E.C. Griffiths

Mechanism of neurotensin degradation by rat brain peptidases.

Neurotensin is degraded by peptidases present in soluble and particulate (25000 x g) fractions of rat hypothalamus, thalamus, cortex and pituitary, the soluble fraction of the hypothalamus having the highest activity. High performance liquid chromatography and amino acid analysis were used to identify the degradation pathway. The main product in both fractions was [1-8]neurotensin and the corresponding C-terminal fragment [9-13]neurotensin was identified. [1-10]Neurotensin was also identified, proportionately more of this peptide being produced by the particulate rather than the soluble fraction. In the presence of dithiothreitol, [1-7]neurotensin and [1-10]neurotensin were major products particularly in the soluble fraction. These results suggest that the main sites of cleavage of neurotensin by rat brain peptidases are the Arg8-Arg9, Pro10-Tyr11 and Pro7-Arg8 bonds.

Further characterization of the substrate specificity of a TRH hydrolysing pyroglutamate aminopeptidase from guinea-pig brain.

In this study the substrate specificity of a pyroglutamate aminopeptidase from synaptosomal membranes of guinea-pig brain was investigated. The enzyme was found to be specific for tripeptides, tripeptide-amides and tetrapeptides which possess the N-terminal sequence Glp-His and as such is specific for Thyrotropin Releasing Hormone or only very closely related peptides. The enzyme was found not to hydrolyse a number of analogues of Thyrotropin Releasing Hormone which have been shown to have therapeutical value in certain neuronal disorders.

Antinociceptive effects of thyrotrophin-releasing hormone and its analogues in the rat periaqueductal grey region.

Thyrotrophin-releasing hormone (TRH) and its analogues were injected into the periaqueductal grey (PAG) area of brain in conscious rats and the response time (tail-flick) to a thermal stimulus was recorded. TRH and some of the analogues had an antinociceptive action which, in the case of TRH, was prevented by naltrexone. TRH may interact with endogenous opioid systems in the PAG.

Body temperature effects of opioids administered into the periaqueductal grey area of rat brain

The ability of opioids to influence rectal temperature after injection into the periaqueductal grey region (PAG) of rat brain was investigated. Both morphine and beta-endorphin caused a dose-dependent increase in rectal temperature of up to 2 degrees C. By using selective ligands of the subclasses of opiate receptor such as [D-Ala2,D-Leu5]enkephalin for delta-receptors and ethylketocyclazocine, dynorphin(1-17) and dynorphin(1-8) for kappa-receptors, it was possible to show that neither the delta- nor the kappa-opiate receptor was involved in the hyperthermic response. However, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), a mu-receptor ligand, did produce a dose-dependent hyperthermia. The ability of naltrexone, an opiate receptor antagonist, to reverse the hyperthermia induced by beta-endorphin and DAGO suggests that the opioid-stimulated increase in body temperature via the PAG is mediated through the mu-opiate receptor. Since the application of opioids to the PAG produces a hyperthermic response, it is possible that this brain site may have a role in the peptidergic control of body temperature.

Biotransformation of neuropeptides

The ability of neuropeptides to act as precursors for smaller, biologically active fragments is discussed in terms of their biotransformation. This process may involve cleavage of the parent peptide by peptidase enzymes to produce shorter polypeptides with defined biological activity, though other enzymic processes such as sulphation and acetylation may be implicated. Detection of the specific fragments in vivo, their release, receptor-binding and biological actions may confirm biotransformation of the parent peptide. Control of biotransformation will depend upon the localization, both regional and subcellular, and the specificity of the enzymes involved. This process may give an additional degree of flexibility to the biological effects of neuropeptides.

Mechanism of luteinizing hormone-releasing hormone degradation by subcellular fractions of rat hypothalamus and pituitary.

The pathway of LH-RH degradation by two subcellular fractions (a soluble fraction and a 25 000 X g particulate fraction) of rat hypothalamus, pituitary and cerebral cortex has been studied using high performance liquid chromatography and amino acid analysis to identify the breakdown products. The primary cleavage point in the Tyr5-Gly6 bond giving [1-5] LH-RH and [6-10] LH-RH. In the presence of dithiothreitol, cleavage of LH-RH also occurred at the Pro9-Gly10 bond giving [1-9] LH-RH. The fragment [1-5] LH-RH is further degraded sequentially from the C-terminus and [1-4] LH-RH, [1-3] LH-RH, tyrosine and tryptophan were identified. The other major fragment, [6-10] LH-RH, is rapidly broken down, the only intermediate product positively identified being Arg-Pro.

Inactivation of thyrotropin-releasing hormone (TRH) and (3Me-HIS) TRH by brain peptidases studied by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) has been used to separate and identify the metabolites formed from thyrotrophin-releasing-hormone (TRH) and its hyperactive analogue, (3Me-His)TRH, by subcellular fractions from rat cortex, hypothalamus and thalamus. Deamidation by the proline endopeptidase and formation of histidylproline diketopiperazines by the pyroglutamyl aminopeptidase were found to be the major mechanisms of brain inactivation of both peptides; (3Me-His)TRH was slightly more stable than TRH in the presence of the brain peptidases, and with enhanced receptor binding affinity, this could explain its increased biological activity. The HPLC system used may be applicable to determining the mechanisms of brain inactivation of other TRH analogues and could also be used to define the pathways for inactivation of larger neuropeptides as well.

The effects of opioids in the periaqueductal grey region of rat brain on hind-limb muscle tone

A mechanical apparatus was used to measure hind limb resistance to flexion in rats injected with opioid peptides in the brain periaqueductal grey region (PAG). (D-Ala2, D-Leu5) enkephalin (delta agonist) and dynorphin1-8 and ethylketocyclazocine (kappa agonists) had no effects on hind limb tone. The mu agonist (D-Ala2, MePhe4, Gly-ol5) enkephalin induced limb rigidity when injected into the PAG. beta-Endorphin caused a dose-related limb rigidity that persisted for up to 2h. beta-Endorphin rigidity was prevented by pretreatment of rats with the serotonin depletor 5,7-dihydroxytryptamine. It is concluded that mu receptors and serotonin mechanisms in the PAG can mediate opiate rigidity.

The hypothermic action of carbachol in the rat brain periaqueductal grey area may involve neurotensin.

1 Neurotensin (NT) and carbachol both caused hypothermia when injected into the periaqueductal grey area (PAG) of rat brain. 2 Atropine prevented carbachol‐ but not NT‐induced hypothermia. 3 NT‐induced hypothermia was unaffected by various neurotransmitter agonists and antagonists in the PAG. 4 Both NT antibodies and thyrotrophin releasing hormone prevented carbachol hypothermia. 5 It is concluded that the hypothermic action of carbachol in the PAG is mediated via endogenous NT.

Effects of mammalian and avian neurotensins and neurotensin fragments on wet-dog shaking and body temperature in the rat

The ability of mammalian and avian neurotensins and some neurotensin fragments to reduce wet-dog shaking (WDS) induced by thyrotrophin-releasing hormone (TRH) and to influence rectal temperature was tested after their injection into the periaqueductal grey region of male rats. Both neurotensins inhibited TRH-induced WDS and reduced rectal temperature by 2 degrees C; this latter effect was prevented by prior TRH administration. Of the four neurotensin fragments tested, both (1-8)- and (8-13)-neurotensin reduced WDS but only (8-13)-neurotensin reduced rectal temperature significantly. (1-6)- and (1-11)-neurotensin were without effect in either test system. From the activity of the various peptides, further examples of the mutual antagonism between TRH and neurotensin have been demonstrated. It is suggested that there is a possible role for neurotensin in controlling body temperature via the periaqueductal grey and that this may be one function of neurotensin in avian species; there may also be more than one receptor system binding neurotensin in the brain.