Specific Radioligand Research Articles

Detailed distribution of neuropeptide FF receptors (NPFF1 and NPFF2) in the rat, mouse, octodon, rabbit, guinea pig, and marmoset monkey brains: A comparative autoradiographic study

The distribution of neuropeptide FF receptors (NPFF(1) and NPFF(2)) was analyzed throughout the central nervous system of rodents (rat, mouse, Octodon degus, and guinea pig), rabbit, and marmoset monkey brains, representing three orders of mammals. Quantitative in vitro receptor autoradiography with [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)) and [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) as specific radioligands for NPFF(2) and NPFF(1) receptors, respectively, was used. The NPFF(2) receptor is predominantly expressed in all species, except in the central nervous system of Octodon degus, in which it is undetectable. The density of the NPFF(1) subtype is low in rat and mice, moderate in octodon, rabbit, and monkey, and relatively high in the guinea pig. The present study reveals prominent species differences in the NPFF receptors expression in the brain. The distribution pattern of NPFF(2) receptors in the diencephalon and the superficial layers of the spinal cord is consistent with a hypothesized potential role for NPFF in the modulation of sensory input and opioid analgesia. In contrast, the constant presence of NPFF(1) receptors in the septum, the nucleus of the tractus solitarius, and the hypothalamus suggest its participation in neuroendocrine functions.

Antinociceptive effect of morphine, but not mu opioid receptor number, is attenuated in the spinal cord of diabetic rats.

The mechanisms of decreased analgesic potency of mu opioids in diabetic neuropathic pain are not fully known. The authors recently found that G protein activation stimulated by the mu opioid agonist is significantly reduced in the spinal cord dorsal horn in diabetes. In the current study, they determined potential changes in the number and binding affinity of mu opioid receptors in the spinal cord in diabetic rats. Rats were rendered diabetic with an intraperitoneal injection of streptozotocin. The nociceptive withdrawal threshold was measured before and after intrathecal injection of morphine by applying a noxious pressure stimulus to the hind paw. The mu opioid receptor was determined with immunocytochemistry labeling and a specific mu opioid receptor radioligand, [3H]-(D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin ([3H]-DAMGO), in the dorsal spinal cord obtained from age-matched normal and diabetic rats 4 weeks after streptozotocin treatment. The antinociceptive effect of intrathecal morphine (2-10 microg) was significantly reduced in diabetic rats, with an ED50 about twofold higher than that in normal rats. However, both the dissociation constant (3.99 +/- 0.22 vs. 4.01 +/- 0.23 nm) and the maximal specific binding (352.78 +/- 37.26 vs. 346.88 +/- 35.23 fmol/mg protein) of [3H]-DAMGO spinal membrane bindings were not significantly different between normal and diabetic rats. The mu opioid receptor immunoreactivity in the spinal cord dorsal horn also was similar in normal and diabetic rats. The reduced analgesic effect of intrathecal morphine in diabetes is probably due to impairment of mu opioid receptor-G protein coupling rather than reduction in mu opioid receptor number in the spinal cord dorsal horn.

3H]PK11195 binding sites in human neutrophils: effect of fMLP stimulation and modulation in rheumatic diseases

Objective: The objectives of this study were to evaluate the [ 3H]PK11195 binding parameters in a model of acute inflammation, the N-formylmethionine-leucine-phenylalanine (fMLP)-stimulated neutrophil cell membranes, and to analyze if alterations of peripheral-type benzodiazepine receptor (PBR) characteristics occurred in neutrophil cell membranes of patients affected by osteoarthritis (OA), rheumatoid arthritis (RA), and psoriasic arthritis (PA). Design and methods: Neutrophils were obtained from 15 patients with OA, 15 patients with RA, and 15 patients with PA. fMLP stimulation was performed to aliquots of neutrophils from six healthy individuals. Evaluation of kinetic parameters of PBR was performed using [ 3H]PK11195, as specific radioligand compared with 15 healthy volunteers. Results: The results showed a significant decrease of K d and B max in fMLP-stimulated neutrophil membranes. Moreover, an increase of PBR binding sites and affinity value was observed in neutrophils membranes from PA patients. Conclusions: Our data suggested a fMLP modulation on [ 3H]PK11195 binding in human neutrophils. Moreover, our results showed an up-regulation of PBR in neutrophils of PA patients.

Na +-dependent high affinity binding of [ 3H]LY515300, a 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine opioid receptor inverse agonist

Analogues of 3,4-dimethyl-4-(3-hydroxyphenyl)piperidines are high affinity inverse agonists for μ-, δ- and κ-opioid receptors. To characterize inverse agonist binding, we synthesized a high specific activity radioligand from this series, [ 3H]LY515300 (3-[1-((3-cyclohexyl-[3,4- 3H 2])-3( R, S)-hydroxypropyl)-3( R),4( R)-dimethylpiperidin-4-yl]phenol). In membranes expressing cloned human opioid receptors, [ 3H]LY515300 binding was saturable and exhibited low nonspecific binding. [ 3H]LY515300 bound with high affinity to the μ- ( K d=0.07 nM), δ- ( K d=0.92 nM) and κ-( K d=0.45 nM) opioid receptors. High affinity [ 3H]LY515300 binding to all opioid receptors was Na +-dependent, a characteristic of inverse agonists. Displacement by standard opioid compounds yielded K i values consistent with their known opioid receptor affinities. Autoradiographic localization of specific [ 3H]LY515300 binding in rat and guinea pig brain was high in areas known to express high levels of opioid (particularly μ-opioid receptor) binding sites including the caudate, nucleus accumbens, and nucleus tractus solitarius. Thus, [ 3H]LY515300 is the first radiolabeled opioid receptor inverse agonist useful for the study of opioid receptors in cell lines and native tissues.

Antibodies against astrocyte M1 and M2 muscarinic cholinoceptor from schizophrenic patients' sera

We demonstrated the presence of circulating antibodies from schizophrenic patients able to interact with cultured astrocytes activating muscarinic acetylcholine receptors (mAChRs). Sera and purified IgG from 15 paranoid schizophrenic and 15 age-matched normal subjects were studied by indirect immunofluorescence (IFI), flow cytometry, dot blot, enzyme immunoassay (ELISA), and radioligand competition assays. Astrocyte membranes and/or a synthetic peptide, with identical amino acid sequence of human M(1) and M(2) mAChR, were used as antigens. By IFI and flow cytometry procedures, we proved that serum purified IgG fraction from schizophrenic patients, reacted to astrocyte cell surface. The same antibodies were able to inhibit the binding of the specific mAChR radioligand (3)H-QNB. Using synthetic peptide for dot blot and ELISA, we demonstrated that these antibodies reacted against the second extracellular loop of human cerebral M(1) and M(2) mAChR. Also, the corresponding affinity-purified antipeptide antibody displayed an agonistic-like activity associated to specific M(1) and M(2) mAChR activation, increasing inositol phosphates accumulation and decreasing cyclic AMP production, respectively. This article gives support to the participation of an autoimmune process in schizophrenia disease.

Aging-related alterations in the contractile responses to acetylcholine, muscarinic cholinoceptors and cholinesterase activities in jejunum and colon of the male Fischer 344 rats

In an attempt to examine whether the muscarinic receptor-activated intestinal function is altered by aging, we studied the changes in (1) contractile responses to acetylcholine (Ach), (2) muscarinic cholinoceptors and (3) cholinesterase (ChE) activities, in jejunum and colon of the young (2–3 months) and aged (24–28 months) Fischer 344 rats. In the physiological contraction experiments of jejunum and colon, Ach concentration-dependently increased the force of contraction, and the contractile responses to Ach were not affected by aging. In addition, the true- and pseudo-ChE activities were not significantly changed by aging. The Ach-induced contraction was competitively inhibited by muscarinic M3-selective antagonist hexahydro-sila-difenidolhydrochloride p-fluoroanalog ( p-F-HHSiD), suggesting that the contractile responses in the rat jejunum and colon were mediated through M3-cholinoceptor. Age-related changes in muscarinic cholinoceptors of jejunum and colon were determined with the use of specific muscarinic radioligand [ 3H]-quinuclidinylbenzilate (QNB). The [ 3H]QNB saturation binding experiments revealed that the maximal binding ( B max) was increased only in aged jejunum without changes in K D values. These results suggest that aging may not attenuate the Ach-induced intestinal contraction via muscarinic M3 receptor, although the expression of muscarinic cholinoceptor is differentially modulated in jejunum and colon.

New features of PDE5 as revealed by a potent catalytic-site specific radioligand

New features of PDE5 as revealed by a potent catalytic-site specific radioligand

Atomic force microscopy of ionotropic receptors bearing subunit-specific tagsprovides a method for determining receptor architecture

We have developed an atomic force microscopy (AFM)-basedmethod for the determination of the subunit architecture ofionotropic receptors, and tested the method using the GABAAreceptor as a model system. The most common form of the GABAA receptor probablyconsists of 2α1-,2β2- and1γ2-subunits.We show here that the arrangement of subunits around the central Cl−ion channel can be deduced by AFM of receptors tagged withsubunit-specific antibodies. Transfection of cells with DNA encoding α1-, β2- and γ2-subunitsresulted in the production of receptors containing all three subunits, as judged by bothimmunoblot analysis and the binding of [3H]-Ro15-1788,a specific radioligand for the GABAA receptor. AHis6-tagon the α1-subunitwas used to purify the receptor from membrane fractions oftransfected cells. After incubation with anti-His6immunoglobulin G, some receptors became tagged with either one or twoantibody molecules. AFM analysis of complexes containing two bound antibodiesshowed that the most common angle between the two tags was 135°, close to the value of144° expected ifthe two α-subunitsare separated by a third subunit. This method is applicable to thecomplete elucidation of the subunit arrangement around the GABAAreceptor rosette, and can also be applied to other ionotropic receptors.

Peripheral-type benzodiazepine receptors in human mononuclear cells of patients affected by osteoarthritis, rheumatoid arthritis or psoriasic arthritis

Objectives The objective of this study was to evaluate the kinetic parameters at equilibrium of peripheral benzodiazepine receptors (PBR) in human mononuclear cells from patients affected by osteoarthritis (OA), rheumatoid arthritis (RA) and psoriasic arthritis (PA). Design and methods Mononuclear cells were obtained from 10 patients with OA, 10 patients with RA and 10 patients with PA. Evaluation of kinetic parameters of PBR was performed using [ 3H]PK 11195, a specific radioligand for this receptor, and compared with 10 healthy controls. Results The results show a statistically significant decrease (37.5%, as an absolute percentage) in the maximal number of binding sites (B max) of patients with OA, compared with healthy controls; however, the values of the dissociation constant (K d) at equilibrium do not show any statistically significant variations. Conclusions These data further confirm the presence of peripheral biochemical alterations in OA. As peripheral benzodiazepine receptors appear to be involved in the immune function, and in the protection of hematopoietic cells against oxygen radical damage, the observed decrease in B max might be related to cellular protection.

Peripheral Benzodiazepine Binding Sites in Platelets of Patients Affected by Mitochondrial Diseases and Large Scale Mitochondrial DNA Rearrangements

The peripheral-type benzodiazepine receptors (PBR) are localized on the outer mitochondrial membrane, as a constituent of mitochondrial permeability transition (MPT)-pore. Among its hypothesized functions, the regulation of the mitochondrial respiratory chain and apoptosis have been suggested; in addition alterations of PBR site density have been shown in some neuropathologic conditions with putative mitochondrial involvement. The aim of this work has been to evaluate PBR kinetic binding parameters in platelets from patients affected by mitochondrial disorders (MD) with large-scale mitochondrial DNA deletions and reduced cytochrome c oxidase activity. Using the specific PBR radioligand [(3) H] PK 11195, the kinetic binding parameters of PBR sites were determined in platelet membrane of 15 healthy subjects and 11 patients affected by different form of MD. Significant changes of dissociation constant (K(d)) and maximal number of binding sites (B(max)) values were evidenced in platelets of patients versus controls. In all patients the B(max) values were decreased (2,387.0 +/- 305.6 fmol/ mg proteins versus 4889.0 +/- 357.8 fmol/mg proteins, p< 0.05), whereas the K(d) values were higher in patients than controls (13.18 +/- 2.06 nM versus 5.63 +/- 0.46 nM, p< 0.05). These data suggest that the kinetic binding parameters of PBR are altered in MD and that the observed changes might be related to the mitochondrial dysfunction associated with MD.

Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor.

Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A(4) receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.

The presence of delta and mu-, but not kappa or ORL 1 receptors in bovine pinealocytes

Physicians have noted since antiquity that their patients complained of less pain and required fewer analgesics at night-time. In humans, the circulating levels of melatonin, a pineal substance with analgesic and hypnotic properties, exhibit a pronounced circadian rhythm with serum levels being high at night and low during day-time. Moreover, pinealectomy abolishes the analgesic effects of melatonin, and naloxone disrupts the day–night rhythm of nociception. In this study, we have attempted to identify and characterize the nature and types of opioid receptor in bovine pinealocyte membranes, using a radioligand binding technique with the selective radioligands [ 3H]DAMGO, [ 3H]DPDPE, [ 3H]U69593 and [ 3H]orphanin-FQ (OFQ) for identifying mu (μ)-, delta (δ)-, kappa (κ)- and opioid receptor-like (ORL 1) receptors, respectively. The saturation experiments on bovine pinealocyte membranes for [ 3H]DPDPE binding provided B max and K d values of 553±24 fmol/mg protein and 1.3±0.6 nM; and for [ 3H]DAMGO binding provided B max and K d values of 6.3±1.3 fmol/mg protein and 1.2±0.4 nM, respectively. On the other hand, the specific radioligands ([ 3H]U69593 and [ 3H]OFQ) binding of κ and ORL 1 receptors were undetectable in bovine pinealocyte membranes. Furthermore, competitive experiments with opioid agonist and antagonist and related compounds confirmed the presence of μ- and δ-opioid binding sites in bovine pinealocyte membranes. These results indicate that neither κ nor ORL 1 receptors are present on the pinealocytes, and the majority of opioid receptors found in the bovine pineal gland are δ (possibly, both δ 1 and δ 2) types, with a minority being μ type, and that both are primarily located on the bovine pinealocyte membranes. These opioid receptors, by stimulating the activity of N-acetyltransferase, enhance the synthesis of melatonin.

Reconsideration of 5-hydroxytryptamine (5-HT)(7) receptor distribution using [(3)H]5-carboxamidotryptamine and [(3)H]8-hydroxy-2-(di-n-propylamino)tetraline: analysis in brain of 5-HT(1A) knockout and 5-HT(1A/1B) double-knockout mice.

The characterization and anatomical distribution of 5-hydroxytryptamine (5-HT)(7) receptor binding sites in brain tissue has been hampered by the lack of a specific radioligand. In the present autoradiographic study, we took advantage of 5-HT(1A) knockout and 5-HT(1A/1B) double-knockout mice to revisit the pharmacological characterization and anatomical localization of 5-HT(7) binding sites in mouse brain using [(3)H]5-carboxamidotryptamine (5-CT) and [(3)H]8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT). The distribution pattern of [(3)H]5-CT binding sites (2 nM) in the brain of mice lacking the 5-HT(1A/1B) receptor was scarce and confined to the septum, globus pallidus, thalamus, hypothalamus, amygdala, cortex, and substantia nigra. The low densities of [(3)H]5-CT binding sites detected in septum, thalamus, hypothalamus, amygdala, and cortex were displaced by 10 microM of the selective 5-HT(7) receptor antagonist (R)-3-(2-(2-(4-methylpiperidin-1-yl) ethyl)pyrrolidine-1-sulfonyl) phenol (SB-269970). The SB-269970-insensitive [(3)H]5-CT binding sites detected in globus pallidus and substantia nigra of 5-HT(1A/1B) knockout mice were displaced by N-[3-(2-dimethylamino)ethoxy-4-methoxy-phenyl]-2'-methyl-4'- (5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-carboxamide hydrochloride (SB-216641) (1 microM), demonstrating the 5-HT(1D) nature of these binding sites. In contrast to the low densities of [(3)H]5-CT binding sites, high-to-moderate densities of [(3)H]8-OH-DPAT binding sites (10 nM) were found throughout the brain of 5-HT(1A) and 5-HT(1A/1B) knockout mice (olfactory system, septum, thalamus, hypothalamus, amygdala, CA3 field of the hippocampus, cortical mantle, and central gray). These [(3)H]8-OH-DPAT binding sites were displaced by 10 microM SB-269970, risperidone, and methiothepin but not by pindolol, N-tert-butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropanamide (WAY- 100135), or citalopram. We conclude that despite its high affinity for the 5-HT(7) receptor in tissue homogenates, [(3)H]5-CT is not a good tracer for measuring 5-HT(7) receptor binding sites autoradiographically. Also, the lower affinity ligand [(3)H]8-OH-DPAT is a much better tracer for autoradiographic studies at the 5-HT(7) receptor binding sites.

Pharmacological evaluation of a Br-76 analog of epibatidine: a potent ligand for studying brain nicotinic acetylcholine receptors.

[(76)Br]-Norchlorobromoepibatidine ([(76)Br]BrPH) is a specific and high affinity radioligand for the nicotinic acetylcholine receptors (nAChRs). In vitro, on rat thalamus membranes [(76)Br]BrPH bound to two sites with apparent affinities of 8 pM and 3 nM. The density of binding sites were 1.9 and 70 fmol/mg protein, respectively. In vivo, in biodistribution and autoradiographic studies in rats the regional distribution of [(76)Br]BrPH paralleled the neuroanatomical localization of nAChRs. Two hours postinjection, the highest concentration in the brain was found in thalamus and colliculi (4% ID/g). Competition experiments with specific nicotinic, muscarinic, dopaminergic, and serotoninergic drugs confirmed that the in vivo binding of [(76)Br]BrPH was consistent with neuronal nicotinic receptors. PET imaging of [(76)Br]BrPH in baboon demonstrated a rapid and high uptake in the brain. Peak uptake occurred at 30-40 min for the thalamus. Due to the constant washout in the cerebellum, the thalamus to cerebellum ratio was 5 at 2 h postinjection. Subcutaneous injection of cytisine (1 mg/kg), 3 h postinjection of [(76)Br]BrPH reduced the radioactivity concentration in thalamus and cortex by 58 and 50%, respectively, as observed 1 h later. Cytisine pretreatment (5 mg/kg s.c.) inhibited completely the radioligand accumulation in the thalamus. Chronic MPTP pretreatment resulted in reduction of [(76)Br]BrPH uptake in all brain regions except in cerebellum. These preliminary results suggest that [(76)Br]BrPH has the potential to be a useful radioligand for studying the pharmacology of nicotinic acetylcholine receptors in preclinical experiments.

Pharmacologic and autoradiographic evidence for an up-regulation of kinin B(2) receptors in the spinal cord of spontaneously hypertensive rats.

1. The effects of intrathecally (i.t.) injected kinin B(1) and B(2) receptor agonists and antagonists were measured on mean arterial pressure (MAP) and heart rate (HR) of conscious unrestrained spontaneously hypertensive rats (SHR of 16 weeks old) and age-matched normotensive Wistar Kyoto (WKY). Quantitative in vitro autoradiographic studies were also performed on the thoracic spinal cord of both strains with specific radioligands for B(2) receptors, [(125)I]-HPP-Hoe 140, and B(1) receptors, [(125)I]-HPP-[des-Arg(10)]-Hoe140. 2. Bradykinin (BK) (0.81 - 810 pmol) increased MAP dose-dependently with increases or decreases of HR. The pressor response to BK was significantly greater in SHR. The cardiovascular response to 8.1 pmol BK was reversibly blocked by 81 pmol Hoe 140 (B(2) antagonist) but not by 81 - 810 pmol [des-Arg(10)]-Hoe 140 (B(1) antagonist) in both strains. 3. The B(1) receptor agonist, des-Arg(9)-BK (8100 pmol) produced either no effects or increased MAP with variable effects on HR. These responses were similar in both strains and were reversibly blocked by 81 pmol Hoe 140. Inhibition with 8100 pmol [des-Arg(10)]-Hoe 140 was not specific to B(1) agonist-mediated responses. 4. [(125)I]-HPP-Hoe 140 specific binding sites were predominantly located to superficial laminae of the dorsal horn and were significantly higher in SHR. Low levels of [(125)I]-HPP-[des-Arg(10)]-HOE 140 specific binding sites were found in all laminae of both strains. 5. It is concluded that the hypersensitivity of the cardiovascular response to BK is due to an increased number of B(2) receptors in the spinal cord of SHR and that B(1) receptors are unlikely involved in spinal cardiovascular regulation in SHR.

(+)-[ 76Br]A-69024: a non-benzazepine radioligand for studies of dopamine D1 receptors using PET

(+)-[ 76Br]A-69024 is a specific and enantioselective dopamine D1 receptor radioligand. The Bmax of (+)-[ 76Br]A-69024 measured in vitro on rat striatum membranes was 320 ± 25 fmoles/mg protein with an apparent dissociation constant of Kd = 0.6 ± 0.1 nM. The inactive enantiomer, (−)-[ 76Br]A-69024, displayed no affinity in the same assay. In vivo, the biodistribution (+)-[ 76Br]A-69024 in rats showed a rapid and high uptake in the striatum (1% ID/g), followed by a slow wash out. The striatum/cerebellum concentration ratio (index of specific binding) reached a maximum value of 10 at 60 minutes post injection. A tissue to cerebellum ratio of 2.8 and 1.5 was also observed for frontal and posterior cortex respectively. With the pharmacologically inactive enantiomer, (−)-[ 76Br]A-69024, the brain uptake was determined to be non specific since a striatum/cerebellum ratio of approximately 1 was observed throughout the time course of the experiment. The selectivity of (+)-[ 76Br]A-69024 uptake was demonstrated in competition experiments. The specific uptake in the striatum and cortical regions was completely prevented after administration of the D1 antagonist SCH 23390. Pre-treatment of rats with unlabelled (+)A-69024 also displayed the same regional inhibition of (+)-[ 76Br]A-69024 uptake. Pre-administration of rats with spiperone (D2) and ketanserin (5-HT 2/5-HT 2C) showed no inhibitory effect on (+)-[ 76Br]A-69024 uptake in any brain region. Using (+)-[ 76Br]A-69024, PET study in baboon demonstrated a preferential accumulation of the radioactivity in the striatum, frontal and posterior cortex which was displaced to the level of the cerebellum by SCH 23390. These results suggest that (+)-[ 76Br]A-69024 may deserve further investigation as a potential radioligand for studying striatal and cortical dopamine D1 receptors using PET.

Early cerebral activities of the environmental estrogen bisphenol A appear to act via the somatostatin receptor subtype sst(2).

Recently, considerable interest has been aroused by the specific actions of bisphenol A (BPA). The present investigation represents a first study dealing with the interaction of BPA with the biologically more active somatostatin receptor subtype (sst(2)) in the rat limbic circuit. After treating pregnant female Sprague-Dawley rats with two doses (400 microg/kg/day; 40 microg/kg/day) of BPA, the binding activity of the above receptor subtype was evaluated in some limbic regions of the offspring. The higher dose proved to be the more effective one, as demonstrated by the elevated affinity of sst(2) with its specific radioligand, [(125)I]-Tyr(0)somatostatin-14. The most dramatic effects of BPA on sst(2) levels occurred at the low-affinity states of such a subtype in some telencephalic limbic areas of postnatal rats (10 days of age; postnatal day [PND] 10). These included lower (p < 0.05) sst(2) levels in the gyrus dentate of the hippocampus and basomedial nucleus of the amygdala; significantly higher (p < 0.01) levels were observed only for the high-affinity states of the periventricular nucleus of the hypothalamus. A similar trend was maintained in PND 23 rats with the exception of much lower levels of the high-affinity sst(2) receptor subtype in the amygdala nucleus and ventromedial hypothalamic nucleus. However, greater changes produced by this environmental estrogen were reported when the binding activity of sst(2) was checked in the presence of the two more important selective agonists (zolpidem and Ro 15-4513) specific for the alpha-containing Gamma-aminobutyric acid (GABA) type A receptor complex. In this case, an even greater potentiating effect (p < 0.001) was mainly obtained for the low-affinity sst(2) receptor subtype in PND 10 animals, with the exception of the high-affinity type in the ventromedial hypothalamic nucleus and gyrus dentate. These results support the contention that an sst(2) subtype alpha-containing GABA type A receptor system might represent an important neuromediating station capable of promoting estrogenlike mechanisms of BPA, especially during the early developmental phases.

Increased incidence of anti-LKM autoantibodies in a consecutive cohort of hepatitis C patients from central Greece.

In Greece, there are insufficient data regarding the presence of non-organ and liver-related autoantibodies in hepatitis C patients. This study in a consecutive cohort of 39 such patients from central Greece investigates (1) the prevalence of non-organ and liver-related autoantibodies, and (2) the reactivity of anti-liver-kidney microsomal type 1 antibodies (in the case of positivity with at least one of the methods used) against their molecularly defined antigens. All serum samples were tested by standard and molecular assays for the presence of anti-nuclear antibodies, smooth muscle antibodies, anti-liver-kidney microsomal type 1 antibodies, antibodies against parietal cells, anti-CYP2A6, anti-CYP1A2 and anti-CYP2D6 autoantibodies. Indirect immunofluorescence, competitive enzyme-linked immunosorbent assays, immunoblotting and novel radioligand assays based on immunoprecipitation of [35S]-methionine labelled recombinant CYP2A6, CYP1A2 and CYP2D6 His-taq fusion proteins produced by in vitro transcription/translation were used. Seven out of 39 patients (17.9%) tested positive for smooth muscle antibodies, 2/39 (5.1%) tested positive for anti-nuclear antibodies, 1/39 (2.5%) tested positive for parietal cell antibodies, and 4/39 (10.3%) were found to be anti-liver-kidney microsomal positive (with at least one of the methods used). All sera were negative for anti-CYP2A6 and anti-CYP1A2 autoantibodies. Three out of four anti-liver-kidney microsomal positive samples had the typical liver-kidney microsomal staining pattern shown by indirect immunofluorescence. However, none tested positive for anti-CYP2D6 autoantibodies using the competitive CYP2D6 enzyme-linked immunosorbent assay, the specific CYP2D6 radioligand assay, and western blot using either human microsomes or recombinant CYP2D6. The fourth patient tested negative for anti-liver-kidney autoantibodies by either indirect immunofluorescence or the competitive enzyme-linked immunosorbent assay, but was repeatedly positive for anti-CYP2D6 autoantibodies by the sensitive and specific radioligand assay. Western blot experiments using human microsomes in this patient serum revealed two bands of 50 kDa and 55 kDa that documented as anti-CYP2D6 and anti-uridine triphosphate glucuronosyltransferase autoantibodies when recombinant CYP2D6 and recombinant uridine triphosphate glucuronosyltransferase autoantigens were used for immunoblot, respectively. A relatively high incidence of anti-liver-kidney microsomal autoantibodies (10.3%) was found in a consecutive sample of Greek patients with hepatitis C. The expanded panel of assays, however, failed to document CYP2D6 as the target autoantigen of anti-liver-kidney microsomal autoantibodies in most patients. We report for the first time the detection of parietal cell antibodies and both anti-CYP2D6 (anti-liver-kidney microsomal type 1) and anti-uridine triphosphate glucuronosyltransferase (anti-liver-kidney microsomal type 3) autoantibodies in patients who were hepatitis C positive/hepatitis D negative. Further studies are needed to confirm our findings and to determine whether these preliminary results have a clinical importance or not.

The role of beta(3)-adrenoceptors in mediating relaxation of porcine detrusor muscle.

1. beta-adrenoceptors mediate relaxation of bladder detrusor smooth muscle. This study investigates the contribution of beta(3)-adrenoceptors to relaxation of the pig urinary bladder. 2. Cell membranes were prepared from detrusor muscle of the pig bladder dome and competition experiments with [(3)H]-dihydroalprenolol (DHA), a non-selective beta-adrenoceptor antagonist was used as a specific radioligand to determine the presence of beta-adrenoceptor subtypes. In functional experiments, isolated detrusor muscle strips were used to determine the potency of agonists and the affinity of antagonists. 3. In competition binding experiments, CGP20712A (beta(1)-adrenoceptor selective) displaced [(3)H]-DHA from a single binding site with a low affinity. In contrast, displacement data for ICI 118551 (beta(2)-adrenoceptor antagonist) and SR59230A (beta(3)-adrenoceptor antagonist) best fitted a two-site model suggesting a predominant (70%) population of beta(3)-adrenoceptors. 4. In functional studies, isoprenaline and salbutamol (beta(2)-adrenoceptor agonist) relaxed KCl precontracted muscle strips with high potency (pEC(50) 7.7 and 7.2, respectively), whilst CGP12177 and BRL37344 (beta(3)-adrenoceptor agonists) had low potency and were partial agonists. CGP20712A and atenolol (beta(1)-adrenoceptor antagonists) antagonised responses with a low affinity. ICI118551 antagonized responses to isoprenaline and salbutamol with a high affinity (pK(B)=7.8 and 8.7, respectively), but the Schild slopes were low suggesting that responses were mediated by more than one beta-adrenoceptor. The Schild plot for SR59230A was biphasic, apparent pK(B) values for 3 - 10 nM SR59230A being 8.6 and those for 30 nM - 1 microM being 7.7. 5. These data suggest that beta(3)-adrenoceptors are the predominant beta-adrenoceptor subtype present in the pig bladder and that beta-adrenoceptor mediated responses of this tissue are mediated via both the beta(2)- and beta(3)-adrenoceptor subtypes.

In vivo measures of dopaminergic radioligands in the rat brain: equilibrium infusion studies.

The application of an equilibrium infusion method for measuring specific in vivo radioligand binding in the conscious rat brain was evaluated for two ligands of the dopaminergic system, (+)-alpha-[(11)C]dihydrotetrabenazine (DTBZ) and d-threo-[(11)C]methylphenidate (MePhen). Both radioligands can be successfully utilized to reach equilibrium distributions in rat brain within 1 h; combinations of tritiated and carbon-11-labeled radiotracers can furthermore be used to obtain simultaneous measures of the neuronal membrane dopamine transporter (using [(3)H]MePhen) and vesicular monoamine transporter (using [(11)C]DTBZ) in the same animal. These studies provided quantitative measures of distribution volume ratios, which represent specific radioligand binding. Stereospecificity of in vivo binding was demonstrated using equilibrium infusions of the low-affinity isomers of each ligand, (-)-alpha-[(11)C]dihydrotetrabenazine (DTBZ) and l-threo-[(11)C]methylphenidate, both of which produced uniform brain distributions and no specific binding. Specific binding of (+)-alpha-[(11)C]dihydrotetrabenazine was blocked by co-infusion of tetrabenazine, but was unaffected by administration of methylphenidate, haloperidol, or apomorphine. Specific binding of d-threo-[(11)C]methylphenidate, conversely, was blocked with unlabeled methylphenidate but not affected by tetrabenazine or the dopamine receptor ligands. Equilibrium measures of in vivo radioligand binding, as utilized in this study, offer a quantitative means to evaluate acute and chronic drug effects on in vivo radioligand binding in the rat brain.