Christopher R. Snell

Metabolism of the analgesic peptide lipotropin C-Fragment in rat striatal slices

A series of morphinomimetic peptides has been isolated from brain or pituitary [l-6]. Comparison of their abilities to displace specifically bound [?I] naloxone from brain opiate receptors showed that C-Fragment (lipotropin 61-9 1) is much more potent than C’-Fragment (61-87), rendorphin (61-77), ar-endorphin (61-76) or methionine enkepe [7]. lnvestigation of the analgesic properties revealed that the shorter peptides are at most weakly active whereas C-Fragment produces profound and long lasting analgesia [8-l 11. In addition, the C-Fragment alone exhibits other central activities, including the stimulation of grooming [12], inhibition of neurotransmitter release [ 131 and the production of hyperglycaemia [14]. The high potency of C-Fragment in its central actions would seem to imply that this is the physiologically significant agent in brain. In the present investigation we have shown that the proteolytic degradation of C-Fragment in striatal slices takes place in a specific manner with the formation of products corresponding to the range of opiate active peptides that have been isolated from brain.

Autoradiographic visualization of binding sites for [3H]somatostatin in the rat brain

[4-3H][Phe6]somatostatin-14 was used to localize somatostatin binding sites in the rat brain by tritium-film autoradiography. The distribution of binding sites using 0.7 nM [3H]somatostatin confirmed that previously described for iodinated tyrosyl analogues of somatostatin, with highest densities of sites in the cerebral cortex (particularly in laminae III-V), amygdala, lateral septal nucleus, hippocampus and claustrum. Investigation of the pharmacological specificity of the binding sites showed that somatostatin-28, but not its N-terminal dodecapeptide, somatostatin-28 (1-12) or des-Ala1[Gly2,Lys4,Asn5,Thr12,Ser13]somatostatin displaced [3H]somatostatin. Further examination of the binding inhibition characteristics, using a homogenate assay, suggested the presence of two classes of binding sites in the cerebral cortex, hippocampus, midbrain and striatum. The existence of sub-populations of somatostatin binding sites in the rat brain has implications for future studies on the physiological and pharmacological significance of somatostatin receptors in the central nervous system.

Regional distribution of high-affinity [3H]somatostatin binding sites in the human brain

The distribution of high-affinity binding sites for [3H]somatostatin has been studied in membrane preparations from a number of regions of normal human brain. The highest densities of binding sites (greater than 48 fmol/mg protein) were found in the cerebral and cerebellar cortices and the hippocampus, with intermediate binding densities (30-46 fmol/mg protein) being present in the basal ganglia, amygdala, septum and claustrum. The lowest densities of binding sites (less than 14 fmol/mg protein) were observed in the hypothalamus, thalamus and substantia nigra. The binding of [3H]somatostatin in both the frontal cortex and cerebellar cortex demonstrated pharmacological specificity, since somatostatin-28, but not somatostatin-28(1-12) or Des AA1,2,4,5,12,13, D-Trp8-somatostatin, competed for the binding sites. Scatchard analysis of the binding in both frontal cortex and cerebellar cortex revealed the presence of two classes of high-affinity binding sites.

Preparation and characterisation of a specific antiserum to the C-fragment of lipotropin.

The recent discovery that a series of lipotropin fragments possess opiate activity [l-3] has led to extensive investigation of the central properties of these peptides and discussion of their possible physiological roles. It has been suggested that each of the peptides is elaborated to perform a distinct function, methionine enkephalin (lipotropin 61--65) as a neurotransmitter [4], y-endorphin and cu-endorphin (61-77 and 61-76) in the maintenance of behavioural states [5] and C-Fragment (61-91) in pain perception [6]. However, the unique potency exhibited by C-Fragment in its central activities [7] would seem to imply that this is the physiologically significant peptide. A recent immunofluorescent study provided evidence that the enkephalins occur in the synaptic regions of nerve tracts in brain [8] but cross reactivity of the antisera with the longer peptides was not examined and the evidence could indicate that C-Fragment or y-endorphin is present at the nerve junctions. Furthermore it has been shown that the enkephalins and endorphins can be formed by extracellular proteolysis of C-Fragment [9] and it is possible that the occurrence of the shorter peptides may reflect the occurrence of C-Fragment. This communication describes the preparation and study of an antiserum which has a high specificity for human lipotropin and for C-Fragment. It does not react significantly with y-endorphin or methionine enkephalin. The availability of this antiserum will open the way to study of the distribution of C-Fragment in specific regions of brain.

Human cerebellar cortex possesses high affinity binding sites for [3H]somatostatin

Somatostatin binding sites have been identified in the human brain using [4-3H-(Phe6)]-somatostatin-14. In contrast to that of the rat, the human cerebellar cortex possesses a high density of somatostatin binding sites, comparable to that found in either the rat or human cerebral cortex. Autoradiographic localisation of somatostatin binding sites in the human cerebellum reveals that the highest density is associated with the granule cell layer.

A molecular basis for the interaction of corticotropin with opiate receptors

Earlier investigations have shown corticotropin (ACTH) to produce pharmacological effects previously thought to be specific for opioid peptides and alkaloids. Corticotropm peptides and opioid peptides both produce characteristic grooming behaviour in rats on intraventricular injection [ 11, and have also been shown to inhibit the electrically evoked contraction of the mouse vas deferens in vitro [2] in a naloxone reversible manner. More recently, corticotropin related peptides were found to produce similar analgesic effects to /I-endorphin on injection directly into the periaqueductal gray of the rat [3]. These observations along with the report [4] that corticotropin l-24 can displace [3H]naloxone from opiate receptors in rat brain membranes strongly suggests that corticotropin peptides can interact with the opiate receptor. This paper describes a structure activity study of the opiate receptor interaction of corticotropin and related peptides using [3H]dihydromorphine and [ ‘Hlnaloxone as labelled ligands. The data is correlated with the predicted receptor conformation of the enkephalins and corticotropin and explains the molecular basis for the interaction of corticotropin with the opiate receptor, suggesting that the receptor environment induces the N-terminal region of corticotropin into an o-helical conformation. ology [6]. The peptide resins, BOC.Ser(Bzl).Tyr(Bzl). Ser(Bzl).Met.Glu(Bzl).His.Phe.Arg(Tos).Trp.Gly-resin and the corresponding peptidoresins for the 2-10 and 3-l 0 analogs, were treated with anhydrous methanol saturated with ammonia to remove the peptide from the resin as the protected Gin’ amides. The protected peptides were purified by gel filtration through Sephadex LH-20 eluted with dimethylformamide. The purified peptides were deprotected by treatment with trifluoroacetic acid followed by sodium in liquid ammonia reduction in the presence of excess free tryptophan. The peptides were finally purified by gel filtration through Sephadex G-25 eluted with 50% acetic acid. The resulting peptides had the required ammo acid compositions after hydrolysis in 6 M HCl containing 1% phenol.

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Abstract Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [ 3 H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K d s 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A 2 subtype. It is concluded that [ 3 H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.