Endogenous Enkephalins Research Articles

Subcellular distribution of enkephalin-dipeptidyl carboxypeptidase (enkephalinase) in rat brain

The distribution of enkephalinase activity i.e. of the peptidase activity cleaving the Gly 3-Phe 4 bond of enkephalins, has been established in subcellular fractions of rat cerebral cortex and striatum and compared with that of opiate receptors labelled with [ 3H] dAla 2-Met-enkephalinamide. The fractions were obtained by differential and density gradient centrifugations and their purity assessed by measuring levels of a series of biochemical markers. A major part of enkephalinase activity is associated with the P 2 fraction containing isolated nerve-endings and the subfractions of lysed P 2 containing synaptosomal membranes display the highest enrichment. Another important part of enkephalinase activity is associated with the microsomal P 3 fraction, whereas the enzyme activity is hardly detectable in cytoplasm or synaptic vesicle fractions. The distribution of enkephalinase activity between the various fractions and subfractions strikingly parallels that of enkephalin receptors. These data support the hypothesis that enkephalinase is selectively located in neuronal membranes in the vicinity of enkephalin receptors, as suggested by previous regional distribution and lesion studies, and that it might be responsible for the inactivation of endogenous enkephalins.

Multiple opiate receptor sites on primary afferent fibres.

In addition to their actions at supraspinal sites, opiates can act directly at the spinal cord level to produce analgesia1–3. Opiate receptors and enkephalins are found in abundance in the dorsal horn of the spinal cord, in the region of termination of small-diameter primary afferents4–6. Furthermore, there is evidence that exogenously administered opiates or endogenous enkephalins act both on postsynaptic receptors in dorsal horn and presynaptically to block transmitter release from nociceptive primary afferent terminals7–10. The possiblity of these two distinct sites of opiate action in dorsal horn is of particular interest in view of recent evidence for the existence of multiple opiate receptors11–13. On the basis of differences in the rank orders of potency of opiate alkaloids and opoid peptides observed in different tissues, Lord et al.14 have proposed two categories of opiate receptor, μ and δ, and these can be differentiated in binding studies, provided that selective radioglands are used in the appropriate experimental conditions15–18. Here we have used the selective radioligands 3H-morphine (μ sites) and 3H-D-Ala2, D-Leu5-enkephalin (δ sites) to examine directly in rat the distribution and binding characteristics of opiate receptors on dorsal root and in various regions of the adjacent spinal cord. Primary afferent tissue (dorsal root) and dorsal horn were found to contain μ and δ opiate binding sites with a relatively high proportion of μ sites. Partial destruction of small-diameter primary afferents after cutting the sciatic nerve led to a significant reduction in both μ and δ binding sites on dorsal roots, suggesting that both types of opiate receptors may exist on small-diameter primary afferents.

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.

Inhibition of PGE1 induced intestinal secretion by the synthetic enkephalin analogue FK 33-824.

1. The effects of the enkephalin analogue, FK 33-824 were compared with those of morphine and naloxone on the prostaglandin E1-induced increase in intestinal fluid volume and unanaesthetized and pithed rats. 2. FK 33-824 inhibits the prostaglandin E1-induced increase in intestinal fluid volume both in unanaesthetized and pithed rats. 3. Naloxone abolished the inhibitory effect of FK 33-824 in both experimental models. 4. It can be concluded from the experiments in pithed rats that the inhibitory effect of FK 33-824, like that of morphine, on prostaglandin E1-induced secretion is caused by a peripheral action. 5. It is assumed that endogenous enkephalins protect against prostaglandin-mediated loss of fluid into the gut lumen. This protective effect can be mimicked by an enkephalin analogue and blocked by naloxone.

Blockage of the ether-induced surge of prolactin by naloxone in male rats.

Department of Physiology, Abramsky Hall, Queen's University, Kingston, Ontario, Canada, K7L 3N6 (Received 17 July 1978) It is well established that the stress of ether anaesthesia raises the level of prolactin in the circulation of the rat (Wakabayashi, Arimura & Schally, 1971; Krulich, Hefco, Illner & Read, 1974). Morphine or the endogenous morphine-like peptides, endorphins and enkephalins, can stimulate the secretion of prolactin in vivo (Ojeda, Harms & McCann, 1974; Martin, Audet & Saunders, 1975; Bruni, Van Vugt, Marshall & Meites, 1977; Rivier, Vale, Ling, Brown & Guillemin, 1977; Chihara, Arimura, Coy & Schally, 1978); naloxone, however, blocks the stimulatory effects of these substances (Bruni et al. 1977; Rivier et al. 1977). It was recently demonstrated that naloxone inhibits the secretion of prolactin induced by stress due to heat or immobilization (Van Vugt, Bruni & Meites, 1978). The restraintinduced stress produced an immediate increase in the concentration of prolactin

Enkephalins and nigrostriatal function

Unilateral lesions of the substantia nigra were made with 6-hydroxydopamine in rats. In this model, drugs such as naloxone, which block endogenous enkephalin receptors, potentiated agents with postsynaptic dopaminergic actions, while antagonizing agents with presynaptic dopaminergic actions. Drugs which increase brain enkephalin content (d-phenylalanine or methionine-enkephalin) antagonized postsynaptically active agents and potentiated presynaptic agents. Naloxone also reversed reserpine-induced parkinsonism in rats. Separate pre- and postsynaptic enkephalinergic neurons thus seem to modulate nigrostriatal function.

Met-enkephalin: Rapid separation from brain extracts using high-pressure liquid chromatography, and quantitation by binding assay

Met-enkephalin can be rapidly separated with high recovery from leu-enkephalin, beta-endorphin, and enkephalin metabolites by high-pressure liquid chromatography on a Dupont SCX column. The technique permits separation of endogenous enkephalins from brain extracts for measurement by opiate-receptor binding assay, immunoassay, or for study of the incorporation of radiolabeled amino acids into enkephalin. Using this technique, met-enkephalin was separated from striatal extracts of rats killed by focused microwave irradiation and quantitated by the opiate-receptor binding assay. The concentration of met-enkephalin in the column eluate was 1.3 micron/g tissue. This value is comparable to that obtained by radioimmunoassay without purification of the extracts.

Epileptic properties of leucine- and methionine-enkephalin: Comparison with morphine and reversibility by naloxone

Morphologically similar epileptic seizures were recorded from the cortex of rats after injections into the lateral ventricle of 100 μg of leucine-enkephalin, methionine-enkephalin, and morphine. Seizures were either greatly attenuated or blocked completely by prior systemic administration of naloxone (10 mg/kg). These findings suggest that such seizures result from an interaction of these compounds with opiate receptors in the brain. The epileptogenic potency of the enkephalins was illustrated by the observation that seizures and other pathological manifestations could still be elicited by doses as low as 10 μg. Leucine-enkephalin was seen to have greater epileptic potency than methionine-enkephalin. At doses of 1 μg both enkephalins typically evoked cortical spindles resembling those seen in drowsy animals. Enkephalin-induced analgesia was seen in only one animal at the 100 μg dose. Results obtained with repeated injections of morphine suggest that the epileptogenic effect of opiates may be subject to either tolerance or potentiation, depending on the prior occurrence of seizures. A synthesis of the present findings with several other lines of evidence suggests both that endogenous enkephalins play some role in normal mechanisms of reward, and that, when regulatory processes are disturbed, they may contribute as well to the elaboration of certain epileptic phenomena.

Analgesic by enkephalins injected into the nucleus reticularis gigantocellularis of rat medulla oblongata

Methionine-enkephalin (0.2–20 μgåt) and leucine-enkephalin (1–20 μg/rat) produced a dose-related and naloxone-antagonizable analgesia in the tail-pinch test, when microinjected into the nucleus reticularis gigantocellularis (NRGC) and nucleus reticularis paragigantocellularis (NRPG) of the medulla oblonga of the rat. The median analgesic doses were 1.4 and 4.8 μg/rat for methione- leucine-enkephalin, respectively. The possibility that the endogenous enkephalins play a part as pain control substanaces of modulators in the NRGC and NRPG was discussed.

Morphine and enkephalin: analgesic and epileptic properties.

Systemic and intracerebroventricular administration of analgesic doses of morphine resulted in large increments of spontaneous multiple unit activity in the periaqueductal gray matter of the awake rat. Intracerebroventricular injection of methionine enkephalin gave analgesia in only 8 of 19 rats, but in all 8, and in no others, increased periaqueductal multiple unit firing was also seen. These findings support the view that the periaqueductal gray matter is actively involved in endogenous mechanisms of analgesia. A striking observation was that enkephalin caused electrographic and behavioral epileptic phenomena in most animals. This observation together with other recent findings suggests that endogenous enkephalin may play some role in epileptogenesis.

Effects of microiontophoretically applied methionine-enkephalin on single neurones in rat brain.

THE identification of a specific opiate receptor in mammalian brain1, and also of an endogenous morphine-like factor, enkephalin, which possesses potent opiate agonist activity peripherally2, raises the question of the possible physiological significance of this substance in the central nervous system. There is evidence from a number of studies suggesting that the brainstem is an important site for antinociceptive action3–5. In previous studies8, morphine applied iontophoretically to single neurones in the rat brainstem, has been found to cause either an increase or a decrease in firing rate, but only the depressant response showed stereospecificity and could be blocked by the specific opiate antagonist, naloxone7,8. Similar findings on cortical neurones have been reported9. We have therefore investigated the effects of iontophoretically applied methionine-enkephalin (met-enkephalin), the more potent peripherally of the two pentapeptides, on the activity of single neurones in the rat brain stem and compared these effects with those of morphine, and also of another narcotic analgesic, etorphine, which is considerably more potent. Since naloxone has been shown to block peripheral effects of enkephalin10, we have examined its effects on the neuronal responses to met-enkephalin, morphine and etorphine. Our findings support the concept of a physiological role for endogenous enkephalin in the central nervous system.

Cross tolerance between morphine and methionine-enkephalin.

TOLERANCE and dependence are characteristic symptoms in man during prolonged use of opiates. We have pointed out1 that for an understanding of these phenomena the interaction between endogenous enkephalin and exogenous opiates is of importance. Normally, enkephalin can be assumed to control inhibitory mechanisms determining the rate of neurotransmitter release. If, however, opiates are administered with the intent of increasing the effects of these inhibitory mechanisms, then control will pass from the endogenous enkephalin to the exogenous opiates. Since tachyphylaxis is a pharmacological characteristic of opiates, a state of tolerance will arise in which increasing amounts of opiates will be required to maintain the desired effect. As an important step in the elucidation of the mechanism of action of enkephalin, it was necessary to establish whether cross tolerance between enkephalin and opiates is found in morphine-tolerant animals. This point was tested in two morphine-sensitive models, the myenteric plexus-longitudinal muscle preparation from the guinea pig ileum2,3 and the mouse vas deferens4. As natural enkephalin consists of about 75% methionine–enkephalin and 25% leucine–enkephalin5, the relative potencies of synthetic methionine–enkephalin and morphine were compared in preparations obtained from animals with different degrees of tolerance. Cross tolerance between morphine and methionine–enkephalin was shown to occur in the two preparations obtained from animals implanted with morphine pellets.

Some thoughts on the significance of enkephalin, the endogenous ligand

Enkephalin is compared to morphine and its congeners with particular regard to physico-chemical properties, biological stability and sensitivity to narcotic antagonists. Possible sites of action are considered. Interactions between endogenous enkephalin and exogenous opiate at the opiate receptor are discussed as a possible basis of tolerance and dependence caused by chronic administration of opiates.