Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.593487
Title: Mechanisms of biosynthesis and release of enkephalin in the myenteric plexus preparation of the guinea-pig ileum
Author: Sosa, Roberto P.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1979
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Abstract:
In this thesis, evidence has been presented that the two penta-peptides Met- and Leu-enkephalin are synthesized locally in the myenteric plexus longitudinal muscle preparation of the guinea-pig ileum. The local synthesis of Met- and Leu-enkephalin probably occurs through a mechanism involving ribosomal assembly of the peptides at the 80 S ribosomes of the rough endoplasmic reticulum in the neurone perikarion. The ribosomal assembly did not involve 70 S ribosomes present in mitochondria. That the incorporation of {3H}-tyrosine into enkephalins was inhibited when the antibiotic puromycin was present during the labelling and chase period, that such an effect was absent when the antibiotic was present only during the chase period and the presence of a lag period of 1-2 h followed by a linear increase in the appearance of radiolabelled Met- and Leu-enkephalin during the time course of incorporation of the labelled amino acid into the peptides, all suggest that the enkephalins are synthesized at the ribosomes in an inactive form or precursor of higher molecular weight. This is later processed to the final product, enkephalin, through a maturation mechanism that seems to require 1-2 h. The nature of this pro-enkephalin polypeptide and the processing or post-translational modification of it still remain unknown. Levorphanol but not dextrorphan at relatively low concentration (27 nM) decreased the in vitro incorporation of {3H}-tyrosine into Met- and Leu-enkephalin in the myenteric plexus preparation. This effect was not due to an inhibition of protein synthesis at the ribosomal level. However, a higher concentration of levorphanol (108 nM) did not produce the same effect. Naloxone, 100 nM; D-Ala2 - Met5-enkephalin, 80 nM; tetrodotoxin, 1.5 M and hexamethonium, 70 M did not affect the incorporation of labelled tyrosine into enkephalin or proteins of the myenteric plexus preparation. An increase in potassium concentration (50 muM) produced release of "enkephalin-like material" from the superfused crude synaptosomal fraction of rat brain; the amount of enkephalin released represented 2.3% of the total tissue content measured at the end of the super-fusion. The release of "enkephalin-like material" was calcium dependent, since the release of enkephalin was abolished when calcium was omitted and EDTA was added to the superfusing fluid. Release of enkephalin could not be detected by direct measurement after electrical stimulation of the guinea-pig small intestine, possibly due to the rapid inactivation of the enkephalins. When an indirect method to measure the release of enkephalin from the guinea-pig myenteric plexus preparation was developed, it was shown that electrical stimulation does release enkephalin from this tissue. The release of enkephalins from the myenteric plexus preparation depends on the total number of pulses rather than the frequency used. The amount released was 4-5 fmol g-1 per pulse and the fractional release per pulse was 1.1 x 10-5 and 1.3 x 10-5 for frequencies of 1 and 10 Hz, respectively. The release evoked by electrical stimulation was blocked by tetrodotoxin. The amount of enkephalin present in the myenteric plexus preparation after electrical field stimulation, carried out in the presence and absence of an inhibitor of the synthesis of enkephalins was used to calculate the enkephalin turnover. The myenteric plexus preparation showed a turnover of about 310 pmol g-1 Met-enkephalin during electrical stimulation for 2 h at 1 Hz and 190 pmol g-1 during stimulation for 1 h at 10 Hz. The acute in vivo administration of naloxone or morphine or the chronic exposure to morphine did not change the total enkephalin content of the mouse brain. Thus the present results provide additional direct evidence that Met- and Leu-enkephalin may act as neurotransmitters in the central and peripheral nervous system.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.593487  DOI: Not available
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