scholarly journals Opioid Receptor Protein Expression During Development of the Rat Brainstem

2021 ◽  
Author(s):  
◽  
Bronwyn Maree Kivell
Keyword(s):  
Confocal Microscopy ◽  
Opioid Receptor ◽  
Glial Cells ◽  
Opioid Receptors ◽  
Cultured Cells ◽  
Expression Patterns ◽  
Receptor Expression ◽  
Developmental Expression ◽  
Receptor Protein ◽  
Western Blots

<p>Few satisfactory protocols exist for primary culture of postnatal brainstem neurons, and commonly used procedures often give poor survival rates in older foetal (>E16) and early postnatal brainstem cultures. The present study describes the first reliable method for establishing stable in vitro cultures of foetal and postnatal brainstem neurons up to six days postnatal age in a defined, serum-free culture medium. This novel culture method was used to study opioid receptor expression and distribution in developing brainstem cells. Opioids play an important role in brainstem functions, being involved in respiratory and cardiovascular modulation and pain control (Olsen et al., 1995; Olson et al., 1997; Vaccarino et al., 1999; Vaccarino and Kastin, 2001). These brainstem functions are particularly important for survival at birth, and opioid receptor distribution patterns and sensitivities to opioid ligands change during development. Using cultured cells and frozen sections of brainstem tissue, mu (MOR) and delta (DOR) opioid receptor localisation in neuronal and glial cells at different stages of foetal and postnatal development in the rat were examined by immunocytochemistry and confocal microscopy. Bipolar and multipolar neurons showed similar immunoreactivities; whereas, glial cells were more lightly stained than neurons. Developmentally advanced stages were more intensely stained for MOR (P<0.006, Mann-Whitney test); whereas, DOR immunoreactivity did not change during development. These developmental expression patterns observed in culture for MOR were similar to those obtained from Western blots of electrophoreses brainstem lysates. DOR, however, decreased in expression in brainstem lysates with increased developmental age, even though there was no difference in DOR expression in cultured cells. MOR and DOR were colocalised in specific brainstem regions and in the cerebellum of foetal and postnatal animals, although the distribution of both opioid receptors in the foetal brain was more diffuse than in the older animals. The intracellular distributions of MOR and DOR were investigated by confocal microscopy. In addition to plasma membrane staining, a population of internalised cytoplasmic receptors was present in neurons. MOR was down-regulated after exposure of either cultured brainstem cells or transfected or non-transfected SH-SY5Y neuroblastoma cells to the MOR agonist DAMGO. From the above investigation, it was concluded that opioid receptors are developmentally regulated during maturation of the brainstem of the rat, and that primary cell culture, immunocytochemistry, and immunoblotting of cell lysates are suitable techniques for investigating opioid systems in the foetal, postnatal, and adult rat.</p>

scholarly journals Opioid Receptor Protein Expression During Development of the Rat Brainstem

2021 ◽  
Author(s):  
◽  
Bronwyn Maree Kivell
Keyword(s):  
Confocal Microscopy ◽  
Opioid Receptor ◽  
Glial Cells ◽  
Opioid Receptors ◽  
Cultured Cells ◽  
Expression Patterns ◽  
Receptor Expression ◽  
Developmental Expression ◽  
Receptor Protein ◽  
Western Blots

<p>Few satisfactory protocols exist for primary culture of postnatal brainstem neurons, and commonly used procedures often give poor survival rates in older foetal (>E16) and early postnatal brainstem cultures. The present study describes the first reliable method for establishing stable in vitro cultures of foetal and postnatal brainstem neurons up to six days postnatal age in a defined, serum-free culture medium. This novel culture method was used to study opioid receptor expression and distribution in developing brainstem cells. Opioids play an important role in brainstem functions, being involved in respiratory and cardiovascular modulation and pain control (Olsen et al., 1995; Olson et al., 1997; Vaccarino et al., 1999; Vaccarino and Kastin, 2001). These brainstem functions are particularly important for survival at birth, and opioid receptor distribution patterns and sensitivities to opioid ligands change during development. Using cultured cells and frozen sections of brainstem tissue, mu (MOR) and delta (DOR) opioid receptor localisation in neuronal and glial cells at different stages of foetal and postnatal development in the rat were examined by immunocytochemistry and confocal microscopy. Bipolar and multipolar neurons showed similar immunoreactivities; whereas, glial cells were more lightly stained than neurons. Developmentally advanced stages were more intensely stained for MOR (P<0.006, Mann-Whitney test); whereas, DOR immunoreactivity did not change during development. These developmental expression patterns observed in culture for MOR were similar to those obtained from Western blots of electrophoreses brainstem lysates. DOR, however, decreased in expression in brainstem lysates with increased developmental age, even though there was no difference in DOR expression in cultured cells. MOR and DOR were colocalised in specific brainstem regions and in the cerebellum of foetal and postnatal animals, although the distribution of both opioid receptors in the foetal brain was more diffuse than in the older animals. The intracellular distributions of MOR and DOR were investigated by confocal microscopy. In addition to plasma membrane staining, a population of internalised cytoplasmic receptors was present in neurons. MOR was down-regulated after exposure of either cultured brainstem cells or transfected or non-transfected SH-SY5Y neuroblastoma cells to the MOR agonist DAMGO. From the above investigation, it was concluded that opioid receptors are developmentally regulated during maturation of the brainstem of the rat, and that primary cell culture, immunocytochemistry, and immunoblotting of cell lysates are suitable techniques for investigating opioid systems in the foetal, postnatal, and adult rat.</p>

IP3, IP3receptor, and cellular senescence

2000 ◽  
Vol 278 (4) ◽  
pp. F576-F584 ◽  
Author(s):  
Ming-Shyan Huang ◽  
Olugbenga A. Adebanjo ◽  
Emmanuel Awumey ◽  
Gopa Biswas ◽  
Antoliy Koval ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cellular Senescence ◽  
Replicative Senescence ◽  
Receptor Expression ◽  
Receptor Protein ◽  
Western Blots ◽  
Human Diploid Fibroblasts ◽  
Store Depletion ◽  
Population Doublings

Herein we demonstrate that replicative cellular senescence in vitro results in sharply reduced inositol 1,4,5-trisphosphate (IP3) receptor levels, reduced mitogen-evoked IP3formation and Ca2+release, and Ca2+store depletion. Human diploid fibroblasts (HDFs) underwent either 30 mean population doublings [mean population doublings (MPDs) thymidine labeling index (TI) >92% (“young”) or between 53 and 58 MPDs (TI < 28%; “senescent”)]. We found that the cytosolic Ca2+release triggered by either ionomycin or by several IP3-generating mitogens, namely bradykinin, thrombin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), was attenuated markedly in senescent HDFs. Notably, the triggered cytosolic Ca2+transients were of a smaller magnitude in senescent HDFs. However, the response latency seen with both PDGF and EGF was greater for senescent cells. Finally, a smaller proportion of senescent HDFs showed oscillations. In parallel, IP3formation in response to bradykinin or EGF was also attenuated in senescent HDFs. Furthermore, senescent HDFs displayed a sharply diminished Ca2+release response to intracellularly applied IP3. Finally, to compare IP3receptor protein levels directly in young and senescent HDFs, their microsomal membranes were probed in Western blots with a highly specific anti-IP3receptor antiserum, Ab40. A ∼260-kDa band corresponding to the IP3receptor protein was noted; its intensity was reduced by ∼50% in senescent cells. Thus, we suggest that reduced IP3receptor expression, lowered IP3formation, and Ca2+release, as well as Ca2+store depletion, all contribute to the deficient Ca2+signaling seen in HDFs undergoing replicative senescence.

scholarly journals Microfibril-associated Glycoprotein-2 (MAGP-2) Is Specifically Associated with Fibrillin-containing Microfibrils but Exhibits More Restricted Patterns of Tissue Localization and Developmental Expression Than Its Structural Relative MAGP-1

1998 ◽  
Vol 46 (8) ◽  
pp. 871-885 ◽  
Author(s):  
Mark A. Gibson ◽  
Merran L. Finnis ◽  
Jaliya S. Kumaratilake ◽  
Edward G. Cleary
Keyword(s):  
Skeletal Muscle ◽  
Expression Patterns ◽  
Fetal Lung ◽  
Northern Blotting ◽  
Developmental Expression ◽  
Mrna Levels ◽  
Western Blots ◽  
Fibrillin 1 ◽  
Significant Expression ◽  
Nuchal Ligament

SUMMARY We developed an affinity-purified anti-MAGP-2 peptide antibody that specifically identified MAGP-2 on Western blots of purified matrix proteins and extracts of nuchal ligament. Immunolocalization studies on tissues from a 210-day-old fetus and a mature bovine showed that MAGP-2 was located in similar regions to MAGP-1 and fibrillin-1 but that the distribution of MAGP-2 was more restricted. In fetal nuchal ligament, skeletal muscle, and spleen the distribution of MAGP-2 was indistinguishable from that of MAGP-1. In contrast to MAGP-1, MAGP-2 was not detected in the medial layer of fetal thoracic aorta and in much of the peritubular matrix of fetal and mature kidney and in the mature ocular zonule. Some differences in the immunolocalization patterns were also evident in fetal lung, cartilage, skin, and heart. Immunoelectron microscopy confirmed that MAGP-2 was specifically associated with fibrillin-containing microfibrils in nuchal ligament, dermis, adventitia of aorta, glomerular mesangium and perimysium. Northern blotting of RNA from tissues of a 210-day-old fetus indicated that steady-state MAGP-2 mRNA levels were highest in nuchal ligament. Significant expression was also detected in lung, heart, skeletal muscle, skin, and Achilles tendon. The tissue pattern of MAGP-2 expression differed significantly from that of MAGP-1. MAGP-2 expression appeared to be higher in nuchal ligament, heart, and skeletal muscle and lower in aorta and kidney. In nuchal ligament, MAGP-2 mRNA expression appeared to peak around 180 days of fetal development, which correlates with the period of onset of elastinogenesis in this tissue. Overall, the immunolocalization and expression patterns of MAGP-2 appeared to be distinct from those of other microfibrillar components. This is consistent with the view that MAGP-2 plays a unique role in the biology of the microfibrils, perhaps by mediating their interaction with cell surfaces at specific stages of development and differentiation.

scholarly journals Developmental expression of retinoic acid receptors (RARs)

2009 ◽  
Vol 7 (1) ◽  
pp. nrs.07006 ◽  
Author(s):  
Pascal Dollé
Keyword(s):  
Retinoic Acid ◽  
Receptor Gene ◽  
Expression Patterns ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Retinoic Acid Receptors ◽  
Developmental Expression ◽  
Specific Expression ◽  
Functional Studies ◽  
Organ Systems

Here, I review the developmental expression features of genes encoding the retinoic acid receptors (RARs) and the ‘retinoid X’ or rexinoid receptors (RXRs). The first detailed expression studies were performed in the mouse over two decades ago, following the cloning of the murine Rar genes. These studies revealed complex expression features at all stages of post-implantation development, one receptor gene (Rara) showing widespread expression, the two others (Rarb and Rarg) with highly regionalized and/or cell type-specific expression in both neural and non-neural tissues. Rxr genes also have either widespread (Rxra, Rxrb), or highly-restricted (Rxrg) expression patterns. Studies performed in zebrafish and Xenopus demonstrated expression of Rar and Rxr genes (both maternal and zygotic), at early pre-gastrulation stages. The eventual characterization of specific enzymes involved in the synthesis of retinoic acid (retinol/retinaldehyde dehydrogenases), or the triggering of its catabolism (CYP26 cytochrome P450s), all of them showing differential expression patterns, led to a clearer understanding of the phenomenons regulated by retinoic acid signaling during development. Functional studies involving targeted gene disruptions in the mouse, and additional approaches such as dominant negative receptor expression in other models, have pinpointed the specific, versus partly redundant, roles of the RARs and RXRs in many developing organ systems. These pleiotropic roles are summarized hereafter in relationship to the receptors’ expression patterns.

scholarly journals Regulation of oxytocin receptor in the placentome capsule throughout pregnancy in the ewe: the possible role of oestradiol receptor, progesterone receptor and aromatase

1998 ◽  
Vol 158 (2) ◽  
pp. 173-181 ◽  
Author(s):  
ST Leung ◽  
TS Reynolds ◽  
DC Wathes
Keyword(s):  
Progesterone Receptor ◽  
Hormonal Regulation ◽  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Receptor Expression ◽  
Receptor Protein ◽  
Receptor Mrna ◽  
Oxytocin Receptors ◽  
Hybridisation Analysis

The hormonal regulation of uterine oxytocin receptors (OTR) during the establishment of pregnancy and at parturition has been studied extensively, but little information is available during mid-pregnancy. This study investigated the localisation of OTR mRNA in the ovine placentome throughout gestation and related this to expression patterns for the putative regulatory agents aromatase, oestradiol receptor, progesterone receptor and oxytocin. Placentomes were collected at regular intervals throughout pregnancy for in situ hybridisation analysis and immunocytochemistry (oestradiol receptor only). Results were quantified by optical density measurements of autoradiographs. Progesterone receptor mRNA was localised to the caruncular tissues on day 30 but became undetectable by day 34. Aromatase mRNA appeared in the fetal villi at days 34-40, with concentrations peaking at days 52-55 and again at days 132-137. Oestradiol receptor mRNA was localised to the caruncular tissues from days 13 to 30 and found in the maternal villi and placentome capsule from days 45 to 70. Oestradiol receptor protein was barely detectable in either tissue. OTR mRNA was localised to the placentome capsule at days 34-40, remaining high at day 45 and declining to basal levels by days 132-137. Oxytocin mRNA was not detected in the placentome. In conclusion: (1) progesterone acting via its receptor may suppress the expression of aromatase and OTR in early pregnancy; (2) the up-regulation of OTR expression in the capsule may not involve the oestradiol receptor; (3) there is a differential regulation between different regions of the uterus as the increase in the placentome capsule occurs at a time when concentrations in the rest of the endometrium and myometrium remain low; (4) oestradiol receptor expression in the placentome may be regulated at the translational level; and (5) there is no local production of oxytocin in the sheep placenta. The role of ORTs in the capsule during mid-pregnancy remains to be determined.

scholarly journals Wet cupping therapy improves mu opioid receptor expression and pain threshold in animal models of inflammation

2020 ◽  
Author(s):  
Imam Subadi ◽  
Martha Kurnia Kusumawardani ◽  
Mohammad Fathul Qorib ◽  
Imam Susilo ◽  
Hanik Badriyah Hidayati
Keyword(s):  
Opioid Receptor ◽  
Opioid Receptors ◽  
Pain Threshold ◽  
Threshold Value ◽  
Mu Opioid Receptor ◽  
Cupping Therapy ◽  
Receptor Expression ◽  
Control Group ◽  
Mu Opioid Receptors ◽  
Mu Opioid

Background & Objectives: Treatment of chronic pain using NSAIDs, steroids, opioids, and herbs has been associated with many complications with the long-term use. Wet cupping therapy (WCT) has been used to reduce pain, by triggering mu opioid receptor expression. We conducted this study to compare the effectiveness between WCT with oral opioids for pain management. Methodology: It was an experimental study with randomized control group post-test only design. Thirty two male white rats of strain Wistar were divided into four groups: (1) Group-NC; mice in this group were given nothing as a negative control group, (2) Group-CFA; group that was given Complete Freund’s Adjuvant (CFA) only as a positive control group, (3) Group-WCT; mice were given CFA and WCT, and (4) Group-O was given CFA and oral opioids. The measured variables were pain threshold value and mu opioid receptors. Statistical analysis was done us(ing SPSS software (version 22.0, Chicago, IL). Results: The results showed no significant differences in the expression of mu opioid receptors between Group-NC and Group-CFA (p = 0.061). There were significant differences in the expression of opioid receptors between Group-CFA and Group-WCT (p < 0.001), and also between WCT group and Group-O (p = 0.002). The differences of pain threshold value were only significant between Group-NC (p = 0,006) and Group-CFA (p = 0,013) with Group-O. Conclusion: Wet cupping therapy triggers the expression of mu opioid receptors. Wet cupping therapy as effective in relieving pain as opioids. Citation: Subadi I, Kusumawardani MK, Qorib MF, Susilo I, Hidayati HB. Wet cupping therapy improves mu opioid receptor expression and pain threshold in animal models of inflammation. Anaesth pain & intensive care 2019;23(4):__   Received: 6 February 2019; Reviewed: 16 February 2019, 2 August 2019; Revised: 9 September 2019; Accepted: 15 September 2019

PDGF-β receptor expression and ventilatory acclimatization to hypoxia in the rat

2000 ◽  
Vol 279 (5) ◽  
pp. R1625-R1633 ◽  
Author(s):  
Oscar A. Alea ◽  
Marc A. Czapla ◽  
Joseph A. Lasky ◽  
Narong Simakajornboon ◽  
Evelyne Gozal ◽  
...  
Keyword(s):  
Brain Stem ◽  
Late Phase ◽  
Receptor Expression ◽  
Receptor Protein ◽  
Hypoxic Exposure ◽  
Sprague Dawley ◽  
Western Blots ◽  
Term Administration ◽  
Β Receptor ◽  
Over Time

Activation of platelet-derived growth factor-β (PDGF-β) receptors in the nucleus of the solitary tract (nTS) modulates the late phase of the acute hypoxic ventilatory response (HVR) in the rat. We hypothesized that temporal changes in PDGF-β receptor expression could underlie the ventilatory acclimatization to hypoxia (VAH). Normoxic ventilation was examined in adult Sprague-Dawley rats chronically exposed to 10% O2, and at 0, 1, 2, 7, and 14 days, Northern and Western blots of the dorsocaudal brain stem were performed for assessment of PDGF-β receptor expression. Although no significant changes in PDGF-β receptor mRNA occurred over time, marked attenuation of PDGF-β receptor protein became apparent after day 7 of hypoxic exposure. Such changes were significantly correlated with concomitant increases in normoxic ventilation, i.e., with VAH ( r: −0.56, P < 0.005). In addition, long-term administration of PDGF-BB in the nTS via osmotic pumps loaded with either PDGF-BB ( n = 8) or vehicle (Veh; n = 8) showed that although no significant changes in the magnitude of acute HVR occurred in Veh over time, the typical attenuation of HVR by PDGF-BB decreased over time. Furthermore, PDGF-BB microinjections did not attenuate HVR in acclimatized rats at 7 and 14 days of hypoxia ( n = 10). We conclude that decreased expression of PDGF-β receptors in the dorsocaudal brain stem correlates with the magnitude of VAH. We speculate that the decreased expression of PDGF-β receptors is mediated via internalization and degradation of the receptor rather than by transcriptional regulation.

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