Vitamin B6 Metabolism and Binding to Proteins in the Blood of Alcoholic and Nonalcoholic Men

Blood obtained from nonalcoholic and alcoholic subjects was incubated with 100 nm [³H]pyridoxine to study its uptake and metabolism by erythrocytes and the binding of vitamin B₆ metabolites to proteins in plasma and erythrocytes. Erythrocytes of the alcoholics accumulated tritium faster than those of the controls; however, they contained the same total amount of tritiated compounds by 15 min. After incubation for 30 min, the erythrocytes had converted most of the pyridoxine to pyridoxal phosphate and pyridoxal. Pyridoxal-P remained in the erythrocytes, and ?40% of the pyridoxal diffused into the plasma. [³H]Pyridoxal and [³H]pyridoxal-P levels in the erythrocytes and plasma of the alcoholics were similar to those in the controls. However, dialyzed hemolysates of the alcoholics had more [³H]pyridoxal and a lower percentage of [³H]pyridoxal-P than those of the controls. The total concentration of plasma pyridoxal-P was lower in the alcoholics than in the controls and did not change upon incubation of whole blood with pyridoxine or upon dialysis. The erythrocytes of the alcoholics and controls had similar concentrations of pyridoxal-P that increased 2.5-fold upon incubation of whole blood with pyridoxine for 30 min and returned to the initial concentrations upon dialysis. The amount of [³H]pyridoxal and [³H]pyridoxal-P bound to protein was assessed by treating hemolysate and plasma samples with borohydride before dialysis. More ³H was bound to protein in the erythrocytes than in the plasma. The amount of protein-bound ³H in the erythrocytes of the alcoholics was lower than that of the controls, whereas the amount of protein-bound ³H in plasma was similar in both groups. It is concluded that less of the pyridoxal-P and pyridoxal was protein bound, and the pyridoxal-P was more susceptible to hydrolysis to pyridoxal by phosphatases in the erythrocytes of the alcoholics compared with the controls.     

L-glutamate and γ-aminobutyric acid uptake in synaptosomes from the cerebral cortex of dogs with congenital chronic hepatic encephalopathy

High affinity [³H]-aminobutyric acid (GABA) and [³H]L-glutamate uptake were determined in synaptosomes prepared from the cerebral cortex of dogs with congenital hepatic encephalopathy and control dogs. The K_m value for GABA uptake was increased by 35% but there was a concomitant 34% increase in V_max suggesting that GABA uptake capacity was not changed in HE dogs. In contrast, mean V_max for glutamate uptake in HE dogs was 85% greater than mean V_max in control dogs; mean K_m was increased by 25% in HE dogs. Therefore, overall synaptosomal high affinity glutamate uptake capacity was increased in HE dogs compared to controls.     

Expression of functional Y1 receptors for neuropeptide Y in human Ewing's sarcoma cell lines

Summary In the human Ewing's sarcoma cell line WE-68, saturation analysis using³H-labelled neuropeptide Y ([³H]NPY) as the radioligand disclosed a homogeneous population of binding sites with a dissociation constant (K _d ) of 4.5 nM and maximal binding capacity (B _max) of 712 fmol/mg cell protein. Besides the WE-68 cell line, ten other human Ewing's sarcoma cell lines (FM-62, HS-80, HT-78, HT-M1-78, NT-68, RM-82, RS-63, VH-64, WE-M1-68, WE-M2-68) were also found to display NPY receptors withK _d varying from 3.5 nM to 10.7 nM andB _max=247–3744 fmol/mg cell protein. NPY, its natural analogues and the Y₁-receptor-specific peptide ligand [Leu³¹, Pro³⁴]NPY inhibited [³H]NPY binding in the potency order: [Leu³¹,Pro³⁴]NPYhuman NPYpeptide YY (PYY)>> salmon pancreatic polypeptide (PP) > human PP>porcine NPY_13–36NPY_22–36. In the Ewing's sarcoma cell lines NPY provoked inhibition of forskolin-stimulated cyclic AMP formation by up to 98%. Pertussis toxin alleviated the cyclic-AMP-inhibitory response to NPY. In isolated Ewing's sarcoma plasma membranes pertussis toxin [³²P]ADP-ribosylated a 41-kDa protein. The ability of NPY and analogues to inhibit cyclic AMP accumulation paralleled their potencies in displacing radioligand binding. By contrast, a cell line derived from an atypical form of Ewing's sarcoma did not express specific and functional NPY receptors. These results demonstrate that conventional Ewing's sarcoma cells possess G_i-potein-coupled NPY receptors of the Y₁ type, which upon interaction with NPY, PYY, and PP mediate inhibition of cyclic AMP generation.Abbreviations NPY neuropeptide Y - PP pancreatic polypeptide - PYY peptide YY - VIP vasoactive intestinal peptide     

Binding of the calcium channel blocker nitrendipine to its receptor in purified sarcolemma from canine cardiac ventricle

High affinity binding of the 1,4-dihydropyridine calcium channel blocker [³H]nitrendipine was found in cardiac sarcolemma but not cardiac sarcoplasmic reticulum or mitochondria. Sarcolemmal binding of [³H]nitrendipine was saturable and reversible, with a maximum (B_max) of approximately 1 pmol/mg protein and a K_d of approximately 0.14 nm. Displacement of sarcolemma-bound [³H]nitrendipine by other nifedipine analogs was stereospecific. The K_d for nitrendipine binding was approximately three orders of magnitude lower than the IC₅₀ for the negative inotropic effect of this drug on isolated cat myocardium.     

Decreases in Dopamine Receptors but not in Dopamine Transporters in Alcoholics

It has been hypothesized that ethanol's actions on the dopamine (DA) system may participate in addiction. The purpose of this study was to evaluate the DA system in the brain of alcoholics. We evaluated 10 alcoholics and 17 nonalcoholics using positron emission tomography and [¹¹C]raclopride to measure DA D2 receptors. In addition, in 5 of the alcoholics and 16 of the nonalcoholics, we also measured DA transporters with [¹¹C]d-threo methylphenidate. The ratio of the distribution volumes in striatum to that In cerebellum, which corresponds to B_max/K_d+ 1, was used as model parameter of DA D2 receptor and transporter availability. Dopamine D2 receptor availability (B_max/K_d) was significantly lower in alcoholics (2.1 ± 0.5) than in nonalcoholics (2.7 ± 0.6) (p < 0.05) and was not correlated with days since last alcohol use. Alcoholics showed DA transporter values similar to those in nonalcoholics. The ratio of DA D2 receptor to transporter availability was significantly higher in nonalcoholics (1.4 ± 0.1) than in alcoholics (1.1 ± 0.1) (p < 0.005). Alcoholics showed significant reductions in D2 receptors (postsynaptic marker) but not in DA transporter availability (presynaptic marker) when compared with nonalcoholics. Because D2 receptors in striatum are mainly localized in γ-aminobutyric acid (GABA) cells these results provide evidence of GABAergic involvement in the dopaminergic abnormalities seen in alcoholics.     

Binding of [3H]ouabain to endothelial cells derived from various vascular beds

Summary Binding experiments were performed with [³H]ouabain on plasma membranes derived from several types of isolated and cultivated endothelial cells. Identical saturation curves for [³H]ouabain binding to endothelial cells form pig aorta, caval vein, and pulmonary artery were obtained with a dissociation constant (K_D) of 3.29±0.31 nmol/l and a binding capacity (B_max) of 5.22±0.12 pmol/mg protein. On guinea-pig coronary endothelial cells, saturation of [³H]ouabain revealed much lower affinity (K_D 95±15 nmol/l, B_max 2.08±0.09 pmol/mg protein). All Scatchard plots were linear, indicating a homogeneous class of binding sites. In competition experiments, cardiac glycosides and their aglycons displaced the radioligand with a structure-activity relationship typical for interaction with Na⁺/K⁺-ATPase (proscillaridin A>ouabain>digoxin>g-strophanthidin>digoxigenin>dihydrodigoxin); in particular, removal of the sugar moiety results in considerable reduction of affinity. Furthermore, K⁺ displayed a steep inhibition curve with a half-maximal inhibitory constant of 2 mmol/l. All these findings suggest the presence of endothelial ouabain receptors linked to Na⁺/K⁺-ATPase. However, direct measurement of this enzyme was not possible due to an extremely high Mg²⁺-ATPase activity.     

TRH receptor binding in avian pituitary and brain

The pituitary gland of the domestic fowl binds [³H]-[3-methyl-His²]thyrotropin-releasing hormone ([³H]MeTRH) with properties very similar to those exhibited by mammalian TRH receptors, including affinity (K_D = 4.9 nM), density of binding sites (B_max = 5.1 pmol/g), and pharmacology for eight TRH analogs. Chicken brain appears to contain similar binding sites, but the level of binding was too low for detailed characterization.     

Characterization of a [3H]glutamate binding site in rat pineal gland: Enhanced affinity following superior cervical ganglionectomy

Abstract: Glutamate, an excitatory neurotransmitter/neuromodulator involved in cell-to-cell communication within the central nervous system, is now believed to play a role in neuroendocrine function. In this study we describe a single, saturable, stereospecific, and temperature-, time-, and pH-dependent binding site for glutamate in the pineal gland of the rat (K_d= 612 ± 23 nM, B_max= 3.17 ± 0.33 pmol/mg protein). After removal of the sympathetic innervation to the pineal gland, [³H]glutamate binding displayed a higher apparent affinity (K_d= 412 ± 28 nM) (P < 0.05) without a change in binding site number (B_max= 3.60 ± 0.24 pmol/mg protein). No difference in [³H]glutamate binding site number was observed in pineal glands obtained from animals sacrificed during the middle of the light and dark periods. These data suggest a possible modulatory role for a glutamate binding site in pineal gland function.     

Mechanism of Action of Acamprosate. Part I. Characterization of Spermidine-Sensitive Acamprosate Binding Site in Rat Brain

It has been suggested that the anticraving drug, acamprosate, acts via the glutamatergic system, but the exact mechanism of action is still unknown. The aim of this study was to characterize [³H]acam-prosate binding and establish whether this showed any relation to sites on the NMDA receptor complex. We found saturable specific binding of [³H]acamprosate to rat brain membranes with a KD of 120 μM and a B_max of 450 pmol/mg of protein. This acamprosate binding site was sensitive to inhibition by spermidine (IC₅₀: 13.32 ± 1.1 μM; Hill coefficient = 1.04), and arcaine and glutamate both potentiated the inhibitory effect of spermidine. Acamprosate binding to the acamprosate binding site was also sensitive to inhibition by divalent cations (Ca2⁺, Mg2⁺, and Sr²⁺). Conversely, acamprosate displaced [¹⁴C]spermidine binding from rat brain membranes with an IC₅₀, of 645 μM and a Hill coefficient = 1.74. This inhibitory effect of acamprosate was not affected by arcaine, and was associated with a significant reduction in B_max and binding affinity for spermidine, suggesting an allosteric interaction between acamprosate and a spermidine binding site. These data are consistent with an effect of acamprosate on the NMDA receptor protein complex, and acamprosate was also found to alter binding of [³H]dizocilpine to rat brain membranes. When no agonists were present in vitro (minimal NMDA receptor activation), acamprosate markedly potentiated [³H]dizocilpine binding at concentrations in the 5 to 200 μ range. However, under conditions of maximal receptor activation (100 μM glutamate, 30 μM glycine), acamprosate only inhibited [³H]dizocilpine binding (at concentrations concentrations > 100 μM). When these binding studies were performed in the presence of 1 μM spermidine, the enhancing effects of acamprosate on [³H]dirocilpine binding were inhibited. The results show that acamprosate binds to a specific spermidine-sensitive site that modulates the NMDA receptor in a complex way. Together, with data from al Quatari et al. (see next paper), this work suggests that acamprosate acts as “partial co-agonist“ at the NMDA receptor, so that low concentrations enhance activation when receptor activity is low, whereas higher concentrations are inhibitory to high levels of receptor activation. This may be relevant to the clinical effects of acamprosate in alcohol-dependent patients during abstinence.     

The effects of Ca-free perfusion and the calcium paradox on [I] endothelin-1 binding to rat cardiac membranes

The binding characteristics of [¹²⁵I]endothelin-1 (ET-1) to cardiac membranes isolated from rat hearts subjected to Ca²⁺-free perfusion or the Ca²⁺ paradox were examined. The effect of treatment with 2, 3 butanedione monoxime (BDM), which inhibits the tissue damage associated with the calcium paradox, was also investigated. Membranes from rat hearts perfused under control conditions bound [¹²⁵I]ET-1 to a single population of sites with a B_max of 107·7 ± 3.7 fmol/mg protein and an affinity (K_D) of 153 ± 12 pM. Ten minutes of Ca²⁺-free perfusion resulted in a significant (P < 0.01) increase in B_max to 167.5 ± 8.3 fmol/mg protein without change in K_D. Ca²⁺ repletion following Ca²⁺-free perfusion tended to increase further the B_max (180.6 ± 10.4 fmol/mg protein) without change in K_D. Treatment with BDM attenuated but did not prevent the rise in B_max following Ca²⁺-free perfusion. Following Ca²⁺ repletion, however, B_max returned to control levels in the BDM treated group. These changes were not associated with changes in the ability of ET-1 and ET-3 to inhibit [¹²⁵I]ET-1 binding. The results demonstrate that Ca²⁺-free perfusion is associated with an increase in the binding site density of [¹²⁵I]ET-1 which is maintained or further increased upon Ca²⁺ repletion. If, however, the tissue damage associated with the Ca²⁺ paradox is prevented with BDM, Ca²⁺ repletion is associated with a reversal of the increase due to Ca²⁺-free perfusion.     

The Electronic Structure and Secondary Pyroelectric Properties of Lithium Tetraborate

We review the pyroelectric properties and electronic structure of Li₂B₄O₇(110) and Li₂B₄O₇(100) surfaces. There is evidence for a pyroelectric current along the [110] direction of stoichiometric Li₂B₄O₇ so that the pyroelectric coefficient is nonzero but roughly 10³ smaller than along the [001] direction of spontaneous polarization. Abrupt decreases in the pyroelectric coefficient along the [110] direction can be correlated with anomalies in the elastic stiffness C33D contributing to the concept that the pyroelectric coefficient is not simply a vector but has qualities of a tensor, as expected. The time dependent surface photovoltaic charging suggests that surface charging is dependent on crystal orientation and doping, as well as temperature.     

Treatment of cirrhotic rats with epidermal growth factor and insulin accelerates liver DNA synthesis after partial hepatectomy

Prevention of postoperative hepatic failure is important after hepatic resection. In patients with cirrhosis, impaired liver function and regenerative capacity after major hepatic resection are associated with increased morbidity and mortality. In this study, a combination of epidermal growth factor (EGF) and insulin were used as hepatotrophic factors in an attempt to stimulate DNA synthesis after 70% hepatectomy (HTX). Regenerative capacity was evaluated in normal and cirrhotic rat liver by measuring DNA synthesis in vivo. Micronodular liver cirrhosis was established by the simultaneous oral administration of CCl₄ and phenobarbital. Epidermal growth factor plus insulin was injected subcutaneously immediately after and 12 h after HTX or sham operation was performed. Rats were killed 24 h after the operation and liver regeneration was estimated by [³H]-thymidine incorporation into DNA as well as an autoradiographic nuclear labelling index. Hepatectomy increased [³H]-thymidine incorporation significantly in both normal and cirrhotic rats. In cirrhotic rats, [³H]-thymidine incorporation after HTX was significantly lower than in normal rats and administration of a combination of EGF and insulin after HTX enhanced [³H]-thymidine incorporation. In conclusion, DNA synthesis 24 h after HTX is decreased in cirrhotic rats compared with normal rats and EGF supplementation with insulin accelerates DNA synthesis in hepatectomized cirrhotic rats. The data suggest that administration of combinations of exogenous hepatotrophic factors may play a useful role in the treatment of cirrhotic patients undergoing major hepatic resection.     

Alterations of GTP-binding Proteins (Gsα and Gq/11α) in Gastric Smooth Muscle Cells from Streptozotocin-Induced and WBN/Kob Diabetic Rats

We investigated possible impairment of the signal transduction system in gastric myocytes of streptozotocin-induced diabetic (STZ) and spontaneous diabetic WBN/Kob (WBN/Kob) rats. Gastric motility 10 weeks after the onset of diabetes mellitus was significantly reduced in both diabetic rats compared with control, and the decreased motility was not recovered by the administration of insulin to maintain normal blood glucose levels. There was no significant difference between both types of diabetic rats and control rats in total number of [³H]quinuclidinyl benzilate ([³H]QNB) binding sites (B _max: 545–587 fmol/mg protein) on gastric smooth muscle cell membranes or in the affinity of [³H]QNB for the binding sites(K _d : 0.06–0.07 nM). Immunoblot analysis using polyclonal anti-G-protein antibodies indicated increased expression of G_s in gastric smooth muscle cell membranes, but no significant change in G_i or G_q/₁₁ expression in STZ rats, and decreased expression of G_q/₁₁ with no significant change in G_s and G_i in WBN/Kob rats. The cAMP production in gastric smooth muscle cell membranes was augmented in the absence and presence of 100 M isoproterenol, and 100 M forskolin in STZ rats, whereas no significant change of cAMP production was observed in WBN/Kob rats irrespective of the presence of the stimulants. These findings suggest that long-standing diabetes may induce alterations in signal transduction at downstream receptors in gastric myocytes, resulting in the impairment of gastric motility, although the mechanism of reduced contractile activity may differ between STZ and WBN/Kob rats.     

In vitro studies on the relative potency of bronchodilator agents

This study describes a rapid in vitro assay for the order of potency of bronchodilator drugs using specific binding of (−)-[³H] dihydroalprenolol ([³H]DHA) to rat lung membranes. Under linear conditions with respect to tissue, specific binding of [³H]DHA showed saturability, rapid kinetics of association and dissociation of radioligand, and sterospecificity. Nanomolar (nM) concentrations for 50% inhibition (IC₅₀±SE) for the bronchodilator drugs examined were as follows: albuterol, 1485±170; isoproterenol, 136±53; procaterol, 162±28; terbutaline, 3310±934; and zinterol, 51±8.3. A comparison of binding studies using rat lung tissue membranes and similar preparations of rat heart and skeletal muscle demonstrated that lung tissue had 7 to 8 times more receptor sites (B_max) for [³H]DHA than heart or skeletal muscle. Adenyl cyclase activit of the rat lung membrane preparation almost doubled in the presence of (−)-isoproterenol. Displacement of specific (³H)DHA binding in membrane preparations may provide useful data for evaluating bronchodilator compounds.     

Autoradiographic Localization of Dopamine D2-Like Receptors in the Rat Adrenal Gland

The pharmacological profile and the anatomical localization of dopamine D₂-like receptors were studied in sections of the rat adrenal gland using combined radioligand binding and autoradiographic techniques with [³H]-spiroperidol as a ligand. [³H]-Spiroperidol was bound to sections of the rat adrenal gland in a manner consistent with the labelling of dopamine D₂-like receptor sites. The binding was time-, temperature-and concentration-dependent and of high affinity with a dissociation constant (K_s) value of 1.6 ± 0.04 nM and a maximum density of binding sites (B_max) of 60 ± 3.6 fmol/mg tissue. Experiments on the pharmacological specificity of [³H]-spiroperidol binding to sections of the rat adrenal gland suggest the labelling of dopamine D₃ and/or D₄ receptors. The presence of dopamine D₃ and D₄ receptors in the rat adrenal gland was confirmed by the demonstration of a specific binding for the D₃ radioligand [³H]-7-hydroxy-N,N-di-n-propyl-2-aminotetralin (DPAT) and for the D₄ radioligand [³H]-clozapine. Light microscope autoradiography showed the highest accumulation of silver grains which correspond to [3H]-spiroperidol binding sites in the rat adrenal medulla. In the adrenal cortex, where density of silver grains is about 40% lower than in the medulla, the radioligand is accumulated primarily in the zona glomerulosa and to a lesser extent in the zona reticularis. These findings suggest that dopamine D2-like receptor sites in the rat adrenal gland cortex are primarily involved in the modulation of catecholamine secretion from the medulla and of aldosterone secretion from the cortex. The possible relevance of the occurrence of dopamine D3 and D4 receptor subtypes in the adrenal gland is discussed.     

Gamma-aminobutyric acid (GABAA) receptor-function in a rat model of hepatic encephalopathy

The functional activity of the gamma-aminobutyric acid (GABA_A) receptor-chloride ionophore complex was studied in rats with hepatic encephalopathy (HE) secondary to thioacetamide-induced fulminant hepatic failure (FHF). Muscimol stimulation and benzodiazepine potentiation of GABA receptor-mediated³⁶Cl^– uptake into cerebral cortical synaptoneurosomes was compared in HE and control rats. [³H]Flumazenil binding assays were conducted to determine whether the levels of endogenous benzodiazepine-like ligands in extracts of cortex were increased with stages of encephalopathy in this animal model of HE. In both control and HE rats maximal uptake of³⁶Cl^– via the GABA_A receptor complex occurred at muscimol concentrations of 30M. Potentiation of muscimol-stimulated³⁶Cl^– uptake into synaptoneurosomes by diazepam (5M) was equivalent in both groups. Aqueous extracts of proteolytically digested homogenates of cerebral cortices prepared from control and HE rats were effective in stimulating³⁶Cl^– uptake into synaptoneurosomes. Alkaline organic extracts of proteolytically digested homogenates of cerebral cortices from HE rats were more effective than corresponding extracts from controls at inhibiting the binding of [³H]flumazenil. Inhibition of [³H] fumazenil binding by organic extracts derived from the cerebral cortices of HE rats did not increase with progression of encephalopathy. The results show that muscimol-stimulated³⁶Cl^– uptake into synaptoneurosomes and, consequently, GABA^A receptor-mediated chloride channel function are not significantly altered in the model of HE studied and are consistent with the hypothesis that HE results in an increased availability of one or more endogenous ligands which can augment GABA receptor-gated chloride conductance.     

Type I and type II insulin-like growth factor receptors and their function in human Ewing's sarcoma cells

Summary Binding studies using recombinant human¹²⁵I-labelled insulin-like growth factor I ([¹²⁵I]IGF-I) revealed IGF-I receptors in three Ewing's sarcoma cell lines withK _d ranging from 74×10^–12 M to 100×10^–12 M andB _max=36–63 fmol/mg cell protein. [¹²⁵I]IGF-I binding was displaced by IGF-I, IGF-II and insulin with IC₅₀ values of 1.5 nM, 6.3 nM and 0.7 M respectively. Recombinant human [¹²⁵I]IGF-II radioligand-binding assays in the cell lines disclosed specific binding sites for IGF-II withK _d=(110–175)×10^–12 M andB _max varying from 21 fmol/mg to 72 fmol/mg cell protein. Neither IGF-I nor insulin displaced [¹²⁵I]IGF-II binding. IGF-I was found to increase basal glucose transport by maximally 1.5 times with EC₅₀=0.9 nM IGF-I. The efficacy and potency of IGF-II on glucose uptake were comparable to those of IGF-I whereas insulin was ineffective. IGF-I and IGF-II also provoked stimulation of glycogen synthesis in Ewing's sarcoma cells. The maximal glycogenic response was reached at 0.01 M IGF-I and 0.1 M IGF-II, the EC₅₀ value being approximately 1 nM IGF-I and 2 nM IGF-II. Insulin did not significantly influence glycogen formation. IGF-I and IGF-II but not insulin increased DNA synthesis in Ewing's sarcoma cells. The maximal mitogenic response was obtained with 10 nM IGF-I or IGF-II with an EC₅₀ value of about 0.7 nM for both peptides. -IR-3, a monoclonal antibody specific for the IGF type I receptor, effectively blocked IGF-I- and IGF-II-mediated metabolic responses. In conclusion, the data show that IGF-I and IGF-II induce rapid and longterm biological responses in Ewing's sarcoma cells exclusively through interaction with IGF type I receptors.     

CNQX binding to non-NMDA glutamate receptors in canine cerebro-cortical crude synaptosomal membranes: Pharmacological characterization and comparison of binding parameters in dogs with congenital portosystemic encephalopathy and control dogs

The pharmacological profile and binding characteristics of the non-NMDA antagonist of glutamate receptors [³H]6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), were investigated in triton-washed crude synaptosomal membranes prepared from canine cerebral cortex. [³H]CNQX binding was inhibited by various glutamate agonists and antagonists, the rank order of potency being CNQX>-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)=quisqualate=kainate>glutamate. Two binding sites for [³H]CNQX were apparent when non-specific binding (NSB) was defined with unlabelled CNQX. In contrast, when NSB was defined with saturating concentrations of unlabelled AMPA and kainate, only one binding site was identified which corresponded to the high affinity site identified when CNQX was used to define NSB. No physiologically relevant differences were found in binding parameters for [³H]CNQX membranes from dogs with congenital portosystemic encephalopathy (PSE) when compared with control dogs. The affinity constant (K_i of AMPA displacement of [³H]CNQX binding was not significantly different in PSE dogs compared with control dogs. These results suggest that the antagonist site on cortical non-NMDA receptors is not perturbed in dogs with congenital PSE.     

Involvement of eicosanoids and macrophage-like cells in cytokine-mediated changes in rat myenteric nerves

Proinflammatory cytokines alter function in enteric nerves, but little is known about underlying mechanisms. This study was designed to investigate the roles of prostanoids and of macrophage-like cells in cytokine-induced suppression of [³H]norepinephrine release from rat myenteric plexus. The release of ³H from jejunal longitudinal muscle-myenteric plexus preparations that had been loaded with [³H]norepinephrine was measured. Measurements of ³H release as well as concentrations of prostaglandin E₂ and leukotriene were made in preparations exposed to interleukin 1β plus interleukin 6 and in the presence or absence of piroxicam, 5-lipoxygenase inhibitor MK886, cycloheximide, or cyclosporin A. An ultrastructural analysis was also performed to investigate the presence of macrophage-like cells in the myenteric plexus. Interleukin 1β plus interleukin 6 suppressed ³H release and caused an increase in tissue prostaglandin E₂ butnot leukotriene E₄. Piroxicam and cycloheximide but not MK886 attenuated the cytokine-induced increase in prostaglandin E₂ and the suppression of [³H]norepinephrine release. Ultrastructural analysis showed macrophage-like cells in the plexus, and the cytokine effects were inhibited by cyclosporin A. Prostanoids but not leukotrienes mediate the cytokine-induced suppression of norepinephrine release, and the results of this study suggest that macrophage-like cells are also involved.     

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.