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

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.     

Thyrotropin-releasing hormone: its distribution and metabolism during development in bullfrog (Rana catesbeiana)

The development changes in the metabolism of thyrotropin-releasing hormone (TRH), cyclo (His-Pro) formation from TRH, and the levels of endogenous TRH in frog brain and skin were determined. The results indicated that TRH concentrations were considerably higher in brain than in skin, and in both of these structures TRH content increased significantly following metamorphosis to adulthood. This increase in TRH concentration is probably a reflection of a marked decrease in TRH-metabolism in adult frogs compared to tadpoles. However, the formation of cyclo (His-Pro) from TRH increased during the developmental period reaching to a maximum in adulthood. The possible role of cyclo (His-Pro) in the amphibian developmental process is discussed in relation to our recent observation showing cyclo (His-Pro) inhibition of prolactin secretion.     

Hypothalamic regulation of histidyl-proline diketopiperazine binding sites in the rat liver

The effects of hypothalamic hormones and electrolytic lesioning of the ventromedial hypothalamic nuclei (VMH) on histidyl-proline diketopiperazine (cyclo(His-Pro] binding in the rat liver were studied. VMH-lesioning markedly decreased cyclo(His-Pro) binding in the liver. Scatchard analysis revealed that the loss of cyclo(His-Pro) binding induced by VMH lesioning was due to a decrease in the number and affinity of binding sites. Somatostatin (SS) administration decreased cyclo(His-Pro) binding. The SS-induced changes in cyclo(His-Pro) binding were due to changes in the binding affinity. On the other hand, the administration of TRH or LH-RH did not affect cyclo(His-Pro) binding in the liver, although cyclo(His-Pro) has been proposed to be a metabolite of TRH. These findings suggest that the hypothalamus may regulate the cyclo(His-Pro) binding sites in the liver probably by controlling pancreatic SS secretion, since a VMH-lesion is reported to cause hypersecretion of pancreatic SS.     

Thyrotropin releasing hormone but not histidyl-proline diketopiperazine is depleted from rat spinal cord following 5,7-dihydroxytryptamine treatment

Histidyl-proline diketopiperazine (His-Pro DKP) has been proposed as a metabolite of thyrotropin releasing hormone (TRH). Since spinal cord TRH arises from serotoninergic (5-HT) neurons in the brainstem, a 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), was injected into the lateral ventricle of 7 rats, and the levels of TRH and His-Pro DKP in the spinal cord were studied 5 weeks later. In comparison to the saline treated controls, 5,7-DHT treated animals showed marked depletion of TRH throughout the spinal cord, especially in the lumbosacral area where almost 90% disappeared, (0.28 +/- 0.02 vs. 2.46 +/- 0.01 ng/mg protein; P less than 0.0001). In contrast, His-Pro DKP showed no significant change in any region. Since 5,7-DHT lowers spinal cord TRH by destroying TRH perikarya in the medulla, we conclude that spinal cord His-Pro DKP is not derived from the same neurons as TRH.     

Localization of immunoreactive thyrotropin releasing hormone in the lower brainstem of the rat

The distribution of thyrotropin releasing hormone (TRH) in individual nuclei of the rat brainstem was examined by radioimmunoassay. TRH was detectable in 36 of 40 brainstem nuclei investigated and the localization of TRH in the brainstem was unlike other known brainstem neuropeptides. By far the highest concentration of TRH in the brainstem (1.2 ng/mg protein) is present in the nucleus of the solitary tract. The concentration of TRH was relatively high in the motor nuclei of the IIIrd, Vth, VIIth, Xth and XIIth cranial nerves, and less high in the area postrema, nucleus gracilis, locus coeruleus, lateral reticular nucleus (A1-catecholamine cell group), dorsal raphe and central gray matter. Cerebellum and pontine nuclei contained very low levels (<0.03ng/mg protein) of TRH.     

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

Cyclo (His-Pro) [C(HP)] has been measured by radioimmunoassay in perchloric acid extracts of human gastrointestinal (GI) tract structures derived from autopsy sources and fresh colonic biopsies. C(HP) was identified in all regions of the human GI tract, ranging in concentrations from 599 +/- 102 pg/mg protein in stomach, to 127 +/- 26 pg/mg protein in esophagus. The mean concentration of C(HP) from colonic biopsies was 335 +/- 30 pg/mg protein, statistically similar to values derived from postmortem sources. Since C(HP) concentrations are within the range of other gut peptide modulators, cyclo (His-Pro) is speculated to play a role as a new paracrine modulator of human GI tract function(s).     

The biological activities of the neuropeptide histidyl-proline diketopiperazine

Histidyl-proline diketopiperazine [cyclo(His-Pro)] is a recently discovered neuropeptide which is produced by the metabolism of thyrotropin releasing hormone, TRH. This peptide mediates a number of central nervous system activities, some of which can also be observed with TRH, some of which are opposed by TRH and some which are not affected by TRH. Cyclo(His-Pro) also inhibits release of prolactin from pituitary by a process inhibited by TRH.     

Distribution of vasoactive intestinal polypeptide (VIP) in the rat brain stem nuclei

The distribution of vasoactive intestinal polypeptide (VIP) in the individual nuclei of the rat brain stem was examined. Highest VIP concentrations in brain stem (> 1 ng/mg protein) are confined to dorsal regions, including periaqueductal gray and structures under the fourth ventricle. VIP concentrations are moderately high (0.8–1 ng/mg protein) in gracile nucleus, area postrema, nucleus of the solitary tract, motor nucleus of the XIIth, locus ceruleus and dorsal tegmental nucleus. Cerebellum and pontine nuclei contained only very low levels (< 0.2 ng/mg protein) of VIP.     

Autoradiographic distribution of thyrotropin-releasing hormone receptors in the African lungfish Protopterus annectens

We used quantitative autoradiography to examine the distribution of thyrotropin-releasing hormone (TRH) receptors in the central nervous system (CNS) of the African lungfish Protopterus annectens. We found that the distribution of TRH receptors throughout the CNS of the lungfish was heterogeneous with the highest concentrations (500-800 fmol/mg protein) in the olfactory bulb and telencephalon, moderately high concentrations (200-500 fmol/mg protein) in the diencephalon, and moderate (50-200 fmol/mg protein) to low (less than 50 fmol/mg protein) concentrations in the brainstem and spinal cord. Except for the motor nuclei of the cranial nerves and spinal cord, TRH receptors were concentrated in the acellular regions. In the telencephalon and diencephalon, the receptor density was inversely related to cellular density. These results provide a neuroanatomic and neuropharmacologic basis for further investigations of TRH in the African lungfish.     

Distribution of immunoreactive metorphamide (adrenorphin) in discrete regions of the rat brain: Comparison with met-enkephalin-Arg-Gly-Leu

The distribution of immunoreactive (ir)-metorphamide (adrenorphin) in 101 microdissected rat brain and spinal cord regions was determined using a highly specific radioimmunoassay. The highest concentration of metorphamide in brain was found in globus pallidus (280.1 fmol/mg protein). High concentrations of ir-metorphamide (>120 fmol/mg protein) were found in 9 nuclei, including central amygdaloid nucleus, lateral preoptic area, anterior hypothalamic nucleus, hypothalamic paraventricular nucleus, interpeduncular nucleus, periaqueductal grey matter and nucleus of the solitary tract. Moderate concentrations of the peptide (between 60 and 120 fmol/mg protein) were found in 47 brain nuclei such as nucleus accumbens, bed nucleus of stria terminalis, several septal and amygdaloid nuclei, most of the hypothalamic nuclei, ventral tegmental area, red nucleus, raphe nuclei, lateral reticular nucleus, area postrema and others. Low concentrations or ir-metorphamide (<60 fmol/mg protein) were measured in 41 nuclei, e.g., cortical structures, hippocampus, caudate nucleus, thalamic nuclei, supraoptic nucleus, substantia nigra, vestibular nuclei, cerebellum (nuclei and cortex). The olfactory bulb has the lowest metorphamide concentration (5.8 fmol/mg protein). Spinal cord segments exhibit very low peptide concentrations.     

Distribution of immunoreactive metorphamide (adrenorphin) in discrete regions of the rat brain: comparison with Met-enkephalin-Arg6-Gly7-Leu8

The distribution of immunoreactive (ir)-metorphamide (adrenorphin) in 101 microdissected rat brain and spinal cord regions was determined using a highly specific radioimmunoassay. The highest concentration of metorphamide in brain was found in globus pallidus (280.1 fmol/mg protein). High concentrations of ir-metorphamide (greater than 120 fmol/mg protein) were found in 9 nuclei, including central amygdaloid nucleus, lateral preoptic area, anterior hypothalamic nucleus, hypothalamic paraventricular nucleus, interpeduncular nucleus, periaqueductal grey matter and nucleus of the solitary tract. Moderate concentrations of the peptide (between 60 and 120 fmol/mg protein) were found in 47 brain nuclei such as nucleus accumbens, bed nucleus of stria terminalis, several septal and amygdaloid nuclei, most of the hypothalamic nuclei, ventral tegmental area, red nucleus, raphe nuclei, lateral reticular nucleus, area postrema and others. Low concentrations or ir-metorphamide (less than 60 fmol/mg protein) were measured in 41 nuclei, e.g., cortical structures, hippocampus, caudate nucleus, thalamic nuclei, supraoptic nucleus, substantia nigra, vestibular nuclei, cerebellum (nuclei and cortex). The olfactory bulb has the lowest metorphamide concentration (5.8 fmol/mg protein). Spinal cord segments exhibit very low peptide concentrations.     

Distribution of immunoreactive dynorphin A1–8 in discrete nuclei of the rat brain: Comparison with dynorphin A

The distribution of immunoreactive (ir)-dynorphin A1-8 (Dyn A1-8) in 78 microdissected rat brain areas as well as in the neurointermediate lobe of pituitary gland was determined using a highly specific radioimmunoassay. The highest concentrations of Dyn A1-8 in brain were found in substantia nigra (673.8 fmol/mg protein) and lateral preoptic area (565.1 fmol/mg protein). High concentrations of ir-Dyn A1-8 (greater than 240 fmol/mg protein) were found in 5 nuclei: ventral premamillary nucleus, anterior hypothalamic nucleus, dorsomedial nucleus, arcuate nucleus, and medullary reticular nuclei. Moderate concentrations of the peptide (between 120 and 240 fmol/mg protein) were found in 55 brain nuclei such as septal and amygdaloid nuclei, most diencephalic structures, mesencephalic nuclei, pons and medulla oblongata nuclei and others. Low concentrations of ir-Dyn A1-8 (less than 120 fmol/mg protein) were found in 16 regions, e.g. frontal cortex, hippocampus, caudate-putamen cortical amygdaloid nucleus, several thalamic nuclei, mamillary body superior and inferior colliculi, cerebellar nuclei and others. The posterior thalamic nucleus has the lowest ir-Dyn A1-8 concentration (62.0 fmol/mg protein). The neurointermediate lobe of the pituitary gland is extremely rich in ir-Dyn A1-8 (4063.0 fmol/mg protein).     

High affinity specific binding of the thyrotrophin releasing hormone metabolite histidylproline to rat brain membranes

Histidylproline a metabolite of Thyrotrophin Releasing Hormone specifically binds to both high and low affinity sites in fresh rat brain membranes. Characterisation of the high affinity site under optimal conditions demonstrated an equilibrium dissociation constant (KD) of approximately 9nM and maximum binding capacity of approximately 120 fmols/mg protein. Kinetic analysis of [3H]-His-Pro binding is limited by low binding density, instability of the high affinity site and rapid degradation of the radioligand. The thiol blocking reagent pHydroxymercuriphenylsulphonic acid (HMPS) inhibited [3H]-His-Pro degradation but also reduced binding of the peptide to membranes. The results are discussed with reference to the lack of specific binding sites in brain for the proposed neuropeptide and TRH metabolite cyclo(His-Pro).     

Distribution of immunoreactive dynorphin B in discrete areas of the rat brain and spinal cord

The distribution of immunoreactive (ir)-dynorphin B in 101 microdissected rat brain and spinal cord regions was determined using a specific radioimmunoassay. The highest concentration of dynorphin B in brain was found in the substantia nigra (1106.2 fmol/mg protein). Very high concentrations of ir-dynorphin B (> 400 fmol/mg protein) were also found in the lateral preoptic area, parabrachial nuclei and globus pallidus. Relatively high concentrations of ir-dynorphin B (250–400 fmol/mg protein) were found in 19 nuclei, including the periaqueductal gray matter, anterior hypothalamic nucleus, median eminence, nucleus accumbens and hippocampus. Moderate levels of the peptide (between 100 and 250 fmol/mg protein) were found in 42 brain nuclei such as the perifornical nucleus, nucleus of the diagonal band, medial forebrain bundle, and dorsal premamillary nucleus. Low concentrations of ir-dynorphin B (< 100 fmol/mg protein) were found in 28 brain areas, e.g. cerebral cortical structures (parietal, cingulate, frontal), claustrum, olfactory bulb, lateral and periventricular thalamic nuclei. The cerebellar cortex has the lowest dynorphin B concentration (53.7 fmol/mg protein). Spinal cord segments exhibit low or moderate (cervical segment) levels of the peptide. The neurointermediate lobe of the pituitary gland is extremely rich in ir-dynorphin B (11,047.1 fmol/mg protein).     

Neuropeptide-dopamine interactions. II. Cyclo (His-Pro) augmentation of amphetamine- but not apomorphine-induced stereotypic behavior

Histidyl-proline diketopiperazine (cyclo [His-Pro]) not only exists in the basal ganglia of rodents, monkeys, and humans, but also exhibits a variety of biologic activities, some of which appear to be mediated via dopaminergic mechanisms. We investigated the potential modulation by cyclo (His-Pro) of amphetamine- and apomorphine-induced stereotypic behavior, a behavior that is associated with the activation of postsynaptic dopamine receptor. Administration of amphetamine to rats resulted in a dose-dependent increase in stereotypic behavior that was further augmented if animals were pretreated with cyclo (His-Pro). Although apomorphine also led to a dose-related progression in the stereotypic behavior, the apomorphine effects were not modified by cyclo (His-Pro) pretreatment. We conclude that cyclo (His-Pro) either acts indirectly at the presynaptic dopamine site or modulates other neurotransmitters to potentiate actions of amphetamine.     

Cyclo(His-Pro) and the development of tolerance to the hypothermic effect of ethanol

Acute intraperitoneal administration of ethanol to rats causes a dose-dependent transient hypothermia. On repeated exposure, however, rats develop tolerance to hypothermic effects of ethanol. Cyclo(His-Pro), an endogenous brain peptide, modifies both acute and chronic themomodulatory effects of alcohol. For example, a) acute pretreatment of rats with increasing amounts of cyclo(His-Pro) produces a progressive decrease in ethanol hypothermia, and b) chronic cyclo(His-Pro) administration augments the development of tolerance to hypothermic effects of alcohol. While the mechanism of cyclo(His-Pro) action is not clear, these data are interpreted to suggest that this peptide may play important roles in ethanol intoxication, preference, tolerance, and/or addiction.     

Metabolism of thyrotropin-releasing hormone in human cerebrospinal fluid. Isolation and characterization of pyroglutamate aminopeptidase activity

Pyroglutamate aminopeptidase, which catalyzes metabolism of thyrotropin-releasing hormone (TRH) to cyclo(His-Pro), is the major enzyme of TRH metabolism in human CSF. The partially purified CSF pyroglutamate aminopeptidase has a pH optimum between 6.0 and 7.4, and a Km of 15.9 +/- 3.1 microM. A number of potential competitive inhibitors of the enzymatic activity were examined, of which luteinizing hormone-releasing hormone and bombesin were the most effective. An examination of the structure of various peptides that inhibit pyroglutamate aminopeptidase activity indicated that the enzyme generally prefers a substrate having amino-terminal pyroglutamic acid (pGlu) and a COOH-terminal that is either blocked or distant from amino-terminal pGlu. Heavy metals, EDTA and reducing agents inactivated the enyzme, whereas benzamidine, phenylmethylsulfonylfluoride, trypsin inhibitor and alkylating agents had little or no effect on the enzymatic activity. Thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid), however, considerally inhibited the enzymatic activity. We hypothesize that CSF pyroglutamate aminopeptidase may play a role in the biologic actions of TRH.     

Neuropeptide-dopamine interactions. I. Dopaminergic mechanisms in cyclo(His-Pro)-mediated hypothermia in rats

Administration of cyclo(His-Pro) to rats produces a dose-dependent hypothermia that is attenuated by dopaminergic antagonists. Chronic treatment with cyclo(His-Pro) potentiates hypothermia induced by apomorphine. These results suggest that cyclo(His-Pro) acts via a dopaminergic mechanism to modulate body temperature.