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J C Shih

Publications and source records attributed to J C Shih.

At least 145 records · Page 8Linked to original sources
PubMed1987

Evidence for two conformational states of 5-hydroxytryptamine carrier in rat cortical synaptosomes.

The effects of SABA ((3-beta-(4-azidobenzamidino)ethyl)-5-hydroxyindole), and fluoxetine, on the uptake of 5-hydroxytryptamine (5-HT; serotonin) by cerebral cortical synaptosomes of the rat were studied under normal (147 mM) and small (29 mM) concentrations of sodium. In the dark, both compounds competitively inhibited the uptake of 5-HT. However, in the presence of a small concentration of sodium (29 mM), the Ki(nM) for SABA decreased from 128 +/- 7 (n = 3) to 93 +/- 7 (n = 3) (P less than 0.05), whereas the Ki for fluoxetine increased from 30 +/- 2 (n = 3) to 59 +/- 6 (n = 3) (P less than 0.05). The maximum irreversible photoinactivation of the uptake of 5-HT by 1 microM SABA was increased by about 20% when the concentration of sodium ions [( Na+]) was decreased. This is consistent with the Ki data indicating an increase in the affinity of SABA for the uptake site for 5-HT when [Na+] was decreased. The results suggest that there may be two conformational states of the carrier for 5-HT which can be interconverted by changing the concentration of Na+. The affinity of SABA and fluoxetine for these two conformational states is different.

Animals↗
PubMed1987

Photoaffinity labeling of adenylate cyclase-linked serotonin receptors in Aplysia neurons.

Serotonin stimulated adenylate cyclase in Aplysia neurons with a Kact of 0.7 microM. Under the same conditions, 1-[2-(4-aminophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine stimulated adenylate cyclase with a Kact of 20 microM. The azido derivative of this compound, 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine, or of serotonin, (4-amino, 3-nitrophenylazido-serotonin), also stimulated the cyclase in the dark, but with lower efficiency (Kact greater than 10(-4) M). Irradiation of the membranes in the presence of 100 microM 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine abolished 75% of the cyclase activity stimulated by 5 microM serotonin. Under the same conditions, 100 microM 4-amino, 3-nitrophenylazido-serotonin did not inhibit serotonin-stimulated adenylate cyclase activity. When [3H]1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine (20 microM) was irradiated with membranes for 5 min at 4 degrees C, a dozen peptides were labeled, as revealed by a fluorogram of sodium dodecyl sulfate-polyacrylamide gels. Among them, the labeling of five polypeptides (molecular weights of 45,000, 55,000, 63,000, 80,000, and 94,000) was protected by the presence of 0.2 mM serotonin during photolysis. These peptides may be related to serotonin receptors.

Adenylyl Cyclases↗
PubMed1987

Solubilization of serotonin1a and serotonin1b binding sites from bovine brain.

Serotonin1 (5-hydroxytryptamine1, 5-HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1% Nonidet P-40 and 0.3% digitonin in a low-salt buffer containing 0.1% ascorbic acid. The affinity of [3H]5-HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane-bound binding sites (2.1 nM). [3H]8-Hydroxy-2-(di-n-propylamino)tetralin ([3H]DPAT), a selective 5-HT1a radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5-HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5-HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5-HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5-HT1a and 5-HT1b binding sites have been cosolubilized. [3H]5-HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone-specific binding was not detectable in this preparation, suggesting that 5-HT2 sites were not cosolubilized.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1987

Performance, methanogenesis and nitrogen metabolism of finishing steers fed monensin and nickel.

Sixty-four Angus steers initially averaging 354 kg were allotted to a 2 X 2 factorial arrangement of treatments to determine the effects of dietary Ni (0 or 5 mg/kg supplemental), monensin (0 or 33 mg/kg) and their possible interaction on performance, methane production and N metabolism. The basal diet was a high energy, corn-cottonseed hull based diet containing 10.2% crude protein and .30 mg/kg Ni on a dry matter basis. Monensin reduced (P less than .05) feed intake, did not affect average daily gain and improved (P less than .05) feed conversion over the 102-d study. Nickel supplementation did not significantly alter or interact with monensin to affect steer performance. However, steers fed Ni tended to have higher average daily gains and improved feed conversions. Monensin decreased (P less than .05) in vitro methane production, altered several carcass traits, increased (P less than .05) molar proportion of ruminal propionate and decreased (P less than .05) molar proportion of ruminal acetate. Nickel did not alter methane production, carcass characteristics or ruminal volatile fatty acid proportions. Both monensin and Ni increased (P less than .05) ruminal fluid urease activity when samples were obtained before feeding. A significant monensin X Ni interaction was found to affect ruminal epithelial urease activity. Monensin increased ruminal epithelial urease in steers not receiving supplemental Ni, but had no effect on ruminal epithelial urease activity in steers fed supplemental Ni. Ruminal fluid protein and ammonia-N were decreased (P less than .05) by monensin. Results of this study indicate that Ni may interact with monensin to affect ruminal epithelial urease activity but not performance or methane production in finishing steers.

Animals↗
PubMed1987

Composition and phosphorus bioavailability of a solid by-product from anaerobically digested waste from caged layer hens.

A poultry waste digester was constructed at the North Carolina State University Poultry Research Farm to produce methane from the waste of caged layer hens. A solid by-product (SBP) from the digester was recovered and dried for chemical and biological analysis as a feed supplement. Dried SBP was chemically analyzed for minerals, trace elements, and certain vitamins. The SBP was evaluated as a phosphorus (P) supplement in broiler diets. Biological availability of P in the SBP was determined by a slope ratio assay with femur bone ash as the criterion of P availability. Dietary treatments consisted of graded levels of standard available P (AP) supplemented as to provide .2, .3, .4, and .6% AP and graded levels of SBP added to provide 1.5, 2.5, 3.5, and 4.5% SBP to a corn-soy basal diet. Two treatments, containing 1.5% and 2.5% SBP added to an adequate diet (.4% AP) were used to determine if other factors in SBP were contributing to femur bone ash. The P in SBP was determined to be 90% available to the growing broiler chick. Femur bone ash did not respond to SBP in the diets already adequate in AP. Body weight gain, feed consumption, and feed conversion were not significantly different (P less than .05) among treatments with similar AP levels. The study concluded that SBP is a safe, effective feed supplement that supplies P, Ca, and total minerals.

Anaerobiosis↗
PubMed1986

Necessity of ascorbic acid in the radioligand binding assay for [3H]5-hydroxytryptamine.

Evidence is presented to suggest that ascorbic acid is required in the radioligand binding assay for [3H]5-hydroxytryptamine ([3H]5-HT, [3H]serotonin). In the absence of ascorbic acid, oxidation of [3H]5-HT occurred if the radioligand solution was left on ice for 1 or 3 hr. The oxidative products were detectable by thin-layer chromatography. They increased the binding significantly, although there was only slight oxidation (less than 1%) of the [3H]5-HT. When ascorbic acid was not used in the radioligand binding assay for [3H]5-HT, even though [3H]5-HT was prepared immediately before incubating with the membranes from the cortex of the rat, the binding also increased. The increased binding of [3H]5-HT had a low affinity (Kd = 14 nM) and high B max (1180 fmol/mg protein), compared to that in the presence of ascorbic acid (Kd = 5 nM; B max = 210 fmol/mg protein). However, the increased binding was not receptor-related because the additional radioactivity was not displaceable by excess of unlabelled 5-HT (10 microM) or d-LSD (d-lysergic acid iethylamide) and anomalous "specific binding" occurred in boiled membranes from cortex, in which the binding sites for 5-HT were destroyed. These results suggest that oxidation of [3H]5-HT may occur during the incubation with membranes and that ascorbic acid is therefore required as an antioxidant.

Animals↗
PubMed1986

Photoaffinity labeling of the 5-hydroxytryptamine 1A receptor in rat hippocampus.

1-[2-(4-Azidophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (p-azido-PAPP) inhibits [3H]5-hydroxytryptamine [( 3H]5-HT) binding to 5-HT1A and 5-HT1B sites in rat brain with equilibrium dissociation constants (KD) of 0.9 nM and 230 nM, respectively. [3H]p-Azido-PAPP was synthesized and its reversible and irreversible binding properties to the hippocampal 5-HT1A site characterized. [3H]p-Azido-PAPP labeled a single class of sites in rat hippocampal membranes with a KD of 1 nM and a maximal binding density of 370 fmol/mg protein. The pharmacological profile of [3H]p-azido-PAPP binding was consistent with the radioligand's selective interaction with the 5-HT1A receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes preincubated with [3H]p-azido-PAPP and irradiated showed a major band of incorporation of radioactivity at approximately 55,000 daltons. This incorporation could be blocked when membranes were incubated with 1 microM of several agents that have high affinity for 5-HT1A sites [5-HT, 8-hydroxy-2-(di-n-propylamino)tetraline, TVX Q 7821, spiperone, buspirone, d-lysergic acid diethylamide, metergoline]. The results indicate that on photolysis [3H]p-azido-PAPP irreversibly labels a polypeptide that is, or is a subunit of, the 5-HT1A receptor in rat hippocampus.

Affinity Labels↗
PubMed1986

[3H]1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine: a selective radioligand for 5-HT1A receptors in rat brain.

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1985

5-HT-1a and 5-HT-1b selectivity of two phenylpiperazine derivatives: evidence for 5-HT-1b heterogeneity.

The ability of m-trifluoromethylphenylpiperazine (TFMPP) and an N-substituted derivative, LY 165163 (p-NH2-PE-TFMPP), to discriminate 5-HT-1 binding sites labelled by [3H]5-HT is compared in rat corpus striatum and rat cortex. TFMPP displays at least a 30 fold selectivity for the 5-HT-1b subtype. Furthermore, TFMPP reveals heterogeneity within the 5-HT-1b binding sites. TFMPP displays a Kd of 6 nM for approximately two-thirds of the 5-HT-1b binding sites and a Kd of 273 nM for the remaining one third of the 5-HT-1b sites. p-NH2-PE-TFMPP, on the other hand, discriminates the 5-HT-1 sites in a manner similar to spiperone, displaying a 110 fold selectivity for the 5-HT-1a sites. p-NH2-PE-TFMPP displays a Kd of about 3 nM for the 5-HT-1a sites. p-NH2-PE-TFMPP does not discriminate subtypes within the 5-HT-1b binding sites. The significance of the selectivity of these two compounds as well as structurally related compounds is discussed.

Animals↗
PubMed1985

4-Fluoro-3-nitrophenyl azide, a selective photoaffinity label for type B monoamine oxidase.

The effects of 4-fluoro-3-nitrophenyl azide (FNPA) on types A and B monoamine oxidase in rat brain cortex were studied using serotonin and phenylethylamine as substrates respectively. FNPA competitively inhibited the oxidative deamination of both serotonin (Ki = 3 microM) and phenylethylamine (Ki = 0.78 microM) in the dark. Upon photoirradiation in the presence of FNPA, a photodependent inhibition of type B MAO activity resulted. This photodependent inhibition was apparently irreversible since there was no recovery of activity upon washing of the photolyzed FNPA-enzyme mixture. Additional evidence for the photoinduced covalent binding of FNPA to type B MAO is that non-competitive inhibition kinetics resulted after photolysis. The specificity of the photodependent incorporation of FNPA to type B MAO was shown by the protective effect of phenylethylamine and by decreased [3H]pargyline labeling after the enzyme was photolyzed with FNPA. Under the same experimental conditions, only minimal photodependent inhibition of type A MAO by FNPA was found. The observed difference in the efficiencies of the photodependent inactivation of the two types of MAO by FNPA suggests that there is a conformational or a structural difference in the active sites of the two types of MAO. The active site of type B MAO could be characterized by utilizing FNPA as a photoaffinity labeling probe.

Affinity Labels↗
PubMed1985

Inhibition of monoamine oxidase by phenyl azides.

We had previously shown that 4-fluoro-3-nitrophenyl azide (FNPA) is a competitive inhibitor of both types of monoamine oxidase (MAO) in the dark, but it is a preferential photoaffinity label for only the type B MAO (MAO-B). Recently we synthesized a number of arylazido compounds with structures related to FNPA and determined the effects of these compounds on the two types of MAO in rat brain cortex. We found that the fluoro group of FNPA was not required for the inhibition of MAO activities because neither the presence nor the position of the fluoro group affected its inhibition of MAO. On the other hand, both the nitro and the azido groups of FNPA were shown to be important for FNPA inactivation of two types of MAO. The inhibitory potency was significantly lower for compounds without either group. Furthermore, we found that all nitrophenyl azide isomers except 2-nitrophenyl azide were photodependent inhibitors of MAO-B. Under the same experimental conditions none of the compounds photoinactivated MAO-A. On the basis of these findings, mechanisms for FNPA inhibition of the two types of MAO are discussed.

Aniline Compounds↗
PubMed1985

A trifluoromethylphenyl piperazine derivative with high affinity for 5-hydroxytryptamine-1A sites in rat brain.

The inhibition of [3H]5-hydroxytryptamine [( 3H]5-HT) binding in rat brain by 1-[2-(3-bromoacetamidophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine (BrAcTFMPP) and that by spiperone were compared. Spiperone inhibition of [3H]5-HT binding in cortex was consistent with displacement from two sites with dissociation constants (KD) of 24 nM (5-HT-1A site) and 19 microM (5-HT-1B site) for spiperone. BrAcTFMPP also discriminated two subpopulations of [3H]5-HT binding sites with dissociation constants of 0.5 nM and 146 nM for the compound. The proportion of high-affinity sites for each compound represented about 35% of the specific [3H]5-HT binding. In the presence of 1 microM spiperone, a concentration that saturates the 5-HT-1A sites while having a minimal effect on 5-HT-1B sites, BrAcTFMPP displaced [3H]5-HT from a single site with a KD for BrAcTFMPP of 145 nM. The inhibition of [3H]5-HT binding by spiperone in the presence of 30 nM BrAcTFMPP was best fit by a single-site model with a KD of 21 microM for spiperone. In corpus striatum, 5-HT-1A sites, as defined with spiperone, represented 15% of the specific [3H]5-HT binding and 30 nM BrAcTFMPP also blocked about 15% of the binding. A significant difference between spiperone and BrAcTFMPP was their affinity for 5-HT-2 receptors. BrAcTFMPP (KD = 41 nM) had an 80-fold lower affinity for these sites than spiperone (KD = 0.5 nM). Thus, BrAcTFMPP and spiperone discriminate the same two subpopulations of [3H]5-HT binding sites and BrAcTFMPP displays a high affinity and a selectivity for 5-HT-1A sites versus both 5-HT-1B and 5-HT-2 sites.

Animals↗
PubMed1985

Photoinactivation of serotonin uptake by an arylazido derivative of 5-hydroxytryptamine.

A potential photoaffinity probe for the substrate-binding polypeptide of the neuronal serotonin uptake system has been synthesized. Under dark conditions, 3-(beta-(4-azidobenzamidino)ethyl-5-hydroxyindole (serotonin azidobenzamidine (SABA) was found to inhibit competitively [3H]5-hydroxytryptamine uptake by rat cortical synaptosomes with a K1 of 130 nM. The selectivity of this action was indicated by SABA's much lower potency as an inhibitor of synaptosomal [3H]norepinephrine uptake (K1 = 7 microM). When synaptosomes were irradiated in the presence of SABA, serotonin uptake was irreversibly inhibited in a concentration-dependent fashion with the maximum effect occurring at 1 microM SABA. At this concentration, approximately 40% of the serotonin uptake activity could not be recovered upon repeated washing of the synaptosomes. This inhibition was determined not to result from the production of a potent inhibitory photolysis product of SABA. The photoinactivation of serotonin transport by SABA was found to depend on the time of irradiation and could be prevented by the presence of agents that interact with the uptake system. Serotonin, p-chloroamphetamine, fenfluramine, and alaproclate protected the serotonin carrier against SABA's irreversible effects in a concentration-dependent manner. The presence of high concentrations of Tris or p-aminobenzoic acid, two nitrene-scavenging agents, did not reduce the level of photoinactivation of serotonin uptake by SABA, indicating that the irreversible inhibition is a result of true photoaffinity labeling of the carrier.

Affinity Labels↗
PubMed1984

Interaction of N-(2-nitro-4-azidophenyl)-serotonin with two types of monoamine oxidase in rat brain.

The effects of N-(2-nitro-4-azidophenyl) serotonin (NAP-5-HT) on types A and B monoamine oxidase (MAO) in rat brain cortex were studied. In the dark this compound acted as a competitive inhibitor for both types A and B MAO (Ki values of 0.19 microM and 0.21 microM for types A and B MAO, respectively). Upon photolysis, NAP-5-HT became an irreversible inhibitor for only type B MAO. A 50% inhibition was obtained by irradiation of the enzyme in the presence of 35 nM NAP-5-HT. Furthermore the inhibition of type B MAO could be protected by including its substrate phenylethylamine during the irradiation. Under the same photolytic conditions photodependent inhibition of type A MAO by NAP-5-HT was not clearly observed. These results provide further evidence that there is a fundamental difference in the active site of the two types of MAO in brain. NAP-5-HT may be a useful photoaffinity probe for characterizing the active site of type B MAO.

Affinity Labels↗
PubMed1983

Atherosclerosis in Japanese quail and the effect of lipoic acid.

The Japanese quail (Coturnix coturnix japonica) is a useful laboratory animal for the study of atherosclerosis. It is small, omnivorous, easy to maintain, and susceptible to either spontaneous or cholesterol-induced atherosclerosis, and it has a low feed consumption and short life cycle. It develops atheromatous lesions with the characteristic lipid deposition and myofibroblastic proliferation in the aorta and sometimes in the coronary artery. Japanese quail can be genetically bred into lines highly susceptible and resistant to atherosclerosis. A nutritional study has indicated that a high intake of soy protein prevents the disease in the quail. Contradictory results of studies with rabbits were reported in the early literature on the prevention of atherosclerosis by lipoic acid. Recently the effect of lipoic acid on atherosclerosis was reevaluated in the quail. A preventive effect of this compound was demonstrated when it was implanted s.c. and slowly released in the animal.

Animals↗
PubMed1983

Differential effect of cholesterol on two types of 5-hydroxytryptamine binding sites.

The specific binding of [3H]5-hydroxytryptamine ([3H]5-HT, [3H]serotonin) to rat cerebral cortex is increased approximately 1.5 to 2.0 fold by cholesterol hydrogen succinate (CHS) and is solubilized into the supernatant fraction by 12 mM CHS. [3H]5-HT binding sites can be constituted by incubating the supernatant fraction obtained from CHS-treated cerebral cortex with cerebellum in which no significant [3H]5-HT binding is detectable. The constituted [3H]5-HT binding could be displaced by unlabeled 5-HT, d-lysergic acid diethylamide (d-LSD) and spiperone as could the binding to cortex membranes. Unlabeled 5-HT, d-LSD and spiperone each inhibited specific [3H]5-HT binding to constituted binding sites by 50% at about 1 X 10(-9) M. Specific [3H]spiperone binding was not detectable in the constituted membranes. Stearic acid which is reported to have similar effects on membrane fluidity as cholesterol also increased specific [3H]5-HT binding in cortical membranes. Stearic acid does not affect specific [3H]spiperone binding. These results suggest that [3H]5-HT and [3H]spiperone binding sites are affected differently by membrane fluidity.

Animals↗
PubMed1983

Genetic selection, general characterization, and histology of atherosclerosis-susceptible and -resistant Japanese quail.

A new animal model of atherosclerosis has been developed through genetic selection of Japanese quail (Coturnix coturnix japonica) into susceptible (SUS) and resistant (RES) lines. Characterization of the selected quail has shown that the RES birds were resistant to the disease and developed little atherosclerosis on a diet containing 1% cholesterol. The SUS birds were sensitive and developed severe atherosclerosis in 8-9 wks on a diet containing only 0.5% cholesterol. The histology of the progression of atherosclerosis in the SUS quail was studied. It bore a morphological similarity to that of human arterial atherosclerosis. The atherosclerotic plaque was characterized by intimal thickening, the presence of foam cells, the proliferation of smooth muscle cells and/or fibroblasts, and the formation of scar with collagen deposition. We believe that these two lines of quail may serve as a valid animal model for the study of the genetic and biochemical basis of cholesterol-induced atherosclerosis.

Animals↗