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.     

Cyclo (His-Pro): Mapping hypothalamic sites for its hypothermic action

Hypothalamic loci of Sprague-Dawley rats were individually injected with cyclo (His-Pro) to determine the sites where that metabolite of thyrotropin-releasing hormone acts to produce hypothermia. There was almost always a positive hypothermic response in the preoptic-anterior hypothalamic area (POA/AHA); injection into the posterior or middle hypothalamic areas or into the hippocampus caused no significant decrease in core temperature. The fact that only injection into the POA/AHA evoked hypothermia suggests that this area is a major hypothalamic site of action of cyclo (His-Pro) in modulating thermoregulation in the rat.     

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.     

The distribution of Histidyl-Proline Diketopiperazine [cyclo(His-Pro)] in discrete rat hypothalamic nuclei

The distribution of Histidyl-Proline Diketopiperazine [cyclo(His-Pro)], a metabolite of thyrotropin-releasing hormone (TRH), was determined by specific RIA in Palkovits micropunch pools derived from discrete hypothalamic nuclei. Highest concentrations of cyclo(His-Pro) were identified in the anterior nucleus (3.5 ng/mg protein) and the paraventricular nucleus (2.95 ng/mg protein), while lower concentrations of cyclo(His-Pro) were seen in the other 6 nuclei. In contrast, TRH concentrations were highest in the ventromedial nucleus pars medialis (3.2 ng/mg protein) and arcuate nucleus (2.7 ng/mg protein). This qualitatively different distribution of cyclo(His-Pro) from that of TRH suggests that not all of cyclo(His-Pro) is derived exclusively from TRH.     

Neuropeptide-dopamine interactions. III. Cyclo(His-Pro) and persistence of dopaminergic supersensitivity on withdrawal from one year continuous neuroleptic treatment

When rats are given dopaminergic antagonists (neuroleptics) continuously for several days behavioral supersensitivity to dopaminergic agonists results. This supersensitivity, however, declines rapidly on neuroleptic withdrawal. The data presented here, however, are the first to show that the supersensitivity persists for a longer duration (greater than 100 days) if the neuroleptic is administered continuously for 1 year. Oral administration of 0.76 +/- 0.13 mg/kg/day cyclo(His-Pro), a peptide exhibiting dopaminergic agonist-like properties, during neuroleptic withdrawal did not change the course of dopaminergic supersensitivity reversal.     

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.     

Failure of cyclo (His-Pro) to exhibit natriuretic activity

Administration of exogenous cyclo (His-Pro) to dogs has been reported to elicit natriuresis. In contrast, our data fail to show any natriuretic activity of cyclo (His-Pro) in dogs or rats. The possible reasons underlying this discrepancy are discussed.     

Cyclo(His-Pro): its distribution, origin and function in the human

Cyclo(His-Pro), or histidyl-proline diketopiperazine, is a cyclic dipeptide endogenous to blood, cerebrospinal fluid (CSF), semen, brain, spinal cord, and gastrointestinal tract of humans. Although a part of cyclo(His-Pro) clearly appears to be derived from the limited proteolysis of thyrotropin-releasing hormone by Pyroglutamate aminopeptidase, the biosynthetic origin of the remainder of the peptide can only be speculated. The levels of this peptide in blood and CSF fluctuate in health and disease in a manner appropriate for a physiologically active endogenous molecule.     

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 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).     

Cyclo (His-Pro), d-amphetamine and striatal dopamine: a microdialysis study

Extracellular levels of dopamine (DA) and its metabolites (DOPAC and HVA) were monitored in the striatum of rats using in vivo microdialysis, in an attempt to elucidate the mechanism of cyclo (His-Pro) (histidyl-proline-diketopiperazine, CHP) on dopaminergic activity. Pretreatment with CHP (0.5 mg/kg SC) (n = 5) or the equivalent volume of saline (n = 5) was followed 30 min later by 5 mg/kg IP of d-amphetamine. Dialysate samples were collected and analyzed by high performance liquid chromatography with electrochemical detection (HPLC-EC). Following the initial increase in DA caused by d-amphetamine, DA levels of CHP-treated rats were significantly lower than saline-treated rats across time (p less than 0.05). No difference was observed for DOPAC or HVA. It is therefore unlikely that CHP interferes with the d-amphetamine-induced inhibition of DA reuptake. Other neurotransmitter systems may be involved in the CHP-induced augmentation of amphetamine's behavioral effects. Our data, as well as previous findings, suggest that attenuation of the dopaminergic response to d-amphetamine might be best explained on the basis of striatal DA depletion, possibly via tyrosine hydroxylase (TH) inhibition. This study also indicates that a dissociation may exist between the behavioral and the striatal DA response to acute amphetamine. The data support the hypothesis that amphetamine releases DA from a newly synthesized, extravesicular cytoplasmic pool, and that intracellular striatal DA is present in considerable excess relative to the extracellular DA.     

Increased cerebrospinal fluid cyclo(His-Pro) content in schizophrenia.

Cyclo(His-Pro) (CHP) is a peptide endogenous to human brain and cerebrospinal fluid (CSF). In animal studies administration of exogenous CHP augments dopaminergic neurotransmission. To explore the role of this peptide in schizophrenia, a disease characterized by a hyperdopaminergic state, we have measured CSF CHP levels in control, never-medicated schizophrenics and medicated schizophrenics. Our data show a 53% increase in CSF levels of CHP in never-medicated schizophrenics (p = 0.015), and a 25% increase in medicated schizophrenics when compared to controls. We speculate that CHP may contribute to the expression of hyperdopaminergic symptoms in schizophrenia.     

Could dietary proteins serve as cyclo(His-Pro) precursors?

Cyclic dipeptides or diketopiperazines are readily generated during in vitro hydrolysis of proteins and polypeptides. This led us to examine whether cyclo(His-Pro) (CHP), a diketopiperazine containing histidine and proline, could be formed in vivo from dietary proteins. The data presented here show that at least in rat, neither urinary nor plasma concentration of CHP is elevated by consumption of a diet rich in proteins. Several dietary supplements derived from casein and/or soy protein hydrolysates, however, contain high levels of CHP-LI. Oral intake of one such supplement led to a sharp increase in the plasma level of CHP-LI.     

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.     

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).     

Increased survival of embryonic nigral neurons when grafted to hypothermic rats

Hypothermia can reduce neuronal death caused by ischemia and traumatic brain injury. We therefore investigated whether mild hypothermia in rats receiving a transplant of embryonic mesencephalic rat tissue increases survival of the implanted dopaminergic neurons. Mild hypothermia (32-33 degrees C) during graft implantation and for the following 90 min significantly increased the survival of transplanted dopaminergic neurons to 171% of control values in normothermic (37 degrees C) rats. This demonstrates that treatment of the graft recipient for a relatively short period during and after surgery has a favorable effect on the survival of grafted dopaminergic neurons. These findings may be of importance for clinical neural transplantation trials which are in need of procedures that improve transplant survival.     

Acute alterations of cyclo(His-Pro) levels after oral ingestion of glucose

We analyzed the response of plasma cyclo(His-Pro) (CHP) (N = 14), insulin (N = 8), and glucose (N = 8) to oral ingestion of 75 grams of glucose in normal volunteers. Mean Fasting CHP levels prior to glucose were 614 +/- 112 pg/ml (+/-SE). After glucose ingestion there occurred an acute rise of plasma CHP within 15 minutes (12/14) followed by a fall in plasma concentrations to below baseline values (11 of 14). The mean of highest levels within the first 15 minutes after glucose ingestion was 1035 +/- 300 pg/ml (+/-SE), significantly above baseline (p = 0.002). The mean of lowest values below baseline was 490 +/- 94 pg/ml (+/-SE), p less than 0.001. We conclude that levels of CHP are acutely and reversibly altered by the ingestion of glucose. The possible significance of these perturbations in CHP concentrations is discussed.