Biomedical subjects

C Abbadie

Publications and source records attributed to C Abbadie.

At least 19 recordsLinked to original sources

PubMed2004

Immunohistochemical study of the expression of exon11-containing mu opioid receptor variants in mouse brain.

The cloned mu opioid receptor MOR-1 undergoes alternative splicing. Extensive 3'-splicing downstream from exon 3 leads to a number of C-terminal splice variants that are differentially expressed within the CNS. Recently, 5'-splicing has been observed with eight additional variants containing exon 11, a new exon located approximately 10 kb upstream from exon 1 that is under the control of a different promoter located even further upstream. Three of these variants generate the same protein as MOR-1 itself, but under the control of the new exon 11 promoter. Three variants in which exon 11 is translated have been identified within the brain, including MOR-1G, MOR-1M and MOR-1N. The present paper defines immunohistochemically the distribution of these variants using an exon 11-specific antiserum. The expression of exon 11-like immunoreactivity (-LI) was seen primarily in the olfactory tubercle, caudate-putamen, globus pallidus and substantia nigra. We did not observe exon 11-LI in a number of regions expressing MOR-1. Within the caudate-putamen, the general pattern of labeling was diffuse, in contrast to the pattern seen with an exon 4-generated antiserum that labels MOR-1 itself. However, we did observe in the caudate-putamen co-expression of exon 4- and exon 11-LI in cells that were apposed to dopaminergic terminals. These results provide new insights regarding the potential physiological significance of these exon 11-containing variants.

Alternative Splicing↗

PubMed2002

Anatomical and functional correlation of the endomorphins with mu opioid receptor splice variants.

The present study characterizes the relationship between the endogenous mu opioid peptides endomorphin-1 (EM-1) and endomorphin-2 (EM-2) and several splice variants of the cloned mu opioid receptor (MOR-1) encoded by the mu opioid receptor gene (Oprm). Confocal laser microscopy revealed that fibers containing EM-2-like immunoreactivity (-LI) were distributed in close apposition to fibers showing MOR-1-LI (exon 4-LI) and to MOR-1C-LI (exons 7/8/9-LI) in the superficial laminae of the lumbar spinal cord. We also observed colocalization of EM-2-LI and MOR-1-LI in a few fibers of lamina II, and colocalization of EM-2-LI and MOR-1C-LI in laminae I-II, and V-VI. To assess the functional relevance of the MOR-1 variants in endomorphin analgesia, we examined the effects of antisense treatments that targeted individual exons within the Oprm1 gene on EM-1 and EM-2 analgesia in the tail flick test. This antisense mapping study implied mu opioid receptor mechanisms for the endomorphins are distinct from those of morphine or morphine-6beta-glucuronide (M6G).

Alternative Splicing↗

PubMed2001

cRel induces mitochondrial alterations in correlation with proliferation arrest.

We have previously shown that overexpressing cRel, a transcription factor of the Rel/NF-kappa B family, concomitantly inhibits proliferation of HeLa cells and makes them resistant against TNF alpha-induced apoptosis. Both effects rely on the upregulation of the manganese superoxide dismutase (MnSOD), a mitochondrial enzyme that converts O(2)(*-) in H(2)O(2). Here we describe additional alterations induced by cRel, namely mitochondrial clustering and accumulation of dense dark granules near the nucleus. These changes preferentially occur in cells that display a sustained cRel expression in the nucleus and that are cell-cycle arrested. As the cell-cycle arrest, these changes are reproduced by directly overexpressing MnSOD or by treating cells with H(2)O(2), suggesting they are due to MnSOD induction and ensuing H(2)O(2) accumulation. We propose that mitochondria cluster because they are damaged by the H(2)O(2) they overproduce. They would then be autophagocytosed and degraded in secondary lysosomes. In support of this scenario, we documented the occurrence of oxidative damage and the presence of lysosomes in the area of mitochondrial clustering. In addition, we identified the dense dark granules as lipofuscin, based on their autofluorescence. Lipofuscin could directly originate from the mitochondrial degradation products that would aggregate and become indigestible because of the presence of H(2)O(2) in the secondary lysosomes. Altogether, our findings show that cRel overexpression in HeLa cells creates, via the induction of MnSOD, an oxidative injury that culminates in mitochondrial degeneration, proliferation blockage, and resistance against TNF alpha-induced apoptosis.

Cell Cycle↗

PubMed2001

Differential in vivo internalization of MOR-1 and MOR-1C by morphine.

The mu opioid receptor MOR-1 is internalized by many mu agonists, but not morphine. To see whether differences in the intracellular carboxy terminus influences internalization, we examined internalization of a splice variant of the mu opioid receptor, MOR-1C, in the lateral septum of the mouse in vivo. Following intracerebroventricular (i.c.v.) saline treatment, MOR-1C-like immunoreactivity (LI) within neurons in naive mice was found predominantly in clusters close to the plasma membrane. Following either intracerebroventricular [d-Ala2, MePhe4,Gly(ol)5]enkephalin (DAMGO) or morphine, MOR-1C-LI clustered into endosomes in the cytoplasm. This effect was suppressed by prior administration of the opioid antagonist naloxone. In contrast, only DAMGO, and not morphine, internalized MOR-1-LI. These results illustrate differences in internalization between two MOR-1 variants that have alternative splicing at the COOH terminus.

Alternative Splicing↗

PubMed2001

Differential contribution of substance P and neurokinin A to spinal cord neurokinin-1 receptor signaling in the rat.

Although the tachykinins substance P (SP) and neurokinin A (NKA) are coreleased from primary afferent nociceptors and act via neurokinin (NK) receptors, their differential effects in vivo are not known. Despite pharmacological evidence that NKA preferentially binds NK-2 receptors, this receptor is not found in spinal cord neurons. Thus, in the present studies, we compared the extent to which SP and NKA contribute to spinal nociceptive processing via the NK-1 receptor. We found that SP and NKA induce NK-1 receptor internalization with identical dose dependence and induce increases in intracellular calcium at the same concentrations, suggesting that SP and NKA equally activate the NK-1 receptor. We found, however, that the selective NK-1 receptor antagonist GR 205171 blocked NKA but not SP-induced NK-1 receptor internalization in the rat spinal cord in vivo and in embryonic day 19 rat spinal neurons in vitro. Using this selectivity of GR 205171 for NKA-induced NK-1 receptor activation, we examined the relative contribution of SP and NKA to noxious stimulus-induced activation of spinal NK-1 receptors. We estimate that NKA contributes to at least 50% of the NK-1 receptor activation in lamina I. Under inflammatory conditions, all noxious stimulus-induced NK-1 receptor internalization in deep dorsal horn neurons was blocked by GR 205171, suggesting that it is entirely NKA-mediated. Substance P-mediated NK-1 receptor internalization was focused at the site of termination of stimulated nociceptors but NKA also activated NK-1 receptors at more distant sites. We conclude that NKA not only targets the NK-1 receptor but may be a predominant pronociceptive primary afferent neurotransmitter.

Animals↗

PubMed2001

Rel/NF-kappaB transcription factors protect against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by up-regulating the TRAIL decoy receptor DcR1.

Rel/nuclear factor (NF)-kappaB transcription factors play a major role in the regulation of programmed cell death. A few anti-apoptotic Rel/NF-kappaB target genes have been characterized; they act either downstream in the apoptotic pathway or upstream, for example at the tumor necrosis factor (TNF) receptor level. We found using DNA arrays, reverse transcription-polymerase chain reaction, and immunofluorescence that Rel/NF-kappaB factors up-regulate DcR1, a receptor for TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF family that induces apoptosis in tumor cells. Four related receptors bind TRAIL, two death receptors (DR4 and DR5) that signal apoptosis and two decoy receptors (DcR1 and DcR2) that act as dominant negative inhibitors of TRAIL-mediated apoptosis. DcR1 is devoid of an intracellular domain and is anchored at the cell surface membrane by a glycophospholipid. Our results indicate that overexpression of cRel or activation of endogenous Rel/NF-kappaB factors by TNFalpha in HeLa cells up-regulates DcR1 without changing the expression of DcR2, DR4, and DR5 and makes cells resistant against TRAIL-induced apoptosis. This resistance is a consequence of DcR1 up-regulation, because it was abolished when DcR1 was removed from the cell surface by a phosphatidylinositol phospholipase C. Therefore, Rel/NF-kappaB transcription factors could regulate the sensitivity of cells to TRAIL, by controlling the ratio of TRAIL-decoy to -death receptors.

Apoptosis↗

PubMed2001

Antiproliferative and antiapoptotic effects of crel may occur within the same cells via the up-regulation of manganese superoxide dismutase.

Rel/nuclear factor kappaB transcription factors were shown to have either pro- or antiapoptotic as well as pro- or antiproliferative functions, and it is often assumed that the outcome of their activation depends on the cell type or cellular context. Inconsistent with this assumption, we show here that cRel is able in one cell type to inhibit proliferation, protect against apoptosis induced by tumor necrosis factor alpha (TNF-alpha) + cycloheximide (CHX), and increase the basal rate of apoptosis. Both the effects of proliferation inhibition and protection against TNF-alpha + CHX-induced apoptosis are massive and occur in the same cells. Using reverse transcription-PCR, Western blot and immunofluorescence, and transactivation assays, we found that the manganese superoxide dismutase (MnSOD), an enzyme that converts O2*- in H2O2, is up-regulated by cRel through a kappaB site in intron 2. Inhibition of MnSOD induction by antisense oligonucleotides and overexpression of MnSOD respectively reverts and mimics both the antiproliferative and antiapoptotic effects of cRel, suggesting that they both occur via the induction of this gene. On one hand, MnSOD could improve the efficiency of cRel-overexpressing cells in eliminating toxic O2*- produced on TNF-alpha treatment, explaining why they escape TNF-alpha-induced apoptosis. On the other hand, cRel-overexpressing cells should accumulate H2O2. We present evidence linking this H2O2 accumulation to the proliferation arrest induced by cRel. Therefore, different effects on proliferation and apoptosis could arise from the induction of MnSOD and thus coexist in cRel-overexpressing cells.

Apoptosis↗

PubMed2001

Presynaptic localization of the carboxy-terminus epitopes of the mu opioid receptor splice variants MOR-1C and MOR-1D in the superficial laminae of the rat spinal cord.

Opioids inhibit nociceptive transmission at the level of the spinal cord, possibly through inhibition of neurotransmitter release by presynaptic mu opioid receptors (MORs) thus preventing the activation of ascending pathways and the perception of pain. Most nociceptive primary afferents are unmyelinated fibers containing peptides such as substance P and/or calcitonin gene-related peptide. However, few terminals contain both substance P and MOR. Recently, we identified new carboxy-terminal MOR splice variants that are localized in the superficial laminae of the dorsal horn. We now report the precise cellular distribution of two of these MOR-1 variants, MOR-1C (exon 7/8/9 epitope) and MOR-1D (exon 8/9 epitope), at the ultrastructural level. In the superficial laminae of the dorsal horn, the majority of the labeling of MOR-1C and MOR-1D was found in unmyelinated axons. This distribution contrasts with that of MOR-1 (exon 4 epitope), in which labeling is equally found in dendrites and soma, as well as in axons. The presence of dense core vesicles in many of the MOR-1C-like immunoreactive terminals implies that this splice variant might be involved in presynaptic inhibition of transmitter release from peptide-containing afferents to the dorsal horn. Consistent with this finding, confocal microscopy analyses showed that many MOR-1C profiles in laminae I-II also contained calcitonin gene-related peptide, whereas fewer MOR-1 profiles contained either substance P or calcitonin gene-related peptide in this same region. From these findings we suggest that there are differential distributions of MOR-1 splice variants as well as distinct peptide colocalizations in the dorsal horn.

Afferent Pathways↗

PubMed2000

Postsynaptic signaling via the [mu]-opioid receptor: responses of dorsal horn neurons to exogenous opioids and noxious stimulation.

Although both pre- and postsynaptic mechanisms have been implicated in the analgesia produced by mu-opioids at the spinal cord, it is not known under what conditions these different controls come into play. Because the mu-opioid receptor (MOR) can be visualized in individual lamina II excitatory interneurons and internalizes into endosomes on ligand binding, we tested whether MOR internalization could be monitored and used to measure postsynaptic MOR signaling. To test whether endogenous opioids modulate these lamina II interneurons during noxious stimulation, we next assessed the magnitude of postsynaptic MOR internalization under a variety of nociceptive conditions. As observed in other systems, we show that MOR internalization in dorsal horn interneurons is demonstrated readily in response to opioid ligands. The MOR internalization is dose-dependent, with a similar dose-response to that observed for opioid-induced increases in potassium conductance. We demonstrate that MOR internalization in lamina II neurons correlates precisely with the extent of analgesia produced by intrathecal DAMGO. These results suggest that MOR internalization provides a good marker of MOR signaling in the spinal cord and that postsynaptic MORs on lamina II interneurons likely participate in the analgesia that is produced by exogenous opioids. We found, however, that noxious stimuli, under normal or inflammatory conditions, did not induce MOR internalization. Thus, endogenous enkephalins and endomorphins, thought to be released during noxious peripheral stimuli, do not modulate nociceptive messages via postsynaptic MORs on lamina II interneurons. We suggest that any endogenous opioids that are released by noxious stimuli target presynaptic MORs or delta-opioid receptors.

Analgesics, Opioid↗

PubMed2000

Immunohistochemical localization of the carboxy terminus of the novel mu opioid receptor splice variant MOR-1C within the human spinal cord.

The present study examined the distribution in the human spinal cord of a unique carboxy terminus sequence contained within MOR-1C, one of the recently described splice variants of the cloned mu opioid receptor gene MOR-1. Although MOR-1-like immunoreactivity (LI) and delta opioid receptor-like immunoreactivity were observed only in the superficial laminae, MOR-1C-LI was abundant in the superficial laminae of the dorsal horn and around the central canal. In the substantia gelatinosa, MOR-1C-LI was found in small diameter axonal elements, the cytoplasm and the plasmalemma of small spinal neurons and dendrites in inner lamina II and in some fibers within Lissauer's tract. These studies imply the presence of MOR-1C in human spinal cord and its distribution suggests that it plays a role in the control of pain processing.

Animals↗

PubMed2000

Differential distribution in rat brain of mu opioid receptor carboxy terminal splice variants MOR-1C-like and MOR-1-like immunoreactivity: evidence for region-specific processing.

The present study examined immunohistochemically the regional distribution of the mu opioid receptor splice variant MOR-1C by using a rabbit antisera generated against the C-terminal peptide sequences and compared it with MOR-1. Overall, the distribution of MOR-1C-like immunoreactivity (-LI) differed from MOR-1-LI. Both MOR-1C-LI and MOR-1-LI were prominent in a few central nervous system regions, including the lateral parabrachial nucleus, the periaqueductal gray, and laminae I-II of the spinal trigeminal nuclei and the spinal cord. In the striatum, hippocampal formation, presubiculum and parasubiculum, amygdaloid nuclei, thalamic nuclei, locus coeruleus, and nucleus ambiguous MOR-1-LI predominated, whereas MOR-1C-LI was absent or sparse. Conversely, MOR-1C-LI exceeded MOR-1-LI in the lateral septum, the deep laminae of the spinal cord, and most hypothalamic nuclei such as the median eminence, periventricular, suprachiasmatic, supraoptic, arcuate, paraventricular, ventromedial, and dorsomedial nuclei. Double-labeling studies showed colocalization of the two receptors in neurons of the lateral septum, but not in the median eminence or in the arcuate nucleus, even though both MOR-1 isoforms were expressed. Because both MOR-1 and MOR-1C are derived from the same gene, these differences in regional distribution represent region-specific mRNA processing. The regional distributions reported in this study involve the epitope seen by the combinations of exons 7, 8, and 9. However, if other MOR-1 variants containing exons 7, 8, and 9 exist, the antisera would not distinguish between them and MOR-1C.

Alternative Splicing↗

PubMed2000

Comparative immunohistochemical distributions of carboxy terminus epitopes from the mu-opioid receptor splice variants MOR-1D, MOR-1 and MOR-1C in the mouse and rat CNS.

The present study examined immunohistochemically the CNS distributions of a splice variant of the mu-opioid receptor, MOR-1D, in both rats and mice. In MOR-1D, exon 4 of MOR-1 is replaced by two additional exons that code for seven amino acids. Using rabbit antisera, we compared immunohistochemically the regional distribution of a C-terminal epitope of MOR-1D to that of a C-terminal epitope from MOR-1 and a C-terminal epitope from another splice variant, MOR-1C. The general distribution of MOR-1D-like immunoreactivity was similar in both mouse and rat. MOR-1D-like immunoreactivity was seen in the dentate gyrus and in the mossy fibers of the hippocampal formation, the nucleus of the solitary tract and the area postrema, the inferior olivary nucleus, the nucleus ambiguous, the spinal trigeminal nucleus and the spinal cord. MOR-1D-like immunoreactivity was not observed in some regions containing dense MOR-1-like immunoreactivity, such as the striatum or the locus coeruleus. In regions containing MOR-1, MOR-1C and MOR-1D, the pattern of each variant was unique.MOR-1D and MOR-1C are splice variants of the cloned mu-opioid receptor MOR-1. Although they differ only at the tip of the carboxy terminus, they show marked differences in their regional distributions, as determined immunohistochemically by epitopes in their unique carboxy termini. Since the splice variants are derived from the same gene, these differences in regional distribution imply region-specific messenger RNA processing.

Animals↗

PubMed1999

Spinal opioid analgesia: how critical is the regulation of substance P signaling?

Although opioids can reduce stimulus-evoked efflux of Substance P (SP) from nociceptive primary afferents, the consequences of this reduction on spinal cord nociceptive processing has not been studied. Rather than assaying SP release, in the present study we examined the effect of opioids on two postsynaptic measures of SP release, Fos expression and neurokinin-1 (NK-1) receptor internalization, in the rat. The functional significance of the latter was first established in in vitro studies that showed that SP-induced Ca(2+) mobilization is highly correlated with the magnitude of SP-induced NK-1 receptor internalization in dorsal horn neurons. Using an in vivo analysis, we found that morphine had little effect on noxious stimulus-evoked internalization of the NK-1 receptor in lamina I neurons. However, internalization was reduced when we coadministered morphine with a dose of an NK-1 receptor antagonist that by itself was without effect. Thus, although opioids may modulate SP release, the residual release is sufficient to exert maximal effects on the target NK-1 receptors. Morphine significantly reduced noxious stimulus-induced Fos expression in lamina I, but the Fos inhibition was less pronounced in neurons that expressed the NK-1 receptor. Taken together, these results suggest that opioid analgesia predominantly involves postsynaptic inhibitory mechanisms and/or presynaptic control of non-SP-containing primary afferent nociceptors.

Analgesia, Epidural↗

PubMed1999

Neurons in the dorsal column white matter of the spinal cord: complex neuropil in an unexpected location.

It is common to think of gray matter as the site of integration in neural circuits and white matter as the wires that connect different groups of neurons. The dorsal column (DC) white matter, for example, is the spinal cord axonal pathway through which a topographic map of the body is conveyed to the somatosensory cortex. We now describe a network of neurons located along the midline of the DCs. The neurons are present in several mammals, including primates and birds, and have a profuse dendritic arbor that expresses both the neuron-specific marker, microtubule-associated protein-2, and the neurokinin-1 receptor, a target of the neuropeptide, substance P. Electron microscopy and double immunostaining for synaptophysin and a marker of gamma-aminobutyric acid-ergic terminals documented a rich synaptic input to these neurons. Finally, injection of a gamma-aminobutyric acid type A receptor antagonist or of substance P into the cerebrospinal fluid of the rat spinal cord induced Fos expression and internalization of the neurokinin-1 receptor in these neurons, respectively, indicating that the DC neurons are under tonic inhibitory control and can respond to neurotransmitters that circulate in the cerebrospinal fluid.

Animals↗

PubMed1999

Expression of transcription factor c-Rel and apoptosis occurrence in polydactylous and syndactylous limb buds of the talpid3 mutant chick embryo.

The chicken proto-oncogene c-rel encodes a transcription factor of the Rel/NF-kappa B family. We have previously shown that c-rel mRNAs accumulate in different types of apoptotic cells of the chick embryo, especially in mesenchymal cells within the four cell death areas of the limb bud: the anterior and posterior necrotic zones, the opaque patch and the interdigital necrotic zones. This study aimed to further establish the involvement of c-Rel in apoptosis of the developing limb by investigating its expression in the talpid3 mutant which was originally shown to be defective in apoptosis. However, our preliminary examinations highlighted the apparent presence of apoptotic cells in talpid3 embryos. Hence, we performed a systematic study of the occurrence of apoptosis in mutant and control embryos by the TUNEL method. The results revealed that apoptosis does occur in talpid3 embryos but with altered spatial and temporal patterns. This suggests that the talpid3 mutation does not affect a gene involved in apoptosis per se but rather in the determination of the pattern of apoptosis. Neither the expression of c-Rel nor that of its I kappa B alpha inhibitor are grossly modified in talpid3 limb buds, suggesting that the talpid3 mutation does not affect any of these genes. They are mostly expressed in epidermal, endodermal and striated muscle cells in control and in talpid3 limb buds as well. C-Rel was also detected in some scarce mesenchymal cells that could be apoptotic, in both control and mutant embryos. The only slight difference between control and talpid3 limbs lies in the perichondrium which is not fully differentiated in talpid3 embryos: c-Rel and I kappa B alpha are only faintly expressed in talpid3 perichondrial cells, whereas they are both detected in control perichondrial cells. Taken together, these results suggest that c-Rel could participate in several developmental processes, especially in the differentiation of perichondrial cells, besides its already documented involvement in apoptosis and haematopoeisis.

Journal Article↗

PubMed1999

Identification and characterization of three new alternatively spliced mu-opioid receptor isoforms.

We have identified four new mu-opiod receptor (MOR)-1 exons, indicating that the gene now contains at least nine exons spanning more than 200 kilobases. Replacement of exon 4 by combinations of the new exons yields three new receptors. When expressed in Chinese hamster ovary cells, all three variants displayed high affinity for mu-opioid ligands, but kappa and delta drugs were inactive. However, there were subtle, but significant, differences in the binding profiles of the three variants among themselves and from MOR-1. Immunohistochemically, the major variant, MOR-1C, displayed a regional distribution quite distinct from that of MOR-1. Region-specific processing also was seen at the mRNA level. Antisense mapping revealed that the four new exons were all involved in morphine analgesia. Together with two other variants generated from alternative splicing of exon 4, there are now six distinct MOR-1 receptors.

Alternative Splicing↗

PubMed1998

The contribution of capsaicin-sensitive afferents to the dorsal root ganglion sprouting of sympathetic axons after peripheral nerve injury in the rat.

Transection of the sciatic nerve leads to sprouting of sympathetic efferent, noradrenergic axons and terminals around large cell bodies in the dorsal root ganglion. Here we examined whether injury to unmyelinated afferents contributes to the sprouting. Neonatal treatment with the C-fiber neurotoxin capsaicin increased sprouting after nerve injury. We conclude that injury to large, rather than small diameter fibers, triggers the sprouting of sympathetic efferents after nerve injury.

Animals↗

PubMed1998

The avian transcription factor c-Rel is expressed in lymphocyte precursor cells and antigen-presenting cells during thymus development.

Transcription factors of the Rel/NF-kappaB family are widely involved in the immune system. In this study, we investigate the in vivo expression of the avian protein c-Rel in the T-cell lineage during thymus development. The majority of thymocytes do not express the c-Rel protein. However, lymphocyte precursor cells that colonize the thymus express the c-Rel protein shortly after their homing in the organ and before they begin to differentiate. c-Rel is also detected in different subsets of antigen-presenting cells such as epithelial cells, dendritic cells, and macrophages. In vitro studies have shown that Rel/NF-kappaB proteins are sequestered in an inactive form in the cytoplasm by interaction with the IkappaBalpha inhibitory protein. By immunocytochemistry, we show that in vivo c-Rel is localized in the cytoplasm of antigen-presenting cells but in both the cytoplasm and nucleus of lymphocyte precursor cells. The cytoplasmic localization of c-Rel in antigen-presenting cells correlates with a high expression of IkappaBalpha, whereas the nuclear localization of c-Rel in lymphocyte precursor cells correlates with a much lower expression of IkappaBalpha. These results suggest that c-Rel might be constitutively activated in lymphocyte precursor cells.

Animals↗