Expression Of Functional Receptors Research Articles

Attenuated vasodilator effectiveness of protease-activated receptor 2 agonist in heterozygous par2 knockout mice.

Studies of homozygous PAR2 gene knockout mice have described a mix of phenotypic effects in vitro and in vivo. However, there have been few studies of PAR2 heterozygous (wild-type/knockout; PAR2-HET) mice. The phenotypes of many hemi and heterozygous transgenic mice have been described as intermediates between those of wild-type and knockout animals. In our study we aimed to determine the effects of intermediary par2 gene zygosity on vascular tissue responses to PAR2 activation. Specifically, we compared the vasodilator effectiveness of the PAR2 activating peptide 2-furoyl-LIGRLO-amide in aortas of wild-type PAR2 homozygous (PAR2-WT) and PAR2-HET mice. In myographs under isometric tension conditions, isolated aortic rings were contracted by alpha 1-adrenoeceptor agonist (phenylephrine), and thromboxane receptor agonist (U46619) and then relaxation responses by the additions of 2-furoyl-LIGRLO-amide, acetylcholine, and nitroprusside were recorded. A Schild regression analysis of the inhibition by a PAR2 antagonist (GB-83) of PAR2 agonist-induced aortic ring relaxations was used to compare receptor expression in PAR2-WT to PAR2-HET. PAR2 mRNA in aortas was measured by quantitative real-time PCR. In aortas contracted by either phenylephrine or U46619, the maximum relaxations induced by 2-furoyl-LIGRLO-amide were less in PAR2-HET than in the gender-matched PAR2-WT. GB-83 was 3- to 4-fold more potent for inhibition of 2fly in PAR2-HET than in PAR2-WT. PAR2 mRNA content of aortas from PAR2-HET was not significantly different than in PAR2-WT. Acetylcholine- and nitroprusside-induced relaxations of aortas from PAR2-HET were not significantly different than in PAR2-WT and PAR2 knockout. An interesting secondary finding was that relaxations induced by agonists of PAR2 and muscarinic receptors were larger in females than in males. We conclude that the lower PAR2-mediated responses in PAR2-HET aortas are consistent with evidence of a lower quantity of functional receptor expression, despite the apparently normal PAR2 mRNA content in PAR2-HET aortas.

P-Rex1 Cooperates with PDGFRβ to Drive Cellular Migration in 3D Microenvironments

Expression of the Rac-guanine nucleotide exchange factor (RacGEF), P-Rex1 is a key determinant of progression to metastasis in a number of human cancers. In accordance with this proposed role in cancer cell invasion and metastasis, we find that ectopic expression of P-Rex1 in an immortalised human fibroblast cell line is sufficient to drive multiple migratory and invasive phenotypes. The invasive phenotype is greatly enhanced by the presence of a gradient of serum or platelet-derived growth factor, and is dependent upon the expression of functional PDGF receptor β. Consistently, the invasiveness of WM852 melanoma cells, which endogenously express P-Rex1 and PDGFRβ, is opposed by siRNA of either of these proteins. Furthermore, the current model of P-Rex1 activation is advanced through demonstration of P-Rex1 and PDGFRβ as components of the same macromolecular complex. These data suggest that P-Rex1 has an influence on physiological migratory processes, such as invasion of cancer cells, both through effects upon classical Rac1-driven motility and a novel association with RTK signalling complexes.

The trafficking and targeting of P2X receptors

The functional expression of P2X receptors at the plasma membrane is dependent on their trafficking along secretory and endocytic pathways. There are seven P2X receptor subunits, and these differ in their subcellular distributions because they have very different trafficking properties. Some are retained within the endoplasmic reticulum (ER), while others are predominantly at the cell surface or within endosomes and lysosomes. Changes in recruitment of receptors to and from the plasma membrane provides a way of rapidly up- or down-regulating the cellular response to adenosine triphosphate (ATP). An additional layer of regulation is the targeting of these receptors within the membranes of each compartment, which affects their stability, function and the nature of the effector proteins with which they form signaling complexes. The trafficking and targeting of P2X receptors is regulated by their interactions with other proteins and with lipids and we can expect this to vary in a cell-type specific manner and in response to changes in the environment giving rise to differences in receptor activity and function.

Comparative functional expression of nAChR subtypes in rodent DRG neurons

We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70–80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15–30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca2+ transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.

Steroid resistance in leukemia

There are several types of leukemia which are characterized by the abnormal growth of cells from the myeloid or lymphoid lineage. Because of their lympholytic actions, glucocorticoids (GCs) are included in many therapeutic regimens for the treatment of various forms of leukemia. Although a significant number of acute lymphoblastic leukemia patients respond well to GC treatment during initial phases; prolonged treatments sometimes results in steroid-resistance. The exact mechanism of this resistance has yet not been completely elucidated, but a correlation between functional GC receptor expression levels and steroid-resistance in patients has been found. In recent years, several other mechanisms of action have been reported that could play an important role in the development of such drug resistances in leukemia. Therefore, a better understanding of how leukemic patients develop drug resistance should result in drugs designed appropriately to treat these patients.

Spinal Cord FAAH in Normal Micturition Control and Bladder Overactivity in Awake Rats

We assessed whether spinal inhibition of the cannabinoid degrading enzyme FAAH would have urodynamic effects in normal rats and rats with bladder overactivity induced by partial urethral obstruction or prostaglandin E2. We also determined the expression of FAAH, and the cannabinoid receptors CB1 and CB2 in the sacral spinal cord. We used 44 rats for functional (cystometry) and Western blot experiments. The FAAH inhibitor oleoyl ethyl amide (3 to 300 nmol) was administered intrathecally (subarachnoidally) or intravenously. The expression of FAAH and CB1/CB2 receptors was determined by Western blot. Oleoyl ethyl amide given intrathecally affected micturition in normal rats and rats with bladder overactivity but effects were more pronounced in the latter. In normal rats oleoyl ethyl amide only decreased micturition frequency, while it decreased frequency and bladder pressures in rats with bladder overactivity. Intravenous oleoyl ethyl amide (3 to 300 nmol) had no urodynamic effect. FAAH and CB1/CB2 receptors were expressed in the rat sacral spinal cord. The expression of CB1/CB2 receptors but not FAAH was higher in obstructed than in normal rats. FAAH inhibition in the sacral spinal cord by oleoyl ethyl amide resulted in urodynamic effects in normal rats and rats with bladder overactivity. The spinal endocannabinoid system may be involved in normal micturition control and it appears altered when there is bladder overactivity.

The dominant functional nicotinic receptor in progenitor cells in the rostral migratory stream is the α3β4 subtype.

Addition of newly generated neurons into mature neural circuits in the adult CNS responds to changes in neurotransmitter levels and is tightly coupled to the activity of specific brain regions. This postnatal neurogenesis contributes to plasticity of the olfactory bulb and hippocampus and is thought to play a role in learning and memory, context and odor discrimination, as well as perceptual learning. While acetylcholine plays an important role in odor discrimination and perceptual learning, its role in adult neurogenesis in the olfactory bulb has not been elucidated. In this study, I have examined the functional expression of nAChRs in progenitor cells of the rostral migratory stream (RMS) in the adult olfactory bulb of mice. I show that most of these cells in the RMS exhibit large nAChR-mediated calcium transients upon application of acetylcholine (ACh). Unlike in the hippocampus, the predominant functional nAChRs on progenitor cells are of α3β4 subtype. Interestingly, functional receptor expression is lost once progenitor cells mature, and are incorporated into the granule cell layer. Instead, nAChRs are now expressed on some presynaptic terminals and modulate glutamate release onto granule cells. My results imply that ACh is a part of the permissive niche and likely plays a role in development of progenitor cells.

MicroRNA-574-5p Was Pivotal for TLR9 Signaling Enhanced Tumor Progression via Down-Regulating Checkpoint Suppressor 1 in Human Lung Cancer

Accumulating data suggested that functional expression of Toll-like receptors (TLRs) in tumor cells was involved in tumor progression. Our previous study demonstrated that TLR9 signaling could enhance the tumor progression of human lung cancer cells in vitro and in vivo. We further showed that miR-574-5p was the mostly up-regulated miRNA in human lung cancer cells under TLR9 signaling by miRNA array analysis. Here we characterized the potential role of miRNA-574-5p in enhanced tumor progression induced by TLR9 signaling in human lung cancer. We confirmed that TLR9 signaling effectively elevated the expression of miR-574-5p in human lung cancer cells. Notably, we found that down-regulation of miRNA-574-5p using miR-574-5p inhibitor in vitro or miR-574-5p sponge in vivo significantly abrogated the enhanced tumor progression induced by TLR9 signaling. Further studies showed that miR-574-5p was an important player associated with enhanced tumor progression of human lung cancer cells. Notably, we identified checkpoint suppressor 1 (Ches1) as the dominant direct target for miRNA-574-5p to confer the TLR9 signaling enhanced tumor progression. We revealed that over-expression of Ches1 significantly inhibited the cell cycle entry of human lung cancer cells. Finally, we revealed that the expression of miR-574-5p was positively correlated with TLR9 and reversely correlated with Ches1 in lung cancer patients. Our findings not only facilitated the further understanding of the crosstalk between miRNAs and TLRs in tumor biology, but also provided novel potential candidates for treatment of cancer.

Type I Interferon Signaling Protects Mice From Lethal Henipavirus Infection

Hendra virus (HeV) and Nipah virus (NiV) are closely related, recently emerged paramyxoviruses that form Henipavirus genus and are capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. However, in contrast to many other species and despite expression of functional virus entry receptors, mice are resistant to henipavirus infection. We report here the susceptibility of mice deleted for the type I interferon receptor (IFNAR-KO) to both HeV and NiV. Intraperitoneally infected mice developed fatal encephalitis, with pathology and immunohistochemical features similar to what was found in humans. Viral RNA was found in the majority of analyzed organs, and sublethally infected animals developed virus-specific neutralizing antibodies. Altogether, these results reveal IFNAR-KO mice as a new small animal model to study HeV and NiV pathogenesis, prophylaxis, and treatment and suggest the critical role of type I interferon signaling in the control of henipavirus infection.

Reelin-dependent ApoER2 downregulation uncouples newborn neurons from progenitor cells

SummaryReelin and its receptor machinery are well known to be required for the migration and positioning of neocortical projection neurons. More recently, reelin has been shown both necessary and sufficient to determine the rate of neocortical neurogenesis. The molecular links underlying its seemingly distinct proliferative and post-proliferative functions remain unknown. Here we reveal an enriched expression of functional reelin receptors, largely of Apolipoprotein E Receptor 2 (ApoER2), in radial glia basal processes and intermediate progenitor cells during mid/late cortical development. In vivo, ApoER2 overexpression inhibits neuronal migration. In contrast, precluding excessive levels of ApoER2 in reelin-deficient cortices, by either ApoER2 knock-down or the transgenic expression of reelin in neural progenitor cells, improves neuronal migration and positioning. Our study provides groundwork for the highly orchestrated clearance of neocortical neurons from their birth site, suggesting that a reelin-dependent ApoER2 downregulation mechanism uncouples newborn neurons from progenitor cells, thereby enabling neurons to migrate.

Differential expression of the P2X7 receptor in ovarian surface epithelium during the oestrous cycle in the mouse

Purinergic signalling has been proposed as an intraovarian regulatory mechanism. Of the receptors responsible for purinergic transmission, the P2X7 receptor is an ATP-gated cationic channel that displays a broad spectrum of cellular functions ranging from apoptosis to cell proliferation and tumourigenesis. In the present study, we investigated the functional expression of P2X7 receptors in ovarian surface epithelium (OSE). P2X7 protein was detected in the OSE layer of the mouse, both in situ and in primary cultures. In cultures, 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) activation of P2X7 receptors increased [Ca(2+)]i and induced apoptosis. The functionality of the P2X7 receptor was investigated in situ by intrabursal injection of BzATP on each day of the oestrous cycle and evaluation of apoptosis 24h using the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end-labelling (TUNEL) assay. Maximum effects of BzATP were observed during pro-oestrus, with the effects being blocked by A438079, a specific P2X7 receptor antagonist. Immunofluorescence staining for P2X7 protein revealed more robust expression during pro-oestrus and in OSE regions behind the antral follicles, strongly supporting the notion that the differences in apoptosis can be explained by increased receptor expression, which is regulated during the oestrous cycle. Finally, P2X7 receptor expression was detected in the OSE layer of human ovaries, with receptor expression maintained in human ovaries diagnosed with cancer, as well as in the human ovarian carcinoma SKOV3 cell line.

Xenopus laevis RIC-3 enhances the functional expression of the C. elegans homomeric nicotinic receptor, ACR-16, in Xenopus oocytes.

RIC-3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC-3 may be cell-type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric-3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC-3 shares 52% amino acid identity with human RIC-3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR-16, to compare the ability of RIC-3 from three species to enhance receptor expression. In the absence of RIC-3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr-16 to X. laevis ric-3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric-3 cRNAs were co-injected with acr-16 cRNA (1 : 1 ratio), 100 μM acetylcholine induced larger currents in oocytes expressing X. laevis RIC-3 compared with its orthologues. This provides further evidence for a species-specific component of RIC-3 activity, and suggests that X. laevis RIC-3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.

Expression of complement factors and functional C5a receptors in human embryonic stem cells, and induced pluripotent stem cells

Expression of complement factors and functional C5a receptors in human embryonic stem cells, and induced pluripotent stem cells

Expression of Genomic Functional Estrogen Receptor 1 (Esr1) in Mouse Sertoli Cells.

Expression of Genomic Functional Estrogen Receptor 1 (Esr1) in Mouse Sertoli Cells.

Possible neuroprotective property of nicotinic acetylcholine receptors in association with predominant upregulation of glial cell line‐derived neurotrophic factor in astrocytes

The underlying mechanisms are still unclear for the neuroprotective properties of nicotine to date, whereas we have shown functional expression of nicotinic acetylcholine receptors (nAChRs) responsible for the influx of extracellular Ca(2+) in cultured rat cortical astrocytes. In this study, we investigated the possible involvement of astrocytic nAChRs in the neuroprotection by this agonist. Exposure to nicotine predominantly induced mRNA expression of glial cell line-derived neurotrophic factor (GDNF) among the different neurotrophic factors examined in cultured astrocytes, in a manner sensitive to nAChR antagonists, nifedipine, and aCa(2+) chelator. Nicotine significantly increased GDNF in a concentration-dependent manner in cultured astrocytes but not in neurons or neural progenitors even at the highest concentration used. In cultured astrocytes, a transient increase was seen in the expression of mRNA and corresponding protein for GDNF during sustained exposure to nicotine for 24 hr. Cytotoxicity mediated by oxidative, calcium, mitochondrial, or endoplasmic reticulum stress was invariably protected against in cortical neurons cultured with conditioned medium from astrocytes previously exposed to nicotine, and preincubation with the anti-GDNF antibody reduced the neuroprotection by conditioned medium from astrocytes exposed to nicotine. Intraperitoneal administration of nicotine transiently increased the number of cells immunoreactive for both GDNF and glial fibrillary acidic protein in rat cerebral cortex. These results suggest that astrocytic nAChRs play a role in the neuroprotection against different cytotoxins after predominant upregulation of GDNF expression through a mechanism relevant to the acceleration of extracellular Ca(2+) influx in rat brain in a particular situation.

Characterization of two neuronal subclasses through constellation pharmacology

Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology."

Morphine-induced nitric oxide production in PC12 cells.

The role of nitric oxide (NO) in many well-known effects of morphine is well defined. NO is involved in the signaling pathway of the N-methyl-D-aspartate (NMDA) receptor, which is proposed to mediate some of morphine's effects. This research studies the effect of morphine and NMDA on lipopolysaccharide (LPS)-stimulated NO production by clonal rat pheochromocytoma (PC12) cells. We used the Griess reaction to measure NO concentrations in cell culture medium. PC12 cells that were incubated for 24 h with varying concentrations of morphine (0.1, 1, 10, 100, and 1000µM) plus LPS (1 µg/ml) did not significantly alter the concentration of NO in the medium. However, NO production increased when cells were treated for both 48 h with 100 and 1000 µM morphine and for 72 h with 10,100, and 1000 µM of morphine. After 72 h, 1 µM naloxone significantly decreased NO concentration. Naloxone, at doses of 0.1, 1, and 10µM prevented NO production by 1000 µM of morphine. NMDA (0.1, 1, and 10 µM) did not alter NO concentrations after 24, 48 or 72 h. Morphine (1 µM)-induced NO production was inhibited by 10 µM NMDA after 48 h. Inhibition of NO was also noted with1 and 10 µM concentrations of morphine after 72 h. Chronic treatment of PC12 cells with morphine significantly increases LPS-stimulated NO production via naloxone-sensitive receptors.The cells seem to release endogenous morphine in medium. NMDA does not affect NO production, which may be due to the lack of functional NMDA receptor expression in PC12 cells.

Human Psychometric and Taste Receptor Responses to Steviol Glycosides

Steviol glycosides, the sweet principle of Stevia Rebaudiana (Bertoni) Bertoni, have recently been approved as a food additive in the EU. The herbal non-nutritive high-potency sweeteners perfectly meet the rising consumer demand for natural food ingredients in Europe. We have characterized the organoleptic properties of the most common steviol glycosides by an experimental approach combining human sensory studies and cell-based functional taste receptor expression assays. On the basis of their potency to elicit sweet and bitter taste sensations, we identified glycone chain length, pyranose substitution, and the C16 double bond as the structural features giving distinction to the gustatory profile of steviol glycosides. A comprehensive screening of 25 human bitter taste receptors revealed that two receptors, hTAS2R4 and hTAS2R14, mediate the bitter off-taste of steviol glycosides. For some test substances, e.g., stevioside, we observed a decline in sweet intensity at supra-maximum concentrations. This effect did not arise from allosteric modulation of the hTAS1R2/R3 sweet taste receptor but might be explained by intramolecular cross-modal suppression between the sweet and bitter taste component of steviol glycosides. These results might contribute to the production of preferentially sweet and least bitter tasting Stevia extracts by an optimization of breeding and postharvest downstream processing.

Astrocytes as GABA-ergic and GABA-ceptive Cells

GABA (gamma-aminobutyric acid) is considered to be the major inhibitory neurotransmitter that is synthesized in and released from GABA-ergic neurons in the brain. However, recent studies have shown that not only neurons but astrocytes contain a considerable amount of GABA, which can be released and activate the receptors responsive to GABA. In addition, astrocytes are themselves responsive to GABA by expressing GABA receptors. These exciting new findings raise more questions about the origin of GABA, whether it is synthesized or taken up, and about the role of astrocytic GABA and GABA receptors. In this review, we propose several potential pathways for astrocytes to accumulate GABA and discuss the evidence for functional expression of GABA receptors in astrocytes.

Expression of functional follicle-stimulating hormone receptor and luteinizing hormone/chorionic gonadotrophin receptor in oviduct and uterus in prepubertal gilts

The presence of the FSHR and LH/CGR in nongonadal tissues of reproductive tracts during estrus has been reported. But localization study for FSHR in the endometrium has not been performed. Moreover, the expression of FSHR and LH/CGR in the nongonadal tissues of prepubertal gilts is also unclear. Our objectives were to demonstrate the presence of active and functional LH/CGR and FSHR and to localize the two receptors in uterus and oviducts in prepubertal gilts. Fifteen prepubertal crossbred (Landrance/Yorkshine) gilts were used in the study. RT-PCR revealed that both uterus and oviducts contained a 145bp LH/CGR transcript and a 103bp FSHR transcript. Western blotting revealed that the two tissues also contained a 75KDa immunoreactive LHR protein and a 75KDa immunoreactive FSHR protein. Immunocytochemistry demonstrated that the LHR and FSHR were expressed both in the uterus and oviducts. In the oviducts, the two receptors were all present in the mucosal epithelium, but not detected in the myosalpinx and the serosa. In the uterus, they were present in the luminal and glandular epithelium of the endometrium. Furthermore, the vascular smooth muscle also expressed the LHR protein. Injection of eCG to ovariectomized gilts and intact gilts increased both the concentrations of FSH and LH in serum and expressions of FSHR and LH/CGR proteins in uterus and oviducts. In summary, active FSHR and LH/CGR are present in the uterus and oviducts of prepubertal gilts.