Potential mechanism(s) underlying the fasting-associated rise in hypothalamic cyclo(His-Pro) content was explored by examining the effects of 24-hour fasting on: (i) cyclo(His-Pro) synthesis from TRH, (ii) cyclo(His-Pro) metabolism, and (iii) cyclo (His-Pro) secretion by hypothalamic tissue in vitro. The data presented here show that none of these three variables were altered due to fasting. Two additional potential changes that could cause cyclo(His-Pro) elevations during fasting are suggested. These include an in vivo decrease in hypothalamic cyclo(His-Pro) secretion that may not be apparent in vitro, and/or an increase in the synthesis of cyclo(His-Pro) from a precursor(s) other than TRH.

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone and pyroglutamate aminopeptidase activity was examined in the CSF of human and a number of other mammalian species. Cyclo(His-Pro)-like immunoreactivity was present in the CSF of all species examined, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentration in CSF had no significant correlation with CSF TRH or pyroglutamate aminopeptidase.

S-adenosyl-L-methionine-dependent methylation of membrane phosphatidylethanolamine to phosphatidylcholine has been shown to exist in a number of tissues including pituitary gland and to play important roles in receptor-mediated functions. The possible role of this phospholipid methylation reaction in pituitary hormone secretion has been studied. To this end, the ability of thyrotropin-releasing hormone (TRH) to release thyrotropin (TSH) and prolactin and the ability of luteinizing hormone-releasing hormone (LH-RH) to release luteinizing hormone (LH) were evaluated after inhibition of pituitary phospholipid methylation. Both TRH and LH-RH stimulated the release of their corresponding pituitary hormone in a dose-dependent manner and this stimulatory effect was inhibited in the presence of phospholipid methylation inhibitors. Non-specific stimulation of TSH release by 55 mM KCl or 0.1 mM veratridine, however, was not affected by the methylation inhibitors. The data suggest that phospholipid methylation may participate in receptor-mediated release of pituitary hormones.

Thyrotropin-releasing hormone (TRH) is unevenly distributed throughout the rat central nervous system including spinal cord, where exogenous TRH elicits profound pharmacological effects. [Pro-3H]TRH binds to 30,000g pellet fraction from the spinal cord saturably, reversibly, and with high affinity (apparentKd = 24 − 25nM). This binding is displaced by TRH and related biologically active, but not inactive, peptides. TRH binding is evenly distributed throughout the rat spinal cord. Characteristics of this binding suggest an association with a physiologically relevant TRH receptor.

Rat pituitary extracts catalyze methylation of phosphatidylethanolamine to phosphatidylcholine using S-adenosyl-L-methionine as the methyl donor. In vitro incubation of hemipituitaries with 1 mM 2-methylaminoethanol led to a dose dependent decrease in phosphatidylethanolamine methyltransferase activity as well as the incorporation of the radioactivity from [3H-methyl]-L-methionine into phosphatidyl-choline. The inhibitory effect of 2-methylaminoethanol was selective for phospholipids methylation since protein carboxymethyltransferase and catechol-o-methyltransferase activities were not affected.

The distribution of cyclo (His-Pro)-like immunoreactivity in frog skins from seven frog species was examined. The chromatographic elution profile of cyclo (His-Pro)-like immunoreactivity in amphibian skins measured by radioimmunoassay corresponded precisely to that of [3H-Pro]-cyclo (His-Pro) after DEAE-Cellulose, Sephadex G-25 and high-pressure liquid chromatography. The concentrations of cyclo (His-Pro) in frog skins were much higher than the concentrations of TRH previously observed in skin and the concentrations of cyclo (His-Pro) in both brain and gastrointestinal tract.

The specific binding of thyrotropin-releasing hormone (TRH) by 30,000g pellet fraction was ubiquitously distributed throughout various rat brain regions including cerebellum. Although the cerebellum had the lowest apparent density of specific TRH binding sites found in any of the brain regions studied, it represented a single class of high afinity receptor (KD=37.73±4.88 nM,Bmax=156.0±5.7 fmol/mg protein,n=4). Furthermore, the cerebellar synaptic plasma membrane fractions were richly endowed with TRH-binding, two other membrane fractions (light-synaptic plasma membrane and microsomal) exhibited high TRH-binding whereas nuclear, mitochondrial or myelin fractions were devoid of significant binding activity. These data show for the first time the existence of specific TRH-binding in cerebellum, and thus suggest that TRH may modulate cerebellar synaptic functions by acting through a specific high affinity-receptor.

The specific binding of [3H-Pro]cyclo (His-Pro) to a 27,000 g pellet fraction from rat adrenal gland was studied. Binding sites were exclusively localized in the cortical region of the adrenal gland and exhibited a binding affinity (Kd) of 1.79 +/- 0.21 microM and maximal binding capacity (Bmax) of 123.4 +/- 10.2 pmol/mg protein. In vivo administration of cyclo (His-Pro) to neonatal rats led to the downregulation of cyclo (His-Pro)-binding sites with a decrease in Bmax but not Kd. Such changes were not observed after similar treatments of adult rats. These data suggest a receptor-like characteristic of cyclo (His-Pro) binding sites.

Phospholipid methyltransferase activity is unevenly distributed throughout the contractile and the specialized regions of rat heart. Chronic alcohol treatment stimulated the incorporation of [3H-methyl]-methionine but not [3H-methyl]-choline into phosphatidylcholine by left ventricular slices. The increase in the incorporation of [3H-methyl]-group from [3H-methyl]-methionine into phosphatidylcholine was due to an increase in phospholipid methyltransferase activity.

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and pyroglutamate aminopeptidase activity was examined in the rat gastrointestinal (GI) tract. Cyclo(His-Pro)-like immunoreactivity was present in the following order of distribution (fmoles/mg protein): caecum > colon = jejunum = ileum > stomach = duodenum = rectum, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentrations showed significantly positive correlations with TRH concentrations, but not with pyroglutamate aminopeptidase activities, in most tissues of the GI tract, suggesting a precursor role of TRH for gut cyclo(His-Pro). These data suggest that cyclo(His-Pro) may be involved in regulating rat GI functions.

Rat pituitary extracts contain two methyltransferases that catalyze stepwise methylation of phosphatidyl-ethanolamine to phosphatidylcholine using S-adenosylmethionine as the methyl donor. The activities of both of these enzymes were stimulated by 40 μM lysine or arginine vasopresin but not oxytocin, arginine vasotocin and Pro-Leu-Gly NH 2 . The concentration of lysine-vasopressin required for the half-maximal stimulation of phospholipid methylation was 27 μM. A comparison of the chemical structure of different peptides with their ability to stimulate phospholipid methylation suggests that the stimulatory activity resides in the covalent ring structure (pressinoic acid) of the vasopressin molecule.