Biomedical subjects
Publications and source records attributed to C Prasad.
The activity of pyroglutamate aminopeptidase, the major enzyme catalyzing thyrotropin-releasing hormone (TRH) metabolism in human CSF, decreased with age. This decrement is not due to age-dependent appearance of any enzyme inhibitor in CSF. The results of these studies underline the importance of using age-matched controls in assessing abnormalities of TRH metabolism in CSF during disease states.
The effects of dithiothreitol on pyroglutamate aminopeptidase activity in rat serum, spinal cord, brain, liver, kidney, and adrenal extracts were investigated in vitro. Addition of dithiothreitol to the reaction mixture resulted in noncompetitive inhibition of serum (Ki = 2.1 +/- 0.3 mM) and brain (Ki = 3.2 +/- 0.28 mM) enzymes and stimulation of liver, spinal cord, kidney and adrenal enzymes with an increase in Vmax but not Km. The findings of these studies suggest that the properties of Pyroglutamate aminopeptidases vary from tissue to tissue.
The distribution pattern of adrenocorticotropin-like immunoreactivity (ACTH-LI) in cats using the avidin-biotin modification of an immunocytochemical method shows cell bodies containing ACTH-LI in the medial basal hypothalamus, especially in the infundibular nucleus. The fibers from these neurons extended beyond the hypothalamus, into the paraventricular nucleus of the thalamus, rostral amygdala, periaqueductal gray, locus coeruleus, parabrachial nucleus and medial nucleus of the nucleus tractus solitarius. The distribution pattern of the cell bodies and fibers containing ACTH-LI bears several similarities to that seen in rats. The pattern differs from that of rats in the fact that the termination in the amygdala is more extensive and that ACTH-LI was not observed in cell bodies in any location other than the medial basal hypothalamus.
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.
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.
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.
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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.
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) [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).
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 30,000 g pellet fraction from the spinal cord saturably, reversibly, and with high affinity (apparent Kd = 24-25 nM). 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.
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.
The specific binding of thyrotropin-releasing hormone (TRH) by 30,000 g 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 affinity 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.
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.
Synthesis of phosphatidylcholine (PC) by S-adenosylmethionine-dependent methylation of phosphatidylethanolamine has previously been associated with receptor-mediated histamine and pituitary hormone secretion. We investigated stimulation of phospholipid methylation by TSH and its possible role in thyroid hormone secretion. Rat hemithyroids were incubated in Krebs-Henseleit-glucose-BSA buffer and the effect of various treatments on the incorporation of [3H-methyl]L-methionine into PC and T4/T3 secretion was studied. TSH treatment elevated thyroid phosphatidylethanolamine methyltransferase activity, the incorporation of [3H-methyl]methionine into PC, and T4/T3 secretion. The increase in PC synthesis was linear up to 6 h in a dose-dependent fashion (half-maximal stimulation at 2.5 micrograms TSH/ml). Stimulation required protein synthesis, because cycloheximide inhibited the increase in PC synthesis by 77%. Inhibitors of phospholipid methylation (100 microM adenosine + 10 microM L-homocysteine thiolactone + 10 microM erythro-9[2-hydroxy-3-nonyl]adenine) significantly decreased TSH-stimulation of phospholipid methylation but not T3/T4 secretion. In conclusion, stimulation of thyroid phospholipid methylation by TSH is not required for stimulated secretion of thyroid hormones.
To evaluate effects of protein-energy malnutrition on the brain neuropeptides thyrotropin-releasing hormone (TRH) and cyclo (His-Pro) in the cerebellum and the remainder brain, we studied neonatal rats in the lactational period. Protein-energy malnutrition caused an increase in cyclo (His-Pro) concentrations, but not in TRH concentrations, in the neonatal cerebellum. In the remainder brain, a reduction in cyclo (His-Pro) concentrations occurred with an increase in TRH concentrations. These data indicate that protein-energy malnutrition affects both brain TRH and cyclo (His-Pro) in the neonatal rats during lactation.
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.
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