β-Adrenoceptors in the Tree Shrew Brain. II. Time-Dependent Effects of Chronic Psychosocial Stress on [125I]Iodocyanopindolol Binding Sites

1. Stress is known to affect the functioning of the central noradrenergic system in a region-specific manner. The aim of the present investigation was to understand better the consequences of recurrent stressful experiences on central -adrenoceptors.2. Alterations in the central nervous -adrenoceptor system resulting from different periods of psychosocial stress (PSS) were analyzed in male tree shrews (Tupaia belangeri) which were submitted to subordination stress for varying time periods.3. In the first experiment, the whole number of -adrenoceptors was analyzed in the forebrains of subordinate animals and controls by in vitro autoradiography using ¹²⁵I-iodocyanopindolol (¹²⁵ICYP), while nonspecific binding of the radioligand to serotonin receptors was blocked with 100 M 5HT.4. PSS affects -adrenoceptors in a time-dependent manner. A decrease in receptor affinity occurred after just 21 days of PSS in cortical areas and in the hippocampus, indicating stress effects on the conformation of -adrenoceptors. After 30 days of PSS,the numbers of -adrenoceptors were significantly decreased in several cortical regions and in the olfactory area.5. In the second experiment, we investigated the influence of PSS on both ₁- and ₂-adrenoceptors separately. ¹²⁵ICYP binding was quantified in the presence of either ICI188.551 to block ₂-adrenoceptors or in the presence of CGP20712A to block ₁-adrenoceptors.6. After 2, 10, 21,and 28 days of PSS, it become apparent that the two -adrenoceptor subtypes are regulated independently. ₁-Adrenoceptors were transiently down-regulated after 2 days of PSS in the prefrontal cortex and in the olfactory area and were decreased after 28 days of PSS in the parietal cortex and the hippocampus. A transient up-regulation of ₁-adrenoceptors occurred in the pulvinar nucleus after 10 days of PSS. ₂-Adrenoceptors were transiently down-regulated after 2 days of PSS in the prefrontal cortex and up-regulated in the pulvinar nucleus after 28 days of PSS.7. These data demonstrate that chronic psychosocial stress in subordinate tree shrews leads to time-dependent changes in the central nervous -adrenoceptors system.8. The high regional variability in stress-induced -adrenoceptor regulation is supposed to be due to the complex mechanisms of intracellular -adrenoceptor sequestration, which includes down-regulation and/or reinsertion of receptors into the plasma membrane. These mechanisms may be important components of the regulatory apparatus which enables the individual to adapt to situations of recurrent stressful experiences by balancing the central nervous adrenoceptor number.     

Kinetic and Equilibrium Studies of (-)125Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) over a large concentration range (1.5-600pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (B_max1=1000±400 sites/cell, K_d1= 2.1±0.9 pmol/l for intact MNL and B_max1=550±190 sites/cell, K_d1=4.1±0.9 pmol/l for MNL membranes)and a low affinity binding site (B_max2=9150±3590 binding sites/cell, K_d2=440±50 pmol/l for intact MNL and B_max2=11560±4690 sites/cell, K_d2=410±70 pmol/l for MNL membranes). Dissociation of (-)¹²⁵ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k_-1=(0.5±0.2)x10^?3 min^?1 for intact MNL and k_-1=(1.0±0.1)x10^?3min^?1 for MNL membranes) and a fast dissociating component (k_-2=(80±20)x10^?3min^?1 for intact MNL and k_-2=(60±10)x10^?3min^?1 for MNL membranes). In dissociation experiments started after equilibration with various (-)¹²⁵ICYP concentrations k_-1 and k_-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)¹²⁵ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)¹²⁵ICYP on MNL.     

Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

The binding of [³⁵S]GTPS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 M. The specific binding was 98%. Different guanine nucleotides were all able to compete with [³⁵S]GTPS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [³⁵S]GTPS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [³⁵S]GTPS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPS, did not displace [³⁵S]GTPS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [³⁵S]GTPS binding was strongly Mg²⁺-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [³⁵S]GTPS in the presence of GTP may reflect functional G-protein activity.     

Characterization of Imido [8-3H] Guanosine 5′-Triphosphate Binding Sites to Rat Brain Membranes

Besides their well-defined intracellular roles in transmembrane signals transduction, guanine derivatives play important roles by acting from the outside of neural cell membranes. These roles are mediated by two different pool sites in cell membranes: G proteins, which bind to specific (GDP and GTP) intracellular guanine derivatives, and sites that bind to extracellular guanine derivatives. In this study we investigated some methodological characteristics of both guanine derivatives binding sites (intracellular and extracellular) in rat brain neural membranes. By investigating the binding of a poorly hydrolyzed GTP analogue and the adenylate cyclase activity in neural membranes, we observed some distinctiveness of guanine derivatives binding sites: stability to washing procedures (extracellular) and modulation of adenylate cyclase activity (intracellular). These results allow dealing with each site separately, which could be useful for discriminating the roles of extracellular and intracellular guanine derivatives in the central nervous system.     

Labelling of Rat Brain ß-Adrenoceptors: (3H)CGP-12177 or (125I)Iodocyanopindolol?

Abstract

Binding of (¹²⁵I) iodocyanopindolol (ICYP) and (³H) CGP-12177 to rat brain homogenates was characterized and compared. ICYP was shown to bind to both ß-adrenergic and serotonin1B (5HT_1B) receptors whereas (³H)CGP-12177 only labelled the first ones. The addition of 10 μM serotonin (5HT) prevented ICYP binding to 5HT receptors and under these experimental conditions both ligands labelled a similar total number of ß-adrenoceptors in the different rat brain regions. ICYP displayed a higher affinity for cerebellar (mainly ß₂-subtype) than for cerebral cortex ß-adrenoceptors (mainly ß-subtype) suggesting a subtype selectivity. A multiple displacement binding approach using CGP-20712A, a ß₁-subtype ligand, as competitor revealed a 2.6 fold selectivity of ICYP for the ß₂-adrenoceptor subtype. On the other hand, (³H)CGP-12177 binds only to ß-adrenoceptors and is not subtype selective in the rat brain homogenate. Considering both its high specificity and its lack of subtype selectivity (³H)CGP-12177 seems to be a more suitable ligand than ICYP to non-selectively label ß-adrenoceptors in rat brain.     

Orcadian Rhythm of Serotonin Binding in Rat Brain—I. Effect of the Light-Dark Cycle

Moving rapidly from a supine to a standing posture is a common daily activity, yet a significant physiological challenge. Syncope can result from the development of initial orthostatic hypotension (IOH) involving a transient fall in systolic/diastolic blood pressure (BP) of >40/20?mm Hg within the first 15 s, and/or a delayed orthostatic hypotension (DOH) involving a fall in systolic/diastolic BP of >20/10?mm Hg within 15?min of posture change. Although epidemiological data indicate a heightened syncope risk in the morning, little is known about the diurnal variation in the IOH and DOH mechanisms associated with postural change. The authors hypothesized that the onset of IOH and DOH occurs sooner, and the associated cardiorespiratory and cerebrovascular changes are more pronounced, in the early morning. At 06:00 and 16:00?h, 17 normotensive volunteers, aged 26?±?1 yrs (mean?±?SE), completed a protocol involving supine rest, an upright stand, and a 60° head-up tilt (HUT) during which continuous beat-to-beat measurements of middle cerebral artery velocity (MCAv), mean arterial BP (MAP), heart rate, and end-tidal Pco₂ (P_ETco₂) were obtained. Mean MCAv was ～12% lower at baseline in the morning (p?≤?.01) and during the HUT (p?<?.01), despite a morning elevation in P_ETco₂ by ～2.2?mm Hg (p?=?.01). The decline in MAP during initial standing (morning vs. afternoon: 50%?±?4% vs. 49%?±?3%) and HUT (39%?±?3% vs. 38%?±?3%) did not vary with time-of-day (p?>?.30). In conclusion, although there is a marked reduction in MCAv in the morning, there is an absence of diurnal variation in the onset of and associated physiological responses associated with IOH and DOH. These responses, at least in this population, are unlikely contributors to the diurnal variation in orthostatic tolerance. (Author correspondence: N.C.Lewis@2004.ljmu.ac.uk)     

Specific Binding and Characteristics of 18β-Glycyrrhetinic Acid in Rat Brain

18β-Glycyrrhetinic acid (GA) is the aglycone of glycyrrhizin that is a component of Glycyrrhiza, and has several pharmacological actions in the central nervous system. Recently, GA has been demonstrated to reach the brain by crossing the blood-brain barrier in rats after oral administration of a Glycyrrhiza-containing traditional Japanese medicine, yokukansan. These findings suggest that there are specific binding sites for GA in the brain. Here we show evidence that [³H]GA binds specifically to several brain areas by quantitative autoradiography; the density was higher in the hippocampus, moderate in the caudate putamen, nucleus accumbens, amygdala, olfactory bulb, cerebral cortex, thalamus, and mid brain, and lower in the brain stem and cerebellum. Several kinds of steroids, gap junction-blocking reagents, glutamate transporter-recognized compounds, and glutamate receptor agonists did not inhibit the [³H]GA binding. Microautoradiography showed that the [³H]GA signals in the hippocampus were distributed in small non-neuronal cells similar to astrocytes. Immunohistochemical analysis revealed that immunoreactivity of 11β-hydroxysteroid dehydrogenase type-1 (11β-HSD1), a defined molecule recognized by GA, was detected mainly in neurons, moderately in astrocytes, and very slightly in microglial cells, of the hippocampus. These results demonstrate that specific binding sites for GA exist in rat brain tissue, and suggest that the pharmacological actions of GA may be related to 11β-HSD1 in astrocytes. This finding provides important information to understand the pharmacology of GA in the brain.     

Brain Serotonin Transporter Binding of [123I]ADAM: Within‐Subject Variation between Summer and Winter Data

The neurotransmitter serotonin (5‐HT) controls several physiological functions, and a disturbance of the 5‐HT system is implicated in many psychiatric conditions. Seasonal variation has been suggested in the 5‐HT system. We investigated within‐subject seasonal variation in brain serotonin transporter (SERT) binding with the SERT‐ligand [¹²³I]ADAM and single photon emission computed tomography (SPECT) in 12 healthy individuals. No systematic variation was found in the midbrain or thalamus areas between scans done in summer and winter. Our results suggest that factors other than season are more important in causing within‐subject variation of brain SERT binding between summer and winter. (Author correspondence: anu.koskela@helsinki.fi)     

Incorporation of [3H] uridine into the ribonucleic acid of rat uterus during pseudopregnancy and in the presence of I.C.I. 46474 [trans-1-(p-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1 -ene]

1. The incorporation of [(3)H]uridine into RNA of rat uterine tissue has been measured during pseudopregnancy and in rats receiving different doses of an anti-implantation compound [trans-1-(p-beta-dimethylaminoethoxyphenyl)-1,2-diphenylbut- 1-ene, I.C.I. 46474]. 2. In the uterus of the pseudopregnant rat uridine incorporation into RNA increased markedly on day 3 of pseudopregnancy, remained high until day 5 and decreased sharply by day 6, rising again by day 9. 3. This pattern of change was similar to, but not identical with, that previously found in the pregnant rat. 4. Rats receiving I.C.I. 46474 at a dose concentration below that preventing implantation showed a pattern of RNA synthesis similar to that found in untreated control rats. 5. Rats treated with doses of I.C.I. 46474 sufficient to inhibit implantation revealed a totally different pattern of incorporation of [(3)H]uridine into uterine RNA. 6. The results are discussed in terms of previous findings with the normally pregnant rat. It is concluded that the increasing uterine RNA synthesis found on day 3 in the pregnant rat is an important requirement for the occurrence of the subsequent implantation.     

LOBAR PNEUMONIA—The Distribution of Sites in Infants and Children

In a review of roentgenograms of 228 pediatric patients with segmental pneumonia, the left upper lobe was observed to be involved in only 4 per cent of cases. The right upper lobe was involved most frequently—over eight times as often as the left upper lobe and twice as often as the right middle or lower lobe. It is suggested that this distribution is related to the fact that the angle of the left bronchus in relation to the median plane is more acute than the angle of the right bronchus, the sharper angle perhaps serving as protection against aspiration.     

Binding of [125I]iodocyanopindolol by rat Harderian gland crude membranes: Kinetic characteristics and day—night variations

The Harderian glands are innervated by sympathetic fibers originating in the superior cervical ganglia. The aim of this study is to characterize the -adrenergic receptors in the rat Harderian gland. The characteristics of -adrenergic receptors were determined in crude membrane preparations from rat Harderian gland, using [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP) as radioligand. The binding of the ligand to the receptor is rapid, reversible, saturable, specific and dependent on time, temperature and membrane concentration. At 30 °C, stoichiometric data suggest the presence of one binding site with a K_d value of 0.29 nM and B_max of 32 pmol/L. The interaction shows a high degree of specificity for -adrenergic agonists and blockers, as suggested by competitive displacement experiments with isoproterenol (IC₅₀=19.1 nM), propranolol (IC₅₀=28.1 nM), and norepinephrine (IC₅₀=96.3 nM). Clonidine, yohimbine, methoxamine, and prazosin are ineffective at concentrations up to 1 M. In the other hand, binding of [¹²⁵I]CYP by Harderian gland membranes exhibits day—night variations. Binding values are low during the daytime and increase progressively late in the evening to reach a maximum at 2200 h (2 h after the onset of dark period), but decreased to the end of the dark period (0600 h). In conclusion, the results presented in this paper show the functional and pharmacological characterization of -adrenergic receptors in the rat Harderian gland. This neurotransmitter may play a physiological role at this level regulating, at least, processes such as a thyroid hormone metabolism.     

Non-Adrenergic Binding Sites for the “α-Antagonist” [3H] Idazoxan in Rabbit Urethral Smooth Muscle

Abstract

In the present study, we have provided evidence that [³H] rauwolscine and [³H] idazoxan bind to different sites in rabbit urethra. The [³H] idazoxan capacity and affinity was 215 ± 14 fmol/mg protein and 1.59 ± 0.16 nM while [³H] rauwolscine binding parameters were 45.9 ± 3.4 fmol/mg protein and 2.39 ± 0.27 nM. [³H] idazoxan specific binding was inhibited only by compounds possessing an imidazoli(di)ne or a guanidinium moiety, while [³H] rauwolscine specific binding was inhibited by phenylethanolamines and classical α-antagonists. [₃H] idazoxan was inhibited by KCI in a competitive and by MnCI₂ in a non-competitive way, while other cations such as Na⁺, Li⁺ and Mg²⁺ did not inhibit [³H] idazoxan binding. Moreover, we investigated the regional distribution of [³H] idazoxan and [³H] rauwolscine along the rabbit urethra using quantitative autoradiography. Analysis of the films revealed a different distribution of these two binding sites on the urethral sections.     

Circadian Rhythm of Serotonin Binding in Rat Brain—II. Influence of Sleep Deprivation and Imipramine

Sleep deprivation (SD) modified the circadian rhythm of specific high affinity serotonin (5-HT) binding to rat brain membranes. In control rats a 24-hr rhythm was evident with a trough at 1000-1200 and a nadir at 0000. During the last 26 hr of a 49 hr SD period, trough and peak values were delayed by 4-6 hr. The 24-hr mean binding was significantly (P < 0.001) different from that of controls. If sleep deprivation was followed by recovery sleep (RS), the normal rhythm of 5-HT binding was obtained already within 1 hr after SD. The effects of SD and RS were ascertained by plasma ACTH and corticosterone assay. No significant change in the hormone rhythms were observed though the mean plasma level of ACTH and corticosterone were enhanced to about 180 and 150%, respectively. Chronic treatment with the antidepressant imipramine resulted in a decrease of the 24-hr mean 5-HT binding by about 50% and a 2-hr delay of peak and trough values. Imipramine treatment decreased the peak valueof 5-HT concentration at 1000 to about 65% and appears to abolish the rhythm of 5-HT concentration.     

Primer extension studies on α-amylase mRNAs in barley aleurone. I. Characterization and quantification of the transcripts

Primer extension was used to characterize alpha-amylase mRNAs from aleurone tissue of barley (Hordeum vulgare L. cv. Himalaya) grains. Two synthetic oligonucleotides, specific for the low-pI and high-pI alpha-amylase groups, were used as primers for synthesis of cDNA from total RNA preparations. Between them, these two oligonucleotides appear to account for all major alpha-amylase mRNAs as judged by hybrid-arrested translation of alpha-amylase mRNAs in a cell-free system. Reconstruction experiments indicated that the levels of extended primers (determined by scintillation counting) were directly proportional to the level of input mRNA over a wide range. This indicates that the technique is suitable for quantification of relative levels of individual alpha-amylase from approximately 2% to 100% of maximal levels. The nucleotide sequences of extended primers defined two different alpha-amylase mRNAs in each of the low-pI and high-pI groups, and possibly a third mRNA in the high-pI group.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Characterization and Evaluation of Key Sites in the Peptide Inhibitor of TAB1/p38α Interaction

A combination of computation techniques and peptide mutants have been used to determine the binding site and amino acid residues on the inhibitor peptide that are critical for binding to Mitogen-activated protein kinase 14 (p38α). In our previous research work, the functional peptide, named as PT5, target to p38α, was obtained based on the theoretical complex structure of p38α and [transforming growth factor-β (TGF-β)-activated protein kinase 1 (TAK1)-binding protein 1] (TAB1). Based on the computer-guided ab initio modeling method, the inhibitor peptide PT5 and its mutants were modeled. Furthermore, the 3-D complex structures of PT5 or its mutants and p38α were constructed using molecular docking and dynamics simulation methods. The key residues in the peptide PT5 involved in binding interaction to p38α were predicted. According to the 3-D theoretical complex structure PT5/ p38α, the interaction binding mode between PT5 and p38α was analyzed using distance geometry technology. Mutants of the peptide PT5 was used to evaluate the bio-function when the critical residues were mutated. The mutant experimental results identified the key residues in PT5, i.e. Thr¹¹ and Asp¹² and determined the core sequence of PT5 binding to p38α. Based on the results, optimized peptides compounds could be developed for treating myocardial ischemia/reperfusion (I/R) injury in clinical.