Platelet-derived growth factor (PDGF-BB) and brain-derived neurotrophic factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions.

The effects of i.c.v. infused platelet-derived growth factor and brain-derived neurotrophic factor on cell genesis, as assessed with bromodeoxyuridine (BrdU) incorporation, were studied in adult rats with unilateral 6-hydroxydopamine lesions. Both growth factors increased the numbers of newly formed cells in the striatum and substantia nigra to an equal extent following 10 days of treatment. At 3 weeks after termination of growth factor treatment, immunostaining of BrdU-labeled cells with the neuronal marker NeuN revealed a significant increase in newly generated neurons in the striatum. In correspondence, many doublecortin-labeled neuroblasts were also observed in the denervated striatum following growth factor treatment. Further evaluation suggested that a subset of these new neurons expresses the early marker for striatal neurons Pbx. However, no BrdU-positive cells were co-labeled with DARPP-32, a protein expressed by mature striatal projection neurons. Both in the striatum and in the substantia nigra there were no indications of any newly born cells differentiating into dopaminergic neurons following growth factor treatment, such that BrdU-labeled cells never co-expressed tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. In conclusion, our results suggest that administration of these growth factors is capable of recruiting new neurons into the striatum of hemiparkinsonian rats.

The effect of amiodarone on the beta-adrenergic receptor is due to a downregulation of receptor protein and not to a receptor-ligand interaction.

Downregulation of beta adrenergic receptors (beta-AR) by amiodarone (Am) have been reported in several studies both in vivo and in vitro. The mechanism underlying the antiadrenergic effect of Am is, however, still unclear. The aim of this study was to characterize whether the antiadrenergic effect of amiodarone is due to binding to the beta-AR or to downregulation of the beta-AR receptor protein. All experiments were performed on confluent mouse AT-1 cardiomyocytes cultured for 6 days. In acute experiments, equilibrium binding with [3H]-CGP-12177 to beta-AR was not directly inhibited by Am and the equilibrium binding constant did not change during prolonged exposure up to 72 hours. After Am exposure for 48 hours beta-AR density was decreased by 26% (p<0.005). T3 partially prevented the downregulation elicited by Am (p<0.05). A Western blot analysis with beta1-AR antibodies revealed a decreased signal intensity in cells treated with Am for 48 h as compared to control (p<0.05). Isoproterenol-provoked cAMP response did not change after acute exposure to Am. After incubation for 48 hours with Am there was, however, a 20% decrease in cAMP response as compared to control (p<0.05). This study shows that the effect of Am on beta-AR is due to a downregulation of the beta-AR protein and not to a competitive or non-competitive receptor-ligand interaction. This indicates a new pharmacological mechanism for modulation of beta-AR, which probably is transcriptionally regulated.

Desethylamiodarone prolongation of cardiac repolarization is dependent on gene expression: a novel antiarrhythmic mechanism.

Desethylamiodarone (DEA) is the major metabolite of amiodarone and has similar electrophysiologic effects with prolongation of the repolarization that is reversed by thyroid hormone (T3). Some of the electrophysiologic effects are probably due to antagonism of T3 at the receptor level. Such effects of T3 are mediated by modulation of gene transcription. The aim of this study was to investigate whether cycloheximide (Cy), an inhibitor of protein synthesis, and actinomycin D (ActD), a RNA-synthesis inhibitor, block DEA-induced prolongation of the repolarization and whether DEA takes part in the autoregulation of the nuclear thyroid hormone-receptor subtypes (ThR). Corrected monophasic action potentials (MAPc) and QTc were measured in Langendorff-perfused guinea pig hearts for 1 h. The hearts were continuously perfused with (a) vehicle, (b) 7.5 microM Cy, (c) 5 microM DEA, (d) 5 microM DEA + 7.5 microM Cy, (e) 1 microM T3, (f) 5 microM DEA + 1 microM T3, (g) 1.5 microM ActD, and (h) ActD + DEA. A potassium channel blocker with class III antiarrhythmic effects, 0.5 microM almokalant, was used as a control, separately and together with Cy. Western blot analysis for the ThR subtypes alpha, beta1, and beta2 was performed on vehicle- and DEA-treated hearts. DEA increased MAPc by 19% (p < 0.0005) and QTc by 18% (p < 0.0005). There was no effect on MAPc or QTc when Cy, ActD, or T3 was added with DEA. Almokalant increased MAPc by 14% (p < 0.005) and QTc by 13% (p < 0.0005). When Cy was present, almokalant still induced a similar prolongation of MAPc by 14% (p < 0.005) and QTc by 17% (p < 0.0005). Western blot analysis revealed no change in the expression of the ThR protein. In conclusion, the prolongation of the cardiac repolarization by DEA, but not almokalant, can be totally blocked by Cy and ActD. This indicates that the class III action of DEA is at least in part dependent on transcription rather than a direct effect on cell-membrane channels or receptors. The action of DEA could be reversed by T3, indicating an antagonism between DEA and T3. These results suggest a new antiarrhythmic mechanism dependent on gene expression.

Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta.

The rat estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ER alpha and rat ER beta protein revealed a single binding component for 16 alpha-iodo-17 beta-estradiol with high affinity [dissociation constant (Kd) = 0.1 nM for ER alpha protein and 0.4 nM for ER beta protein]. Most estrogenic substances or estrogenic antagonists compete with 16 alpha-[125I]iodo-17 beta-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.

Application of a novel method for the comparison of DNA binding parameters of the two human thyroid hormone receptor subtypes hTR alpha 1 and hTR beta 1.

DNA-binding characteristics of the two human thyroid hormone receptors alpha 1 and beta 1 (hTR alpha 1 and hTR beta 1) were studied by applying the recently developed solid-phase scintillation technique. Biotinylated double stranded oligonucleotides containing thyroid hormone response elements (TRE) were immobilized to streptavidin coated scintillating microtiter plates. The TRE:s consisted of variants of the consensus core sequence AGGTCA as monomers or as dimers in direct repeats. Equilibrium binding of radioactive labelled hTR alpha 1 and hTR beta 1 were studied. Metabolically 35S-labelled hTR (in vitro translated cDNA) as well as hTR expressed in the baculovirus-system and labelled with 125I-triiodothyronine (125I-T3) were used. In binding saturation experiments, the affinity for the TRE:s investigated did not differ greatly between hTR alpha 1 and hTR beta 1. No significant effects of T3 on the amplitude of DNA binding of either hTR alpha 1 or hTR beta 1 to the single site response elements could be demonstrated. Receptor binding to direct repeats was stimulated by the hormone in the case of the hTR beta 1. The hTR alpha 1 binding to direct repeats was not significantly altered by T3. The single site octameric variant of a TRE, TAAGGTCA, was observed to bind tighter to the hTR:s as compared to the hexameric variant AGGTCA. In the binding competition format, with one response element immobilized and other (un-biotinylated) added to the reaction mixture, there was a larger dynamic range for the affinity constants (IC50) as compared to the affinity constants (Kd) obtained in the binding saturation experiments. The present quantitative results confirm previous reports obtained with qualitative methods like gel shift assays. The method described here is applicable in basic research concerning characterisation of DNA binding of nuclear receptors. It also lends itself to automatization in high capacity formats.

Quantitative determination of DNA-binding parameters for the human estrogen receptor in a solid-phase, nonseparation assay.

Binding of transcription factors to specific sequences of DNA has been studied for more than a decade and has become a very productive field of research. This paper describes the application of the recently developed technique of scintillating microtitration plates in the study of protein-DNA interactions. A DNA sequence containing the classical consensus estrogen response element derived from the 5' upstream regulatory region of the frog vitellogenin gene was immobilized to scintillating microtitration plates. Equilibrium binding studies and kinetic studies were performed with [3H] estradiol labeled human estrogen receptor. The observed equilibrium dissociation constant (Kd) was 2.0 +/- 0.3 nM and the observed Hill coefficient of 2.0 indicated a positive cooperativity. Two association rate constants were observed, one slower of 0.3 x 10(6) M-1 min-1 for lower concentrations of estrogen receptor and one faster of 6.3 x 10(6) M-1 min-1 for higher concentrations. The dissociation rate was 0.005 min-1. The technique described has a potential in basic research concerning characterization of DNA binding. It is also well suited to applied research as a tool in high-throughput screening of compound libraries in the search of agents inhibiting transcription factor binding to DNA.

Amiodarone is a dose-dependent noncompetitive and competitive inhibitor of T3 binding to thyroid hormone receptor subtype beta 1, whereas disopyramide, lignocaine, propafenone, metoprolol, dl-sotalol, and verapamil have no inhibitory effect.

The cardiovascular and electrophysiological effects of amiodarone resemble those of hypothyroidism. The drug has a structural resemblance to thyroid hormone (T3). Previous studies indicate that amiodarone exerts its major effect through antagonism of T3, probably as a result of inhibition of ligand binding to the thyroid hormone receptor (ThR). There are five subtypes of ThR, of which the beta 1 is the most prominent in the human heart. Our first aim was to investigate whether ThR is involved in a general antiarrhythmic mechanism for antiarrhythmic drugs or whether this action is specific for amiodarone. Therefore, we studied the affinity of one antiarrhythmic drug from every Vaughan-Williams group on T3 binding to human ThR beta 1 (hThR beta 1). Second, we wished to investigate whether amiodarone is a competitive or noncompetitive inhibitor. hThR beta 1, expressed in insect cells using a recombinant baculovirus, was used in regular binding competition assays. Disopyramide, lignocaine, propafenone, metoprolol, dl-sotalol, and verapamil had no effect on T3 binding to hThR beta 1. Amiodarone showed a noncompetitive binding pattern at low concentrations (0.25-2 microM) and a competitive binding at high concentrations (2-8 microM). Among the antiarrhythmics tested, only amiodarone had affinity for hThR beta 1. This may represent a novel type of antiarrhythmic mechanism. The finding that amiodarone, in concentrations corresponding to therapeutic range in plasma, shifts from a noncompetitive to a competitive inhibitor, is of clinical interest in comparisons of low- and high-dose treatment.

Evidence for the presence of functional thyrotropin receptor in cardiac muscle.

There is increasing evidence that the membrane-bound thyrotropin receptor (TSHR) may be mediating clinically important direct effects of thyrotropin (TSH) and of TSHR antibodies (TSHRab) in extra-thyroidal tissues. TSHR mRNA has formerly been detected in thyroid, retroorbital muscle and fibroblasts, peripheral lymphocytes and rodent fat. It is well known that thyroid disease may aggravate or induce heart disease, but the pathophysiological role of TSH and TSHRab is not clear. The aim of this study was to investigate if TSHR is present in cardiac muscle. Reverse transcriptase polymerase chain reactions revealed TSHR in human heart and Northern blot on extracted RNA showed a RNA species of 4.4 kb. TSH stimulation of cultured mouse AT-1 cardiomyocytes elevated the levels of intracellular second messenger 3',5'-cyclic AMP. This effect of TSH could be inhibited by TSHR antibodies. In solution hybridization levels of TSHR mRNA in AT-1 cells were 50% of mRNA in crude mouse heart. In conclusion functional TSHR is present in cardiomyocytes.

Beta-adrenergic receptor function in cultured AT-1 cardiomyocytes.

AT-1 cells are highly differentiated, contracting cardiomyocytes derived from atrial tumours in transgenic mice. The aim of this study was to characterize beta-adrenergic receptor function and associated intracellular calcium regulation in AT-1 cells. Equilibrium binding experiments with [3H]-CGP-12177 showed a Kd = 0.30 +/- 0.08nM and a Bmax = 2.25 +/- 0.47 fmol/10(5) cells. Competition binding experiments with CGP-20712A showed presence of predominantly beta 1-adrenoreceptors. S-(-)propranolol, atenolol and R-(+)propranolol showed a competitive inhibition of binding with successively lower affinity. Isoproterenol, 2 microM, for 48 hours down-regulated the number (p < 0.05) of beta-adrenergic receptors/cell by about 50%; 10 microM for one hour increased the cAMP concentration (p < 0.05) by about 100%. Cytosolic [Ca2+] was measured flourimetrically in spontaneously and synchronously beating AT-1 cells. The resting cytosolic concentration was 94 +/- 10 nM. The observed sinusoidal Ca2+ oscillation frequency increased after addition of 10 microM isoproterenol (p < 0.02). This effect was antagonized by 10 microM alprenolol (p < 0.01). In conclusion, AT-1 cells have functional beta-adrenoreceptor signalling pathways and constitute an important tool in cardiac biology.

Scintillating microtitration plates as platform for determination of [3H]estradiol binding constants for hER-HBD.

A novel approach to direct determination of ligand binding constants for the human estrogen receptor hormone binding domain was developed. Recombinantly produced human receptor in yeast extracts was attached to scintillating microtitration plates. Radioligand binding to receptors was determined in a multi-detector scintillation counter designed for the microtitration plate format. The method was employed in equilibrium binding experiments, in binding competition tests and in determination of kinetic rate constants. The results obtained show that the methodology is valid in comparison to previously published data regarding hormone binding characteristics of estrogen receptors. Furthermore, the methodology offers several advantages over previous binding assays because the scintillating microtitration plates constitute both the binding reaction vial and the scintillant for the detection of bound radioactivity.

Equilibrium hormone binding to human estrogen receptors in highly diluted cell extracts is non-cooperative and has a Kd of approximately 10 pM.

It is generally accepted that the Kd for hormone binding to estrogen receptors in extracts ranges between 0.1-1 nM and that binding displays positive cooperativity due to formation of homodimers. After carefully optimizing assay procedures, to diminish ligand depletion phenomena and to fully control recoveries, we find a single class of non-interacting high affinity hormone binding sites with a Kd of approx. 10 pM. Ligand depletion was avoided by decreasing receptor concentrations to 5-8 pM. We were therefore obliged to employ radioiodinated estradiol as a probe as the specific radioactivity of tritiated estradiol was too low to maintain the accuracy of the binding assay. Human estrogen receptor extracted from the MCF7 cell line and recombinantly produced (in yeast) wild-type human receptor have identical equilibrium hormone binding characteristics.

The human estrogen receptor hormone binding domain dimerizes independently of ligand activation.

High level expression of biochemically active human estrogen receptor hormone binding domain (hER-HBD) was achieved using a Saccharomyces cerevisiae expression system. Using dissociation kinetic analysis, density gradient centrifugation and cross-linking studies, a spontaneous dimerization activity of hER-HBD independent of the presence of the DNA binding domain, ligand, and of elevated temperature is demonstrated.

Human parathyroid hormone (1-34) and (1-84) increase the mechanical strength and thickness of cortical bone in rats.

An anabolic effect on bone of intermittent parathyroid hormone (PTH) treatment has been found in patients with osteoporosis and also in experimental animals. Controversies exist, however, about whether the positive effect on the trabecular bone balance occurs at the expense of the cortical bone. We examined the biomechanical quality of cortical bone after intermittent treatment with different doses of PTH and, furthermore, compared the effects of PTH-(1-34) and PTH-(1-84). Groups of rats were treated with biosynthetic human PTH-(1-34) or PTH-(1-84), 1.1, 3.3, 10, or 30 nmol/kg/day for 30 days. No changes in the body weights and no changes in the lengths of the femora were observed after the PTH treatments. The biomechanical properties were analyzed by means of a materials-testing machine. A dose-related increase in the bending strength and stiffness of the femora was found, and this increase in mechanical strength corresponds with a 9-12% increase in the cross-sectional area of the femoral diaphyses. The deflection capability and energy absorption were not influenced by any of the PTH treatments. No differences were found between the effects of PTH-(1-34) or PTH-(1-84) on the biomechanical properties of the femora. Consequently, intermittent treatment with biosynthetic PTH-(1-34) or PTH-(1-84) increased the formation of cortical bone, and the biomechanical competence of the femora was found to be preserved.

Basal and ATP-stimulated phosphoinositol metabolism in fusing rat skeletal muscle cells in culture.

A considerable rise in inositol phosphates was observed at the beginning of myoblast fusion. Extracellular ATP, through P2-purinergic receptors, induced inositol phosphate accumulation before and after fusion; however, no effect of ATP on phosphoinositol levels could be detected during the period of fusion. The possibility of ATP being a fusion signal is discussed.

Receptor-triggered polyphosphoinositide turnover produces less cytosolic free calcium in cultured dysgenic myotubes than in normal myotubes.

Myotubes prepared from mice with muscular dysgenesis (mdg) were used to further elucidate the putative role of inositol triphosphate (InsP3) in excitation-contraction (E-C) coupling of skeletal muscle. The mdg mutation is characterized by an uncoupling of the E-C coupling. InsP3 production in normal and mdg/mdg myotube cultures and its relation to the levels of cytosolic free calcium were analyzed. Basal and ATP-stimulated levels of InsP3 were equal in normal and mdg/mdg myotube cultures. In contrast, the transient increases of cytosolic free calcium in mdg/mdg myotubes in culture were generally much lower than those in normal ones. This suggests that the defect in dysgenic myotubes does not rest on the InsP3 formation but on the InsP3-triggered transduction of excitation and/or the induction of calcium release from internal stores.

"On-line" measurement of acetylcholinesterase release from the substantia nigra of the freely-moving guinea-pig.

Acetylcholinesterase is released from dopaminergic cells within the substantia nigra. The functional significance of this phenomenon has been studied in the freely-moving animal by a novel system for measuring acetylcholinesterase release from the substantia nigra "on-line" and in vivo. In the unanaesthetized guinea-pig the amount of acetylcholinesterase released was significantly greater than during anaesthesia, and release occurred in a more pulsatile manner. In addition, release of acetylcholinesterase could be evoked by either pharmacological or physiological manipulations, i.e. (1) a depolarizing concentration of potassium ions administered locally; (2) metamphetamine, administered systematically, which also resulted in increased locomotor activity; (3) drinking behaviour, elicited by presentation of a water bottle. Although all three treatments were accompanied by an increase in acetylcholinesterase release within the substantia nigra, potassium-evoked release did not cause any detectable change in behaviour. It is therefore suggested that release of the protein acetylcholinesterase within the substantia nigra is not necessarily a direct cause of locomotor activity: rather, it reflects diverse sensorimotor events.

Measurement of cholinesterase activity released from the brain "on-line" and in vivo.

In this study we describe for the first time measurement of cholinesterase release from the brain "on-line" in the living animal, i.e. in a flow circuit incorporating a continuous supply of perfusate of a specific brain area, the substantia nigra. The basic approach has been to adapt a now well-established chemiluminescent assay. The different optimal physico-chemical conditions for various reaction stages of the assay were modified such that exogenous acetylcholinesterase, administered in pulses of short duration, could be detected above spontaneous hydrolysis of substrate in concentrations as low as 0.1 mU/ml. When exogenous enzyme was replaced by a constant delivery to the circuit of perfusate of the substantia nigra, a fluctuating signal was recorded, corresponding to spontaneous release of cholinesterase. In addition, potassium-evoked release of cholinesterase was seen to occur in a pulsatile fashion which would have been masked by the poor time resolution of any of the "off-line" methods. The potential value of this novel technique is discussed.

P2-purinoceptor-stimulated phosphoinositide turnover in chick myotubes. Calcium mobilization and the role of guanyl nucleotide-binding proteins.

ATP, a trigger of P2-purinoceptor-mediated polyphosphoinositide (PI) turnover in cultured myotubes, increased cytosolic calcium levels in a time- and dose-dependent manner (quin2 fluorescence). The calcium was released from intracellular stores, as acute addition of 5 mM EGTA was without significant effect. Adenosine 5'-(3-thiotriphosphate) and 5'-adenylyl imidodiphosphate also increased intracellular levels of inositol phosphates (InsP) and cytosolic calcium levels. Treatment with cholera or pertussis toxin of myotube cultures did not affect the P2-purinoceptor-mediated InsP increase although PI turnover in permeabilized myotubes was stimulated by guanosine 5'-(3-thiotriphosphate). The results suggest that myotube P2-purinoceptors trigger PI turnover and increase intracellular free calcium levels, via a mechanism insensitive to ADP-ribosylation, by cholera or pertussis toxin of guanyl nucleotide-binding (G) proteins. However, the presence of a phospholipase C-coupled G-protein was otherwise demonstrated.

Adenosine and neuropeptide Y enhance alpha 1-adrenoceptor-induced accumulation of inositol phosphates and attenuate forskolin-induced accumulation of cyclic AMP in rat vas deferens.

The action on alpha 1-adrenoceptor-coupled polyphosphoinositide breakdown of two modulators of adrenergic neurotransmission, adenosine and neuropeptide Y (NPY), was studied in pieces of rat vas deferens. Both adenosine and NPY dose-dependently increased the accumulation of inositol phosphates induced by phenylephrine (50 microM), but neither of the two compounds had any effect alone on inositol phosphate accumulation. In parallel experiments, adenosine and NPY dose-dependently decreased forskolin-induced cyclic AMP accumulation without affecting resting levels. Phenylephrine slightly increased forskolin (1 microM)-induced cyclic AMP accumulation, whereas treatment with agents that increase or mimic cyclic AMP (forskolin, prostaglandin E2, 8-Br-cyclic AMP) had no significant effect on phenylephrine-induced inositol phosphate accumulation. The previously described potentiation of the alpha 1-adrenoceptor-mediated contractile responses of the rodent vas deferens by adenosine and NPY is suggested to result from an enhanced alpha 1-adrenoceptor-induced accumulation of inositol triphosphate. Possible mechanisms are discussed.