Enkephalin Amide Research Articles

Enkephalin dimers: Regulation of cyclic AMP levels in NG108-15 cells

Intracellular cyclic AMP levels were determined for dimeric and monomeric enkephalins interacting with PGE 1-stimulated NG108-15 cells. The dimeric pentapeptide enkephalin (DPE 2), [D-Ala 2, Leu 5 -NH-CH 2] 2, displaying very high affinity (K = 4.2 ± 0.3 nM −1) for the δ-opiate receptor, inhibited cyclic AMP production by 70%. Its IC 50-value was between 0.1 and 0.2 nM, similar to that of the potent δ-agonist [D-Ala 2, D-Leu 5] enkephalin (DADLE) with K = 1.0 ± 0.1 nM −1. [D-Ala 2, Leu 5] enkephalin amide (DALEA), which is the monomer of DPE 2, showed an IC 50 = 4 nM. The dimeric tetrapeptide enkephalin (DTE 12), [D-Ala 2, des-Leu 5-NH-(CH 2) 6] 2 and its monomer [D-Ala 2, desLeu 5] enkephalin amide (DAPEA) showed IC 50 = 2 and 20 nM, respectively. These results indicate that the DPE 2 and DTE 12 enkephalin dimers are potent δ-agonists.

Differential effects of GTP and cations on binding of labeled dimeric and monomeric enkephalins to neuroblastoma-glioma cell delta opiate receptors

Binding of radio-labeled enkephalin monomers [D-Ala 2,Met 5]Enkephalin Amide (DAMEA) and [D-Ala 2,D-Leu 5]Enkephalin (DADLE) and a dimer of [D-Ala 2,Leu] Enkephalin Amide (DPE 2) to neuroblastoma-glioma (NG108-15) cells was examined in the presence and absence of GTP and/or cations. We found that: (1) binding occurs to a single class of homogeneous and non-interacting membane sites; (2) the affinity of the enkephalin dimer is reduced 50% in the presence of Mn 2+ and 65% in the presence of both Mn 2+ and GTP; (3) GTP alone either increases or does not change affinity of DPE 2; (4) Na + and GTP significantly decrease the affinities of monomers, but not that of the dimer; and (5) a higher concentration (0.1 mM) of GTP increases the binding of DPE 2 but significantly decreases binding of monomers. Conclusion: Changes in binding of a dimeric enkephalin by Na +, Mn 2+ and GTP are significantly and qualitatively different than those occurring for monomers.

Dimeric tetrapeptide enkephalins display extraordinary selectivity for the delta opiate receptor.

The μ and δ opiate receptors, postulated on the basis of pharmacological observations, have been well characterized in ligand-binding studies1–6. The several classes of opiate receptor appear to subserve different functions7–9. Although highly specific μ ligands are available, there has been a paucity of high-affinity ligands with δ specificity that could be used to investigate the functions, properties and spatial distribution of the δ receptor. Previous structure–activity studies of the enkephalins have suggested that a free carboxyl group at the C-terminus is an important determinant of δ selectivity9,10: amidation of Leu5 or Met5 results in a non-selective ligand. Recently, however, we have demonstrated that cross-linking enkephalin amides by a methylene bridge of suitable length produces a dimeric pentapeptide, with greater affinity and selectivity for the δ receptor than is found in the original δ ligand11,12. Evidence that this dimer may interact simultaneously with two δ, but not two μ receptors12 is consistent with independent demonstrations that the δ receptors are clustered in the membrane7,13. When the C-terminal amino acid of enkephalin is removed, the resulting tetrapeptide enkephalin amide H-Tyr-D-Ala-Gly-Phe-NH2 and its analogues are potent and selective ligands for μ opiate sites14,15. If the δ receptors were closely clustered, as suggested by our findings with dimeric enkephalin pentapeptides11,12 and by others7,13, then it might be possible to form a dimeric analogue of the tetrapeptide enkephalin which could interact with two δ receptors but not with two μ receptors. Theoretically, this would confer δ selectivity on the dimer of a μ-selective ligand. We have thus synthesized a series of dimeric analogues of the μ -selective tetrapeptide [D-Ala2, des-Leu5]enkephalin amide by cross-linking at the C-terminus with NH2-(CH2)n-NH2, with n = 2–12. We report here that the dimer with n = 12 has a nearly 1,000-fold increase in δ/μ selectivity ratio, and is thus a δ-selective ligand.

ChemInform Abstract: Synthesis and Biological Properties of Enkephalin‐like Peptides Containing Carboranylalanine in Place of Phenylalanine.

AbstractDie Darstellung der Titelverbindungen (IIIb) und ‐ ähnlich ‐ (IVa), (IVb) erfolgt nach klassischer Methode in Lösung.

Dehydro-enkephalins. IV. Discriminative recognition of delta and mu opiate receptors by enkephalin analogs

We have studied the receptor binding activities of C -terminal free and amidated enkephalins with and without the dehydrophenylalanine 4 residue. For the selective labeling of so-called δ and μ opiate receptors, specific tracers were used at low concentrations in rat brain membranes and neuroblastoma cells containing pure δ receptors. C -Terminal free enkephalins are five to eight times more potent in the assay for δ receptors than in μ assay, while the amides are almost equipotent in both assays. The presence of a C-terminal carboxyl group is a determining factor for selective activity. [D-Ala 2, ΔPhe 4, Met 5]-enkephalin amide is very potent in all of the binding assays examined, and, in particular, twice as active as the saturated amide and the C -terminal free enkephalin in the δ assay. We suggest that the steric arrangement of the dehydrophenylalanine residue in position 4 is very important to the enhanced interaction with the δ receptors.

Synthesis and Biological Properties of Enkephalin‐like Peptides Containing Carboranylalanine in Place of Phenylalanine

AbstractThe syntheses of [D‐Ala2, Car4, Leu5]‐enkephalin, its amide, and of [D‐Ala2, Car4, Met5]‐enkephalin amide are described in detail. The three compounds show pronounced morphine‐like potencies in certain bio‐assays, and were used to investigate the influence of electronic, steric and hydrophobic properties of the side‐chains of amino acids no. 4 and 5 on opioid activity (see [13]).

Dehydro-enkephalins: Receptor binding activity of unsaturated analogs of Leu 5-enkephalin

FEBS LettersVolume 133, Issue 2 p. 269-271 Full-length articleFree Access Dehydro-enkephalins: Receptor binding activity of unsaturated analogs of Leu5-enkephalin Yasuyuki Shimohigashi, Yasuyuki Shimohigashi Department of Chemistry, University of Georgia, Athens, GA 30602, USA Present address: NIH, NICHD, Bethesda, MD 20205, USA Search for more papers by this authorTommaso Costa, Tommaso Costa National Institute of Child Health and Human Development, National Institues of Health, Bethesda, MD 20205, USASearch for more papers by this authorCharles H. Stammer, Charles H. Stammer Department of Chemistry, University of Georgia, Athens, GA 30602, USASearch for more papers by this author Yasuyuki Shimohigashi, Yasuyuki Shimohigashi Department of Chemistry, University of Georgia, Athens, GA 30602, USA Present address: NIH, NICHD, Bethesda, MD 20205, USA Search for more papers by this authorTommaso Costa, Tommaso Costa National Institute of Child Health and Human Development, National Institues of Health, Bethesda, MD 20205, USASearch for more papers by this authorCharles H. Stammer, Charles H. Stammer Department of Chemistry, University of Georgia, Athens, GA 30602, USASearch for more papers by this author First published: October 26, 1981 https://doi.org/10.1016/0014-5793(81)80521-2Citations: 31 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 I. Paper, M.L. English, C.H. Stammer, Biochem. Biophys. Res. Commun., 85, (1978), 780– 782. 2 C.H. Stammer, B. Weinstein Chemistry and Biochemistry of Amino Acids, Peptides and Proteins 6, (1981), in press 3 R.E. Chipkin, J.M. Stewart, C.H. Stammer, Biochem. Biophys. Res. Commun., 87, (1979), 890– 895. 4 E.G. Breitholle, C.H. Stammer, J. Org. Chem., 41, (1976), 1344– 1349. 5 R. Walter, Fed. Proc. FASEB, 36, (1977), 1872– 1878. 6 J.A.H. Lord, A.A. Walerfield, J. Hughes, H.W. Kosterlitz, Nature, 267, (1977), 495– 499. 7 I. Photaki, J. Am. Chem. Soc., 85, (1963), 1123– 1126. 8 M. Bergmann, U. Schmidt, A. Miekeley, Z. Physiol. Chem., 187, (1930), 264– 276. 9 H. Poisel, U. Schmidt, Int. Ed. Engl. Angew. Chem. 15, (1976), 294– 295. 10 Y. Shimohigashi, J.W. Dunning jr, M.D. Grim, C.H. Stammer, J. Chem. Soc. Perkin Trans 2, (1981), in press 11 A. Srinivasan, R. Olsen, Tetrahedron Lett., (1976), 891– 894. 12 C.B. Pert, S.H. Snyder, Mol. Pharmacol., 10, (1974), 868– 879. 13 V.B. Fader, D. Rodbard, 3rd ed Radioimmunoassay Data Processing 1, (1975), Natl. Tech. Infor. Ser. Report no. PB246223 14 C.R. Beddell, R.B. Clark, R.L. Follenfant, L.A. Lowe, F.B. Ubatuba, R.J. Miller, J.A.K. Buisman Biological Actvity and Chemical Structure (1977), Elservier/North-Holland Amsterdam, New York 177– 193. 15 A.Z. Ronai, J.I. Szekely, I. Berzetei, E. Miglecz, S. Bajusz, Biochem. Biophys. Res. Commun., 91, (1979), 1239– 1249. 16 C.B. Pert, A. Pert, J. Chang, B. Fong, Science, 194, (1976), 330– 332. 17 J.M. Hambrook, B.A. Morgan, M.J. Rance, C.F.C. Smith, Nature, 262, (1976), 782– 783. 18 B. Malfroy, J.P. Swerts, A. Guyon, B.P. Roques, J.C. Schwartz, Nature, 276, (1978), 523– 526. 19 K.-J. Chang, P. Cuatrecasas, J. Biol. Chem., 254, (1979), 2610– 2618. 20 M.L. English, C.H. Stammer, Biochem. Biophys. Res. Commun., 83, (1978), 1464– 1467. 21 M.D. Grim, V. Chauhan, Y. Shimohigashi, A.J. Kolar, C.H. Stammer, J. Org. Chem., 46, (1981), 2671– 2673. Citing Literature Volume133, Issue2October 26, 1981Pages 269-271 ReferencesRelatedInformation

β-lipotropin contains two lipolytic sequences

The lipolytic activity of β-lipotropin (β-LPH) is commonly supposed to reside in the heptapeptide sequence, 47–53. However, β-endorphin is also lipolytically active, so a second active sequence must be postulated. In attempts to define this second sequence, porcine β-LPH, β-endorphin, 19 partial sequences of β-LPH, [Leu]enkephalin, and [D-Met 2, Pro 5]-enkephalin amide have been examined for glycerol releasing activity in rabbit adipocytes. An N-terminal fragment of β-LPH lacking the 47–53 heptapeptide was inactive. The message within the C-terminal portion of β-LPH could be localized to a C-terminal sequence greater than 87–91, but probably smaller than 78–91.

Design, synthesis, and biological activity of peptides which release growth hormone in vitro.

Experimental observations showed that the analogs [D-Trp2]- and [D-Phe2]methionine enkephalin amide were weakly active in releasing GH from rat pituitary in vitro. These observations were used to design more active GH-releasing factors. Conformational energy calculations were carried out, and energetically favored conformations of these polypeptides were found. Structural similarities as well as structural differences between active and inactive analogs were examined, and new sequences were predicted. Progressively more active analogs were designed, then synthesized, and tested. This cycle of steps was repeated, each time using structural and chemical concepts as design guides, until a series of very active analogs resulted. The most active analog to date, Tyr-D-Trp-Ala-Trp-D-Phe-NH2, was shown to release GH in vitro at 10-30 ng/ml medium, which is approximately 10(3) times more active than the two starting enkephalin-based analogs. From the structure-activity data, a mechanism for binding at the receptors is formulated, and a comparison is made between the structural relationships of the GH-releasing peptide analogs and the GH inhibitor, somatostatin.

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone.

AbstractEmpirical conformational energy calculations have been carried out for N‐methyl derivatives of alanine and phenylalanine dipeptide models and N‐methyl‐substituted active analogs of three biologically active peptides, namely thyrotropin‐releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone‐releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N‐methylated residue is in the trans configuration show that (1) N‐methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ϕ = −135° ± 5° and ψ = 75° ± 5°, and (3) the αL conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C5 (extended) conformation is second lowest in energy. When the peptide bond (ωi) is in the cis configuration the ith residue is energetically forbidden in the range ϕ = 0° to 180° and ψ = −180° to +180°. Conformations of low energy for ωi = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the αR conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low‐energy conformers are found for [(N‐Me)His2]TRH. These conformers differ only in the ϕ and ψ values at the (N‐Me)His2 residue. Among the different low‐energy conformers found for each of the ENK analogs [D‐Ala2,(N‐Me)Phe4, Met5]ENK amide and [D‐Ala2,(N‐Me)Met5]ENK amide, one low‐energy conformer was found to be common for both analogs with respect to the side‐chain orientations. The stability of the low‐energy structures is discussed in the light of the activity of other analogs. Two low‐energy conformers were found for [(N‐Me)Leu7]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D‐Ala6,(M‐Me)Leu7]LHRH is considered.

Naloxone-reversible effects of d-Ala 2-Met 5-enkephalinamide-induced behavioral activity in rats

In the first experiment, male rats were injected intracerebroventricularly (icv) with either saline, 12.5, 25, 50, or 100 μ g d -Ala 2 -Met 5 -enkephalinamide (ENK). Uncannulated controls were not injected but were handled similarly. The subsequent behavior of each animal was observed by means of a video system, and was recorded automatically with a system of two crossing photocell beams. Quadrant crossing, rearing, and photocell interruptions of the ENK groups decreased initially, but later increased above control levels. Grooming decreased with all doses and head movement decreased with the two highest doses of ENK. Wet-dog shakes were observed for all ENK groups. In the second experiment, icv-cannulated rats were maintained in the activity chambers and injected each morning with icv-saline followed by an ip saline injection 25 min later. They were habituated to the surroundings and injection procedures for 3 days. On the fourth day, half of the 50 rats were injected with 25 μ g ENK in 2.5 μ l, icv, while the other half were injected with saline and returned to their original activity chamber. Half of each group was injected ip 25 min after the icv injection with 5 mg/kg naloxone or saline, and the activity was recorded for about 4 more hr. Four to five rats from each group were treated similarly for the next 3 days. The activity of the ENK-injected group was higher overall than that of the controls. Naloxone reduced the activity of both the ENK-injected and saline-injected rats. Furthermore, the activity of the ENK-treated rats increased over successive days, whereas that of the controls did not. These results suggest that (1) the increased activity was due to the direct effects of ENK, and (2) some naturally occurring activity may involve endorphins.

Effect of D-alanine methionine enkephalin amide on ion transport in rabbit ileum.

The presence of enkephalins in the intestine and the use of opiates to treat diarrheal diseases suggests that enkephalins may affect intestinal ion transport. Using isolated rabbit ileal mucosa, we found that leucine enkephalin, methionine enkephalin, and D Ala2-methionine enkephalin amide (D Ala2-Met E) decreased the short circuit current (Isc) and potential difference although the effect of D Ala2-Met E was more pronounced and prolonged. D Ala2-Met E increased net sodium (+1.27 +/- 0.5 mu eq/cm2h), and chloride absorption (+2.33 +/- 0.4), and increased tissue conductance by 37%. Although the effect of enkaphalin on ion transport is opposite that of cyclic AMP, D-Ala2-Met had no effect on basal or vasoactive intestinal polypeptide-stimulated cyclic AMP levels. The effect of D-Ala2-Met E on Isc was blocked by naloxone, suggesting the involvement of specific opiate receptors. Tetrodotoxin completely blocked the decrease in Isc induced by D-Ala2-Met E but not by epinephrine, inferring that enkephalins are preganglionic neurotransmitters. The effect of D-Ala2-Met E on Isc was not blocked by phentolamine, haloperidol, or pretreatment of animals with 6-hydroxydopamine, suggesting that enkephalin does not affect the Isc by stimulating the release of alpha-adrenergic or dopaminergic agonists. D-Ala2-Met E also decreased the Isc in the presence of carbachol and bethanechol, indicating that enkephalin does not inhibit the release of acetylcholine. Further, up to 10 mu M atropine had no effect on the Isc. These studies demonstrate that enkephalins stimulate intestinal ion transport and may do so by stimulating (or inhibiting) the release of a nonadrenergic, noncholinergic neurotransmitter.

Entry of opioid peptides into the central nervous system.

Cerebrovascular permeability of four modified opioid peptides--[D-Ala2]methionine enkephalin amide, beta-[D-Ala62,14C-Homoarg69]lipotropin 61 -69, alpha-[D-Ala2,14C-Homoarg9]endorphin, and beta-[D-Ala2,14C-Homoarg]endorphin--ranged from 1.4 to 3.9 X 10(-6) centimeters per second in brain regions of the conscous rat. These significant permeabilities should allow the peptides to fill the extracellular brain space with a half time of 3 to 11 minutes, as a result of a step increase in plasma concentration of unbound peptide.

Effects of enkephalins and two enzyme resistant analogues on monoamine synthesis and metabolism in rat brain

Methionine-enkephalin and leucineenkephalin, administered into the lateral ventricle of intact rats, increased the accumulation of DOPA by a naloxone-sensitive mechanism in different brain regions after inhibition of the aromatic l-amino acid decarboxylase. Because of the rapid enzymatic degradation of both enkephalins large doses (500 μg) were required to enhance brain catecholamine synthesis. The two enzyme resistant enkephalin analogues d-Ala2-methionine enkephalin amide (DALA (4–256 μg) and FK 33-824 (0.003–1 μg) also increased the synthesis of DOPA, dose-dependently and by naloxone-sensitive mechanisms, but at much lower dosage level. The enkephalins markedly enhanced the brain tyrosine concentration but this effect was not antagonized by naloxone, probably because the enzymatic cleavage releases tyrosine from the administered peptides. In contrast, neither DALA nor FK 33-824 increased the brain tyrosine concentration. The formation of 5-HTP and the brain tryptophan concentration were also increased by the enkephalins, although these effects were not blocked by naloxone. The enkephalin analogues, however, enhanced the formation of 5-HTP and the brain tryptophan concentration by naloxonesensitive mechanisms. All four peptides accelerated the disappearance of dopamine, noradrenaline and 5-hydroxytryptamine after inhibition of monoamine synthesis. The results suggest that endogenous enkephalins, through the activation of opiate receptors, are involved in the short-term regulation of central monoaminergic systems.

Coupling of opiate receptors to adenylate cyclase: requirement for Na+ and GTP.

Inhibition of the adenylate cyclase activity in homogenates of mouse neuroblastoma-glioma hybrid cells (NG108-15) by the opioid peptide [D-Ala2,Met5]enkephalin amide (AMEA) requires the presence of Na+ and GTP. In this process, the selectivity for monovalent cations is Na+ greater than or equal Li+ greater than K+ greater than choline+; ITP will replace GTP but ATP, UTP, or CTP will not. The apparent Km for Na+ is 20 mM and for GTP it is 1 microM. Under saturating Na+ and GTP conditions, the apparent Ki for AMEA-directed inhibition is 20 nM for basal and 100 nM for prostaglandin E1-activated adenylate cyclase activity. For both cyclase activities, maximal inhibition is only partial (i.e., approximately 55% of control in each case). In intact viable NG108-15 cells, the decrease in basal and prostaglandin E1-stimulated intracellular cyclic AMP concentrations by AMEA is also dependent upon extracellular Na+. The enkephalin-directed reductions in cyclic AMP concentrations are at least 75%. The specificity of the monovalent cation requirement for enkephalin action on intact cells is the same as for enkephalin regulation of homogenate adenylate cyclase activity. Based on these data, a model is presented in which the transfer of information from opiate receptors to adenylate cyclase requires active separate membrane components, which correspond to the sites of action of Na+ and GTP in this process.

Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration.

Sensory neurons grown in dispersed cell culture in the absence of non-neuronal cell types contain immunoreactive substance P that is chemically similar to synthetic substance P. When depolarized in high-K+ media (30-120 mM), the neurons release this peptide by a Ca2+-dependent mechanism. An enkephalin analogue, [D-Ala2]enkephalin amide, at 10 micron inhibits the K+-evoked release of substance P. At the same or lower concentrations, [D-Ala2]enkephalin amide and enkephalin decrease the duration of the Ca2+ action potential evoked and recorded in dorsal root ganglion cell bodies without affecting the resting membrane potential or resting membrane conductance. This modulation of voltage-sensitive channels may account for the inhibition of substance P release.

PH and Solvent titrations of enkephalins by carbon-13 nuclear magnetic resonance spectroscopy: complete assignments of resonances

Carbon-13 magnetic resonances of two enkephalins, [Met5]- and [Leu5]-enkephalin, have been assigned in water, dimethyl sulphoxide, and trifluoroethanol, and assignments of [D-Ala2]-enkephalin-amide have been made in water. The assignments of the Gly2 carbon atoms have been made by comparison with [D-Ala2]-enkephalin amide. pD Titrations were followed to assign all the carbon resonances in D2O first, and then the assignments of resonances in other solvents were obtained by solvent–solvent titrations. An unusual titration behaviour of the Tyr1 aromatic γ-atom at a higher pH was found, and this has been suggested to be due to a conformational change in the Tyr1 Cα–Cβ bond. Conformational features of enkephalins in solution are briefly considered in relation to previous studies.

D-Ala 2, Δ zPhe 4-methionine enkephalin amide, a dehydropeptide hormone

The enkephalin analog, D-Ala 2, Δ zPhe 4 amide, has been prepared and shown to be five times as active as D-Ala 2-methionine enkephalin amide in vitro and the dehydrophenylalanine moiety conferred complete stability to chymotrypsin on the peptide.

Studies on the Interaction of Endorphins, Substance P, and Endogenous Somatostatin in Growth Hormone and Prolactin Release in Rats*

Substance P and various opiate receptor activators were injected into the lateral ventricle of urethane-anesthetized male rats which were pretreated with either normal sheep serum (NSS), sheep antiserum to somatostatin (anti-SS), or rabbit antiserum to substance P (anti-SP). The injection of substance P (10-9 mol) suppressed serum GH levels, but not PRL levels, in both NSS- and anti-SP-pretreated rats, whereas the suppressive effect was not observed in animals pretreated with anti-SS. In both NSS- and anti-SS-pretreated rats, β-endorphin (10-10 and 10-9 mol) and D-[Ala2]enkephalin amide (10-9 mol) stimulated both GH and PRL release, whereas α- and γ-endorphins, [Met5]enkephalin, and morphine sulfate (10-9 mol) induced only a meager GH response, but a significant PRL response. Simultaneous injection of β-endorphin (10-10 or 10-9 mol) and substance P (10-9 mol) caused greater GH and PRL responses than did β-endorphin alone in rats pretreated with either NSS, anti-SS, or anti-SP. Both GH and PRL responses to...

Enkephalins and dorsal horn neurones of the cat: effects on responses to noxious and innocuous skin stimuli.

1. In spinal cats anaesthetized with alpha-chloralose, a study was made of the effects of methionine enkephalin and methionine enkephalin amide on the responses of neurones of spinal laminae IV and V to noxious and innocuous skin stimuli. The enkephalins were ejected from micropipettes either in the region of cell bodies or in the substantia gelatinosa. 2. Administered near cell bodies the enkephalins reduced spontaneous firing and cell responses to both types of skin stimuli. These effects were antagonized by naloxone when administered near cell bodies but not when given intravenously in doses (0.3-0.6 mg/kg) more than adequate to antagonize analgesic doses of morphine. 3. Administered in the substantia gelatinosa the enkephalins were more selective in their action. The predominant effect was a reduction in nociceptive responses with little effect on non-nociceptive responses although spontaneous firing was commonly reduced. Naloxone administered either in the substantia gelatinosa or intravenously (0.1-0.3 mg/kg) reversed these effects of the enkephalins.