The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
David E. Smith - One of the best experts on this subject based on the ideXlab platform.
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Enhanced antinociceptive response to intracerebroventricular Kyotorphin in Pept2 null mice.
Journal of neurochemistry, 2009Co-Authors: Huidi Jiang, Richard F. Keep, David E. SmithAbstract:l-Kyotorphin (l-KTP), an endogenous analgesic neuropeptide, is a substrate for aminopeptidases and a proton-coupled oligopeptide transporter, PEPT2. This study examined the CSF efflux, antinociceptive response, and hydrolysis kinetics in brain of l-KTP and its synthetic diastereomer d-Kyotorphin (d-KTP) in wild-type and Pept2 null mice. CSF clearance of l-KTP was slower in Pept2 null mice than in wild-type animals, and this difference was reflected in greater l-KTP-induced analgesia in Pept2 null mice. Moreover, dose-response analyses showed that the ED50 of l-KTP in Pept2-deficient animals was one-fifth of the value observed in Pept2-competent animals (4 vs. 21 nmol for null vs. wild-type mice, respectively). In contrast, the ED50 of d-KTP was very similar between the two genotypes (9–10 nmol). Likewise, there was little difference between genotypes in slope factor or baseline effects of l-KTP and d-KTP. The enhanced antinociceptive response to l-KTP in Pept2 null mice could not be explained by differences in neuropeptide degradation as Vmax and Km values did not differ between genotypes. Our results demonstrate that PEPT2 can significantly impact the analgesic response to an endogenous neuropeptide by altering CSF (and presumably brain interstitial fluid) concentrations and that it may influence the disposition and response to exogenous peptide/mimetic substrates.
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PEPT2-Mediated Uptake of Neuropeptides in Rat Choroid Plexus
Pharmaceutical Research, 2001Co-Authors: Nathan S. Teuscher, Richard F. Keep, David E. SmithAbstract:Purpose . The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuropeptide uptake into choroid plexus. Methods . Whole-tissue rat choroid plexus uptake studies were performed on GlySar in the absence and presence of neuropeptides and on carnosine. Results . The neuropeptides NAAG, CysGly, GlyGln, Kyotorphin, and carnosine inhibited the uptake of radiolabeled GlySar at 1.0 mM concentrations. In contrast, TRH, [D-Arg^2]-Kyotorphin, glutathione, and homocarnosine did not inhibit GlySar uptake. Kyotorphin, an analgesic, was a competitive inhibitor of GlySar with a Ki of 8.0 μM. The direct uptake of carnosine was also shown to be mediated by PEPT2 in isolated choroid plexus (Km = 39.3 μM; Vmax = 73.9 pmol/mg/min). Radiolabeled carnosine uptake was inhibited by 1.0 mM concentrations of GlySar or carnosine but not homocarnosine, L-histidine, or β-alanine. Conclusions . These findings indicate that PEPT2 mediates the uptake of a diverse group of neuropeptides in choroid plexus, and suggests a role for PEPT2 in the regulation of neuropeptides, peptide fragments, and peptidomimetics in cerebrospinal fluid.
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PEPT2-mediated uptake of neuropeptides in rat choroid plexus
2001Co-Authors: Nathan S. Teuscher, Richard F. Keep, David E. SmithAbstract:Purpose. The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuro-peptide uptake into choroid plexus. Methods. Whole-tissue rat choroid plexus uptake studies were per-formed on GlySar in the absence and presence of neuropeptides and on carnosine. Results. The neuropeptides NAAG, CysGly, GlyGln, Kyotorphin, and carnosine inhibited the uptake of radiolabeled GlySar at 1.0 mM concentrations. In contrast, TRH, [D-Arg2]-Kyotorphin, glutathione, and homocarnosine did not inhibit GlySar uptake. Kyotorphin, an analgesic, was a competitive inhibitor of GlySar with a Ki of 8.0 mM. The direct uptake of carnosine was also shown to be mediated by PEPT2 in isolated choroid plexus (Km 4 39.3 mM; Vmax 4 73.9 pmol/mg/min). Radiolabeled carnosine uptake was inhibited by 1.0 mM concentrations of GlySar or carnosine but not homocarnosine, L-histidine, or b-alanine. Conclusions. These findings indicate that PEPT2 mediates the up-take of a diverse group of neuropeptides in choroid plexus, and sug-gests a role for PEPT2 in the regulation of neuropeptides, peptide fragments, and peptidomimetics in cerebrospinal fluid. KEY WORDS: PEPT2; GlySar; carnosine; neuropeptides; choroid plexus
Hiroshi Takagi - One of the best experts on this subject based on the ideXlab platform.
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Effects of Kyotorphin (l-tyrosyl-l-arginine) on [3H]NG-nitro-l-arginine binding to neuronal nitric oxide synthase in rat brain
Neurochemistry international, 1997Co-Authors: Takashi Arima, Hiroshi Takagi, Yoshihisa Kitamura, Tadashi Nishiya, Takashi Taniguchi, Yasuyuki NomuraAbstract:Abstract l -Tyrosyl- l -arginine (Kyotorphin) is known as an endogenous analgesic neuropeptide. We examined whether Kyotorphin and other arginine-containing neuropeptides were endogenous substrates for neuronal nitric oxide synthase (NOS) in the rat brain. Cytosol fractions of the rat cerebellum contained higher concentrations of neuronal NOS (nNOS) than endothelial NOS. In rat cerebellar cytosol, the binding activity of [ 3 H]N G -nitro- l -arginine (NNA) was inhibited equally by l -arginine ( l -Arg), Kyotorphin, and l -leucyl- l -Arg (a Kyotorphin receptor antagonist). Binding activities were inhibited to lesser degrees by fibronectin active fragments, bradykinin, and dynorphin A, but were not inhibited by l -tyrosyl- d -Arg or substance P. Interestingly, the inhibition of [ 3 H]NNA binding by Kyotorphin was attenuated by inhibitors of Kyotorphin-hydrolyzing peptidases (KTPases) such as bestatin and arphamenine B. These results suggest that Kyotorphin is degraded to l -Arg by KTPases, which in turn may act as substrate for nNOS.
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Kyotorphin (L-tyrosyl-L-arginine) as a possible substrate for inducible nitric oxide synthase in rat glial cells.
Neuroscience letters, 1996Co-Authors: Takashi Arima, Hiroshi Takagi, Yoshihisa Kitamura, Tadashi Nishiya, Yasuyuki NomuraAbstract:Abstract l -Arginine ( l -Arg) is an endogenous substrate for nitric oxide synthase (NOS). In the present study, we examined whether l -tyrosyl- l -Arg (Kyotorphin), an endogenous analgesic neuropeptide, might be a substrate for inducible NOS (iNOS) in the brain. Both Kyotorphin and l -Arg caused an accumulation of nitrites in lipopolysaccharide (LPS)-treated glial cells cultured from infant rat brains. However, such accumulation of nitrites was not induced by NG-nitro-l-Arg ( a NOS inhibitor), l -tyrosyl- d -Arg ( de -Kyotorphin) or d -Arg. l -Leucyl- l -Arg (an antagonist for Kyotorphin receptors) or bestatin (an inhibitor for Kyotorphin-hydrolyzing peptidase) did not inhibit the Kyotorphin-induced accumulation of nitrites in LPS-treated cells. On the contrary, l -Leucyl- l -Arg caused an accumulation of nitrites in a concentration-dependent manner. The results indicate that nitric oxide (NO) is produced in LPS-treated glial cells directly from Kyotorphin through the catalytic action of iNOS.
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Central antinociceptive effect of l-ornithine, a metabolite of l-arginine, in rats and mice
European Journal of Pharmacology, 1996Co-Authors: Atsufumi Kawabata, Katsumi Iwatsubo, Shinji Takaya, Hiroshi TakagiAbstract:Abstract l -Arginine produces central antinociception by acting as a precursor of Kyotorphin ( l -tyrosyl- l -arginine), a [Met 5 ]enkephalin releaser. This study investigated the antinociceptive activity of l -ornithine, a metabolite of l -arginine. l -Ornithine given s.c. at 300–1000 mg/kg suppressed carrageenin-induced hyperalgesia in rats in a naloxone-reversible manner. l -Ornithine and l -arginine, when given i.c.v. at 10–100 μg/mouse, elicited antinociception even in intact mice, the effects being abolished by naloxone or naltrindole, and potentiated by bestatin, an inhibitor of aminopeptidase and Kyotorphinase. The antinociception induced by i.c.v. l -ornithine was also inhibited by i.c.v. l -leucyl- l -arginine, a Kyotorphin receptor antagonist, but was resistant to intracisternal anti-Kyotorphin serum. l -Tyrosyl- l -ornithine, a synthetic dipeptide, (1–10 μg/mouse, i.c.v.), exerted Kyotorphin-like antinociception in mice. These findings suggest that l -ornithine produces l -arginine-like antinociception via Kyotorphin receptors. However, this effect does not appear to be mediated by Kyotorphin itself, but most likely by l -tyrosyl- l -ornithine, a putative dipeptide.
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Kyotorphin SYNTHETASE ACTIVITY IN RAT ADRENAL GLANDS AND SPINAL CORD
Peptides, 1996Co-Authors: Atsufumi Kawabata, Hitomi Muguruma, Maki Tanaka, Hiroshi TakagiAbstract:Abstract Kyotorphin, an endogenous [Met 5 ]enkephalin-releasing antinociceptive dipeptide ( l -Tyr- l -Arg), is formed by Kyotorphin synthetase from its constituent amino acids, l -Tyr and l -Arg, in the brain in an ATP-Mg 2+ -dependent manner. To elucidate the physiological role of Kyotorphin in organs other than the brain, we examined the activity of Kyotorphin synthetase in the rat adrenal glands and spinal cord. By Sephacryl S-300 gel-filtration chromatography of the soluble extracts from both the organs, the enzyme activity forming immunoreactive Kyotorphin from l -Tyr and l -Arg in the presence of ATP and MgCl 2 was detected in the fractions with the molecular mass of 200–300 kDa, being drastically reduced by the omission of ATP and MgCl 2 from the reaction medium. The K m values of the partially purified adrenal and spinal Kyotorphin synthetase for l -Tyr, l -Arg, ATP, and MgCl 2 were close to those of the brain enzyme. The activity of adrenal Kyotorphin synthetase was inhibited by some l -Arg analogues, N G -nitro- l -arginine methyl ester, α-methyl- l -ornithine and d -Arg, but not by N G -nitro- l -arginine and N -iminoethyl- l -ornithine. In the crude soluble extracts from the adrenal glands and spinal cord, Kyotorphin was formed by Kyotorphin synthetase, and also by the enzymatic processing of the precursor proteins, in the presence of physiological concentrations of l -Tyr and l -Arg in addition to ATP and MgCl 2 . Thus, Kyotorphin synthetase resembling that in the brain is present in the rat adrenal glands and spinal cord. The present findings may predict a functional role of the l -Arg-Kyotorphin pathway in these organs.
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l-Arginine exerts a dual role in nociceptive processing in the brain: involvement of the Kyotorphin-Met-enkephalin pathway and NO-cyclic GMP pathway
British journal of pharmacology, 1993Co-Authors: Atsufumi Kawabata, Nahoko Umeda, Hiroshi TakagiAbstract:1. Intracerebroventricular (i.c.v.) administration of L-arginine (L-Arg), at 10-100 micrograms per mouse, produced antinociception in mice, as assessed by the tail flick test; this antinociception was reversed by pretreatment (s.c.) with naltrindole (NTI), a delta-selective opioid antagonist, and by co-administered L-leucyl-L-arginine (Leu-Arg), a Kyotorphin (endogenous Met-enkephalin releaser) receptor antagonist. 2. L-NG-nitroarginine methyl ester (L-NAME), a NO synthase inhibitor, but not D-NG-nitroarginine methyl ester, given i.c.v. at 3-10 micrograms per mouse, exhibited antinociceptive activity that was resistant to naloxone (s.c.), NTI (s.c.) and Leu-Arg (i.c.v.). 3. The L-NAME (i.c.v.)-induced antinociception was not reversed by L-Arg (i.c.v.), which was antinociceptive by itself, but was abolished by combined injection of L-Arg plus Leu-Arg (i.c.v.) or by L-Arg (i.c.v.) after NTI (s.c.). 4. Methylene blue (MB), a soluble guanylate cyclase inhibitor, at 0.1-1 microgram per mouse, produced antinociception by i.c.v. administration. The antinociception induced by MB (i.c.v.) or L-NAME (i.c.v.) was reversed by co-administered dibutyryl cyclic GMP. 5. These findings suggest that L-Arg plays a dual role in nociceptive processing in the brain, being antinociceptive via the Kyotorphin-Met-enkephalin pathway and nociceptive via the NO-cyclic GMP pathway.
Hiroshi Ueda - One of the best experts on this subject based on the ideXlab platform.
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Review of Kyotorphin Research: A Mysterious Opioid Analgesic Dipeptide and Its Molecular, Physiological, and Pharmacological Characteristics
Frontiers in Medical Technology, 2021Co-Authors: Hiroshi UedaAbstract:Tyrosine-arginine (Kyotorphin), an opioid analgesic dipeptide, was discovered more than 40 years ago in 1979. The evidence accumulated during this period has established the physiological significance of Kyotorphin as a neuromodulating peptide, and pharmacological applications. Some of the following important findings have been discussed in this review: (1) Kyotorphin is unevenly distributed in the brain; it is found in high concentrations in the pain pathway, which involves the regions associated with morphine analgesia; (2) Kyotorphin is subcellularly localized in the synaptosome fraction or nerve-ending particles; (3) a specific synthetase generates Kyotorphin from tyrosine and arginine; (4) Kyotorphin may be also processed from calpastatin by a novel calcium-activated neutral protease or calpain; (5) Kyotorphin preloaded into the synaptosome is released by high K+ depolarization in a Ca2+-dependent manner; (6) Kyotorphin has a specific G protein coupled receptor, which mediates the activation of phospholipase C (PLC) and inhibition of adenylyl cyclase through Gi; (7) leucine-arginine works as a specific Kyotorphin receptor antagonist; 8) membrane-bound aminopeptidase or excretion through a peptide transporter, PEPT2, may contribute to the inactivation of Kyotorphin; and (9) Kyotorphin causes increased Met-enkephalin release from brain and spinal slices. It is also known that the opening of plasma membrane Ca2+ channels through a conformational coupling of the InsP3 receptor with the transient receptor potential C1, which is downstream of the Kyotorphin receptor-mediated activation of Gi and PLC, could be a potential underlying mechanism of Met-enkephalin release. Considering these findings, translational research is an exciting domain that can be explored in the future. As Kyotorphin is a small molecule, we could design function-added Kyotorphin derivatives. These studies would include not only the brain-permeable Kyotorphin derivatives but also hybrid Kyotorphin derivatives conjugated with small compounds that have additional pharmacological actions. Further, since there are reports of Kyotorphin being involved in either the etiology or treatment of Alzheimer's disease, epilepsy, inflammation, and chronic pain, studies on the beneficial effects of Kyotorphin derivatives should also be expected in the future.
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Blockade of analgesic effects following systemic administration of N-methyl-Kyotorphin, NMYR and arginine in mice deficient of preproenkephalin or proopiomelanocortin gene.
Peptides, 2018Co-Authors: Hiroyuki Neyama, Yusuke Hamada, Ryoko Tsukahara, Minoru Narita, Kazuhiro Tsukamoto, Hiroshi UedaAbstract:Kyotorphin is a unique biologically active neuropeptide (l-tyrosine-l-arginine), which is reported to have opioid-like analgesic actions through a release of Met-enkephalin from the brain slices. N-methyl-l-tyrosine-l-arginine (NMYR), an enzymatically stable mimetic of Kyotorphin, successfully caused potent analgesic effects in thermal and mechanical nociception tests in mice when it was given through systemic routes. NMYR analgesia was abolished in μ-opioid receptor-deficient (MOP-KO) mice, and by intracerebroventricular (i.c.v.) injection of naloxone and of N-methyl l-leucine-l-arginine (NMLR), a Kyotorphin receptor antagonist. In the Ca2+-mobilization assay using CHO cells expressing Gαqi5 and hMOPr or hDOPr, however, the addition of Kyotorphin neither activated MOPr-mechanisms, nor affected the concentration-dependent activation of DAMGO- or Met-Enkephalin-induced MOPr activation, and Met-enkephalin-induced DOPr activation. NMYR-analgesia was significantly attenuated in preproenkephalin (PENK)- or proopioimelanocortin (POMC)-KO mice. The systemic administration of arginine, which is reported to elevate the level of endogenous Kyotorphin selectively in midbrain and medulla oblongata, pain-related brain regions, caused significant analgesia, and the analgesia was reversed by i.c.v. injection of NMLR or naloxone. In addition, PENK- and POMC-KO mice also attenuated the arginine-induced analgesia. All these findings suggest that NMYR and arginine activate brain Kyotorphin receptor in direct and indirect manner, respectively and both compounds indirectly cause the opioid-like analgesia through the action of endogenous opioid peptides.
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Kyotorphin has a novel action on rat cardiac muscle
Biochemical and biophysical research communications, 2005Co-Authors: Yumiko Saito, Hiroshi Ueda, Masahiko Suzuki, Makoto Endo, Kei MaruyamaAbstract:Several endogenous peptides for G-protein-coupled receptors have been found to play physiological roles in muscle contraction in addition to their well-demonstrated actions in other tissues. To further identify such peptides, we screened over 400 peptides using an isometric tension assay of rat papillary muscle. Here, we report that Kyotorphin, which is known as an analgesic dipeptide, has a cardiac effect. Although Kyotorphin had no effect on the twitch tension itself, it inhibited β-adrenergic agonist isoprenaline-induced increases in twitch tension in a dose-dependent manner. Leu-Arg, a selective antagonist of Kyotorphin, reversed this inhibitory effect. The inhibitory effect was also reversed by naloxone, an opioid receptor antagonist. These results suggest that Kyotorphin may release opioid peptides from rat cardiac muscle and have an indirect regulatory role in β-adrenergic action through cross-talk with opioid receptors.
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An enzymatically stable Kyotorphin analog induces pain in subattomol doses.
Peptides, 2000Co-Authors: Hiroshi Ueda, Makoto Inoue, Grazyna Weltrowska, Peter W. SchillerAbstract:Abstract Intraplantar injection of the enzymatically stable, N -methylated Kyotorphin analog Tyr(NMe)-Arg-OH produced marked and sharp nociceptive flexor responses in a dose-dependent manner. A significant response was observed with this compound at a dose of 0.01 amol (6000 molecules). Tyr(NMe)-Arg-OH-nociception was completely blocked by the Kyotorphin antagonist leucyl-arginine and its enzymatically stable, N -methylated analog, as well as by CP-99994, a specific neurokinin 1 antagonist. These findings suggest that the nociceptive effect produced by Tyr(NMe)-Arg-OH in subattomol doses occurs via specific interaction with the Kyotorphin receptor and that the extraordinary potency observed may result from amplification through local substance P release.
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In vivo signal transduction of nociceptive response by Kyotorphin (tyrosine-arginine) through Galpha(i)- and inositol trisphosphate-mediated Ca(2+) influx.
Molecular pharmacology, 2000Co-Authors: Hiroshi Ueda, Makoto InoueAbstract:Kyotorphin is a dipeptidic neuropeptide (tyrosine-arginine) that has specific receptor coupled to G(i) and phospholipase C and elicits Met-enkephalin release. Here, we attempted to demonstrate the in vivo evidence for the presynaptic mechanism by analyzing its nociceptive responses after peripheral application. Kyotorphin elicited potent nociceptive flexor responses at extremely low doses between 0.1 and 100 fmol after the intraplantar injection into the hind-limb of mice. The site of action of Kyotorphin-induced responses was identified to be on nociceptor endings, because the responses were markedly attenuated by intrathecal pretreatments with Galpha(i1) or Galpha(i2) antisense-oligodeoxynucleotides. Similar mechanisms were observed with histamine-induced nociceptive responses, except for the use of different antagonist and Galpha(q/11) antisense-oligodeoxynucleotide. Both responses were characterized to be mediated through inositol trisphosphate receptor-gated Ca(2+) influx, because they were blocked by xestospongin C, an allosteric antagonist for inositol trisphosphate receptor and EGTA, but not thapsigargin. Because the nociceptive responses by compound 48/80 through histamine-release from mast cells were completely abolished by thapsigargin, it is unlikely that the dose of thapsigargin is not sufficient to block both responses. All of these in vivo findings strongly support our previous view that Kyotorphin elicits Ca(2+) influx through inositol trisphosphate receptor located at presynaptic plasma membranes.
Richard F. Keep - One of the best experts on this subject based on the ideXlab platform.
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Enhanced antinociceptive response to intracerebroventricular Kyotorphin in Pept2 null mice.
Journal of neurochemistry, 2009Co-Authors: Huidi Jiang, Richard F. Keep, David E. SmithAbstract:l-Kyotorphin (l-KTP), an endogenous analgesic neuropeptide, is a substrate for aminopeptidases and a proton-coupled oligopeptide transporter, PEPT2. This study examined the CSF efflux, antinociceptive response, and hydrolysis kinetics in brain of l-KTP and its synthetic diastereomer d-Kyotorphin (d-KTP) in wild-type and Pept2 null mice. CSF clearance of l-KTP was slower in Pept2 null mice than in wild-type animals, and this difference was reflected in greater l-KTP-induced analgesia in Pept2 null mice. Moreover, dose-response analyses showed that the ED50 of l-KTP in Pept2-deficient animals was one-fifth of the value observed in Pept2-competent animals (4 vs. 21 nmol for null vs. wild-type mice, respectively). In contrast, the ED50 of d-KTP was very similar between the two genotypes (9–10 nmol). Likewise, there was little difference between genotypes in slope factor or baseline effects of l-KTP and d-KTP. The enhanced antinociceptive response to l-KTP in Pept2 null mice could not be explained by differences in neuropeptide degradation as Vmax and Km values did not differ between genotypes. Our results demonstrate that PEPT2 can significantly impact the analgesic response to an endogenous neuropeptide by altering CSF (and presumably brain interstitial fluid) concentrations and that it may influence the disposition and response to exogenous peptide/mimetic substrates.
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PEPT2-Mediated Uptake of Neuropeptides in Rat Choroid Plexus
Pharmaceutical Research, 2001Co-Authors: Nathan S. Teuscher, Richard F. Keep, David E. SmithAbstract:Purpose . The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuropeptide uptake into choroid plexus. Methods . Whole-tissue rat choroid plexus uptake studies were performed on GlySar in the absence and presence of neuropeptides and on carnosine. Results . The neuropeptides NAAG, CysGly, GlyGln, Kyotorphin, and carnosine inhibited the uptake of radiolabeled GlySar at 1.0 mM concentrations. In contrast, TRH, [D-Arg^2]-Kyotorphin, glutathione, and homocarnosine did not inhibit GlySar uptake. Kyotorphin, an analgesic, was a competitive inhibitor of GlySar with a Ki of 8.0 μM. The direct uptake of carnosine was also shown to be mediated by PEPT2 in isolated choroid plexus (Km = 39.3 μM; Vmax = 73.9 pmol/mg/min). Radiolabeled carnosine uptake was inhibited by 1.0 mM concentrations of GlySar or carnosine but not homocarnosine, L-histidine, or β-alanine. Conclusions . These findings indicate that PEPT2 mediates the uptake of a diverse group of neuropeptides in choroid plexus, and suggests a role for PEPT2 in the regulation of neuropeptides, peptide fragments, and peptidomimetics in cerebrospinal fluid.
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PEPT2-mediated uptake of neuropeptides in rat choroid plexus
2001Co-Authors: Nathan S. Teuscher, Richard F. Keep, David E. SmithAbstract:Purpose. The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuro-peptide uptake into choroid plexus. Methods. Whole-tissue rat choroid plexus uptake studies were per-formed on GlySar in the absence and presence of neuropeptides and on carnosine. Results. The neuropeptides NAAG, CysGly, GlyGln, Kyotorphin, and carnosine inhibited the uptake of radiolabeled GlySar at 1.0 mM concentrations. In contrast, TRH, [D-Arg2]-Kyotorphin, glutathione, and homocarnosine did not inhibit GlySar uptake. Kyotorphin, an analgesic, was a competitive inhibitor of GlySar with a Ki of 8.0 mM. The direct uptake of carnosine was also shown to be mediated by PEPT2 in isolated choroid plexus (Km 4 39.3 mM; Vmax 4 73.9 pmol/mg/min). Radiolabeled carnosine uptake was inhibited by 1.0 mM concentrations of GlySar or carnosine but not homocarnosine, L-histidine, or b-alanine. Conclusions. These findings indicate that PEPT2 mediates the up-take of a diverse group of neuropeptides in choroid plexus, and sug-gests a role for PEPT2 in the regulation of neuropeptides, peptide fragments, and peptidomimetics in cerebrospinal fluid. KEY WORDS: PEPT2; GlySar; carnosine; neuropeptides; choroid plexus
Hiroshi Tsuji - One of the best experts on this subject based on the ideXlab platform.
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Purification and Characterization of a Major Kyotorphin-Hydrolyzing Peptidase of Rat Brain
Journal of biochemistry, 1995Co-Authors: Kenji Akasaki, Hirohito Shiomi, Hiroko Yoshimoto, Akihiro Nakamura, Hiroshi TsujiAbstract:We purified a major Kyotorphin (L-Tyr-L-Arg)-hydrolyzing peptidase (KTPase) from the rat brain, to electrophoretic homogeneity using conventional chromatographic techniques. KTPase was purified 1,660-fold with a specific activity of 161 mumol/min/mg protein and 6.8% recovery. The purified enzyme was composed of a single polypeptide with a molecular mass of 67 kDa and an isoelectric point (pI) of 5.5. KTPase has the ability to hydrolyze a variety of natural dipeptides. It also liberated NH2-terminal tyrosine from Tyr-Gly-Gly and Tyr-Tyr-Leu. Bestatin and arphamenine B were potent inhibitors of this enzyme, while amastatin and puromycin had little effect. An excess of anti-KTPase antibody raised in a white rabbit precipitated approximately 80% of the Kyotorphin-hydrolyzing activity in the cytosol of rat brain. These data suggested that 67 kDa KTPase has a role in the degradation of Kyotorphin within neuronal cells of the rat brain.
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An enkephalin-degrading aminopeptidase from rat brain catalyzes the hydrolysis of a neuropeptide, Kyotorphin (L-Tyr-L-Arg).
Chemical & pharmaceutical bulletin, 1991Co-Authors: Kenji Akasaki, Hiroshi TsujiAbstract:We studied the hydrolysis of a neuropeptide Kyotorphin (L-Tyr-L-Arg) by an enkephalin-degrading aminopeptidase purified from cytosol of rat brain in vitro. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), gel filtration and isoelectric focusing. The aminopeptidase with an apparent molecular weight (Mr) = 98000 catalyzed the hydrolysis of Leu- and Met-enkephalins with Km values of 125 and 142 microM, respectively. The enzyme activity was inhibited by bestatin, amastatin and puromycin but not by pepstatin, leupeptin and phenylmethanesulfonyl fluoride (PMSF). Kyotorphin was degraded by the aminopeptidase at pH 7.0, and the Vmax and Km values were 9.2 mumol/min/mg protein and 95 microM, respectively. The Km value for Kyotorphin was compatible to those for Leu- and Met-enkephalins. Taken together, these results suggest a possible involvement of the enkephalin-degrading aminopeptidase in cytosolic degradation of Kyotorphin in neuronal cells of rat brain.
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Identification and characterization of two distinct Kyotorphin-hydrolyzing enzymes in rat brain.
Neuropeptides, 1991Co-Authors: Kenji Akasaki, A Nakamura, H Shiomi, Hiroshi TsujiAbstract:Abstract We identified and characterized two Kyotorphin-hydrolyzing peptidases (KTPases) in a soluble fraction of rat brain. When the soluble fraction was chromatographed with DEAE-Sephacel, the enzyme activity was resolved into two peaks, which were designated as KTPases I and II in their order of elution. KTPases I and II accounted for 95% and 5% of the KTPase activity in the soluble fraction, respectively. KTPases I and II hydrolyzed Kyotorphin with Km values of 22 μM and 110 μM, respectively. By gel filtration, Mr values of KTPases I and II were determined to be 55 000 and 98 000, respectively. Immunological analyses of KTPase II with an anti-enkephalin aminopeptidase antibody indicated that KTPase II was identical to an enkephalin aminopeptidase with Mr = 98 000. However, KTPase I was a novel peptidase responsible for the major Kyotorphin-degrading activity in the soluble fraction of rat brain.
