Clinical and biochemical implications of low thyroid hormone levels (total and free forms) in euthyroid patients with chronic kidney disease.

OBJECTIVES: In this study, we explore the associations of decreased thyroid hormone levels with inflammation, wasting and survival in biochemically euthyroid patients with end-stage renal disease (ESRD). DESIGN: After exclusion of 23 patients with thyroid-stimulating hormone (TSH) values outside the normal range (0.1-4.5 mIU L(-1)), 187 clinically and biochemically euthyroid incident ESRD stage 5 patients starting dialysis were followed for a median of 20 (range 1-60) months. Measurements of total and free forms of thyroid hormones, s-albumin, hs-CRP, interleukin (IL)-6, vascular adhesion molecule (VCAM)-1 and insulin-like growth factor 1 (IGF-1) were performed at baseline. RESULTS: In this population, 17 out of 210 patients (8%) were defined as subclinically hypothyroid. Multivariate analysis, according to receiver operating characteristic (ROC) curves, showed that mortality was best predicted by total triiodothyronine (T3). When using the cut-off levels derived from ROC, low T3 levels were associated with increased inflammation (higher hs-CRP, IL-6 and VCAM-1) and lower concentration of both s-albumin and IGF-1. Finally, low T3 but not low free triiodothyronine was associated with worse all-cause (Likelihood ratio = 45.4; P < 0.0001) and cardiovascular mortality (Likelihood ratio = 47.8; P < 0.0001) after adjustment for confounding factors. CONCLUSION: This study showed that low T3 levels are independent predictors of all-cause and also cardiovascular disease mortality in biochemically euthyroid patients, perhaps due to an intimate association with inflammation. Based on these results, the use of T3 levels in studies assessing the relationship between thyroid dysfunction and mortality risk is recommended.

Structure-activity relationships and molecular modeling analysis of flavonoids binding to the benzodiazepine site of the rat brain GABA(A) receptor complex.

The affinities for the benzodiazepine binding site of the GABA(A) receptor of 21 flavonoids have been studied using [(3)H]flumazenil binding to rat cortical membranes in vitro. We show that flavonoids with high affinity for the benzodiazepine receptor in vitro spanning the whole efficacy range from agonists (1q) to inverse agonists (1l) can be synthesized. The receptor binding properties of the flavonoids studied can successfully be rationalized in terms of a comprehensive pharmacophore model recently developed by Cook and co-workers (Drug Des. Dev. 1995, 12, 193-248), supporting the validity of this model. However, in contrast to the requirement by the model that an interaction with the hydrogen bond-accepting site A2 is necessary for compounds to display inverse agonistic activity, 6-methyl-3'-nitroflavone (1l), which cannot engage in such an interaction, nevertheless displays inverse agonism. The analysis of the binding affinities of 3'- and 4'-substituted flavones in terms of the pharmacophore model has yielded new information for the further development of the pharmacophore model.

Mastoparan, a wasp venom peptide, stimulates release of prolactin from cultured rat anterior pituitary cells.

Studies have shown that mastoparan and other amphiphilic peptides induce exocytosis of hormones from anterior pituitary cells. We have studied the effect of mastoparan on the secretion of prolactin from cultured rat anterior pituitary cells and on the concomitant functional status of signal-transducing pathways in lactotroph-enriched cell cultures. Mastoparan stimulation of prolactin secretion was dose-dependent, time-dependent, reversible and required the presence of calcium. Pretreatment of pituitary cell cultures with cholera and pertussis toxin had no effect on the secretory response, whereas encapsulation of guanosine 5-[beta-thio]diphosphate (GDP-beta-S) by reversible electropermeabilization inhibited mastoparan-stimulated secretion. Incubation of mastoparan with myo-[3H]inositol-labelled lactotroph-enriched anterior pituitary cell cultures resulted in increased formation of inositol phosphates compared with control cells, and encapsulation of GDP-beta-S blocked mastoparan-induced inositol lipid hydrolysis. Mastoparan caused translocation of protein kinase C activity from a soluble to a membrane-attached form. Mastoparan was able to increase the intracellular Ca2+ concentration in Fura-2-loaded individual lactotrophs. Omission of Ca2+ from the extracellular medium did not change the Ca2+ response in lactotrophs when stimulated with mastoparan. On the basis of these results it is concluded that mastoparan-induced release of prolactin is preceded by activation of the inositol(1,4,5)trisphosphate/diacylglycerol pathway with resulting translocation of protein kinase activity and increment in intracellular Ca2+. However, other signal-transducing pathways may be involved in the secretory process.

Differential modulation of brain benzodiazepine receptor subtypes by ricinelaidic acid in vitro.

The C-18 hydroxy fatty acids ricinelaidic acid and ricinoleic acid diminish the oleic acid-stimulated agonist benzodiazepine binding in the rat brain in vitro. The oleic acid-induced enhancement of [3H]diazepam binding was completely abolished in membranes from the cerebellum, but only partially decreased in membranes from the hippocampus, cortex and the whole brain from 7-day-old rat pups. Related hydroxy fatty acids as well as hydroxy fatty acid esters had no effect on the oleic acid-stimulated diazepam receptor binding.

Substance P increases intracellular Ca2+ in individual rat pituitary lactotrophs, somatotrophs, and gonadotrophs.

The present study has investigated transients in the intracellular calcium concentration [Ca2+]i in response to substance P (SP) in single pituitary cells. SP raised [Ca2+]i in three subtypes of pituitary cells: lactotrophs, somatotrophs, and gonadotrophs. In all three cell subtypes the [Ca2+]i response to SP was amplitude-modulated and a concentration of 100 nM was necessary to elicit well pronounced two phased [Ca2+]i transients. The first phase was associated with increased generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in all three cell types. In lactotrophs, the second phase, but not the first, was blunted by depletion of extracellular Ca2+ (Ca2+ free EGTA incubation buffer) and by addition of dopamine (1 microM). In somatotrophs, the second phase of the SP-induced [Ca2+]i response was inhibited by depletion of extracellular Ca2+ and by addition of somatostatin (100 nM), while the first phase was unaffected by this treatment. In gonadotrophs, the second phase, but not the first, was inhibited by the Ca2+ channel blocker methoxyverapamil and depletion of extracellular Ca2+. SP was compared with other agonists having an action on lactotrophs, somatotrophs or gonadotrophs. These experiments demonstrated that SP was a weaker agonist in terms of maximal [Ca2+]i response than thyrotropin-releasing hormone (TRH) (in lactotrophs), growth hormone-releasing hexapeptide (in somatotrophs) and GnRH (in gonadotrophs). On the basis of these results it is concluded that SP exerts direct Ca2+ mobilizing effects in single lactotrophs, somatotrophs, and gonadotrophs derived from male peripubertal rats. The first phase in SP-induced [Ca2+]i transients is likely to be brought about by inositol 1,4,5-trisphosphate-mediated Ca2+ release from internal stores while the second phase reflects an influx of calcium through voltage-gated calcium channels.

The use of in vivo antisense oligonucleotide technology for the investigation of brain GABA receptors.

Antisense oligodeoxynucleotides (ODN) can be used as selective inhibitors of in vivo gene expression in the central nervous system (CNS) of experimental animals. The gamma-aminobutyric acid type A (GABAA) receptor is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors. GABAA receptor function is allosterically modulated by several clinically important compounds, e.g. 1,4-benzodiazepines, barbiturates and certain neurosteroids, which recognize binding sites within the receptor complex. GABAA receptor chloride channel complexes are probably pentamers of different polypeptide subunits. The number of known subunit families and isoforms (six alpha s, four beta s, three gamma s, one delta and two rho s) indicates an extensive heterogeneity of GABAA receptors. The gamma 2 subunit is a functionally integral part of the GABAA receptor, necessary for the high affinity binding of benzodiazepines. The infusion of phosphorothioate ODN antisense to the gamma 2 subunit mRNA, but not control sense or mismatch ODN, into the lateral cerebral ventricle or into the hippocampus of rats leads to significant decreases in benzodiazepine receptor radioligand binding. In the hippocampus this is accompanied by a decrease in the number of GABAA receptors and by a loss of neurones, the latter possibly being due to reduced GABAergic inhibitory neurotransmission. Autoradiographic analysis following continuous intrahippocampal infusion of antisense ODN shows the regional extent of the effect on [3H]flunitrazepam binding. The continuous infusion of antisense ODN, but not of mismatch control ODN, into the right lateral cerebral ventricle induced a significant decrease in benzodiazepine binding and [3H]muscimol binding to membranes of the right cortex. Antisense ODN infused into the striatum decreased benzodiazepine binding and binding to the GABA binding site of the GABAA receptor to an extent similar to that found in the hippocampus. It is concluded that the preferred route of administration of antisense ODN for in vivo studies of the GABAA receptor may be by infusion into defined rat brain regions. The reported data support the idea that antisense ODN can be used as a valuable tool for the investigation of the contribution of individual GABAA receptor subunits to the properties of the receptor complex and of mechanisms of receptor subunit assembly.

Arginine residue 120 of the human GABAA receptor alpha 1, subunit is essential for GABA binding and chloride ion current gating.

The effect of mutating the conserved amino acid residue arginine 120 to lysine in the GABAA receptor alpha 1 subunit was studied. In electrophysiological experiments, the arginine 120 lysine (R120K) mutation in the alpha 1 subunit, when co-expressed with beta 2 and gamma 2 subunits in Sf-9 insect cells, induces a 180-fold rightward shift of the GABA dose-response curve compared with wild type alpha 1 beta 2 gamma 2s GABAA receptors. The diazepam potentiation of GABA-gated chloride ion currents was not affected. The binding of the GABAA ligands [3H]muscimol and [3H]SR 95531 to alpha 1 (R120K) beta 2 gamma 2s GABAA receptors was abolished but the binding affinity of the benzodiazepine receptor ligand [3H]flunitrazepam was unchanged. These results suggest that the arginine residue 120 in the alpha 1 subtype of the GABAA receptor is essential for GABA binding.

Diazepam protects against rat hippocampal neuronal cell death induced by antisense oligodeoxynucleotide to GABA(A) receptor gamma2 subunit.

Antisense oligodeoxynucleotides (ODNs) are used for the selective inhibition of gene expression. Antisense ODNs are promising tools for the investigation of physiological implications of proteins in the central nervous system of rodents in vivo. We have previously demonstrated that a phosphorothioate antisense ODN to the GABA(A) receptor gamma2 subunit, but not sense or mismatch control ODNs, induces a decrease in ex vivo benzodiazepine receptor radioligand binding in rat hippocampus when infused into the hippocampus in vivo [Karle et al., Neurosci. Lett., 202 (1995) 97-100]. This effect is parallelled by a decrease in the number of GABA(A) receptors and an extensive loss of hippocampal neurones. There is increasing awareness of risks of toxic 'non-antisense' effects induced by ODNs, and in particular phosphorothioate ODNs. The present experiments were designed to investigate the specificity of effects induced by the gamma2 subunit antisense ODN. The temporal development of changes in [3H]flunitrazepam and [3H]quinuclidinyl benzilate binding as well as in tissue protein levels supports the notion that the antisense ODN primarily acts by blocking the expression of the targeted receptor subunit protein. Furthermore, it is shown that a threshold for the elicitation of neurodegenerative changes exists. Finally, it is demonstrated that diazepam treatment of rats protects against the development of neuronal cell death induced by the antisense ODN. Collectively, the results support the hypothesis that the neurodegeneration induced by the antisense ODN is a consequence of diminished GABAergic inhibitory tonus following a selective down-regulation of gamma2 subunit-containing GABA(A) receptor complexes.

[3H]diazepam specific binding to rat cortex in vitro is enhanced by oleic, arachidonic and docosahexenoic acid isolated from pig brain.

Substances were found in purified fractions from pig brain that enhanced the specific binding of [3H]diazepam to membranes from rat brain in vitro. These substances were identified as oleic acid, arachidonic acid and docosahexenoic acid. Oleic acid (10(-5) - 10(-4)M) increased the affinity for agonists binding to the benzodiazepine receptor, whereas the binding of antagonists to this receptor was only slightly enhanced. The number of [3H]muscimol binding sites was increased, whereas binding of [3H]SR 95331, a GABA receptor antagonist, was unchanged. The effect of oleic acid was additional to the GABA-induced enhancement of [3H]diazepam binding.

Thiomuscimol, a new photoaffinity label for the GABAA receptor.

Thiomuscimol inhibits [3H]muscimol binding to brain GABAA receptors. Exposure of Ag(+)-treated membrane preparations to UV radiation at 254 nm for 40 min in the presence of thiomuscimol (10(-5) M) produced a 20-30% irreversible decrease in high-affinity [3H]muscimol binding sites. The photoaffinity labeling of thiomuscimol was inhibited by GABA (10(-4) M) added prior to exposure to UV light. The data show that thiomuscimol can label the GABAA receptor site and that the ligand can be used as a photoaffinity label for purification and identification of GABA binding sites within the GABAA receptor complex.

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

Antisense oligonucleotide to GABAA receptor gamma 2 subunit induces loss of neurones in rat hippocampus.

The binding site for 1,4-benzodiazepines in the brain is part of the hetero-oligomeric gamma-aminobutyric acid (GABA)A receptor complex which regulates a chloride ion channel. The presence of the gamma 2 subunit in the complex is necessary for the binding of benzodiazepines to their binding site. This study demonstrates a reduction of benzodiazepine receptor radioligand binding by 43% compared to control following infusion of phosphorothioate antisense oligodeoxynucleotide to gamma 2 subunit into rat hippocampus. Reduction of benzodiazepine binding sites was paralleled by a decrease in [35S]tert-butyl-bicyclo-phosphorothionate ([35S]TBPS) binding (51%) and [3H]muscimol binding (37%), indicating a reduction in the number of GABAA receptors. Changed macroscopic appearance, reduced protein content and severe loss of neurones in antisense-treated hippocampi suggests that the reduced formation of GABAA receptors leads to neuronal cell death.

Isolation and identification from Salvia officinalis of two diterpenes which inhibit t-butylbicyclophosphoro[35S]thionate binding to chloride channel of rat cerebrocortical membranes in vitro.

Ethanolic extracts from dried leaves of sage (Salvia officinalis) showed inhibition of [35S]tertiary-butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes in vitro. This ligand is considered to bind to the chloride channel of the GABA/benzodiazepine receptor complex in brain tissue. Substances having inhibitory activity were purified and their chemical structure identified as the diterpenes carnosic acid and carnosol (IC50 values of 33 +/- 3 microM and 57 +/- 4 microM, respectively). The two compounds did not affect binding of the ligands [3H]muscimol and [3H]diazepam to the GABA/benzodiazepine complex in vitro. Saturation experiments of [35S]TBPS binding indicated that carnosic acid decreases the binding affinity.

Characterisation of the furanocoumarin phellopterin as a rat brain benzodiazepine receptor partial agonist in vitro.

Phellopterin, a naturally occurring furanocoumarin found in the roots of Angelica dahurica, inhibits [3H]diazepam and ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4] benzodiazepine-3-carboxylate ([3H]Ro 15-1788) binding to the benzodiazepine site of the rat brain gamma-aminobutyric acidA (GABAA) receptor in vitro with IC50 values of 400 and 680 nM, respectively. Two other naturally occurring furanocoumarins, byakangelicol and imperatorin were significantly less potent, with IC50 values for inhibition of [3H]diazepam binding of 8.0 and 12.3 microM, respectively. Scatchard plot analysis showed that the inhibitory activity of phellopterin was due to competitive inhibition of the benzodiazepine ligand binding. The results of GABA- and t-butylbicyclophosphorothionate (TBPS)-shift assays suggest that phellopterin is a partial agonist of the central benzodiazepine receptors in vitro.

Effects of 5-(4-piperidyl) isoxazol-3-ol (4-PIOL), a GABA(A) receptor partial agonist, on recombinant human GABA(A) receptors.

gamma-Aminobutyric acidA (GABA(A)) gated chloride ion channels were expressed from human recombinant cDNA using the baculovirus/Sf-9 insect cell expression system. The electrophysiological effects in whole-cell currents of 5-(4-piperidyl) isoxazol-3-ol (4-PIOL), a GABA(A) receptor partial agonist, were investigated on GABA(A) receptor complexes of alpha1beta2gamma2S subunits as well as a slightly modified construct of alpha1(valine 121)beta2gamma2S subunits. Here we report that (1)4-PIOL induces an inward whole-cell current in a concentration-dependent manner in both alpha1(val 121)beta2gamma2S and alpha1(ile 121)beta2gamma2S receptor subunit combinations. (2) The 4-PIOL induced whole-cell currents were more pronounced in alpha1(val 121)beta2gamma2S than in alpha1(ile 121)beta2gamma2S receptor subunit combinations. (3) 4-PIOL inhibited GABA-induced responses on alpha1(ile 121)beta2gamma2S and alpha1(val 121)beta2gamma2S receptor combinations with similar potency.

Calcium homeostasis in fibroblasts from patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disorder of the motor system in the CNS characterized by motor neuron death in the spinal cord, brain stem and cortex. Readily available tissues such as fibroblasts from ALS patients can serve as simple model systems to study the molecular mechanisms leading to degenerative disorders. We have used Fura-2 fluorescence microscopy and single-cell imaging to study the spatiotemporal dynamics of intracellular free calcium ([Ca2+]i) in primary cultures of fibroblasts from skin biopsies from ALS and normal subjects. Increases in [Ca2+]i were induced by stimulation with bradykinin (100 nM); neurotensin (50 nM); N-formyl-Met-Leu-Phe (chemotactic peptide) (1 microM); [Arg8]-vasopressin (1 microM) and histamine (10 microM). The levels of [Ca2+]i in 80-120 individual cells per agonist were monitored for 15 min. No significant differences were found in the resting levels of [Ca2+]i in control (102 +/- 4 nM) and ALS (98 +/- 6 nM) fibroblasts and in the maximal [Ca2+]i levels after stimulation with N-formyl-Met-Leu-Phe, [Arg8]-vasopressin, and histamine. Significantly lower [Ca2+]i transients were found in fibroblasts from ALS donors compared to controls when stimulated with neurotensin (p < 0.002) and bradykinin (p < 0.005). The percentage of individual cells reacting to a given agonist (40-100%) was similar in both groups. The molecular basis of the impaired calcium homeostasis in fibroblasts from ALS patients is not known, but a generalized membrane defect can be excluded since the [Ca2+]i responses are defective only when bradykinin or neurotensin are used as agonists.

Pyrrolizidine alkaloid-induced liver disease in horses: an early diagnosis.

Nine adult horses were fed alfalfa hay cubes containing approximately 10% Senecio vulgaris until all horses had consumed approximately the same amount of toxic components of S vulgaris, pyrrolizidine alkaloids (PA). The amount of PA consumed was determined by the amount that induced clinical signs of PA toxicosis in 3 horses. The 6 other horses were given similar amounts per kilogram of body weight. An initial decrease of feed intake was observed when horses' diets were changed from alfalfa cubes to alfalfa/Senecio cubes, and feed intake was decreased further over 89 to 98 days. From 50 to 159 days, body weight decreased in all horses. Liver disease was induced in all 9 horses after they ate an average of 233 +/- 9.2 mg of PA/kg of body weight. Eight horses died or were euthanatized. Treatment with branched chain amino acids had no effect on mortality, but appeared to reduce neurologic problems. Clinical signs of PA-induced liver disease included ataxia, head pressing, and decreased feed intake. Other clinical signs of toxicosis were observed individual horses, but did not develop in most horses. Megalocytic hepatopathy developed. Liver abnormalities proceeded as PA was consumed and were severe in 8 of 9 horses before clinical signs of toxicosis appeared. Sulfobromophthalein sodium clearance did not decrease until PA-induced liver disease was advanced. Bile acid (BA) concentrations increased to greater than or equal to 50 mumol/L, in the 8 horses that died. One horse had hepatopathy and increased BA concentration, but survived. In this horse, BA concentration peaked at 33 mumol/L and then decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and identification of tetrahydrocolumbamine as a dopamine receptor ligand from Polygala tenuifolia Willd.

The isolation and purification of a dopamine receptor ligand, tetrahydrocolumbamine from Polygala tenuifolia Willd is described. Tetrahydrocolumbamine was shown to inhibit the binding of [3H]-SCH23390 and [3H]-spiroperidol to rat striatum membranes in vitro with IC50 values of 0.75 +/- 0.08 mumol.L-1 and 0.92 +/- 0.10 mumol.L-1, respectively. The compound inhibited the binding of [3H]-prazosin (IC50 value of 46 mumol.L-1), while it did not change the binding of the ligands, [3H]-QNB and [3H]-muscimol to rat cortex in vitro. Scatchard plot analysis showed a mixture of competitive and non-competitive inhibition by the compound on both [3H]-SCH23390 and [3H]-spiroperidol binding to membranes from rat striatum.

Inhibition of [3H] flunitrazepam binding to rat brain membranes in vitro by puerarin and daidzein.

Compounds acting on the central nervous system (CNS) have been isolated and identified from plants used for medicinal purposes. Radix Puerariae is used in Chinese traditional medicine for the treatment of drunkenness and alcoholic addiction. Benzodiazepine tranquilizers exert their pharmacological effects by modulating the efficacy of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) at the GABA/benzodiazepine-chloride channel complex in the brain. Since some of the pharmacological effects of ethanol are thought to be mediated via the GABA/benzodiazepine-chloride channel complex, we investigated if extracts from Radix Puerariae contain active substances at the benzodiazepine receptor which could explain its reported usefulness for medical purposes. Therefore, a bioassay-guided purification of active substances from Radix Puerariae was initiated.