Post-Ischemic Alterations in [3H]FK506 Binding in the Gerbil and Rat Brains

We investigated post-ischemic changes in FK506 binding protein (FKBP) in the brain after transient global ischemia in gerbils or transient focal ischemia in rats. [³H]FK506 was used to label FKBP as a immunophilin. In transient global ischemia, [³H]FK506 binding showed a transient reduction in the frontal cortex only 1 h after recirculation. In the striatum, the dorsolateral part exhibited a significant increase in [³H]FK506 binding 5, 24 and 48 h after ischemia. However, the ventromedial part showed a transient elevation in [³H]FK506 binding 24 h after ischemia. Thereafter, the ventromedial part showed no conspicuous change in [³H]FK506 binding up to 7 days after ischemia. The dorsolateral part also showed no significant change in [³H]FK506 binding 7 days after ischemia. In the hippocampus and thalamus, [³H]FK506 binding was unchanged in the stratum radiatum of the hippocampal CA₁ sector, hippocampal CA₃ sector, dentate gyrus and thalamus up to 7 days after ischemia. However, the stratum oriens of the hippocampal CA₁ sector showed a significant reduction in [³H]FK506 binding 48 h and 7 days after ischemia. A histological study showed that transient cerebral ischemia caused a severe damage in the striatum and hippocampal CA₁ sector. In a model of transient focal ischemia, a marked increase in [³H]FK506 binding was also found in the striatum and cerebral cortex where severe infarctions were observed. These results demonstrate that post-ischemic change in [³H]FK506 binding between the striatum and hippocampus may be produced by different mechanisms. Furthermore, our findings suggest that immunophilins may play some role in the pathogenesis of ischemic diseases.     

Chronic Treatment with FK506 Increases p70 S6 Kinase Activity Associated with Reduced Nitric Oxide Synthase Activity in Rabbit Hearts

FK506, an immunosuppressant, modulates phosphorylation of nitric oxide (NO) synthase, and induces cardiac hypertrophy in clinical settings. Having recently reported that chronic treatment with an inhibitor of NO synthase induces cardiac hypertrophy associated with the activation of 70-kD S6 kinase (p70^S6K), which plays an important role in cardiac hypertrophy by regulating protein synthesis, we investigated the effects of chronic administration of FK506 on NO synthase and p70^S6K activities in hearts. Twenty rabbits were divided into four groups: untreated rabbits, those treated with low-dose FK506 (0.10 mg/kg), those treated with medium-dose FK506 (0.20 mg/kg), and those treated with high-dose FK506 (0.40 mg/kg). FK506 was administered intravenously twice a day. After 4 weeks of treatment with FK506, calcium-dependent NO synthase activity in myocardium in the high-dose FK506 group was lower (P < 0.05) than in the untreated group. p70^S6K activity in myocardium in the high-dose group was higher (P < 0.05) than in the untreated group. There was a significant (P < 0.05) inverse correlation between NO synthase and p70^S6K activities in myocardium. However, the endothelial-dependent vasodilation of aortic rings or plasma levels of NO metabolites during experimental protocols did not differ among the groups studied. These findings suggest that chronic treatment of FK506 activates p70^S6K and reduces NO synthase activity in rabbit hearts. Reduced NO synthase and/or activated p70^S6K activities in hearts might contribute to the cardiac hypertrophy observed in some patients receiving FK506.     

Cyclosporine A, FK-506, 40-O-[2-hydroxyethyl]rapamycin and mycophenolate mofetil inhibit proliferation of human intrahepatic biliary epithelial cells in vitro

AIM: To investigate the effect of cyclosporine A (CsA),FK-506, and mycophenolate mofetil (MMF) and 40-0-[2-hydroxyethyl]rapamycin (RAD) on proliferation of human intrahepatic biliary epithelial cells (BECs)in vitro.METHODS: BECs were isolated from six human liver tissuespecimens with the immunomagnetic separation method and treated with different concentrations of CsA, FK-506, RAD, and MMF in vitro. Proliferation of the cells was measured by MTT assay at 24 and 48 h after treatment, respectively. One-way analysis of variance was used to analyze the results. Expression of CK 19in BECs was monitored by flow cytometry and Western blot.RESULTS: Six lines of BECs were established. They survived for 4-18 wk in vitro. Flow cytometry analysis showed that these cells always expressed CK19. CsA,FK-506, RAD, and MMF inhibited proliferation of BECs in a dose-dependent manner. The lowest concentration of CsA, FK-506, RAD, and MMF to inhibit proliferation of BECs (P＜0.05) was 500, 100, 0.25, and 100 μg/L,respectively. However, the expression of CK19 by BECs was not changed.CONCLUSION: CsA, FK-506, RAD, and MMF have an antiproliferative effect on human intrahepatic BECs in vitro, while RAD has the strongest growth-inhibitory effect. Their possible effects on liver regeneration and bile duct injury in transplant patients should be further investigated.     

Cyclosporine A, FK-506, 40-0-[2-hydroxyethyl]rapamycin and mycophenolate mofetil inhibit proliferation of human intrahepatic biliary epithelial cells in vitro

AIM: To investigate the effect of cyclosporine A (CsA), FK-506, and mycophenolate mofetil (MMF) and 40-0-[2-hydroxyethyl]rapamycin (RAD) on proliferation of human intrahepatic biliary epithelial cells (BECs) in vitro. METHODS: BECs were isolated from six human liver tissuespecimens with the immunomagnetic separation method and treated with different concentrations of CsA, FK-506, RAD, and MMF in vitro. Proliferation of the cells was measured by MTT assay at 24 and 48 h after treatment, respectively. One-way analysis of variance was used to analyze the results. Expression of CK 19 in BECs was monitored by flow cytometry and Western blot. RESULTS: Six lines of BECs were established. They survived for 4-18 wk in vitro. Flow cytometry analysis showed that these cells always expressed CK19. CsA, FK-506, RAD, and MMF inhibited proliferation of BECs in a dose-dependent manner. The lowest concentration of CsA, FK-506, RAD, and MMF to inhibit proliferation of BECs (P<0.05) was 500, 100, 0.25, and 100 M9/L, respectively. However, the expression of CK19 by BECs was not changed. CONCLUSION: CsA, FK-506, RAD, and MMF have an antiproliferative effect on human intrahepatic BECs in vitro, while RAD has the strongest growth-inhibitory effect. Their possible effects on liver regeneration and bile duct injury in transplant patients should be further investigated.     

[3H] Melatonin Binding in Membrane and Cytosol Fractions From Rat and Calf Brain

The binding characteristics of the tritiated pineal hormone, [3H] melatonin, were studied in brain tissues using in vitro binding techniques. In synaptosomal membranes prepared from rat hippocampus and subjected to preincubation at 37 degrees C and multiple washings, high-affinity binding of [3H] melatonin significantly exceeds that previously reported for membrane or cytosol fractions from mammalian brain. Scatchard analysis of saturation binding data indicates that binding affinities are similar in membrane (Kd = 15 nM) and cytosol (Kd = 11 nM) preparations. However, binding is about sevenfold greater in membranes than in cytosol prepared by centrifugation of homogenates at 104,000g for 60 min. Specific binding is also present in both particulate and soluble fractions from calf brain. Inhibition experiments, in rat hippocampal membranes, indicate that norepinephrine is the most potent inhibitor of about 55% of total binding. Serotonin also exhibited high affinity for about 25% of total binding, suggesting that [3H] melatonin labels both adrenergic and serotonergic sites in this brain region. Further studies are required to characterize the serotonergic and adrenergic sites labelled by [3H] melatonin and to determine whether these sites are functionally important receptors for melatonin.     

Characterization of the High Affinity [H]Nociceptin Binding Site in Membrane Preparations of Rat Heart: Correlations with the Non-opioid Dynorphin Binding Site

The binding parameters of [³H]nociceptin were examined in membrane preparations of rat heart and compared with those of [³H]dynorphin A-(1-13) ([³H]Dyn A-(1-13)). Scatchard analysis of [³H]nociceptin binding revealed the presence of two distinct sites: a high affinity (K_d: 583 nm) low capacity (B_max: 132 pmol/mg protein) site and a low affinity (K_d: 10 316 nm) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potencyα-neo-endorphin>Dyn A-(2-13)=Dyn A-(3-13)>Dyn A-(5-13)>Dyn A-(1-13)>Dyn A>Dyn B>Dyn A-(6-10)>>Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a K_iof 4.8μmas compared with 0.72μmfor Dyn A. The order of potency of the various peptides in inhibiting [³H]nociceptin binding correlated well (r=0.93) with their ability to compete with the binding of [³H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [³H]nociceptin and non-opioid [³H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mm) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100μm). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs, Gpp(NH)p and GTP-γ-S. Nociceptin (1–50μm) was also shown to inhibit the uptake of [³H]noradrenaline ( [³H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [³H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL₁mRNA and [³H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [³H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [³H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [³H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [³H]NA uptake and may participate to the pathophysiology of hypertension.     

Uptake and metabolism of [3H]ecdysone in cultured ovaries of the silkworm, Bombyx mori

Uptake of ecdysone by ovaries of the silkworm, Bombyx mori, was studied by organ culture. [3H]Ecdysone was transported almost linearly into the ovary for up to 3 h of the incubation. The uptake was proportional to the concentration of the labeled ecdysone, unsaturably, at concentrations ranging up to 10(-6) M. Ecdysone which had been transported into the ovary could usually be removed when the ovary was re-transferred to the ecdysone-free medium. Analysis of the transported compounds by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed the marked conversion of ecdysone into 20-hydroxyecdysone, unknown metabolites and conjugated forms. Physiological significance of the metabolic activity of the ovary is discussed with respect to the accumulation of ecdysteroids in the ovary.     

Evidence for an Increase in [3H]Spiperone Binding in Hypothalamic Nuclei During the Development of Spontaneous Hypertension in the Rat

The in vivo binding of [³H]spiperone was measured in discrete areas of the hypothalamus in 7, 9 and 16 weeks old spontaneously hypertensive rats (SHR) and age matched normotensive Wistar Kyoto (WKY) controls. The specific binding of [³H]spiperone was significantly higher in the four different hypothalamic regions (H₁, H₂, H₃, H₄) that we have tested in 7 or 9 weeks old SHR than in age matched WKY controls. At 16 weeks a significant increase was only present in H₃. These results suggest that dopaminergic hypothalamic neurons might be implicated in the onset of hypertension in the rat.     

Regional Differences in the Effects of Chronic Ethanol Administration on [3H]Zolpidem Binding in Rat Brain

A strong association has been observed between [³H]zolpidem binding and the presence of γ-aminobutyric acid (GABA_A) receptor mRNA for α₁-, β₂-, and γ₂-subunits in specific brain regions. This correlates with observed sensitivity of individual neurons to zolpidem and ethanol in these same regions. Previous studies using homogenate binding approaches showed small alterations in [³H] zolpidem binding levels after chronic ethanol exposure. This study was undertaken to ascertain if there is regional specificity of the effects of chronic ethanol administration on [³H] zolpidem binding levels. Chronic ethanol administration induced small, but significant alterations in [³H] zolpidem (5 nM) binding in the Inferior colliculus, substantia nigra, and the medial septum. [³H]Zolpidem binding was increased in the inferior colliculus and substantia nigra, and decreased in the medial septum. No significant differences in [³H] zolpidem binding were noted in any other brain area analyzed, including the cortex and cerebellum. These findings show that chronic ethanol administration has small effects on [³H] zolpidem binding, although they occur in a site-specific and bidirectional manner. Moreover, there is no correlation between changes in [³H] zolpidem binding and alterations In GABA_A receptor subunit expression.     

Binding Characteristics, Regional Distribution and Diurnal Variation of [125I]-Iodomelatonin Binding Sites in the Chicken Brain

The [125I]-iodomelatonin binding sites in chicken brain membrane preparations were studied. The binding of [125I]-iodomelatonin to the membrane preparations of chicken brain was rapid, stable, saturable, and reversible. The order of pharmacological affinities of [125I]-iodomelatonin binding sites in the chicken brain membrane preparations was: melatonin greater than 6-chloromelatonin greater than N-acetylserotonin greater than 5-hydroxytryptamine greater than tryptamine greater than 5-methoxytryptophol, much greater than 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, L-tryptophan, 5-hydroxytryptophan, 3-acetylindole. Compounds known to act on the receptor of norepinephrine or acetylcholine were inactive as compared to melatonin. Among the various brain regions studied, melatonin binding had maximal level in the hypothalamus, intermediate levels in the mid-brain, ponsmedulla, and telencephalon, and minimum level in the cerebellum. Subcellular fraction studies indicated that 40% of the binding was located in the mitochondrial fraction, 27% in the nuclear, 26% in the microsomal, and 6% in the cytosol fraction. Scatchard analysis of the membrane preparations revealed a dissociation constant (Kd) of 199.6 +/- 17 pM and a total number of binding sites (Bmax) of 16.6 +/- 0.75 fmol/mg protein at midlight. Thus, our results showed the presence of specific melatonin binding sites in the chicken brain membrane preparations. Saturation studies demonstrated that [125I]-iodomelatonin binding capacity in chicken brain membrane preparations were 40% greater at midlight (16.6 +/- 0.75 fmol/mg protein) than at middark (10.6 +/- 0.56 fmol/mg protein), with no significant variation in their binding affinities.     

In vitro and in vivo interactions of [3H]dimethylstilbestrol with the estrogen receptor

The binding of an antiestrogen with the uterine estrogen receptor (R) has been studied directly in vitro using tritiated dimethylstilbestrol (DMS). The affinity of DMS for R as determined at equilibrium was similar to that of estradiol-17beta (E2) (K(D) approximately .3 nN) when taking into account the higher nonspecific binding of DMS. The number of DMS binding sites was constantly found to be inferior to that of E2. The fact that the DMS binding entity specifically bound estrogen and antiestrogen, was destroyed by pronase, displayed an 8S sedimentation constant, and interacted in vitro with DNA, strongly suggested that DMS interacted directly with R. The association of DMS to R was a simple 2nd-order reaction while its dissociation was a 1st-order reaction with 2 slopes. The association and dissociation rate constants of the R-DMS complex were, respectively, slower and higher than those of the R-E2 complex. The rapid dissociation rate of DMS could be responsible for its inability to protect the receptor binding sites against thermo-inactivation. Tritiated DMS was able in vivo to induce the nuclear translocation of the receptor. However, as with other short-acting antiestrogens and contrary to Nafoxidine, the time of nuclear retention of R was short. These results are in agreement with the assumption that the length of the nuclear retention of R is determinant in explaining the weak agonist activity of this compound.     

Alteration of [3H]MK-801 Binding Associated with the JV-Methyl-d-Aspartate Receptor Complex by Acute Ethanol in Rat Cortex and Hippocampus In Vitro

We investigated the effect of ethanol on specific binding of [³H]MK-801 to the intrachannel phencyclidine (PCP) receptor site, as an index of change in the functional response of the N-methyl-d -Aspartate (NMDA)-associated ion channel. Saturation binding experiments were performed on synaptic membrane homogenates from adult rat cortex and hippocampus. [³H]MK-801 binding assays were conducted under conditions of basal, 10 μm glutamate, or 10 μm glutamate + 30 μm d -serine, with and without 50 or 100 mm ethanol. Association experiments of [³H]MK-801 binding (5 nm) were conducted under conditions of 0 or 10 μm glutamate, with varying concentrations of glycine (0.01, 0.10, and 10 μm) with and without 100 mm ethanol. Ethanol (50 and 100 mm ) significantly decreased the percentage of high-affinity (open-channel state) MK-801 receptors with a concomitant increase in percentage of low-affinity receptors, but did not change high- and low-affinity constants of the two binding states. An ethanol-induced increase in the closed-channel receptor density in basal and activated conditions was suggested by the saturation experiments. Association experiments further explained this finding, in that ethanol (100 mm ) significantly decreased fast component (open-channel) [³H]MK-801 binding in conditions of glycine (0.01–10 μm ) only and activated conditions of glutamate + glycine (0.01–0.10 μm ). However, the observed fast and slow kinetic rate constants of [³H]MK-801 binding, as well as total specific binding (fast + slow components), were not altered. Thus, ethanol seems to act as a noncompetitive antagonist upon the gating mechanism of, and ligand access to, the NMDA-coupled ion channel. These findings support previous observations of ethanol selectively reducing NMDA-activated calcium influx, and reducing the frequency and duration of ion channel opening in electrophysiological studies. Similar to previous reports on NMDA-stimulated calcium influx and [³H]MK-801 binding, glycine (at the maximal concentration of 10 μm ), in the presence of 10 μm glutamate, was found to reverse ethanol inhibition of fast component binding.     

Binding of the ligand [3H]MK-801 to the MK-801 binding site of the N-methyl-D-aspartate receptor during experimental encephalopathy from acute liver failure and from acute hyperammonemia in the rabbit

Binding of the ligand [³H]MK-801 to the MK-801 binding site of the N-methyl-D-aspartate (NMDA) receptor population on brain homogenates in rabbits was studied during experimental encephalopathy from acute liver failure and from acute hyperammonemia in the rabbit. Homogenates were prepared from brain cortex, hippocampus and striatum. Hepatic encephalopathy was induced by a two-stage liver devascularization procedure and acute hyperammonemia by a prolonged ammonium-acetate infusion; rabbits receiving a sodium-potassium-acetate infusion served as controls. In these animal models extracellular brain glutamate levels are known to be elevated. However no significant alterations in the number nor the affinity of the MK-801 binding sites of the NMDA receptors were found during acute liver failure and acute hyperammonemia. These findings suggest that the NMDA receptor population remains unaltered in experimental encephalopathy from acute liver failure and acute hyperammonemia, despite alterations in extracellular brain glutamate levels.Abbreviations used in the text NMDA N-methyl-D-aspartate - NH₄Ac ammoniumacetate - NaKAc sodium/potassium-acetate - AMPA amino-3-hydroxy-5-methylisoxazole4-propionic acid     

Differential involvement of signaling pathways in the regulation of growth hormone release by somatostatin and growth hormone-releasing hormone in orange-spotted grouper (Epinephelus coioides)

Somatostatin is the most effective inhibitor of GH release, and GHRH was recently identified as one of the primary GH-releasing factors in teleosts. In this study, we analyzed the possible intracellular transduction pathways that are involved in the mechanisms induced by SRIF and GHRH to regulate GH release. Using a pharmacological approach, the blockade of the PLC/IP/PKC pathway reversed the SRIF-induced inhibition of GH release but did not affect the GHRH-induced stimulation of GH release. Furthermore, SRIF reduced the GH release induced by two PKC activators. Inhibitors of the AC/cAMP/PKA pathway reversed both the SRIF- and GHRH-induced effects on GH release. Moreover, the GH release evoked by forskolin and 8-Br-cAMP were completely abolished by SRIF. The blockade of the NOS/NO pathway attenuated the GHRH-induced GH release but had minimal effects on the inhibitory actions of SRIF. In addition, inhibitors of the sGC/cGMP pathway did not modify the SRIF- or GHRH-induced regulation of GH release. Taken together, these findings indicate that the SRIF-induced inhibition of GH release is mediated by both the PLC/IP/PKC and the AC/cAMP/PKA pathways and not by the NOS/NO/sGC/cGMP pathway. In contrast, the GHRH-induced stimulation of GH secretion is mediated by both the AC/cAMP/PKA and the NOS/NO pathways and is independent of the sGC/cGMP pathway and the PLC/IP/PKC system.