[International expert consensus on gene therapy for hereditary hearing loss: based on clinical trials].
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Journal Article↗
Biomedical subjects
X T Fan
Publications and source records attributed to X T Fan.
Effects of AP-V and bicuculline on somatostatin-positive neurons in hypothalamus of rats subjected to acute hypobaric hypoxia.
AIM: To investigate the effects of 2-amino-5-phosphonovalerate-pharmacology (AP-V) and bicuculline on somatostatin (SST)-positive neurons in hypothalamus of rats subjected to acute hypobaric hypoxia. METHODS: SST-immunoreactivity (IR) and somatostatin mRNA (SS mRNA)-positive neurons were measured by immunohistochemistry and in situ hybridization methods. RESULTS: Compared with control rats, SST-IR and SS mRNA-positive neurons in hypothalamic periventricular nucleus (PeV), paraventricular nucleus (PVN), and arcuate nucleus (ARC) increased after acute hypobaric hypoxia for 6 h (P < 0.01), and these effects were markedly inhibited by AP-V (10 microg, icv), a highly selective N-methyl-D-aspartate (NMDA) receptor antagonist, whereas were strongly enhanced by bicuculline (1.5 mg/kg, ip), a gamma-aminobutyric acid (GABAA) receptor antagonist. CONCLUSION: SST possibly participates in acute hypoxic reaction in hypothalamus, furthermore, glutamate and GABA can affect somatostatin release and synthesis in hypothalamus through NMDA and GABAA receptors respectively.
2-Amino-5-phosphonovalerate↗
[Excitatory amino acid enhance prepro-somatostatin mRNA expression induced by altitude hypoxia in the rat hypothalamus].
AIM AND METHODS: Contents of glutamate (Glu), asparate (Asp) and expression of prepro-somatostatin mRNA (PPS-mRNA) in rat hypothalamus were measured by using imitated altitude hypoxia rat model, amino acid analyzer and in situ hybridization technique. RESULTS: After rats were subjected to altitude hypoxia, contents of Glu and Asp in hypothalamus and PPS-mRNA expression in periventricular nucleus (PeVN), paraventricular nucleus (PaVN) and arcuate nucleus (ArcN) were increased significantly. Ketamine, a NMDA receptor antagonist, could decrease the number of PPS-mRNA neurons in rat hypothalamus evoked by altitude hypoxia, but had no effect on Glu and Asp contents evoled by altitude hypoxia. CONCLUSION: It is suggested that somatostatin maybe paticipate in altitude hypoxia reaction, Glu can enhance PPS-mRNA expression through NMDA receptor.
Altitude Sickness↗
[Ketamine and L-NAME inhibit NOS and somatostatin mRNA expression induced by altitude hypoxia in the rat hypothalamus].
Using altitude hypoxia model, in situ hybridization and NADPH-d histochemistry, we investigated the effects of ketamine and L-NAME (blocker of NOS) on NOS and somatostatin mRNA (SS mRNA) expression in the rat hypothalamus following acute altitude hypoxia. It was revealed that acute altitude hypoxia induced NOS and SS mRNA overexpression in the rat hypothalamus. When pretreated with NMDA receptor antagonist ketamine and L-NAME, NOS and SS mRNA expression were inhibited significantly. These results suggest that NMDA receptor activation participates in the expression of NOS and SS mRNA in the rat hypothalamus subjected to acute altitude hypoxia. Meanwhile, hypothalamic endogenous NO may mediate expression of SS mRNA.
Altitude Sickness↗
Effects of peptidase inhibitors on the enkephalin-induced anti-nociception in rats.
The intra-third-ventricular (i.t.v.) administration of [Met5]-enkephalin (enk) to rats pretreated i.t.v. with three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon, inhibited the tail-flick response. The enk-induced inhibition was augmented by increasing the doses of the three PIs, with the maximum inhibition being attained at the doses of 10 nmol each. The enk-induced inhibition in rats pretreated with any combination of two PIs, however, were markedly smaller than that in rats pretreated with all three PIs, indicating that three kinds of enzymes all played important roles in the inactivation of enk. The inhibitory effect of enk on the tail-flick response in rats pretreated with the three PIs at doses of 10 nmol each was approximately tenfold higher than that of morphine. The relative anti-nociceptive potencies of enk and morphine were similar to the relative inhibitory potencies obtained previously with the isolated guinea pig ileum pretreated with the three PIs, indicating that the hydrolysis of the i.t.v. administered enk was largely prevented by the three PIs. However, the magnitude of the enk-induced inhibition in rats pretreated s.c. with the three PIs indicated that the hydrolysis of enk injected i.t.v. was not largely prevented by the s.c. administration of three PIs at doses up to 10 micromol each/kg.
Animals↗
A method to estimate the potency of the mu-component of an opioid having mixed mu-, and kappa- and/or delta-agonist activities.
The SC administration of either typical mu-agonists such as morphine, pethidine, fentanyl and levorphanol or a mixed mu- and delta-agonist like [D-Ala2, D-Leu5]-enkephalin to 10-day-old rats produced loss of righting reflex. Additionally, the loss of righting reflex induced by these opioid agonists was antagonized by naloxone, an opioid antagonist having a preference for mu-receptors, but by neither nor-binaltorphimine nor naltrindole, a specific kappa- or delta-antagonist, respectively, indicating that the loss of righting reflex was produced by the interaction of an opioid with mu-receptors. Moreover, the potency of each opioid agonist relative to that of morphine estimated by the present in vivo method was similar to that determined by the traditional in vitro isolated preparation. In contrast to mu-agonists, neither typical kappa-agonists such as U-50, 488H, ketocyclazocine, pentazocine and butorphanol, nor a selective delta-agonist like [D-Pen2, D-Pen5]-enkephalin affected the righting reflex of 10-day-old rats, indicating that mu-agonists, but neither kappa- nor delta-agonists, produced the naloxone-reversible loss of righting reflex in infant rats. By employing the present in vivo method to estimate the mu-agonist activity of an opioid with mixed agonist activities, it was indicated that the mu-agonist activity of ethylketocyclazocine, which had been employed as a representative kappa-agonist, was essentially the same as that of morphine, a representative mu-agonist.
Animals↗
Synergy between phorbol esters and retinoic acid in inducing protein kinase C activation.
All-trans retinoic acid (RA) activates brain protein kinase C (PKC) in a unique fashion. Co-factors such as Ca2+ or PtdSer are not required for histone phosphorylation. Binding experiments have provided evidence that RA does not act as a phorbol-ester-like activator. However, phorbol esters synergistically enhance this activation in a dose-dependent manner and increase the reaction rate up to five-fold when combined with 10 microM RA. Phospholipid-interacting drugs such as phenothiazines and 1-N-(6 aminohexyl) 5-chloro-1-naphthalene-sulfonamide (W7), which compete with PtdSer and inhibit phorbol ester/PtdSer-mediated activation, have potentiating effects on the RA-mediated reaction. RA elicits Ca(2+)-dependent PKC autophosphorylation. The activation resulting from the combined treatment with PtdSer and RA is more than additive in the presence of Ca2+, indicating that PtdSer- and RA-binding sites are distinct. RA shares several characteristics of activation with sodium deoxycholate and arachidonic acid. These present results suggest that the direct activation of PKC may have physiological and/or pharmacological relevance in the signaling triggered by retinoids.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase.
Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activates PLD can be functionally coupled. However, assay of membrane preparations at increasing dilutions showed that stimulation of PLD by the compounds studied, with the partial exception of ATP[S], involved diffusible rather than protein-bound intermediates.
Animals↗
Characteristics of arachidonic-acid-mediated brain protein kinase C activation: evidence for concentration-dependent heterogeneity.
Arachidonic acid (AA) activates brain protein kinase C (PKC) in a specific manner, and which differs from that of diacylglycerol (DG)-mediated PKC activation in cofactor Ca2+ and phosphatidylserine (PtdSer) requirements. We presently report that characteristics of AA-mediated activation are heterogenous, and are dependent upon the concentrations of AA. Highly sensitive PKC activation (HS) occurring at concentrations of 20 microM AA can be distinguished from less sensitive PKC activation (LS) requiring concentrations of at least 160 microM AA, on the basis of the effects of phorbol ester TPA or DG, phosphatidylcholine (PtdCho) and sodium deoxycholate (DOC). TPA, like DG suppressed the HS reaction whereas it enhanced the LS reaction. PtdCho, a phospholipid which does not affect DG-mediated activation, also prevented the HS reaction without affecting the LS reaction. This latter was inhibited at 100 microM DOC, a concentration which slightly stimulated the HS reaction. The substrate specificity was also different in the two reactions: the preferential substrate for PKC in HS was histone type VII-S, while it was histone type V-S in LS. Both reactions were similarly affected by PtdSer. In 0.1 mM CaCl2, PtdSer stimulated AA-mediated activation without evoking additive responses while this phospholipid prevented this activation in 0.5 mM EGTA, suggesting that AA and PtdSer bind PKC on the same or related sites. Together these results provide evidence for the existence of different modes of AA-mediated PKC activation with unique characteristics which presumably involve two different binding sites for AA on the same molecule and/or different PKC isoforms.
Animals↗
Bile acids, non-phorbol-ester-type tumor promoters, stimulate the phosphorylation of protein kinase C substrates in human platelets and colon cell line HT29.
Protein kinase C (PKC) is the target for a number of tumor promoters. The mechanism underlying the promoting effects of bile acids in colorectal cancer is not understood. We report that sodium deoxycholate (DOC) triggered activation of PKC in physiological conditions. The biphasic effects of DOC upon PKC activation were Ca(2+)-stimulated and did not require phosphatidylserine (PtdSer) as phospholipid co-factor. The optimal rate of activation was obtained at 0.4 mM DOC and reached approximately half the maximal rate of activation obtained in the presence of PtdSer. Similarly to PtdSer, DOC supported diacylglycerol- as well as phorbol-ester-mediated PKC activation. The reciprocal effects of PtdSer and DOC upon PKC in either 0.5 mM CaCl2 or 0.5 mM EGTA suggest that DOC interacts with the phospholipid-binding domain to elicit PKC activation. DOC-supported enzyme activation exhibited substrate specificity different from that of PtdSer-supported enzyme activation. All tested primary and secondary bile acids activated PKC to various extents, with DOC being the most potent. We suggest that amphipathic bile acids acting in a PtdSer-like manner provide the hydrophobic environment required for PKC activation. Treatment of 32P-labeled platelets and colonic cells HT29 Cl.19A with DOC enhanced the phosphorylation of endogenous substrates for PKC. Colonic cells responsive at 50 microM DOC, appeared to be 10-fold more sensitive than platelets. We suggest that direct or indirect activation of PKC by bile acids may account for the promoting effects of these non-phorbol-ester-type tumor promoters.
Bile Acids and Salts↗
Does protein kinase C require Ca2+ for activation?
Ca2+ requirement for protein kinase C activation is a matter of controversy. In this report we have examined Ca2+ dependency of the reaction in different assay systems and shown that the enzyme response to Ca2+, as well as diacylglycerol, depends upon phospholipid species, protein substrate and lipid conformation (micelles or sonicates). These results emphasize that the enzyme characteristics as defined in reconstituted membrane systems may not have a physiological relevance.
Calcium↗
Arachidonic acid and related methyl ester mediate protein kinase C activation in intact platelets through the arachidonate metabolism pathways.
Unlike unsaturated fatty acids, which almost fully activated purified brain protein kinase C in a phosphatidylserine- and Ca2(+)-free reaction, related methyl esters were poorly active in vitro. In contrast, methyl arachidonate was revealed to be as potent as arachidonic acid in activating protein kinase C in intact platelets. Arachidonic acid-mediated activation peaked at 20 s while methyl arachidonate-mediated activation plateaued at 2 min when both lipids were added at 50 microM. At concentrations higher than 0.3 mM, all tested unsaturated fatty acids and related methyl esters were weak activators of the enzyme, with the exception of linolenic acid and methyl linolenate which evoked strong enzyme activation. However, inhibitors of arachidonate metabolism blocked both arachidonic-acid and methyl-arachidonate-induced responses. At 5 microM arachidonic acid and methyl arachidonate, protein kinase C activation was due to a cyclooxygenase product(s) whereas at 50 microM the lipoxygenase pathway was mostly involved in the reaction. Therefore, arachidonic acid and its methyl ester activate protein kinase C in platelets mainly through action of their metabolites and eicosanoid synthesis. It is suggested that such indirect protein kinase C activation may account for the tumor-promoting activity of unsaturated fatty acids and related methyl esters.
5,8,11,14-Eicosatetraynoic Acid↗
Phorbol esters mediate phospholipid-free activation of rat brain protein kinase C.
The present study provides evidence that rat brain protein kinase C elicits a phosphotransferase activity towards histone and undergoes autophosphorylation in the absence of phosphatidylserine. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate binds to and activates protein kinase C in a phospholipid-free reaction. The apparent activation constant (Ka = 2.7 nM) is not modified by the absence of phospholipid but the maximum velocity is greatly decreased. The phosphotransfer reaction to exogenous substrates occurs in 0.5 mM ethylene-bis(oxyethylenenitrilo)tetraacetic acid, although autophosphorylation in these conditions requires the presence of Ca2+. The protein kinase C inhibitor (1-(5-isoquinolinesulfonyl)-2-methylpiperazine inhibits the reaction, whereas the cAMP-dependent protein kinase inhibitor is ineffective. In contrast to diacylglycerol, which is a poor activator, unsaturated fatty acids potently activate the phospholipid-free reaction. Moreover, the substrate specificity is markedly changed, e.g., myelin basic protein and histone types VI-S and VII-S appear to be relatively better substrates in the phospholipid-free reaction. The data presented indicate that protein kinase C (or some individual isoforms) may function, at least partially, without binding to membrane phospholipid and suggest that this novel characteristic of phorbol esters may account for their tumor-promoting activity.
Animals↗
Benzene-mediated protein kinase C activation.
Extracellular ligands transfer information into the cell through several pathways that operate in an integrated fashion. Protein kinase C, and enzyme that plays a pivotal role in signal transduction, is the molecular target for tumor promoters from the series of phorbol esters. A number of structurally unrelated tumor promoters also enhance protein kinase C, interacting or not interacting with the phorbol ester binding site. Evidence is provided that benzene potently activate protein kinase C in vitro, as well as in intact platelets. The drug does not compete for the phorbol ester binding site and probably affects the hydrophobic environment requires for full enzyme activation. Toluene is equally active. The relevance of the presented findings in the carcinogenic effects of benzene is discussed.
Animals↗