Low thymic output and reduced heterogeneity of alpha/beta,but not gamma/delta,T lymphocytes in infants with ataxia-telangiectasia

Ataxia-telangiectasia, a genetic disease caused by the homozygous mutation of the ATM gene, is frequently associated to a deficit of humoral and cellular immune functions. A decreased thymic output and skewed T cell and B cell receptor repertoires have been recently described in children over 7 years of age and in adults with this disease and have been proposed as a possible explanation for the immunodeficiency. To understand whether T cell defects arise early in life as a consequence of ATM gene mutations, we analysed the extent of thymic function by measuring the number of na?ve T cells and by studying the heterogeneity of T cells by means of heteroduplex analysis, in two children less than 2 years old with a remarkable reduction of T cell count. We found that the thymic output is decreased in babies with ataxia-telangiectasia if compared with that observed in age-matched normal babies. The low production of new T cells is associated to a reduction of the diversity of alpha/beta, but not gamma/delta, T lymphocytes. Our data indicate that ATM mutation limits the generation of a wide alpha/beta T cell repertoire and this feature can be responsible for the immunodeficiency observed in ataxia-telangiectasia babies.     

Localization of GABAA receptor subunits alpha 1, alpha 3, beta 1, beta 2/3, gamma 1, and gamma 2 in the salamander retina

Electrophysiological studies have demonstrated that gamma-aminobutyric acid receptors type A (GABA(A)) mediate important information processing in the retinas of salamander and other vertebrates. The pharmacology and physiology of GABA(A) receptors depend on their subunit composition. We studied the localization of GABA(A) receptor subunit isoforms alpha(1), alpha(3), beta(1), beta(2/3) (antibody BD-17 and 62-3G1), gamma(1), and gamma(2) in salamander retina with immunocytochemical methods. All three beta-subunit antibodies labeled similarly in the outer retina, especially the inner segments and synaptic terminals of rod photoreceptors (identified with protein kinase C). Somatic labeling was observed in cell bodies of some horizontal cells, bipolar cells, amacrine cells, and cells in the ganglion cell layer (GCL). Puncta were present throughout the inner plexiform layer (IPL) for beta(1) and 62-3G1, but not for BD-17. alpha(1)-immunoreactivity (IR) stained a population of presumed OFF rod-dominated bipolar cells, including dendrites, soma, and axon terminals in the distal IPL. A subtype of GABAergic amacrine cell also expressed alpha(1)-IR, with puncta sparsely distributed at the distal and proximal margins of the IPL. Both the OPL and IPL were labeled throughout for alpha(3)-IR, as opposed to the narrow distribution of alpha(1)-IR in the IPL, suggesting that the two alpha-subunits are localized at different synaptic sites. Punctate gamma(1)-IR was observed in the OPL and IPL, whereas gamma(2) was most prominent in cone photoreceptors (identified with calbindin), including the terminal telodendria, in cell bodies of some horizontal cells, amacrine cells, cells in the GCL, and less intensely in the IPL. In addition, several subunits were present in Müller cells. The differential labeling suggests the existence of GABA(A) receptor subtypes with different subunit compositions that mediate multiple GABAergic functions in salamander retina.     

Megakaryocytic cells synthesize and platelets secrete alpha5-laminins, and the endothelial laminin isoform laminin 10 (alpha5beta1gamma1) strongly promotes adhesion but not activation of platelets

Following vascular injury, basement membrane (BM) components of the blood vessels are exposed to circulating cells and may contribute to hemostasis and/or thrombosis. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major laminin isoforms of the endothelial BM, and LN-8 is also secreted by activated platelets. In the present study, we demonstrate synthesis of alpha5-laminins LN-10 and LN-11 (alpha5beta2gamma1) by megakaryocytic cells, and intracellular expression of these laminin isoforms in blood platelets. In contrast to platelet LN alpha4 chain that had an apparent molecular weight of 180 kDa and associated mostly to LNbeta1 chain, platelet LNalpha5 consisted of 300/350 kDa polypeptides and associated mainly to LNbeta2. Both alpha4- and alpha5-laminins were secreted by platelets following stimulation. When compared to recombinant human (rh) LN-8, rhLN-10 was much more adhesive to platelets, though adhesion to both proteins was largely mediated via alpha6beta1 integrin. In spite of their adhesive properties, rhLN-8 and rhLN-10 induced neither P-selectin expression nor cell aggregation, two signs of platelet activation. This study demonstrates synthesis/expression of heterotrimeric alpha5-laminins in hematopoietic/blood cells, and provides evidence for the adhesive, but not activating, role of endothelial laminin isoforms in platelet biology.     

Developmental and cellular expression pattern of epithelial sodium channel alpha, beta and gamma subunits in the inner ear of the rat

Endolymphatic ion composition in the adult inner ear is characterized by high K(+) and low Na(+) concentration. This unique ion composition is essential for proper functioning of sensory processing. Although a lot has been learned in recent years about molecules involved in K(+) transport in inner ear, the molecules involved in Na(+) transport are only beginning to emerge. The epithelial Na(+) channel (ENaC) is a highly selective Na(+) channel that is expressed in many Na(+)-reabsorbing tissues. The aim of our study was to investigate whether ENaC is expressed in inner ear of rats and could account for Na(+) reabsorption from endolymph. We detected mRNA for the three channel-forming subunits (alpha, beta and gamma ENaC) in cochlea, vestibular system and endolymphatic sac. mRNA abundance increased during the first 12 days of life in cochlea and vestibular system, coinciding with decreasing Na(+) concentration in endolymph. Expression was strongest in epithelial cells lining scala media, most notably Claudius' cells. As these cells are characterized by a very negative resting potential they would be ideally suited for reabsorption of Na(+). mRNA abundance in endolymphatic sac decreased during the first 6 days of life, suggesting that ENaC might be implicated in reabsorption of endolymph in the endolymphatic sac of neonatal animals. Together, our results suggest that the epithelial Na+ channel is a good candidate for a molecule involved in Na(+) homeostasis in inner ear.     

Distribution of alpha1, alpha4, gamma2, and delta subunits of GABAA receptors in hippocampal granule cells

GABAA receptors are pentamers composed of subunits derived from the alpha, beta, gamma, delta, theta, epsilon, and pi gene families. alpha1, alpha4, gamma2, and delta subunits are expressed in the dentate gyrus of the hippocampus, but their subcellular distribution has not been described. Hippocampal sections were double-labeled for the alpha1, alpha4, gamma2, and delta subunits and GAD65 or gephyrin, and their subcellular distribution on dentate granule cells was studied by means of confocal laser scanning microscopy (CLSM). The synaptic versus extrasynaptic localization of these subunits was inferred by quantitative analysis of the frequency of colocalization of various subunits with synaptic markers in high-resolution images. GAD65 immunoreactive clusters colocalized with 26.24+/-0.86% of the alpha1 subunit immunoreactive clusters and 32.35+/-1.49% of the gamma2 subunit clusters. In contrast, only 1.58+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.92+/-0.15% of the delta subunit clusters colocalized with the presynaptic marker GAD65. These findings were confirmed by studying colocalization with immunoreactivity of a postsynaptic marker, gephyrin, which colocalized with 27.61+/-0.16% of the alpha1 subunit immunoreactive clusters and 23.45+/-0.32% of the gamma2 subunit immunoreactive clusters. In contrast, only 1.90+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.76+/-0.10% of the delta subunit clusters colocalized with gephyrin. These studies demonstrate that a subset of alpha1 and gamma2 subunit clusters colocalize with synaptic markers in hippocampal dentate granule cells. Furthermore, all four subunits, alpha1, alpha4, gamma2, and delta, are present in the extrasynaptic locations.     

Evidence that smooth pursuit velocity,not eye position,modulates alpha and beta oscillations in human middle temporal cortex

Suppression of 5–25 Hz oscillations have been observed in MT+ during pursuit eye movements, suggesting oscillations that play a role in oculomotor control and/or the integration of extraretinal signals during pursuit. The amplitude of these rhythms appears to covary with head‐centered eye position, but an alternative is that they depend on a velocity signal that lags the movement of the eyes. To investigate, we explored how alpha and beta amplitude changes related to ongoing eye movement depended on pursuit at different eccentricities. The results revealed largely identical patterns of modulation in the alpha and beta amplitude, irrespective of the eccentricity at which the pursuit eye movement was performed. The signals we measured therefore do not depend on head‐centered position. A second experiment was designed to investigate whether the alpha and beta oscillations depended on the direction of pursuit, as opposed to just speed. We found no evidence that alpha or beta oscillations depended on direction, but there was a significant effect of eye speed on the magnitude of the beta suppression. This suggests distinct functional roles for alpha and beta suppression in pursuit behavior. Hum Brain Mapp 36:5220–5232, 2015. © 2015 Wiley Periodicals, Inc.     

Hypothermia inhibits translocation of CaM kinase II and PKC-alpha, beta, gamma isoforms and fodrin proteolysis in rat brain synaptosome during ischemia-reperfusion

To clarify the involvement of intracellular signaling pathway and calpain in the brain injury and its protection by mild hypothermia, immunoblotting analyses were performed in the rat brain after global forebrain ischemia and reperfusion. After 30 min of ischemia followed by 60 min of reperfusion, Ca2+/calmodulin-dependent kinase II (CaM kinase II) and protein kinase C (PKC)-alpha, beta, gamma isoforms translocated to the synaptosomal fraction, while mild hypothermia (32 degrees C) inhibited the translocation. The hypothermia also inhibited fodrin proteolysis caused by ischemia-reperfusion, indicating the inhibition of calpain. These effects of hypothermia may explain the mechanism of the protection against brain ischemia-reperfusion injury through modulating synaptosomal function.     

Contrasting effects of alpha, beta, and gamma interferons on nonspecific suppressor function in multiple sclerosis.

Interferons are biological molecules with antiviral, antiproliferative, and immunomodulatory actions. Interferon alpha (IFN-alpha) and -beta are potentially useful in the treatment of multiple sclerosis (MS). IFN-gamma, in contrast, increases the frequency of exacerbations of MS. In this study, we compared the effect of recombinant human IFN-alpha, -beta, and -gamma on suppressor function in patients with MS. Nonspecific suppressor cell function, measured in a concanavalin A suppressor assay, was significantly decreased in 16 patients with progressive MS (mean percent suppression +/- SEM, 14.4 +/- 5.5 in patients with MS, 33.5 +/- 4.8 in 16 normal subjects; p less than 0.001). Recombinant human IFN-beta augmented suppressor function in MS to 45.4 +/- 5.1% (p less than 0.001) and in control subjects to 56.8 +/- 3.8% (p less than 0.001). Similarly, recombinant human IFN-alpha improved suppression in MS to 43.0 +/- 5.6% (p less than 0.001) and in control subjects to 51.1 +/- 5.9% (p less than 0.001). In contrast, recombinant human IFN-gamma had no effect on suppressor function in patients with MS and in control subjects. This study shows that IFN-alpha and -beta augment deficient suppressor function in MS, whereas IFN-gamma has no effect on suppressor function in the progressive phase of the disease.     

Inhibition of specific adenylyl cyclase isoforms by lithium and carbamazepine,but not valproate,may be related to their antidepressant effect

Objectives: Lithium, valproate, and carbamazepine decrease stimulated brain cyclic‐AMP (cAMP) levels. Adenylyl cyclase (AC), of which there are nine membrane‐bound isoforms (AC1‐AC9), catalyzes the formation of cAMP. We have recently demonstrated preferential inhibition of AC5 by lithium. We now sought to determine whether carbamazepine and valproate also preferentially inhibit specific AC isoforms or decrease cAMP levels via different mechanisms. Methods: COS7 cells were transfected with one of AC1‐AC9, with or without D1‐dopamine receptors. Carbamazepine’s and valproate’s effect on forskolin‐ or D1 agonist‐stimulated ACs was studied. The effect of Mg²⁺ on lithium’s inhibition was studied in membrane‐enriched fraction from COS7 cells co‐expressing AC5 and D1 receptors. AC5 knockout mice were tested for a behavioral phenotype similar to that of lithium treatment. Results: Carbamazepine preferentially inhibited forskolin‐stimulated AC5 and AC1 and all D1 agonist‐stimulated ACs, with AC5 and AC7 being the most sensitive. When compared to 1 or 3 mM Mg²⁺, 10 mM Mg²⁺ reduced lithium‐induced AC5 inhibition by 70%. In silico modeling suggests that among AC isoforms carbamazepine preferentially affects AC1 and AC5 by interacting with the catechol‐estrogen site. Valproate did not affect any forskolin‐ or D1 receptor‐stimulated AC. AC5 knockout mice responded similarly to antidepressant‐ or lithium‐treated wild‐types in the forced‐swim test but not in the amphetamine‐induced hyperactivity mania model. Conclusions: Lithium and carbamazepine preferentially inhibit AC5, albeit via different mechanisms. Lithium competes with Mg²⁺, which is essential for AC activity; carbamazepine competes for AC’s catechol‐estrogen site. Antidepressant‐like behavior of AC5 knockout mice in the forced‐swim test supports the notion that AC5 inhibition is involved in the antidepressant effect of lithium and carbamazepine. The effect of lithium and carbamazepine to lower cAMP formation in AC5‐rich dopaminergic brain regions suggests that D1‐dopamine receptors in these regions are involved in the antidepressant effect of mood stabilizers.     

Estrogen receptor alpha, but not beta, is expressed in the interneurons of the hippocampus in prepubertal rats: an in situ hybridization study

Estradiol is involved in the differentiation and plasticity of hippocampal neurons. In the CA1 region, estrogen treatment increases dendritic spines and synapse density on pyramidal cells. In the adult hippocampus, immunoreactivity for estrogen receptor alpha (ERalpha) has been reported in inhibitory interneurons, but neither in the pyramidal neurons nor in granule cells. Estrogens also mediate aspects of sexual differentiation of the hippocampus. To examine the possibility that an alteration in expression of the two types of estrogen receptors (ERalpha and ERbeta) in the hippocampus underlies different roles of estrogen and/or ERs during development and in adult life, we applied non-isotopic, digoxigenin (dig)-labeled, in situ hybridization histochemistry (ISHH) for the both ER forms and examined the distribution pattern of their messages in serial, frontal sections over the postnatal period and in the adult. ERalpha mRNA expression was found scattered throughout the hippocampus especially in the hilar region of the dentate gyrus, and in the strata radiatum and pyramidale in the cornus ammonis at postnatal days (PND) 14, 21 and 35. In the hilus of the dorsal hippocampus, the density of ERalpha-labelled cells was greater in the rostro-medial aspect, while less in the lateral and the caudal region. In the ventral hippocampus the signals for ERalpha mRNA were also found in relatively high density in the hilus. No significant sex difference in distribution and intensity of the ERalpha mRNA positive cells were detected. The hippocampal distribution of ERalpha mRNA expression at PND 14 remained the same on PND 21 and 35 and in adulthood. As reported for adults, ERalpha mRNA signals appear to be in interneurons of the hippocampus but neither in the pyramidal cells nor in the dentate granular cells based on their size and location. In contrast to the result of ERalpha, no clear signals for ERbeta mRNA were detected in the hippocampus across all ages examined, whereas they were clearly detected in the hypothalamus.     

Localization of the sources of EEG delta, theta, alpha and beta frequency bands using the FFT dipole approximation.

FFT dipole approximation and 3-dimensional dipole modelling were used to determine the locations of the equivalent dipole model sources of the delta, theta, alpha, beta-1 and beta-2 frequency bands in 13 normal subjects during resting. From each subject, 2 successive data sets were analysed, each consisting of 10 epochs of 2 sec randomly collected during 30 min. ANOVAs showed that over subjects, the source locations of EEG frequency bands differed significantly in the vertical and antero-posterior dimensions. Results of data set 2 confirmed those of data set 1. The source of delta was deepest and most anterior, theta more posterior and less deep, alpha most posterior and highest on the vertical dimension, beta-1 deeper and slightly more anterior than alpha, and beta-2 again more anterior and deeper than beta-1. Thus, the depth of source location was not linearly related to temporal frequency. The sources of all 5 bands were oriented in the sagittal direction; delta mean fields had steeper gradients anteriorly, alpha and beta-1 posteriorly. The power map for any frequency was well described by a single phase angle. The results indicate that the different EEG frequency bands during a given EEG epoch are generated by neural populations in different brain locations.     

Pharmacological and Electrophysiological Properties of Recombinant GABAA Receptors Comprising the alpha3, beta1 and gamma2 Subunits

To assess the role of subunits for channel function and drug modulation in recombinant GABA_A receptors, the α3β1γ2 subunits and the dual combinations α3β1, β1γ2 and α3γ2 were expressed by transfection of human embryonic kidney cells and by RNA injection in Xenopus oocytes (α3β1γ2 combination). GABA-induced chloride currents were recorded using the whole-cell configuration of the patch-clamp technique (transfected cells) or the voltage-clamp technique (oocytes). The currents recorded from the α3β1Γ2 subunit combination in transfected cells were reduced by bicuculline and picrotoxin, enhanced by flunitrazepam in a flumazenil-sensitive manner and reduced by β-carboline-3-carboxylic acid methyl ester (β-CCM). The GABA-induced current was reduced by β-CCM in all combinations containing the γ2 subunit, but potentiation by flunitrazepam was only obtained when the 72 subunit was coexpressed in the presence of the α3 subunit (α3β1γ2 or α3γ2). The GABA sensitivities of the receptors were similar when the α3β1γ2 combination was expressed in oocytes (half-maximum effective concentration = 240 μM) or in the kidney cell line (270 μM). However, the currents were less potentiated by flunitrazepam in oocytes (129% of controls) than in transfected cells (189%). These results suggest that the α3β1γ2 subunit combination, which is coexpressed in various brain regions as shown by in situ hybridization histochemistry, may represent a building block of functional GABA_A receptors in situ.     

Development and localisation of GABA(A) receptor alpha1, alpha2, beta2 and gamma2 subunit mRNA in the chick optic tectum

An in situ hybridisation technique was used to analyse the spatial and temporal pattern of expression of the mRNA encoding the four gamma-aminobutyric acid A (GABA(A)) receptor subunits (alpha1, alpha2, beta2, and gamma2) in the developing chick optic tectum. As a rule, layer i, layer h, and transient cell compartment 3 (TCC3) show the highest levels of expression, especially of alpha1, alpha2 and beta2, which undergo striking changes as a function of time. Apart from these common features, the global pattern is highly complex and dynamic. Such complexity derives from the fact that each subunit exhibits a characteristically distinct pattern of expression and the temporal evolution of each differs in the different layers of the tectum. The influence of several developmental cell behaviours such as proliferation, neuronal migration, programmed cell death, and differentiation must be taken into account to understand pattern complexity and dynamics. Our results suggest that differences in the rate of subunit expression, particularly of alpha1, alpha2, and beta2, could have significant consequences on GABA(A) receptor complex subunit composition along development and on the functional properties of the GABA neurotransmitter system.     

Ric-8B interacts with G alpha olf and G gamma 13 and co-localizes with G alpha olf, G beta 1 and G gamma 13 in the cilia of olfactory sensory neurons

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with Gαolf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with Gγ13, a divergent member of the Gγ subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that Gβ1 is the predominant Gβ subunit expressed in the olfactory sensory neurons. Ric-8B and Gβ1, like Gαolf and Gγ13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with Gαolf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction.     

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

Recent studies suggest that nuclear factor NF-kappaB may be involved in excitotoxin-induced cell apopotosis. To analyze the variation of NF-kappaB, levels of NF-kappaB were measured after the rats were subjected to 30 min of four-vessel occlusion and sacrificed in selected reperfusion time points. Induction of NF-kappaB consisting mainly of p65 and p50 subunits was detected by Western blot with anti p65, p50 antibodies, respectively. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our studies indicate that ischemia-induced NF-kappaB nuclear translocation is time-dependent. Inductions or binding activity of NF-kappaB in nucleus increased about 10-fold from 6 to 12 h as compared with that of the control group, then gradually declined in the following 24, 72 h. To further analyze the regulation by ionotropic glutamate receptor and L-type voltage-gated Ca(2+) channel (L-VGCC) in vivo, N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione and L-VGCC antagonist nifedipine were given 20 min prior to 30 min of ischemia. The NF-kappaB nuclear translocation was completely blocked by these three antagonists in a dose-dependent manner after ischemia/reperfusion 6 h. Increased phosphorylation of the NF-kappaB regulatory unit IkappaB-alpha was detected by Western blot. Decrement of IkappaB-alpha was found after 3 h reperfusion in the cytoplasm following global ischemia, which was also blocked by such three antagonists. These results illustrate that glutamate-gated ionotropic NMDA or non-NMDA receptors and voltage-gated Ca(2+) channels are important routes to mediate NF-kappaB activation during brain ischemic injury. Active NF-kappaB may attend the excitotoxin-induced cell death in turn. Our studies also suggest that IkappaB-alpha is an important regulatory unit that controls the activation of NF-kappaB after its phosphorylation and degradation and resynthesis in rat hippocampus following global ischemia.     

Functional characteristics and sites of gene expression of the alpha 1, beta 1, gamma 2-isoform of the rat GABAA receptor

GABAA receptors, the major synaptic targets for the neurotransmitter GABA, constitute gated chloride channels. By their allosteric, drug-induced modulation, they serve as control elements for the regulation of anxiety, vigilance, and epileptiform activity. The structural requirements of fully functional GABAA receptors in the mammalian brain have remained elusive so far. We report here on the cloning of the gamma 2-subunit cDNA of rat brain and its functional analysis by coexpression with the alpha 1- and beta 1-subunits in Xenopus oocytes, and on the sites of gene expression of the 3 subunits in the rat brain. The recombinant receptor displayed GABA-inducible currents (Imax = 6 microA; Ka = 75 microM) which were allosterically modulated by benzodiazepine receptor ligands (enhancement and inhibition by diazepam and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, respectively). In the absence of GABA, pentobarbital elicited a maximal current amplitude similar to that of GABA. A minor population of channels is expressed which is open in the absence of GABA or pentobarbital. Mapping subunit gene expression by in situ hybridization histochemistry suggests that the alpha 1-, beta 1-, and gamma 2-subunits are likely receptor constituents in some neuronal populations, e.g., mitral cells of the olfactory bulb, pyramidal cells of the hippocampus, and granule cells of the dentate gyrus and cerebellum.     

Immunohistochemical localization of interleukin-2 and its receptor subunits alpha, beta and gamma in the main olfactory bulb of the rat

Endogenous interleukin-2 (IL-2) was found in the adult rat brain, however, it has not been reported whether this cytokine is present in the olfactory bulb. Immunohistochemical techniques were used to examine the cellular localization of IL-2 and its receptor subunits in the main olfactory bulb of the rat. Strong IL-2 immunoreactivity was localized in glial cells, specifically in the olfactory nerve layer, glomerular layer and external plexiform layer. IL-2 mRNA was detected in the olfactory bulb by RT-PCR. All three IL-2 receptor subunits also showed distinct laminar distributions. The IL-2Ralpha and IL-2Rbeta immunoreactivity was found both in neurons and glial cells, whereas IL-2Rgamma imunoreactivity was found in glial cells, and thus resembled IL-2 immunostaining. The present results demonstrated a wide distribution of IL-2 and its receptor subunits in the main olfactory bulb of the rat, suggesting that IL-2 might play a role in the olfactory function through autocrine or paracrine pathways. The exclusive high expression of IL-2 in glial cells in distinct laminar structures, where neuron-glia interactions are closely associated with olfactory nerve regeneration, imply that IL-2 might be involved in the process of nerve regeneration in the olfactory bulb.     

The presence of delta and mu-, but not kappa or ORL1 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 (ORL₁) 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 ORL₁ 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 ORL₁ receptors are present on the pinealocytes, and the majority of opioid receptors found in the bovine pineal gland are δ (possibly, both δ₁ and δ₂) 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.     

Lymphomononuclear cells from multiple sclerosis patients spontaneously produce high levels of oncostatin M,tumor necrosis factors alpha and beta,and interferon gamma

Proinflammatory cytokines are deemed to play a pivotal role in the pathogenesis of multiple sderosis (MS). They provide signals for T-cell activation and inflammatory cell recruitment in the brain and might directly alter neuroglial and neuronal cell survival and function. We found that peripheral blood mononuclear cells (PBMCs) from MS patents spontaneously produce high levels of TNFalpha, TNFbeta, IFNgamma, and oncostatin M (oncM), a proinflammatory cytokine actng on cells of neural, vascular, hematopoietic, and lymphoid origin. Spontaneous production of these cytokines was significantly higher (p < 0.01) in PBMC short-term culture supernatants from MS patients than in blood donors (HC). On average, lectin-induced production of these cytokines by PBMC was higher in MS patents than in HC significantly so only for TNFalpha (p = 0.013). Determination of TNFalpha, TNFbeta IFNgamma, and oncM in corresponding sera showed that on average, oncM levels were higher in MS patients than in HC, though the results were not statistically significant whereas levels of TNFalpha, TNFbeta and IFNgamma were below the assay threshold in most patients. The finding that MS PBMCs are primed in vivo to produce and release high levels of proinflammatory cytokines suggests the presence of a basal activation of the immune system which, in turn, may play a role in the complex circuitry of molecular and cellular interactions responsible for neurologic damage in MS.