Postnatal development of brain alpha 1-adrenergic receptors: in vitro autoradiography with [125I]HEAT in normal rats and rats treated with alpha-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase

The postnatal development of brain alpha 1-adrenergic receptors was studied in the rat brain using in vitro autoradiography. In some regions, such as the globus pallidus, receptor-binding sites were present at birth and increased during the first week but then decreased to very low levels by adulthood. In contrast, other regions such as the olfactory bulb and cerebral cortex exhibited little binding at birth, with a subsequent increase in receptors during the second week of life that persisted into the mature stage. Several regions had an intermediate pattern with significant labelling at birth, an increase in the first few weeks and a smaller decrement in binding sites as adulthood was approached. The data suggested that there were two archetypal development patterns, one of which was potentially related to the arrival of noradrenergic nerve projections (olfactory bulb) and the other of which was determined intrinsically by differentiation (globus pallidus). The two patterns could be distinguished by their sensitivity to alpha-difluoromethylornithine, a drug that inhibits ornithine decarboxylase, leading to a slowing of cellular replication, differentiation and migration. Drug treatment dramatically delayed the developmental fall-off of binding in the globus pallidus such that receptor sites remained in high concentration well past the point at which they disappeared in control animals. In the olfactory bulb, however, alpha-difluoromethylornithine had little or no effect on the ontogeny of receptor binding. These studies provide evidence that alpha 1-adrenergic receptors in various brain regions develop at different rates and with at least two characteristic patterns. Autoradiographic techniques provide important insights into receptor development that cannot be garnered from biochemical methods using isolated membrane preparations.     

Growth resistance-sized arteries in response to bladder hypertrophy in the rat: time-course,DNA-synthesis and LDH-isoform pattern

Bladder growth was induced by partial urethral obstruction. Bladder hypertrophy was evident at 53 h after obstruction and continued over a 6 weeks period. Small bladder arteries were taken from fixed anatomical locations of the bladder circulation, mounted in a small vessel myograph and the optimal diameter for maximal isometric force development was determined (L_max, K⁺=125 mm stimulation). Bladder hypertrophy was associated with an enlarged L_max from 53 h onward (compared with sham-operated controls) and L_max continued to increase until 10 days after urethral obstruction. Between 10 days and 6 weeks no further increase of the diameter was observed. Increased diameters in vitro were accompanied by a transiently increased [³H]Thymidine uptake in the small arteries which peaked at 53 h after obstruction but was still above background at 10 days. At this time point, small arterial growth was associated with a significant relative increase in the M isoform of LDH as determined with agarose electrophoresis on tissue homogenates. Thus organ growth induced small vessel growth in the rat is characterized by a rapid onset, increased but transient DNA-turnover and LDH-isoform changes. The latter mimic changes seen in other types of smooth muscle growth.     

Pentachlorophenol potentiates benzo[a]pyrene DNA adduct formation in adult but not infant B6C3F1 male mice

The objective of this study is to determine whether pentachlorophenol (PCP) alters benzo[a]pyrene (B[a]P)-induced DNA adduct formation in infant and adult B6C3F1 male mice. Mice were exposed intraperitoneally to 55 microg B[a]P/g body weight (BW) alone and in combination with several doses of PCP in DMSO. The 32P-postlabeling assay was used to analyze for (+/-) anti-7,8-diol-9,10-epoxide-B[a]P-N(2)deoxyguanosine (BPDE-N(2)G) adducts formed in liver and lung DNA. Hepatic DNA also was analyzed for 8-hydroxy-2'-deoxyguanosine (8-OHdG) base damage in mice exposed to PCP. 8-OHdG was not detected at any dose of PCP in infant or adult mice. PCP exhibited an antagonistic effect on BPDE-N(2)G accumulation in infant mice exposed to B[a]P in combination with 50 microg PCP/g BW at both 12 and 24 hr. Comparatively, BPDE-N(2)G adducts were increased in adult mice exposed to binary mixtures at 24 hr in both hepatic and lung DNA (P < 0.05). Multiple comparison analysis between infant and adult mice revealed that adduct levels in infants exposed to B[a]P alone or in combination with PCP were not different from those observed in adult mice exposed to B[a]P. However, a significant increase in adducts was observed in adult mice exposed to a combination of B[a]P and PCP compared to that in all other treatment groups (P < 0.05). These results suggest that PCP alters the metabolism of B[a]P in both infant and adult mice through different mechanisms, and that infants are not susceptible to the potentiating effects of PCP observed in adult mice.     

FcγRⅡB1介导的信号传导异常与SLE患者B细胞的过度活化

目的:观察系统性红斑狼疮(SLE)患者B细胞表面功能分子表达的特征及其功能状态,评价以FcγRⅡB1(CD32)为代表的B细胞自身抑制调节机制在SLE发病中的作用。方法:采用Ficoll密度梯度离心法分离出人外周血单个核细胞(PBMC),并以免疫磁珠法(MACS)分离纯化B细胞。采用荧光分光光度法检测B细胞受不同激活物刺激后细胞内钙([Ca2 ]i)的反应。用ELISA法检测B细胞与刺激物共同培养后所分泌IgG的量。采用流式细胞术及间接免疫荧光染色法,检测B细胞膜表面CD32、CD19及IgM的表达水平。结果:(1)以羊抗人μ链的F(ab′)2片段及完整IgG分别刺激SLE患者B细胞时,其[Ca2 ]i反应的比值显著低于类风湿性关节炎(RA)患者(P<0.05)及正常人对照(P<0.01)。(2)分别用葡萄球菌A蛋白(SPA)单独刺激与SPA和羊抗人μ链的完整IgG抗体共同刺激SLE患者的B细胞所分泌的IgG的比值,明显低于RA患者及正常人对照组(P<0.05)。(3)SLE患者与RA患者及正常对照组B细胞上CD19、CD32及IgM的表达无统计学意义(P>0.05)。结论:SLE患者B细胞上CD32抑制性信号传导的异常,可能是导致B细胞过度活化的重要机制。     

NMDA receptor mediated dendritic plasticity in cortical cultures after oxygen-glucose deprivation

Dendrites and spines undergo dynamic changes in physiological and pathological conditions. Dendritic outgrowth has been observed in surviving neurons months after ischemia, which is associated with the functional compensation. It remains unclear how dendrites in surviving neurons are altered shortly after ischemia, which might reveal the mechanisms underlying neuronal survival. Using primary cortical cultures, we monitored the dendritic changes in individual neurons after oxygen-glucose deprivation (OGD). Two to four hours of OGD induced approximately 30–50% cell death in 24 h. However, the total dendritic length in surviving neurons was significantly increased after OGD with a peak at 6 h after re-oxygenation. The increase of dendritic length after OGD was mainly due to the sprouting rather than the extension of the dendrites. The dendritic outgrowth after 2 h of OGD was greater than that after 4 h of OGD. Application of NMDA receptor blocker MK-801 abolished OGD-induced dendritic outgrowth, whereas application of AMPA receptor antagonist CNQX had no significant effects. These results demonstrate a NMDA receptor-dependent dendritic plasticity shortly after OGD, which provides insights into the early response of surviving neurons after ischemia.     

Localization of [F]fluorodeoxyglucose in mouse brain neurons with micro-autoradiography

This is the first study of micro-autoradiography (micro-ARG) for [¹⁸F]2-fluoro-2-deoxy- -glucose ([¹⁸F]FDG). The localization of [¹⁸F]FDG was demonstrated in dendrites of neuron and also in the myelinated axon in mouse normal brain in vivo. The nucleolus was relatively free of label. The counted silver grain numbers in autoradiogram were linearly correlated to the ¹⁸F radioactivities in the specimen. The micro-ARG using positron emitting ¹⁸F is a very time-saving technique with 4 hours exposure compared with the conventional method using ³H- or ¹⁴C-labeled tracers.     

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH 2 9 ] vasotocin binding in microdissected tubules

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(mercapto-,-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 4-threonine, 8-ornithine, 9-¹²⁵I-tyrosylamide]vasotocin ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4° C, specific binding sites (expressed at 10^–18 mol ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67±0.49; cortical thick ascending limbs, 2.20±0.80; cortical collecting ducts, 2.39±0.86; outer medullary collecting ducts (OMCD), 2.54±0.53 and inner medullary collecting ducts, 5.33±0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4° C were 1.06×10⁷ M^–1 min^–1 and 1.95×10^–2 min^–1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:ddAVP>AVP>d(CH₂)₅-[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT=AVT=OT>d(CH₂)₅[Tyr(Me)²]AVP=[Thr⁴, Gly⁷]OT>[Phe², Orn⁸]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V₂ vasopressin receptors.     

[3H]-5-Methoxytryptoline is not actively accumulated by rabbit platelets

Summary 5-Methoxytryptoline (5-MeO-TLN, 6-methoxytetrahydro--carboline) inhibits with high affinity [³H]-imipramine binding to the serotonin transporter in platelets. To evaluate whether 5-MeO-TLN is a substrate for the serotonin transporter, the accumulation of [³H]-5-MeO-TLN into rabbit platelets was studied in vitro. At short incubation times (5 min), [³H]-5-MeO-TLN accumulation was temperature-sensitive, but not saturable over a concentration range from 0.06 mol/l to 10 mol/l Moreover, [³H]-5-MeO-TLN uptake was not affected by 100 mol/1 ouabain, its structural analogs tryptoline and 5-hydroxytryptoline, nor by the serotonin uptake inhibitors imipramine and citalopram. After longer incubation times (60 min), [³H]-5-MeO-TLN accumulation at O°C approached that seen at 37°C and temperature-sensitive [³H]-5-MeO-TLN uptake could no longer be observed. It is concluded that temperature-sensitive accumulation of [³H]-5-MeO-TLN is not mediated by the serotonin transporter and most likely represents a passive, diffusional process, the rate of which is temperature-dependent. The present studies thus confirm the hypothesis that 5-MeO-TLN affects [³H]-imipramine binding in platelets through a competitive mechanism and not via an allosteric interaction mediated through the substrate recognition site of the macromolecular complex of the serotonin transporter. Send offprint requests to S. Z. Langer at the above address     

Agonist-mediated conformational changes of β-adrenoceptors could occur independent of functional coupling to Ns

Summary Agonist and antagonist binding characteristics of -adrenoceptors in turkey erythrocyte ghosts were determined at different temperatures ranging between 7°C and 42°C. [³H]-DHA saturation binding experiments revealed that the antagonist-receptor interaction is entropy-driven with a small enthalpic contribution. Isoproterenol/[³H]-DHA competition binding followed the law of mass action at all the investigated temperatures. The agonist-receptor interaction is enthalpy driven with a small unfavorable decrease in entropy. This is consistent with the agonist's ability to favor an endoenergetic transconformation of the receptors.Only part of the agonist-bound receptors can undergo functional coupling to the stimulatory component of the adenylate cyclase system (Ns). This coupling process is associated with locking-in of the agonist and becomes persistent in the presence of the alkylating reagent N-ethylmaleimide. The number of agonist/N-ethylmaleimide-sensitive sites (i.e. coupling-prone receptors) increases with the temperature until it reaches a plateau value of 50% between 27–32°C. Qualitatively similar data were obtained for rat lung and turkey erythrocyte membranes. These observations suggest that the whole receptor population can undergo agonist-mediated conformational changes but that only part of them can couple to Ns.     

Extracellular K+ accumulations and synchronous GABA-mediated potentials evoked by 4-aminopyridine in the adult rat hippocampus

Transient changes in extracellular potassium concentration ([K⁺]₀) and field potentials were evoked by 4-aminopyridine (4-AP; 50–100 M) and recorded with ion-selective microelectrodes in CA1b, CA3b and dentate sectors of adult rat hippocampal slices. Long-lasting field potentials recurred at a frequency of 1/60 s (0.016±0.003 Hz) in association with increases in [K⁺]₀ which were largest and most sustained in the dendritic regions where afferent fibers terminate (dentate>CAl>CA3) and in the hilus. In stratum radiatum of CA1 or stratum moleculare of the dentate these fields had a peak amplitude of 1.4±0.29 mV, duration 8.3±1.6 s, and were accompanied by increases in [K⁺]₀ of 1.8±0.22 mM that lasted 32±5.5 s (n = 17 slices). Interictal epileptiform potentials, which were brief (<0.2 s) and more frequent at 1/3 s (0.30±0.02 Hz) were also present in CA1, CA3 and the hilus and associated with small increases in [K⁺]₀ (0.5 mM, duration 2 s). Interictal activity was blocked by 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX; 5–20 M); the slow, less frequent potentials were resistant to both CNQX and dl-2amino-5-phosphonovaleric acid (APV; 50 M) and reversibly blocked (or attenuated by 80%) by bicuculline methiodide (BMI) (25–100 M). The BMI-sensitive potentials were also abolished by baclofen (100 M), an effect which was reversed by 2-OH-saclofen (100 M). Focal application of KCl or GABA in the absence of 4-AP evoked long-lasting field and [K⁺]₀ potentials which were similar to those evoked by 4-AP but more sustained. The proportional relationship between the amplitudes of field and K⁺ potentials with GABA closely resembled that observed for 4-AP; in contrast the slope of KCl-evoked responses was lower. Our results demonstrate that in the adult rat hippocampus 4-AP induces in many different regions accumulations of [K⁺]₀ in synchrony with the long-lasting field potentials, which are known to correspond to an intracellular long-lasting depolarization of the pyramidal cells. These changes are smaller than those which occur in the immature rat hippocampus — which may be related to differences in Na-K-ATPase and susceptibility to seizures. These events involve the activation of GABA_A receptors, are under the modulatory control of GABA_B receptors, and likely arise from the activity of GABAergic interneurons and/or afferent terminals. The long-lasting field potentials appear to reflect mainly the direct depolarizing actions of GABA and to a much more limited extent the associated accumulation of [K⁺]₀.