John F. Wilber

Distribution and metabolism of Cyclo (His-Pro): A new member of the neuropeptide family

Cyclo (His-Pro) is a biologically active cyclic dipeptide derived from thyrotropin-releasing hormone by its limited proteolysis. We have developed a specific radioimmunoassay for this cyclic peptide and shown its presence throughout rat and monkey brains. The normal rat brain concentration of cyclo (His-Pro) ranged from 35-61 pmols/brain. The elution profiles of rat brain cyclo (His-Pro)-like immunoreactivity and synthetic radioactive cyclo (His-Pro) following gel filtration, ion-exchange chromatography, and high pressure liquid chromatography were similar. An analysis of the regional distribution of cyclo (His-Pro) and TRH in rat and monkey brains exhibited no apparent precursor-product relationship. The possible additional factors determining regional differences in the endogenous cyclo (His-Pro) concentrations are discussed. The endogenous levels of brain cyclo (His-Pro) were elevated when rats were made either hypothyroid by surgical thyroidectomy or forced to drink alcohol for six weeks. These studies demonstrate that cyclo (His-Pro) is present throughout the central nervous system in physiologically relevant concentrations which can be modulated by appropriate physiological and pharmacological manipulations. These data in conjunction with earlier reports of multiple biological activities of exogenous cyclo (His-Pro), suggest that endogenous cyclo (His-Pro) is a biologically active peptide and it may play a neurotransmitter or neuromodulator role in the central nervous system.

Amyotrophic lateral sclerosis Effects of acute intravenous and chronic subcutaneous administration of thyrotropin‐releasing hormone in controlled trials

We performed double-blind crossover trials to assess the effects of thyrotropin-releasing hormone (TRH) on amyotrophic lateral sclerosis patients. For acute intravenous trials, 500 mg TRH or placebo with norepinephrine was given at 1-week intervals (16 patients). CSF TRH concentration increased, and clinical side effects appeared with TRH. For chronic studies, 25 mg TRH and a saline placebo were given subcutaneously every day for 3 months (25 patients). CSF TRH level increased 29-fold after a single TRH injection, and mild transient side effects occurred. Vital signs, respiratory function, semiquantitative and quantitative neurologic function, muscle strength by manual and dynamometer testing, and EMG were studied. With daily TRH, 10 patients noted subjective improvement without objective evidence, and 10 patients complained of worsening of the disease with objective decline after TRH was stopped. Statistical analysis, however, showed no beneficial effects from either acute or chronic TRH trials.

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in the rat gastrointestinal tract

Abstract 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.

Histidyl-proline diketopiperazine cyclo (His-Pro): Identification and characterization in rat pancreatic islets

Measurements of cyclo (His-Pro) in the pancreas were carried out in the rat by a specific radioimmunoassay. Cyclo (His-Pro)-like immunoreactivity was identified in pancreatic islets with a mean concentration of 2023 pg/mg protein, 88-fold higher than that of the whole pancreas. Cyclo (His-Pro) immunoreactivity from pancreatic extracts was indistinguishable immunologically and chromatographically from synthetic cyclo (His-Pro). Insulin-induced hypoglycemia caused a significant, 53% decrease in pancreatic cyclo (His-Pro) concentrations, and FLA-63, a dopamine beta-oxidase inhibitor, also reduced islet cyclo (His-Pro) concentrations 51%. These data indicate that cyclo (His-Pro) is present in rat pancreatic islets and may play a potential role in modulating pancreatic responses to nutrient and pharmacologic stimuli.

Cyclo (His-Pro): a selective inhibitor of rat prolactin secretion in vitro.

Abstract Cyclo (His-Pro) (10 ng/ml), inhibits KCl (59 mM) or thyrotropin-releasing hormone (10 ng/ml) stimulated, but not basal, release of prolactin from rat hemipituitaries in vitro . However, cyclo (His-Pro) has no effect on the basal or stimulated release of thyrotropin and growth hormone. Cyclo (His-Pro) does not inhibit the binding of thyrotropin-releasing hormone to pituitary membrane suggesting that cyclo (His-Pro) inhibition of prolactin release is not mediated via the pituitary TRH-receptor.

Developmental changes in the distribution of rat brain pyroglutamate aminopeptidase, a possible determinant of endogenous cyclo(His-Pro) concentrations

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and Pyroglutamate aminopeptidase activity in adult and developing rat brains were studied. A comparison of the subcellular distribution of Pyroglutamate aminopeptidase activity in hypothalamic and cerebral cortical extracts from adult rats exhibited remarkable differences. In hypothalamus, the enzyme activity was mainly associated with the soluble fraction whereas in cortex it was predominantly associated with the particulate fractions. During postnatal development, the brain concentrations of cyclo(His-Pro) and Pyroglutamate aminopeptidase activities declined with age. These data suggest that Pyroglutamate aminopeptidase activity, but not TRH, plays an active role in determining the levels of endogenous cyclo(His-Pro) concentrations in brain.

Thyrotropin-releasing hormone (TRH) in murine motor neuron disease (the wobbler mouse) ☆

Clinical benefits of thyrotropin-releasing hormone (TRH) were tested in wobbler mice, an animal model of motor neuron disease. After the disease was clinically recognized at 3-4 weeks, the animals were divided into two groups, each group consisting of 5 pairs of wobbler mice and normal littermates. TRH (50 mg/kg) and normal saline (NS) were injected intraperitoneally daily, 6 times per week for 9 weeks, in a double-blind study. Weekly assessments consisted of front paw grip strength, push walking, body weight, and semiquantitative grading. At the end of the trial, the brain and spinal cord were sampled to measure TRH and cyclo (His-Pro) concentrations. Progression of motor neuron disease was evident in wobbler mice, regardless of treatment. Descriptive semiquantitative gradings showed the tendency of improvement in TRH-treated wobbler mice. In saline-injected controls, TRH levels in the cervical spinal cord were significantly increased (P less than 0.01) in wobbler mice compared to littermates. However, with TRH treatment, there was no significant difference in TRH and cyclo (His-Pro) levels in any neural tissue between wobbler and controls. The lack of clinical benefits with TRH in wobbler mice may be due to increased TRH levels found in diseased spinal cord in murine motor neuron disease.

Distribution of histidyl-proline diketopiperazine [cyclo (His-Pro)] and thyrotropin-releasing hormone (TRH) in the primate central nervous system.

Abstract The concentrations of cyclo (His-Pro) and its precursor, thyrotropin-releasing hormone (TRH) were measured in 47 different loci of monkey brain using specific radioimmunoassays. Cyclo (His-Pro) concentrations were higher than those of TRH in all loci excepting the hypothalamus, where TRH concentration was found to be the highest of all the loci and twice those of cyclo (His-Pro). The high levels of cyclo (His-Pro) were seen within the cerebellar system (inferior olivary nucleus>nucleus interpositus>fastigial nucleus>posterior vermis). The great variations in TRH-cyclo (His-Pro) ratios among different loci suggest that other factors in addition to TRH concentration must play roles in determining the unique distribution pattern of cyclo (His-Pro) in the primate brain.

Histidyl-proline diketopiperazine cyclo (His-Pro): Measurement by radioimmunoassay in human blood in normal subjects and in patients with hyper- and hypothyroidism

Abstract An extraction procedure for cyclo (His-Pro) in human blood has been developed, employing sequential extraction with 90% methanol and 0.3 M perchloric acid. Chromatographic elution profiles of immunoreactive cyclo (His-Pro) in blood extracts measured by radioimmunoassay corresponded precisely to those of {3H} cyclo (His-Pro) following separation with Sephadex G-25, DEAE-cellulose, and SP-Sephadex columns, and also HPLC. Concentrations of cyclo (His-Pro) in normal human subjects (5073 ± 730 fmoles/ml) were approximately 50 times greater than respective TRH concentrations measured in identical samples. Cyclo (His-Pro) concentrations in hypothyroid patients (12373 ± 802 fmoles/ml) were significantly higher than those in normal and those in hyperthyroid patients (5949 ± 566 fmoles/ml). TRH concentrations, by contrast, were similar in all 3 groups. Pyroglutamate aminopeptidase activity which catalyzes the conversion of TRH to cyclo (His-Pro), was identified also in peripheral plasma. The plasma enzyme activity in normal subjects was 76 ± 27 fmoles cyclo (His-Pro) formed/min/ml plasma. This activity was not statistically increased in hypothyroid patients, so that the increased concentrations of cyclo (His-Pro) in hypothyroidism could not be attributed to the enhanced conversion of TRH in the peripheral blood.

Exogenous triiodothyronine lowers thyrotropin-releasing hormone concentrations in the specific hypothalamic nucleus (paraventricular) involved in thyrotropin regulation and also in posterior nucleus

Recent evidence indicates that thyroid hormones can regulate thyrotropin secretion in vivo in part by inhibiting thyrotropin-releasing hormone (TRH) secretion itself. Therefore, to explore whether triiodothyronine (T3) interacts with the specific hypothalamic area involved in thyrotropin (TSH) secretory regulation, the paraventricular nucleus (PVN), Palkovitz micropunches from eight nuclear regions were obtained from 1,000-microns frozen coronal brain slices for immunoassay determinations of TRH. Rats were treated either with parenteral L-T3 for 6 days to induce experimental thyrotoxicosis, or 0.15 M saline. The induction of thyrotoxicosis was confirmed by demonstrating that mean plasma TSH concentrations fell from 108 to less than 10 microU/ml (p less than 0.01). TRH concentrations in the PVN were reduced concomitantly after L-T3 from 1.9 to 1.1 ng/mg protein (p less than 0.05). No reductions in TRH concentrations during T3 treatment occurred in other nuclear groupings except in the posterior hypothalamic nucleus. Total TRH content in the median eminence declined also in T3-treated animals from 1.77 to 1.29 ng, representing a 32% reduction (p less than 0.01). No significant change was seen in the median eminence content of the TRH structurally related dipeptide, cyclo(His-Pro). The data herein indicate that experimental thyrotoxicosis in the rat is associated with a selective reduction in TRH concentrations in the PVN, documenting T3 effects upon hypothalamic TRH metabolism per se.