fat a freqm'm'y nmgJ+'g fi'Otll O.117 I0 O, [)0 ltz
41 I') I'~t~ 2

(MetS)-enkepholin

2 x I0 - $ M

Ih," ,+!],+,1 ,,l [th't;J-,,r~,+l,h,Jh,:

h+,t;~ ,,n

+/w t l P . I t! +rap ,,! +h,+,h,¢ d,,um I th'++l r'~&,'lm,:h'+ 2 . i 0 * ~ ,+~ admmlswret Into 4 ml o[the hath fluid m I'+du~'.s Of 40 ~J at the d,~t

enkephalin. [l)-Ala'. i eu~Fenkephaltn. [I)-Ala =. l)-l.eu~]-enkcphalin. ID-Ala ~ MePl-cnkcphalinamide. [ MeP]-cnkephalin and [LeuS]-cnkephalin ~as the same a,+ that obtained in the rabbit deum. Mor¢oxer. the Ke valu~ ~q naloxone ciil~er against [.MeP]-enkephalin or agaia~t [i)Ala =. MeP]-enkephalin x~a,, al~+ e,+~entlall,+ the ,,ame as that obtained in the rab +It ileum Thus. enkephalin receptors In the dog ileum ;ire likeK to I~" the same a,+tht~c in the rabbit ileum. It is tentatively Woposed, therefore, to call the enkephalin receptor found both in the rabbit ileum aqd in the dog ileum the iota reccpt,+r t l r t : n l inte,,tine). in contraq to the rabbit ileulu, the d-+~ ileum is likelx it, contain mu-recept,,P, since the cla,,,+ical non-l~:ptid+ n.:rcotic analge,,ic~ ,,uch as morphine, methadt+nc and l~:thidine haxe slgnifican! inhihih,rx adions on the MP-LM ~trip of the dog ileum, although the+ narcotic arl:ll~e,,io. xxere [e.~s p o t e n t in i n h i b i t i n g the contr;..'-

lions of the ,,trip than opioid t~.'ptid.-'.',. Additional e,,idence supportnlg the presence of nlu-receptors m the do~ ileum i,, the fact that the Ke ~aluc of nah-,one against morphine, obtained in the dog ileum, xxas es.,,cntiall.x the .,ame :is that obtained +n the guinea-pig ileum and t~as approximatcl.~ tell times Io~er thzin the Kc ~alue of nJloxone again.~t [McPl-enkcphalin obtained either in the rabbit ileum, d o g ileum or ill IliOUS¢ va~ d e f e r e n s . M o r e o v e r . tile relative

potency of [D-Ala". MePheLMet(O)5-ol] enkephalin, ahich i,+ a relati~elx +,clectixc mu-rcceptor agonist, t~as higher in the d o g ileum than in the rabbit ileum. Brcmazocinc. a kappa-receptor agonist. alm~ had significant inhibitory actions in the dog ileum, ahhough its relative potenc.~ ~as much less in the dog ilemn than in the rabbit ileum.

330

Enkephalin receptors in the guinea-pig ileum It is well-known that the guinea-pig ileum contains opiate receptors and it has been widely employed as a saitable preparation for the measurement of the activity of classical morphine-like compounds. The guinea-pig ileum has also pla)ed an important role in the discovery of enkephalins as an/n vitro bioassay preparation. The fact that the Ke value of naloxonc against nonpeptide narcotic analgesics was essentially the ,same as that against opioid peptides and was approximately ten times lower than that against [MeP]-cnkephalin obtained either in the mouse vas deferens, rabbit ileum or dog ileum, suggests that opioid peptides inhibit the contractions by interacting mainly with mu-receptors in the guinea-pig ileum. In contrast to guinea-pig ileum, opioid peptides other than [D-Ala 2, MePhe', Met(O)S-ol]-enkephalin inhibited the contractions by interacting mainly with delta-receptors in the mouse vas defcrens, since the Ke value of naloxone against opioid pcptidcs such as [MeP]-enkephalin and [ D-Ala2,MeP]-enkephalin was approximately ten times higher than thal against morphine-like compounds in the mouse vas deferens. The kappa-receptor agonists such as bremazocine and ketocyclazocine were more potent than morphine in inhibiting the contractions of the guinea-pig ileum, The Ke value against ketocyclazocine of Mr 2206 was significa,0?: k,v, er than ihal of naloxone in the guinea-pig ileum, indicating that the ;,uinea-pig ileum also contained the kappa-receptor. Enkephalin receptor in the ih:um of the other animals Naturally occurring enkephalins such as [MeP]-enkephalin and [LetP]-enkephalin were at least ten times more potent than morphine in inhibiting the electricallyevoked contractions of the MP-LM preparation either of the rat ileum or of the mouse ileum. And the Ke value of naloxone against [MeP]-enkephalin obtained in these two animals was approximately the same as that obtained in the rabbit ileum and was approximately ten times higher than that obtained in the guinea-pig ileum. Thus, the characteristic~ of enkephalin receptors either in the rat ileum or in the mouse ileum were similar t,o those in the rabbit ileum and were different from those in the guinea-pig ileum. Reading list ! Hughes, J.. Kosteditz. H, W. and Smith. T. W. (1977) Br. 1. Pharmacol. 61.639--647 2 Paton. W. D. M. and Zar, M. A. ( ! 968)J. PhysioL 194, I .~-33

TIPS

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December 1981

3 ()ka. T, (1980) Br. J. Pharmac(d. 68, I03-1q5 4 Martin, W. R . Eades, C. G., Thompson. J. A., tluppler, R. E. and Gil~'rl. P. E. (1976)J. Phar. macol. Exp. Ther. 197,517-532 5 Lord, J. A. It., Walerfi¢ld, A. A., itughes, J. and Koslerlit& H. W. (It)77) Nature (London) 267, 495.--49 ~ , 6 Lemaire, ~., Magnan, J. and Regoli, D. (1978)Br, J i'harma 0,5 64, 327-329 7 0 k a , T., Nq,'~hi, K, and Suda, M. (in prc.~) in

~euromutwnitte, Receptors: BiochemitvJI Aspect and Physiological 3igniflcam'e (Yamamura, ft, i. and Yoshida, H, eds) John Wiley & Sons, Inc.. New York 8 Chang, K.-J,, Cooper, B.R., Hazum, E, and Cuatrcca~s, P. (1979) Mo/. Pharmacol. 1~, 91-104 Y R6mer. D.. Bii~her, H., Hill, R. C.. Maurcr. R.. Percher, T.J.. Welle, tl. B.A., Bakel, H. C. C. K. and Akkerman. A. M. (1080) life Sci. 27, ~71-~78 I 0 Koslerlitr~ H. W. (1978) in Advam'es in Pharma, ccdogy and Therapeutics (Jacob, J. cd) Vol, I, pp. 15-23, Pergamon Prc,~, New York ii

Tetsuo Oka was born in Okayama City in 1938 and graduated from geio University Medi(~l School in 1063. He then joined the staff o f the Pharmacology Dcpartment. at the Medical Schm.d. under Professor E. itosoya, becoming Assistant Professor in 1072. Between 1007 and 1069, he ,'~t~ a Postdoctoral Fellow sumdying m,uropharmacology at tke Univer,w~" of Michigan under Professor M. H. S,,evt,rs. Sim'e 1074, he lut~ been Professor cat the Department of Pharma. cology at 1oke,: UniveTsity Medwal S('hool.

i

Drugs and the acute porphyrias M. R. Moore, K. E. L. McColl and A. Goldberg Universi~."of Glasgow. Department of Medicine, Western lnfirma~. , Gimgow G I 16NT. U.K.

The acute porphyrias have been described as pharmacogenetic diseases, that is, genetically transmitted diseases in which there is an idiosyncratic reaction to the effects of many commonly used drugs'. They are a group of metabolic disorders in which there are partial deficiencies in the activities of individual haem biosynthet;c ,mzymes (Fig. 1). As a result of this d~,-.,mution in the activity of one of the en: ~rues of the pathway, there is a consequent marked increase in the activity of the initial and rate controlling enzyme of the pathway, deltaaminolaevulinic acid synthetase which normally results in overproduction of porphyrin precursors and porphyrins synthesized prior to the genetic blocks. The porphyrias may be classified into two groups. The first classification is on ti'l~ basis of whether or not the disease has an acute presentation with or without cutaneous manifestati ms. They may secondarily be identified as hepatic or erythropoietic porphyrias dependent on the primary locus of the metabolic defects . These classifications are shown in Fig. 2. The effects of drugs in the porphyrias is o f greatest importance in the acute porphyrias, in which diseases excess concentrations of the porphyrin precursors, deltaaminolaevulinic acid and porphobilinogen are synthesized and excreted in the urine

and the normal presentation of the disease is in acute attack. The various clinical manifestations of an attack of acute porphyria may be explained by neurodysfunction involving the autonomic, central and peripheral nervous systems. Severe abdominal pain, vomiting and constipation, tachycardia and hypertension are the common presenting features of such an attack. In the most severe attacks, the peripheral nervous system becomes involved with potential development of a fatal respiratory paralysis. Mental disturbance and convulsions are found at the height of an attack and illustrate CNS involvement. The non-acute porphyrias are generally a less severe form of disease usually presenting with skin photosensitivity. Drugs are of less importance in the non-acute porphyrias although there is good evidence that various hormone preparations and ethanol may exacerbate cutaneous hepatic porphyriasu . Influence or d r ~ on control of Imem

bk~thesb In order to understand the mode of action of drugs upon the haem biosynthetic pathway, and in consequence upon the porphyrias, it is important to understand that the pathway is controlled by feedback inhibition and repression of the initial © ElkvierlNonh-HollandBiomedicalPress 1981 0165 - 6147/81/00110- 0000/$02.75