Isolation and structural characterization of enkephalins in the brain of the rhynchobdellid leech Theromyzon tessulatum.

This paper reports the purification of four peptides related to enkephalins from the brain of the leech Theromyzon tessulatum. After reverse-phase HPLC purification, the sequence of the enkephalins (YGGFM, YGGFL, FM, FL) was established by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and co-elution experiments in reverse-phase HPLC with synthetic peptides. ELISA titrations performed on each purified peptide indicated that the major amount was borne by the leucine-enkephalin. The ratio of leucine-enkephalin and methionine-enkephalin of 2:1 is in line with previous immunocytochemical data obtained on T. tessulatum brains. The presence of enkephalins in T. tessulatum, an animal belonging to the oldest group of coelomate metazoans (the Annelida) establishes the very ancient phylogenetic origin of opioids and their conservation in the course of evolution.

Delta and mu enkephalins inhibit spontaneous GABA-mediated IPSCs via a cyclic AMP-independent mechanism in the rat hippocampus.

The effects of enkephalins selective for delta and mu opioid receptors on inhibitory postsynaptic currents (IPSCs) mediated by GABA were studied in chloride-loaded CA1 pyramidal neurons in adult rat hippocampal slices. The mu agonist DAMGO (0.1 microM) significantly reduced the amplitudes of evoked monosynaptic IPSCs, recorded following the antagonism of excitatory glutamate receptors, and this effect was reversed by the mu antagonist CTOP (1 microM). The selective delta receptor agonists DPDPE and D-Ala2-deltorphin II (both 0.1-0.5 microM) had no effect on these evoked currents. In contrast, the frequency of tetrodotoxin-resistant spontaneous miniature GABA-mediated currents (m-IPSCs) was significantly reduced by both DPDPE (0.1-0.5 microM) and DAMGO (0.1-0.5 microM), while the amplitudes of these events were unaltered. These effects were reversed by the selective delta antagonist ICI 174,864 (1 microM) and the selective mu antagonist CTOP (1 microM), respectively. To investigate the mechanisms of this mu and delta receptor-mediated modulation of GABA release, and the possible involvement of a cAMP-sensitive K+ conductance, spontaneous action potential-dependent IPSCs (s-IPSCs) were measured following pretreatment with 8-bromo-cAMP (8-Br-cAMP). 8-Br-cAMP (250 microM) had no effect alone on the amplitude or frequency of s-IPSCs, nor did it alter the inhibitory effects of the delta and mu agonists. These results indicate that delta and mu opioid receptor activation inhibits spontaneous GABA release, independently of cAMP, through direct actions at inhibitory nerve terminals, and that delta opioids inhibit spontaneous but not evoked GABA release in the hippocampus.

Involvement of different receptor subtypes for the production of in vitro and in vivo effects in a series of synthetic enkephalin analogues.

Enkephalin analogues of tyrosyl group on the N-terminal and Phe-ol or phenylethylamine (PHA) group on the C-terminal which are connected with different chain length were tested for their activities in vitro and in vivo. The inhibitory effect of the synthetic peptides on the electrically evoked contractions of isolated longitudinal muscle strips of guinea pig ileum were weaker than that of morphine or Leu-enkephalin and tended to decrease by increasing the number of methylene group,-(CH2)n-, n = 1-5, between N- and C-terminal. Compounds with PHA group on the C-terminal, n = 4 and 5, showed the least activity. The effect of peptides with short chains of methylene groups, n = 1 or 2, and Phe-ol on the C-terminal were antagonized by naloxone but others were insensitive to naloxone. Differing from in vitro activity, compounds with PHA on the C-terminal with a chain of 4 methylene groups produced short lasting analgesia after i.c.v. injection as well as the compounds with Phe-ol on the C-terminal and 1 or 2 methylene groups. The analgesic effect of these compounds were completely antagonized by naloxone. At a high i.c.v. dose, all the synthetic peptides, except the one with PHA on the C-terminal with a chain of 4 methylene group, produced convulsions and/or ipsilateral rotation to the injection side. These behavioral effects were not antagonized by naloxone. Thus, minor alterations in the chemical structure of enkephalin analogues resulted in the changes of their receptor selectivity and potencies in vitro and in vivo.

A highly potent 3200-dalton adrenal opioid peptide that contains both a [Met]- and [Leu]enkephalin sequence.

A 3200-dalton adrenal enkephalin-containing peptide, designated peptide E, that exhibits high opiate activity in the guinea pig ileum longitudinal muscle preparation was purified, and its structure was determined. It contains an amino-terminal [Met]enkephalin sequence and a [Leu]enkephalin sequence at the carboxyl terminus. Sequence analysis revealed that peptide E arises from a previously characterized 4900-dalton adrenal enkephalin-containing peptide. Peptide E was shown to be 30 times more potent than [Met]enkephalin in the guinea pig ileum assay, which suggests that the adrenal enkephalin-containing peptide may perform a unique biological function in vivo.