Degradation Of Thyrotropin Releasing Hormone Research Articles

Ontogeny of prolyl endopeptidase and pyroglutamyl peptidase I in rat tissues

Prolyl endopeptidase and pyroglutamyl peptidase I are enzymes which participate in the degradation of thyrotropin-releasing hormone (TRH), a hormone which is thought to play an important role in the development of organs and tissues. Here, we have characterized the ontogeny of TRH degrading enzyme activity in the brain cortex, lung, heart, kidney and liver. Overall, prolyl endopeptidase activity was found to be 2 to 5 fold higher in newborn vs. adult rat tissues, with the exception of the soluble form in the liver and the particulate form in the lung. In contrast, the developmental profile of pyroglutamyl peptidase I activity was found to be more variable and tissue dependent. These results corroborate the idea that both enzymes play important, tissue-specific roles during the development and maturation of rat organs.

Marginal involvement of pyroglutamyl aminopeptidase I in metabolism of thyrotropin-releasing hormone in rat brain.

On thyrotropin-releasing hormone (TRH) metabolism, pyroglutamyl aminopeptidase II (PAP-II), a zinc-dependent ectoenzyme primarily located in the central nervous system, is believed to play a predominant role. Recently we cloned pyroglutamyl aminopeptidase I (PAP-I) which is known for specifically removing a L-pyroglutamate (L-pGlu) residue from the amino terminus of proteins and peptides including TRH. To investigate possible contribution of PAP-I toward TRH metabolism, we conducted biochemical and immunohistochemical characterization using recombinant rat, mouse and human PAP-Is and an antibody raised against rat PAP-I. The Km values toward TRH by the recombinant PAP-Is were about 0.05 mM, being similar value to the reported value of recombinant PAP-II. The L-pGlu-cleaving activities toward TRH in rat brain homogenate were inhibited by a PAP-II specific inhibitor 1,10-phenanthroline, but not inhibited by the antibody against rat PAP-I. Immunohistochemical study in rats revealed heterogeneous distribution of PAP-I in the pituitary, the target tissue of TRH, but the distribution was cytosolic. Taken together, these results suggested that PAP-I might not be dominantly involved in the degradation of TRH in rats. Additionally, we found that PAP-I was localized in the renal proximal tubules. Further investigations are needed for elucidating the function of PAP-I in these restricted sites.

A new biological contribution of cyclo(His-Pro) to the peripheral inhibition of pancreatic secretion.

The tripeptide pyro-Glu-His-Pro-NH2[thyrotropin-releasing hormone (TRH)] was isolated from the hypothalamus as a thyrotropin-releasing factor. It has a broad spectrum of central nervous system-mediated actions, including the stimulation of exocrine pancreatic secretion. TRH is also synthesized in the endocrine pancreas and found in the systemic circulation. Enzymatic degradation of TRH in vivo produces other bioactive peptides such as cyclo(His-Pro). Because of the short half-life of TRH and the stability of cyclo(His-Pro) in vivo, we postulated that at least part of the peripheral TRH effects on the exocrine pancreatic secretion may be attributed to cyclo(His-Pro), which has been shown to have other biological activities. This study determines in parallel the peripheral effects of TRH and cyclo(His-Pro) as well as the putative contribution of other TRH-related peptides on exocrine pancreatic secretion in rats. TRH and its metabolite cyclo(His-Pro) dose dependently inhibited 2-deoxy-D-glucose (2-DG)-stimulated pancreatic secretion. TRH and all the related peptides tested had no effect on the basal and cholecystokinin-stimulated amylase release from pancreatic acinar cells in vitro. These data indicate that cyclo(His-Pro) mimics the peripheral inhibitory effect of TRH on 2-DG-stimulated exocrine pancreatic secretion. This effect is not detected on isolated pancreatic acini. Our findings provide a new biological contribution for cyclo(His-Pro) with potential experimental and clinical applications.

Stability of peptides during iontophoretic transdermal delivery

Degradation of thyrotropin releasing hormone (TRH) and insulin was investigated during iontophoretic transdermal delivery in vitro in varied buffer solutions and under various experimental conditions. A large current density enhanced deterioration of TRH during transdermal iontophoresis. Skin or external electrical potential increased the rate of deterioration, as did elevated temperatures. Peptide transformations took place and were significantly influenced by the physiological and pathological status of the skin and the electrochemical conditions during iontophoresis. Skin metabolism and electrical deterioration during iontophoretic transdermal transport was significant, especially at elevated temperature. Decreased temperature or added agents decreased the rate of degradation.

Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on neuropeptide metabolism in the rat brain

The effect of a novel prolyl endopeptidase (PEP) inhibitor, (S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl] carbonyl]-N-(phenylmethyl)-1-pyrrolidine-carboxamide (JTP-4819), on neuropeptide metabolism was investigated in the rat brain. JTP-4819 exhibited a strong in vitro inhibitory effect on cortical and hippocampal PEP activity, with the IC50 values being approximately 0.58 +/- 0.02 and 0.61 +/- 0.06 nM, respectively. JTP-4819 also inhibited the in vitro degradation of substance P (SP), arginine-vasopressin (AVP), and thyrotropin-releasing hormone (TRH) by rat brain supernatants, with the IC50 values being respectively 3.4, 2.1, and 1.4 nM in the cerebral cortex and 3.3, 2.8, and 1.9 nM in the hippocampus. Oral administration of JTP-4819 at doses of 1 and 3 mg/kg increased SP-like immunoreactivity (LI) and AVP-LI in the cerebral cortex. JTP-4819 also increased hippocampal SP-LI and AVP-LI at doses of 1 and 3 mg/kg, as well as hippocampal TRH-LI at a dose of 3 mg/kg. These findings suggest that JTP-4819 inhibited the degradation of SP, AVP, and TRH in the rat brain secondary to the inhibition of PEP, and thus increased cortical and hippocampal SP-LI and AVP-LI as well as hippocampal TRH-LI.

Serotonergic regulation of the spinal cord content of thyrotropin releasing hormone in the cerebellar ataxia mutant mouse

The effects of serotonin (5-HT) and various serotonin receptor antagonists on the spinal cord thyrotropin releasing hormone (TRH) content were studied in the rolling mouse Nagoya (RMN) and in the unaffected C3H mouse. TRH was extracted from the cervical, thoracic, lumbar, and sacral cord, at 1 h after the intraperitoneal injection of a serotonin precursor, 2 serotonin agonists, and 5 serotonin receptor antagonists. Administration of 5-hydroxytryptophan and 2-methyl-5-hydroxytryptamine resulted in an increase of the spinal cord TRH content in C3H mice, but not in RMN. Parachlorophenylalanine decreased the spinal cord TRH content in C3H mice, while it increased TRH levels in all regions of the RMN spinal cord. The TRH contents were decreased in all regions of the spinal cord after 5,7-dihydroxytryptamine administration in both C3H mice and RMN. In C3H mice, methysergide, mianserin, ketanserin, and spiperone significantly decreased the TRH content in all regions of the spinal cord, while 3α-tropanyl-1H-indole-3-carboxylic acid ester (ICS205–930) did not affect it. These antagonists paradoxically increased TRH levels in the cervical cord in RMN. The degradation of synthetic TRH by cord homogenates and the number and affinity of spinal cord serotonergic receptors (5-HT 1 and 5-HT 2) showed no significant difference between C3H mice and RMN. These results suggest that TRH turnover is abnormally regulated by serotonergic neurons in the RMN and that the dysfunction of the serotonergic nerves is attributable to the serotonin autoreceptor.

Evaluation of some substrates and potential inhibitors of tissue pyroglutamyl aminopeptidase I

A fluorometric enzyme assay was developed to evaluate the ability of a variety of compounds to bind to and/or inhibit pyroglutamyl aminopeptidase I. Among these compounds were a series of chloromethyl ketone analogues of thyrotropin releasing hormone (TRH) which had previously been shown to possess TRH-like activity in the central nervous system and have now been found to be good inhibitors of pyroglutamyl aminopeptidase. Thus, it is suggested that the observed TRH-like CNS activity could derive indirectly from inhibition of endogenous TRH degradation by pyroglutamyl aminopeptidase I.

Thyrotropin-Releasing Hormone-Degrading Enzyme in Human Serum Is Classified as Type II of Pyroglutamyl Aminopeptidase: Influence of Thyroid Status

The characteristics of thyrotropin-releasing hormone (TRH)-degrading enzyme in human serum were studied. Serum was incubated in 0.1 M phosphate buffer containing [proline-3H]TRH at 37 degrees C. A thin layer chromatography analysis of TRH degradation did not show any radioactive peak located in an acid TRH position, but apparent radioactive peaks corresponding to His-Pro and His-ProNH2 occurred in the presence of p-hydroxymercuriphenyl sulfonic acid, an inhibitor of proline dipeptidase. With ion exchange paper chromatography, the formation of 3H-labeled His-Pro and His-ProNH2 was estimated as an end point in the measurement of pyroglutamyl aminopeptidase (pGlu-peptidase) activity. An assay using p-hydroxymercuriphenyl sulfonic acid was developed to sensitively quantitate the pGlu-peptidase. Neither bacitracin nor p-chloromercuribenzoic acid increased the activity of pGlu-peptidase. The addition of EDTA, dithiothreitol, and o-phenanthroline significantly inhibited pGlu-peptidase activity, but neither iodoacetamide nor ethylmaleimide altered its activity. The pGlu-peptidase had a stereotypic specificity for the tripeptide, pGlu-His-ProNH2 of TRH, and its Km was 44.9 microM. The pGlu-peptidase activity was not changed by either hyper- or hypothyroidism. The present data indicate that a TRH-degrading enzyme in human serum possesses a nature identical to type II of pGlu-peptidase which is not altered by thyroid status.

Kinetics and pattern of degradation of thyrotropin-releasing hormone (TRH) in human plasma.

The kinetics and mechanism of degradation of the tripeptide TRH (pGlu-His-Pro-NH2) and its various primary and secondary degradation products (TRH-OH, His-Pro-NH2, and His-Pro) have been determined in human plasma at 37 degrees C. The rates of degradation of both TRH and TRH-OH (pGlu-His-Pro) showed mixed zero-order and first-order kinetics. At low substrate concentrations first-order kinetics occurred, with TRH and TRH-OH degradation half-lives of 9.4 and 27 min, respectively. The initial step in the plasma-catalyzed degradation of TRH is due to hydrolysis of the pGlu-His bond by the TRH-specific pyroglutamyl aminopeptidase serum enzyme, resulting in the exclusive formation of histidyl-proline amide (His-Pro-NH2). Using specific HPLC methods the major degradation route (67%) of this dipeptide in human plasma was hydrolysis of the peptide bond to yield His and Pro-NH2, whereas deamidation to yield His-Pro accounted for 29% of the total degradation. A minor pathway (less than or equal to 4%) was spontaneous cyclization to yield cyclo(His-Pro). Both His-Pro-NH2 and His-Pro degraded by first-order kinetics and faster than TRH, with half-lives of 5.3 and 2.2 min, respectively, in 80% plasma.

Neuropeptide degradation by large vessel and microvessel-derived endothelial cells in vitro: cell surface catabolism of thyrotropin releasing hormone (TRH)

Cell surface ectopeptidase activity of purified, cultured large vessel and microvessel-derived endothelial cells (EC) was studied. Degradation of thyrotropin releasing hormone (TRH), and production of cyclo-His-Pro was significantly increased ( P < 0.001) in large vessel EC compared with microcapillary EC. Since the rate of catabolism in the microvascular capillary bed is 5 times less than that in the large vessel wall, peptide concentrations are likely maintained longer in close proximity to their site of biosynthesis, where they are presumably most active.

Pyroglutamyl peptidase II inhibition specifically increases recovery of TRH released from rat brain slices

Pyroglutamyl peptidase II (EC 3.4.19-) is a highly specific membrane-bound thyrotropin releasing hormone (TRH) degrading enzyme. To study the functional significance of pyroglutamyl peptidase II in TRH degradation, we synthesized the reversible inhibitor N-1-carboxy-2-phenylethyl (N imbenzyl)-histidyl-β-naphthylamide (CPHNA). CPHNA inhibited the enzyme with a Ki of 8 μM, but had no effect no TRH receptors or no prolyl endopeptidase (EC 3.4.21.26). It weakly inhibited cytosolic pyroglutamyl peptidase I (EC 3.4.19.3). CPHNA at a concentration of 10 −4 M increased both the basal and potassium stimulated recovery of TRH released from hypothalamic slices by approximately two-fold. An even higher recovery was observed in slices from brain regions with relatively high levels of pyroglutamyl peptidase II. CPHNA had no effect on the basal recovery of γ-aminobutyric acid or Met-enkephalin released from brain slices but decreased the potassium stimulated recovery of both Met-enkephalin and γ-aminobutyric acid. These data further support the involvement of pyroglutamyl peptidase II in the extracellular inactivation of brain TRH.

Tissue-specific regulation of pyroglutamate aminopeptidase II activity by thyroid hormones.

Among the enzymes capable of degrading thyrotropin-releasing hormone (TRH) in vitro, two pyroglutamate aminopeptidases (PGA) are specific for TRH: thyroliberinase, a seric enzyme and PGAII, a membrane-bound peptidase. The effect of thyroid hormone status on the activity of these enzymes was evaluated in serum and various tissues. Only in adenohypophysis, triiodothyronine treatment increased PGAII to 376% of control; hypothyroidism produced the reverse effect (decrease to 23% of control). As previously reported, similar changes were observed for thyroliberinase. TRH degradation at the adenohypophysis level may participate in the negative feedback control of thyroid hormones.

Adenohypophyseal degradation of thyrotropin releasing hormone regulated by thyroid hormones

Thyrotropin Releasing Hormone (TRH; pyroGlu-His-Pro-NH2) is important in the regulation of adenohypophyseal hormone secretion and also serves neurotropic functions in extra-hypothalamic brain areas, indicating that it is involved in neurotransmission and other forms of cellular communication. This hypothesis is strengthened by the observation that TRH is hydrolysed at the pyroGlu-His bond by a particulate enzyme located in the synaptosomal and adenohypophyseal plasma membrane. Furthermore, this enzyme has been identified as a heterogeneously distributed ectoenzyme which has a high degree of substrate specificity like the TRH-degrading serum enzyme studied previously. In the rat, the activity of the TRH-degrading serum enzyme has been shown to be influenced by the thyroid status of the animals; here I report that the activity of the membrane-bound TRH-degrading enzyme of the anterior pituitary is stringently controlled by thyroid hormones, but that the activity of the brain enzyme is not.

Thyrotropin-releasing hormone (TRH) degradation in NIH:N mouse cerebral cortex and spinal cord

Thyrotropin-releasing hormone (TRH) metabolism in NIH:N mouse cerebral cortex and spinal cord was studied with high pressure liquid chromatography. Degradation of added TRH was significantly ( P<0.001) slower in the spinal cord (2.19 ± 0.46%/min) than in the cerebral cortex (7.54 ± 0.31%/min). Bacitracin, a non-competitive inhibitor of TRH deamidase, inhibited TRH degradation by 61.3 ± 3.7% in the spinal cord but only by 35.9 ± 1.0% in the cerebral cortex ( P<0.001). Catabolism of TRH in the mouse spinal cord proceeds at a slower rate and predominantly via the TRH deamidase pathway.

Delineation of a Particulate Thyrotropin‐Releasing Hormone‐Degrading Enzyme in Rat Brain by the Use of Specific Inhibitors of Prolyl Endopeptidase and Pyroglutamyl Peptide Hydrolase

The degradation of thyrotropin-releasing hormone in rat brain homogenates was studied in the presence of N-benzyloxycarbonyl-prolyl-prolinal and pyroglutamyl diazomethyl ketone, specific and potent active-site-directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin-releasing hormone-degrading enzyme(s). Reports of a thyrotropin-releasing hormone-degrading enzyme with narrow specificity that cleaves the pGlu-His bond of this tripeptide led us to develop a coupled assay using pGlu-His-Pro-2NA as the substrate to measure this activity. Cleavage of the pGlu-His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl-peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o-phenanthroline. The particulate pyroglutamyl-peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin-releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu-His bond of thyrotropin-releasing hormone but is distinct from pyroglutamyl peptide hydrolase.

The effect of inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase on TRH degradation in rat serum

The identity of the enzymes catalyzing the degradation of thyrotropin releasing hormone (TRH) in rat serum was investigated by the use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase. These inhibitors did not protect TRH from degradation, but o-phenanthroline afforded significant protection. The participation of “thyroliberinase”, a metalloenzyme which cleaves TRH at the pyroglutamyl-His bond was implied. A coupled assay using the chromogenic substrate pyroglutamyl-His-Pro-2-naphthylamide and excess diaminopeptidase IV was developed to specifically quantitate “thyroliberinase” activity. Rat serum catalyzed the degradation of 67.5 nmoles substrate/ml serum /h. The data indicate that TRH is degraded in rat serum predominantly by “thyroliberinase” and that prolyl endopeptidase and pyroglutamyl peptide hydrolase do not contribute significantly to this process.

Seasonal variation in the accumulation and degradation of thyrotropin-releasing hormone by frog skin in vitro

The possible seasonal variation of the TRH content in the abdominal skin and blood of frogs ( Rana pipiens) was determined. TRH was quantitated by radioimmunoassay after acidic extraction and column chromatography. It was found that there was considerable variability in the TRH content of the skin and blood among individual frogs and there was no seasonal variation in the TRH content in either tissue. Abdominal skins were capable of TRH accumulation and degradation in vitro. Skins from fall frogs accumulated significantly more exogenously added TRH than skin from frogs collected during the remaining months. The majority of the TRH degrading activity could be accounted for by the activity of TRH deamidase. The deamidated products were released to the incubation medium. The rate of TRH degradation was slightly higher in skins from fall frogs than in those from frogs which had been collected during the other seasons.

TRH degradation rates vary widely between different animal species

A study of the degradation of thyrotropin releasing hormone (TRH) by tissues of a variety of animal species and man has been conducted. Rates of degradation of TRH varied widely between different species and also between different tissues. Except for brain, tissues of dogs appeared devoid of TRH degrading activity. Rates of hydrolysis of TRH by rat plasma were dependent on the animal strain. Constituent amino acids were the major TRH metabolites produced by plasma whereas deamidated TRH was a major product from liver tissues. The cyclic metabolite histidylproline diketopiperazine was not a prominent metabolite in these studies.

Enzymes involved in the degradation of thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH) in bovine brain

As part of an investigation of neuropeptide inactivation mechanisms, we have resolved an enzymatic activity in bovine brain which catalyzes the deamidation of thyrotropin releasing hormone (TRH) and the hydrolysis of the Pro9--Gly10--NH2 bond of luteinizing hormone releasing hormone (LH-RH) from a second LH-RH degrading activity which does not degrade TRH. The former activity is similar, if not identical to, the post-proline cleaving enzyme in kidney as it is active toward the post-proline cleaving enzyme substrate CbzGly--Pro--Leu--Gly and inhibited by CbzPro--Phe and diisopropylfluorophosphate. In addition, products derived from the degradation of TRH and LH-RH by this activity show a specific cleavage on the carboxyl side of a proline residue. The latter activity has not yet been characterized with respect to its site of cleavage of the LH-RH molecule due to the presence of other contaminating peptidases.

Subcellular distribution of brain peptidases degrading luteinizing hormone releasing hormone (LHRH) and thyrotropin releasing hormone (TRH)

The luteinizing hormone and thyrotropin-releasing hormones have been shown to be mostly concentrated in the nerve endings of the median eminence. In contrast, the peptidases reponsible for their degradation present an ubiquitous localization and most of the reports have dealt with the total soluble activity. A detailed study of the subcellular distribution of these enzymes was thus performed in cerebral cortical and hypothalamic preparations of rat brain. The activity of a soluble marker, lactic dehydrogenase, was also measured to control for possible contaminants. The results showed that only 10% peptidase activity was present in the nerve ending preparation. Evidence is provided for a non-negligible membrane-bound enzymatic component responsible for TRH degradation at the synaptosomal level of both cortex and hypothalamus.