Influences of maternal caffeine on the neonatal rat brains vary with the nutritional states

The potential effect of maternal caffeine ingestion upon total brain protein and the concentration of two prototype neuropeptides, thyrotropin-releasing hormone (TRH) and its derivative, cyclo (His-Pro) in neonates was examined during the nursing period in the context of variable maternal protein intake. Maternal caffeine intake (2 mg/100 g body weight) significantly increased the total brain protein of neonates derived from dams fed a 6% casein diet, but not from dams fed a 12%- or 20%-casein diet. Maternal caffeine consumption significantly increased the amount of cyclo (His-Pro) in the neonatal brains in all groups. The percent increments in pups from dams fed 6%, 12%, and 20% casein diets were respectively 137%, 131%, and 120%. By contrast, no significant alterations were observed in TRH concentrations between caffeine and control groups. It is concluded that maternal caffeine can influence neonatal brain protein and cyclo (His-Pro) during nursing under conditions of protein-energy malnutrition.     

Brain TRH and Cyclo (His-Pro) and brain protein in the newborn rat are altered by maternal liquid protein feeding

Liquid protein diet (LPD) has been shown previously to produce maternal and fetal weight loss and fetal congenital anomalies, including cataracts and craniofacial malformations. Therefore, to examine the effects of LPD in pregnancy upon the central nervous system of pups, pregnant dams were fed either a 20% casein diet ad libitum, a 20% LPD, or pair-fed with a 20% casein diet. LPD was associated with significant maternal weight loss, and pups had significantly lower birth weights (5.14 +/- 0.64) than pups from the pair-fed controls (5.70 +/- 0.46, p less than 0.05). Total brain protein content was reduced significantly in pups of both sexes from pregnant fed LPD. Moreover, the concentrations of two brain peptides neurotransmitters, thyrotropin-releasing hormone (TRH), and its biologically active metabolite, histidyl-proline diketopiperazine Cyclo (His-Pro), were elevated in the pups from LPD-fed mothers. In contrast, there was no significant difference in brain protein or brain peptides in pups from pair-fed mothers vs. pups from mothers fed ad libitum. These data suggest that qualitative alterations of the protein component in maternal dietary composition have deleterious effects upon the ontogeny of the rat fetal CNS, as reflected by reduced total protein and elevated concentrations of TRH and Cyclo (His-Pro).     

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.     

Histidyl-proline diketopiperazine: its biological role as a regulatory peptide

Summary Histidyl-proline diketopiperazine [cyclo(His-Pro)] is a metabolic of thyrotropin releasing hormone (TRH). This review summarizes the literature concerning cyclo (His-Pro) and, in addition, some studies dealing with TRH and other peptides that are considered of interest. The enzymes concerned with the metabolism of TRH are discussed. Distribution studies of peptides by immunological methods show that, while TRH is concentrated in synaptosomes, cyclo (His-Pro) is not, suggesting that cyclo (His-Pro) is not a classical neurotransmitter. Rat brain contains approximately three times as much cyclo (His-Pro) as TRH, mainly localized in the pituitary and hypothalamus. While the TRH is found in a free form, the cyclo (His-Pro) is bound to a carrier of molecular weight approximately 70 000. While specific membrane receptors for TRH have been detected in pituitary cells, no such receptors for cyclo (His-Pro) have yet been found in brain or pituitary; however, there is a specific binding of cyclo (His-Pro) to adrenal cortex membranes, Both TRH and cyclo (His-Pro) have effects in the central nervous system or pituitary. These include effects on prolactin release, thermoregulation, CNS depression, stereotypic behavior and cyclic nucleotide levels. Possible mechanisms and interrelations of these effects are discussed.     

SUPPLEMENTATION OF A WHEAT BREAD DIET WITH LYSINE AND THREONINE: EFFECTS UPON INTRAUTERINE GROWTH AND BRAIN BIOCHEMISTRY

Abstract— During pregnancy, rats were fed either a standard chow diet, a wheat bread diet or a wheat bread diet supplemented with lysine and threonine. At birth, the offspring of the dams fed the supplemented diet differed from the chow fed animals only in having a smaller amount of protein in the combined cerebellum and brain stem area while the pups whose dams were fed the bread diet showed lower brain weights, deficits in protein in all brain parts and deficits in DNA in the cerebrum.     

Properties of histidyl-proline diketopiperazine [cyclo(His-Pro)] binding sites in rat liver

Characteristics of cyclo(His-Pro) binding sites in the rat liver were studied using 3H-labeled cyclo(His-Pro). Scatchard analysis suggested that the rat liver membrane had a single binding site with an apparent dissociation constant (Kd) of 7 X 10(-8) M. Pretreatment of membrane preparations with soybean trypsin inhibitor increased cyclo(His-Pro) binding, and the binding activity was sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for cyclo(His-Pro) binding. Thiol reagents reduced binding activity, suggesting that the thiol group might be an important constituent of the cyclo(His-Pro) binding site. Cross-reactivities of TRH, TRH analogues, L-His and L-Pro were very low (0.2-9%). These findings indicate that specific binding sites for cyclo(His-Pro) in the rat liver have similar properties to the receptors for other polypeptides.     

Chronic Alcohol Consumption Increases Cyclo (His-Pro)-Like Immunoreactivity in the Rat Brain

Abstract: Administration of histidyl-proline diketopiperazine (cyclo (HisPro)) to rats attenuates ethanol-induced sleep. To understand the role played by cyclo (His-Pro) in the pathophysiology of prolonged alcohol consumption, we have measured the distribution of this peptide in brains of control and alcohol-treated rats. The data show that prolonged alcohol consumption increases the concentration of cyclo (His-Pro) in hypothalamic as well as extrahypothalamic brain. These changes may reflect a physiologic adaptation of the brain during alcohol consumption.     

Is all cyclo(His-Pro) derived from thyrotropin-releasing hormone?

Cyclo(His-Pro), or histidyl-proline diketopiperazine, is an endogenous cyclic dipeptide that is ubiquitously distributed in tissues and body fluids of both man and animals. This cyclic dipeptide is not only structurally related to thyrotropin-releasing hormone (TRH, pGlu-His-ProNH₂), but it can also arise from TRH by the action of the enzyme pyroglutamate amino-peptidase (pGlu-peptidase). The data on the distribution of TRH, cyclo(His-Pro), and pGlu-peptidase under normal and abnormal conditions are summarized and potential relationships analyzed. We conclude that all of the cyclo(His-Pro) cannot be derived from TRH. Two additional sources of cyclo(His-Pro) are suggested. It is proposed that 29,247 molecular weight TRH prohormone, prepro TRH, which contains 5 copies of TRH sequence, can be processed to yield cyclo(His-Pro). Thus, both TRH and cyclo(His-Pro) share a common precursor, prepro[TRH/Cyclo(His-Pro)].     

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

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and $\text{pyroglutamate aminopeptidase}$ 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 $\text{pyroglutamate aminopeptidase}$ 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.     

Distribution and characterization of cyclo (His-Pro)-like immunoreactivity in anuran (frog) skins

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.     

On the mechanism of fasting-associated elevations in hypothalamic cyclo(His-Pro) content

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.     

Inhibition of abstinence syndrome in opiate defendent mice by cyclo (His-Pro)

Intracerebral administration of cyclo (His-Pro), the postulated metabolite of thyroliberin (TRH, pGlu-His-Pro-NH₂) inhibited the naloxone induced withdrawal responses in morphine dependent mice. Mice were rendered dependent on morphine by the subcutaneous implantation of a pellet (containing 75 mg of morphine free base) for three days. Six hours after pellet removal, the naloxone ED₅₀ for the jumping response was found to be higher in mice injected with cyclo (His-Pro) compared with that of vehicle controls. Similarly, the hypothermic response observed following 50 μg/kg of naloxone given given 6 h after pellet removal or that seen with 100 μg/kg of naloxone given 24 h after pellet removal from morphine-dependent mice was inhibited by cyclo (His-Pro). Previously, we have shown similar results with TRH on the morphine abstinence syndrome. It appears, therefore, that cyclo (His-Pro) may be the active metabolite of TRH and analogs of cyclo (His-Pro) may be useful in blocking the symptoms of the opiate abstinence syndrome.     

Studies on the mechanism of thyrotropin releasing hormone induced inhibition of gastrointestinal transit

Intracerebral administration of thyrotropin releasing hormone (TRH) inhibited gastrointestinal transit in the mouse as determined by the charcoal meal test. A similar inhibitory effect was produced by morphine administered subcutaneously. TRH enhanced morphine-induced inhibition of gastrointestinal transit. Intracerebral injections of cyclo (His-Pro), a postulated metabolite, did not affect gastrointestinal transit either by itself or that produced by morphine. It is suggested that gastrointestinal transit effects of TRH are not mediated via its conversion to cyclo (His-Pro).     

Effect of dietary caffeine and zinc on the activity of antioxidant enzymes,zinc, and copper concentration of the heart and liver in fast-growing rats

The purpose of this study was to determine the relationship between concentrations of Zn and Cu and the activities of superoxide dismutase and glutathione peroxidase in the heart and liver of young rat pups whose dams were fed a diet supplemented with caffeine and/or Zn. Four groups of dams with their newborn pups were fed one of the following diets for 22 d: 20% protein basal diet; the basal diet supplemented with caffeine (2 mg/100 body wt); the basal diet supplemented with Zn (300 mg/kg diet); or the basal diet supplemented with caffeine plus Zn. The Cu levels in the livers of the pups were decreased by maternal intake of the caffeine and Zn diet. The maternal intake of the caffeine diet increased Mn-superoxide dismutase (MnSOD) activity and Cu, Zn-superoxide dismutase (CUZnSOD) in the heart of the pups. On the other hand, the activity of Cu,ZnSOD was significantly reduced in the liver of pups whose dams consumed a caffeine, Zn, or caffeine plus Zn diet. Cu, ZnSOD activity in the liver of the pups seems to be correlated with Cu levels in the tissue. Selenium-dependent glutathione peroxidase (GSH-Px) activities in the heart and liver showed no difference among the groups. The effect of dietary caffeine and/or Zn on the activity of antioxidant enzymes in the heart and liver were different in young rats. The activities of these enzymes in the heart were lower than in the liver of 22-d-old rats. Our experiments indicate that the heart has limited defenses against the toxic effects of peroxides when compared to the liver.     

Unresponsiveness of GH cells to cyclo(histidyl-proline), a metabolite of thyrotropin releasing hormone

Cyclo(Histidyl-Proline) is a metabolite of thyrotropin-releasing hormone. It has been suggested that this peptide plays a role in regulating prolactin secretion in GH cells. An investigation of the effect of cyclo(His-Pro) on GH cells indicated that it does not affect basal prolactin release or accumulation or the levels stimulated by TRH. cAMP levels in GH cells are elevated by TRH or VIP, but not influenced by cyclo(His-Pro). cGMP levels in GH cells are not affected by either TRH or cyclo(His-Pro). While there is specific binding of TRH to receptors in GH cells, no such receptors for cyclo(His-Pro) are detectable. It is suggested that GH cells are unresponsive to cyclo(His-Pro).     

Evidence for the presence of immunoreactive histidyl-proline diketopiperazine [Cyclo (His-Pro)] in the adult human brain

The current study was undertaken to evaluate the presence of cyclo (His-Pro) in adult human brain tissues obtained at autopsy. We found evidence for immunoreactive cyclo (His-Pro), which diluted in parallel to the radioimmunoassay standard curve and which had mobility on HPLC that was similar to synthetic cyclo (His-Pro), in several regions of the adult human brain. Whereas the levels of cyclo (His-Pro) in the pituitary stalk-median eminence were high (2.2 ng/mg protein), the concentrations in the whole hypothalamus were much lower (0.105 ng/mg protein). Among the extrahypothalamic brain regions examined, the levels of cyclo (His-Pro) were highest in the cerebellar hemisphere (0.168 ng/mg protein) and olfactory bulbs (0.180 ng/mg protein) and were lowest in the hippocampus (0.080 ng/mg protein) and occipital cortex (0.079 ng/mg protein). Thus, immunoreactive cyclo (His-Pro) has widespread distribution in the adult human brain and the potential exists for this cyclic diepeptide to play a role in human brain function.     

Prenatal and postnatal protein restriction in the rat: effect on some parameters related to brain development, and prospects for rehabilitation.

Abstract— From the third day of pregnancy rats were fed a diet containing either 7% casein (experimental) or 24% casein (control). During lactation the control dams were fed the 24% casein diet and the experimental dams a 12% casein diet. From 25 to 50 days of age the experimental and control progeny were fed diets containing 7 and 24% casein, respectively. Between 50 and 120 days both groups were fed a diet containing 24% crude protein. Several indications of brain maturation in two brain areas were examined at various stages of development. In addition to retardation of brain growth, protein restriction led to myelin of an immature composition at 25 and 50 days of age. The immature composition was indicated by a low plasmalogen content at 25 days and by a high phospholipid and low galactolipid and plasmalogen contents at 50 days of age. The activity of the myelin marker enzyme, 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP), was significantly lower in the brains (excluding the cerebella) of malnourished rats at 21, 30 and 50 days. At all ages except at 50 days the activity of CNP in the cerebellum was higher in protein-deprived animals than in controls. The activity of glutamic acid decarboxylase (GAD) in the brains (excluding the cerebella) of protein-deprived rats was significantly lower at 21, 25 and 30 days but not at 50 and 65 days of age. As indicated by brain/body ratios, myelin composition and GAD activity, nutritional rehabilitation led to almost complete recovery of brain maturity, but the activity of CNP remained lower in the experimental group after rehabilitation.     

Thyrotrophin-releasing hormone inactivation by human postmortem brain

The mechanisms of inactivation of thyrotrophin-releasing hormone (TRH) by peptidases in several areas of normal human postmortem brain have been investigated by radioimmunoassay and high-performance liquid chromatography. Of the several brain regions studied, the cerebral cortex (Brodman's area, BA10) had the highest TRH-degrading activity in both subcellular fractions. Deamidated-TRH (TRH-OH) was the only product formed by the soluble fraction whereas the histidyl-proline diketopiperazine, cyclo(His-Pro), and a small amount of TRH-OH were formed by the particulate fraction. Several centrally acting TRH analogues showed varying degrees of resistance to degradation by the peptidases in the two fractions, the most stable analogue being RX77368 (pGlu-His-3,3'-dimethyl(ProNH2]. Areas of human postmortem brain appear to contain two of the enzymes capable of degrading TRH, a proline endopeptidase forming TRH-OH and a pyroglutamyl aminopeptidase forming cyclo(His-Pro). The use of the assay procedures in further studies on the inactivation of TRH by peptidases from brain areas of patients with neurological disorders may provide complementary information on the dynamics of TRH in these disorders. The stability of the centrally acting TRH analogues may prove useful in examining their therapeutic potential.     

[3H]cyclo(Histidyl-Proline) in rat tissues: Distribution,clearance and binding

Cyclo(Histidyl-Proline), a metabolite of TRH, has been demonstrated to have a number of biological activities. The clearance, distribution and binding of the peptide in the rat was studied. Cyclo(His-Pro) was cleared from the circulation biphasically ($\text{tl}\text{2}\text{= 1.25 and 33 min}$). Unmetabolized cyclo(His-Pro) appeared rapidly in urine. Accumulation of [³H]cyclo(His-Pro) in adrenal, liver and kidney was demonstrated. Membrane preparations from adrenal and liver, but not from kidney, brain, pituitary, and other tissues were shown to bind cyclo(His-Pro) specifically.     

Lack of effect of histidyl-proline diketopiperazine on basal level and TRH-induced response of cytosolic calcium concentration in rat lactotrophs.

Histidyl-proline diketopiperazine [cyclo(His-Pro)] has recently been shown to inhibit prolactin (PRL) secretion in vitro and in vivo. This peptide is well known as a metabolite of thyrotropin-releasing hormone (TRH), which is one of the endogenous secretagogues of PRL. In this study, we investigated the effect of cyclo (His-Pro) on the cytosolic Ca2+ concentration [[Ca2+]i) in cultured lactotrophs by using a lactotroph-enriched fraction separated from female rat pituicytes by centrifugal elutriation. TRH (10 nM) induced a rapid rise in [Ca2+]i in the lactotrophs, followed by a plateau phase of prolonged increase in [Ca2+]i. In contrast, the addition of 100 microM of cyclo (His-Pro) caused no changes in the basal level or the TRH-induced plateau response of [Ca2+]i. Although pretreatment with cyclo (His-Pro) tended to decrease the biphasic increase in [Ca2+]i induced by TRH, the inhibitory effect was not statistically significant. These results demonstrated that cyclo (His-Pro) has no effect on [Ca2+]i in lactotrophs, and does not affect the TRH-induced increase in [Ca2+]i, indicating that the inhibition of PRL secretion by cyclo (His-Pro) may be primarily mediated by other intracellular messengers such as cyclic nucleotides and secondarily involved in other inhibitory systems including that of dopamine.