Rabi Simantov

Isolation and structure identification of a morphine-like peptide “enkephalin” in bovine brain

Abstract The ability of bovine brain extracts to compete in a selective fashion for opiate receptor binding is attributable to a small peptide. The substance has been purified to homogeneity and identified as comprising two penta-peptides Hue5f8Tyrue5f8Glyue5f8Glyue5f8Pheue5f8Leuue5f8OH (Leucine-enkephalin) and Hue5f8Tyrue5f8Glyue5f8Glyue5f8Pheue5f8Metue5f8OH (methionine enkephalin). Bovine brain contains 4 times as much leucine-enkephalin as methionine-enkephalin in contrast to pig brain in which these ratios are reversed. Competition for opiate receptor binding by leucine-enkephalin is reduced more by sodium and enhanced more by manganese than is the case for methionine-enkephalin, suggesting that leucine-enkephalin may be a “purer” agonist than methionine-enkephalin.

The opiate receptor binding interactions of 3H-methionine enkephalin, an opioid peptide

3H-Methionine enkephalin binds stereospecifically with high affinity to opiate receptors in rat brain membranes. Equilibrium experiments indicate two distinct dissociation constants with KD values of 1.8 and 5.8 nM respectively. 3H-Methionine enkephalin associates and dissociates from the opiate receptor with 8--10 fold slower kinetics than 3H-opiates. Though several opiates have similar affinities for sites labeled by 3H-methionine enkephalin, 3H-dihydromorphine and 3H-naloxone, some opiates such as morphine, dihydromorphine and oxymorphone are only one tenth as potent in competing for 3H-methionine enkephalin as for 3H-dihydromorphine and 3H-naloxone binding. As with other opiate agonists, 5--10 mM sodium selectively decreases the binding of 3H-methionine enkephalin. At 26 degrees C, 0.1--1.0 mM manganese but not magnesium or calcium increases the binding of 3H-methionine enkephalin, while at 0 degrees C manganese decreases the binding of methionine enkephalin.

Gamma-aminobutyric acid (GABA) receptor binding selectively depleted by viral induced granule cell loss in hamster cerebellum

Excitatory and inhibitory synaptic interconnections and related neurotransmitters have been more thoroughly studied in the cerebellum than in any other brain region. Of the 5 intrinsic neuronal types in the cerebellar cortex, four (the Purkinje, Golgi, basket and stellate cells) are inhibitory and appear to utilize GABA as their neurotransmitter4,5,9,11,17-19. The remaining inhibitory element is composed of noradrenergic fibers which arise from the locus coeruleus and comprise about 1 of the climbing fiber populationL There are 3 excitatory elements in the cerebellar cortex, the granule cells which appear to use glutamic acid as their transmitter 21 and the mossy and climbing fibers whose transmitters are unknown. All cerebellar neurons seem to receive GABA containing inhibitory terminals 5. Those on the granule cell dendrites appear to arise predominantly and probably exclusively from the Golgi I! cells. The density of GABA receptors upon various cell types in the cerebellum may regulate the prevalence of various types of inhibitory synaptic interconnections. Recently, we have identified selective binding of GABA to its postsynaptic receptor sites in the brain6,7, 22. To ascertain the density of GABA receptors associated with various cell types in the cerebellum, it would be desirable to destroy individual cell types selectively. Several experimental models have been developed to deplete selectively cerebellar granule cells. In the present study we have examined synaptic receptor binding of GABA in the cerebella of hamsters treated with a parvovirus, rat virus, which selectively destroys the rapidly dividing external germinal cells to produce a cerebellum in which only the granule cell population is depleted. We report a selective decline in synaptic GABA receptor binding which parallels the decrease in granule cell number. Granuloprival hypoplasia of the Syrian hamster cerebellum resulted after intracerebral injection of rat virus strain PRE (HA titer 2 -12) into the left hemisphere 21 of

A morphine-like factor in mammalian brain: Analgesic activity in rats

A partially purified morphine-like peptide enkephalin (PPE) extract from bovine brain elicited pronounced apparent analgesia after injection into the periaqueductal gray matter of rat brain. This analgesia was reversed by the opiate antagonist naloxone in a dose-dependent fashion. Analgesia was more rapid in onset and much shorter in duration after PPE than after morphine administration. Analgesia was elicited only by those ion exchange column fractions of PPE that competed potently for opiate receptor binding. No analgesia could be detected when PPE or morphine injections were administered at a site 2 mm lateral to the periaqueductal gray matter. The potencies of synthetic methionine- and leucine-enkephalin in eliciting analgesia were less than 1% of those of partially purified enkephalin extracts when doses of equivalent ability to compete for opiate receptor binding were compared.

Differential binding properties of some opiates and opioid peptides

The opioid peptide enkephalins were discovered as endogenous substances in the brain which mimic effects of morphine pharmacologically at presumed receptor sites in smooth muscle systems (Hughes, 1975) and compete for [3H] opiate binding to opiate receptor sites in brain membranes (Terenius and Wahlstrom, 1975; Pasternak et al. 1975a). Accordingly, these opiate receptor sites which bind opiates with high affinity and in proportion to their pharmacological activity (Snyder, 1975) presumably serve physiologically to interact with endogenous opioid peptides. In the brain the quantitatively predominant opioid peptides are the two pentapeptides met and leu-enkephalin while lower concentrations of a 39-amino acid opioid peptide β-endorphin may also presumably interact with opiate receptors. However, since the localization of enkephalin throughout the brain corresponds more closely with that of opiate receptors than does localization of β-endorphin (Simantov et al. 1977; Rossier et al. 1977), it is likely that the majority of opiate receptors in the brain normally interact with enkephalins rather than β-endorphin.