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.

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 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>

Hypoxia upregulates lung microvascular neurokinin-1 receptor expression

2006 ◽  
Vol 291 (1) ◽  
pp. L102-L110 ◽  
Author(s):  
Eric D. Zee ◽  
Stacey Schomberg ◽  
Todd C. Carpenter
Keyword(s):  
Substance P ◽  
Pulmonary Edema ◽  
Receptor Expression ◽  
Receptor Protein ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Edema Formation ◽  
Moderate Hypoxia ◽  
Neurokinin 1 ◽  
Subacute Exposure

Subacute exposure to moderate hypoxia can promote pulmonary edema formation. The tachykinins, a family of proinflammatory neuropeptides, have been implicated in the pathogenesis of pulmonary edema in some settings, including the pulmonary vascular leak associated with exposure to hypoxia. The effects of hypoxia on tachykinin receptor and peptide expression in the lung, however, remain poorly understood. We hypothesized that subacute exposure to moderate hypoxia increases lung neurokinin-1 (NK-1) receptor expression as well as lung substance P levels. We tested this hypothesis by exposing weanling Sprague-Dawley rats to hypobaric hypoxia (barometric pressure 0.5 atm) for 0, 24, 48, or 72 h. Hypoxia led to time-dependent increases in lung NK-1 receptor mRNA expression and lung NK-1 receptor protein levels at 48 and 72 h of exposure ( P < 0.05). Immunohistochemistry and in situ NK-1 receptor labeling with substance P-conjugated fluorescent nanocrystals demonstrated that hypoxia increased NK-1 expression primarily in the pulmonary microvasculature and in alveolar macrophages. Hypoxia also led to increases in lung substance P levels by 48 and 72 h ( P < 0.05) but led to a decrease in preprotachykinin mRNA levels ( P < 0.05). We conclude that subacute exposure to moderate hypoxia upregulates lung NK-1 receptor expression and lung substance P peptide levels primarily in the lung microvasculature. We speculate that this effect may contribute to the formation of pulmonary edema in the setting of regional or environmental hypoxia.

Permeability of the microcirculation in area postrema of rat

1988 ◽  
Vol 46 ◽  
pp. 348-349
Author(s):  
Shams M. Ghoneim ◽  
Frank M. Faraci ◽  
Gary L. Baumbach
Keyword(s):  
Brain Stem ◽  
Area Postrema ◽  
Upper Body ◽  
Sprague Dawley ◽  
Sodium Cacodylate ◽  
Acute Hypertension ◽  
Sprague Dawley Rats ◽  
Ultrastructural Characteristics ◽  
The Brain ◽  
Adjacent Brain

The area postrema is a circumventricular organ in the brain stem and is one of the regions in the brain that lacks a fully functional blood-brain barrier. Recently, we found that disruption of the microcirculation during acute hypertension is greater in area postrema than in the adjacent brain stem. In contrast, hyperosmolar disruption of the microcirculation is greater in brain stem. The objective of this study was to compare ultrastructural characteristics of the microcirculation in area postrema and adjacent brain stem.We studied 5 Sprague-Dawley rats. Horseradish peroxidase was injected intravenously and allowed to circulate for 1, 5 or 15 minutes. Following perfusion of the upper body with 2.25% glutaraldehyde in 0.1 M sodium cacodylate, the brain stem was removed, embedded in agar, and chopped into 50-70 μm sections with a TC-Sorvall tissue chopper. Sections of brain stem were incubated for 1 hour in a solution of 3,3' diaminobenzidine tetrahydrochloride (0.05%) in 0.05M Tris buffer with 1% H2O2.

scholarly journals Metabolic activation and colitis pathogenesis is prevented by lymphotoxin β receptor expression in neutrophils

2021 ◽  
Author(s):  
Thomas Riffelmacher ◽  
Daniel A. Giles ◽  
Sonja Zahner ◽  
Martina Dicker ◽  
Alexander Y. Andreyev ◽  
...  
Keyword(s):  
Severe Disease ◽  
Metabolic Activation ◽  
Cell Types ◽  
Receptor Expression ◽  
Neutrophil Accumulation ◽  
Functional Studies ◽  
Mitochondrial Ros ◽  
Tnf Superfamily ◽  
Β Receptor ◽  
Lymphotoxin Beta Receptor

AbstractInflammatory bowel disease is characterized by an exacerbated intestinal immune response, but the critical mechanisms regulating immune activation remain incompletely understood. We previously reported that the TNF-superfamily molecule TNFSF14 (LIGHT) is required for preventing severe disease in mouse models of colitis. In addition, deletion of lymphotoxin beta receptor (LTβR), which binds LIGHT, also led to aggravated colitis pathogenesis. Here, we aimed to determine the cell type(s) requiring LTβR and the mechanism critical for exacerbation of colitis. Specific deletion of LTβR in neutrophils (LTβRΔN), but not in several other cell types, was sufficient to induce aggravated colitis and colonic neutrophil accumulation. Mechanistically, RNA-Seq analysis revealed LIGHT-induced suppression of cellular metabolism, and mitochondrial function, that was dependent on LTβR. Functional studies confirmed increased mitochondrial mass and activity, associated with excessive mitochondrial ROS production and elevated glycolysis at steady-state and during colitis. Targeting these metabolic changes rescued exacerbated disease severity. Our results demonstrate that LIGHT signals to LTβR on neutrophils to suppress metabolic activation and thereby prevents exacerbated immune pathogenesis during colitis.

Increased type II transforming growth factor-β receptor expression in liver cells during cholesterol challenge

2000 ◽  
Vol 152 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Giovanna Baccante ◽  
Gabriella Mincione ◽  
Concetta Di Febbo ◽  
Anna Coppa ◽  
Domenico Angelucci ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Liver Cells ◽  
Receptor Expression ◽  
Type Ii ◽  
Β Receptor

Vagally mediated, nonparacrine effects of cholecystokinin-8s on rat pancreatic exocrine secretion

2007 ◽  
Vol 293 (2) ◽  
pp. G493-G500 ◽  
Author(s):  
Eddy Viard ◽  
Zhongling Zheng ◽  
Shuxia Wan ◽  
R. Alberto Travagli
Keyword(s):  
Brain Stem ◽  
Protein Secretion ◽  
Exocrine Secretion ◽  
Vagal Afferents ◽  
Dependent Manner ◽  
Pancreatic Exocrine Secretion ◽  
Sprague Dawley ◽  
Pancreatic Exocrine ◽  
Vagal Afferent

Cholecystokinin (CCK) has been proposed to act in a vagally dependent manner to increase pancreatic exocrine secretion via actions exclusively at peripheral vagal afferent fibers. Recent evidence, however, suggests the CCK-8s may also affect brain stem structures directly. We used an in vivo preparation with the aims of 1) investigating whether the actions of intraduodenal casein perfusion to increase pancreatic protein secretion also involved direct actions of CCK at the level of the brain stem and, if so, 2) determining whether, in the absence of vagal afferent inputs, CCK-8s applied to the dorsal vagal complex (DVC) can also modulate pancreatic exocrine secretion (PES). Sprague-Dawley rats (250–400 g) were anesthetized and the common bile-pancreatic duct was cannulated to collect PES. Both vagal deafferentation and pretreatment with the CCK-A antagonist lorglumide on the floor of the fourth ventricle decreased the casein-induced increase in PES output. CCK-8s microinjection (450 pmol) in the DVC significantly increased PES; the increase was larger when CCK-8s was injected in the left side of the DVC. Protein secretion returned to baseline levels within 30 min. Microinjection of CCK-8s increased PES (although to a lower extent) also in rats that underwent complete vagal deafferentation. These data indicate that, as well as activating peripheral vagal afferents, CCK-8s increases pancreatic exocrine secretion via an action in the DVC. Our data suggest that the CCK-8s-induced increases in PES are due mainly to a paracrine effect of CCK; however, a relevant portion of the effects of CCK is due also to an effect of the peptide on brain stem vagal circuits.

scholarly journals Imaging and serum biomarkers reflecting the functional efficacy of extended erythropoietin treatment in rats following infantile traumatic brain injury

2016 ◽  
Vol 17 (6) ◽  
pp. 739-755 ◽  
Author(s):  
Shenandoah Robinson ◽  
Jesse L. Winer ◽  
Justin Berkner ◽  
Lindsay A. S. Chan ◽  
Jesse L. Denson ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Diffusion Tensor ◽  
Elevated Serum ◽  
Serum Biomarkers ◽  
Receptor Expression ◽  
Acute Injury ◽  
Chronic Injury ◽  
Western Blots ◽  
Targeted Interventions

OBJECTIVE Traumatic brain injury (TBI) is a leading cause of death and severe morbidity for otherwise healthy full-term infants around the world. Currently, the primary treatment for infant TBI is supportive, as no targeted therapies exist to actively promote recovery. The developing infant brain, in particular, has a unique response to injury and the potential for repair, both of which vary with maturation. Targeted interventions and objective measures of therapeutic efficacy are needed in this special population. The authors hypothesized that MRI and serum biomarkers can be used to quantify outcomes following infantile TBI in a preclinical rat model and that the potential efficacy of the neuro-reparative agent erythropoietin (EPO) in promoting recovery can be tested using these biomarkers as surrogates for functional outcomes. METHODS With institutional approval, a controlled cortical impact (CCI) was delivered to postnatal Day (P)12 rats of both sexes (76 rats). On postinjury Day (PID)1, the 49 CCI rats designated for chronic studies were randomized to EPO (3000 U/kg/dose, CCI-EPO, 24 rats) or vehicle (CCI-veh, 25 rats) administered intraperitoneally on PID1–4, 6, and 8. Acute injury (PID3) was evaluated with an immunoassay of injured cortex and serum, and chronic injury (PID13–28) was evaluated with digitized gait analyses, MRI, and serum immunoassay. The CCI-veh and CCI-EPO rats were compared with shams (49 rats) primarily using 2-way ANOVA with Bonferroni post hoc correction. RESULTS Following CCI, there was 4.8% mortality and 55% of injured rats exhibited convulsions. Of the injured rats designated for chronic analyses, 8.1% developed leptomeningeal cyst–like lesions verified with MRI and were excluded from further study. On PID3, Western blot showed that EPO receptor expression was increased in the injured cortex (p = 0.008). These Western blots also showed elevated ipsilateral cortex calpain degradation products for αII-spectrin (αII-SDPs; p < 0.001), potassium chloride cotransporter 2 (KCC2-DPs; p = 0.037), and glial fibrillary acidic protein (GFAP-DPs; p = 0.002), as well as serum GFAP (serum GFAP-DPs; p = 0.001). In injured rats multiplex electrochemiluminescence analyses on PID3 revealed elevated serum tumor necrosis factor alpha (TNFα p = 0.01) and chemokine (CXC) ligand 1 (CXCL1). Chronically, that is, in PID13–16 CCI-veh rats, as compared with sham rats, gait deficits were demonstrated (p = 0.033) but then were reversed (p = 0.022) with EPO treatment. Diffusion tensor MRI of the ipsilateral and contralateral cortex and white matter in PID16–23 CCI-veh rats showed widespread injury and significant abnormalities of functional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD); MD, AD, and RD improved after EPO treatment. Chronically, P13–P28 CCI-veh rats also had elevated serum CXCL1 levels, which normalized in CCI-EPO rats. CONCLUSIONS Efficient translation of emerging neuro-reparative interventions dictates the use of age-appropriate preclinical models with human clinical trial–compatible biomarkers. In the present study, the authors showed that CCI produced chronic gait deficits in P12 rats that resolved with EPO treatment and that chronic imaging and serum biomarkers correlated with this improvement.

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