scholarly journals Gene and cDNA cloning and characterization of the mouse V3/V1b pituitary vasopressin receptor

1999 ◽  
Vol 22 (3) ◽  
pp. 251-260 ◽  
Author(s):  
MA Ventura ◽  
P Rene ◽  
Y de Keyzer ◽  
X Bertagna ◽  
E Clauser
Keyword(s):  
Inositol Phosphate ◽  
Rank Order ◽  
Single Gene ◽  
Dependent Manner ◽  
Binding Studies ◽  
V1b Receptor ◽  
Competition Binding ◽  
Receptor Cdna ◽  
Dose Dependent

The gene of the mouse V3/V1b receptor was identified by homology cloning. One of the genomic clones contained the entire coding sequence. The cDNA presented high identity with rat (92%) and human (84%) sequences. Southern blot analysis indicated the existence of a single gene. Tissue distribution was studied by RT-PCR. The major site of expression was the pituitary. A faint signal was also present in hypothalamus, brain, adrenal, pancreas and colon. The mouse corticotroph cell line, AtT20, did not express the transcript. In order to confirm the identity of the sequence, the V3/V1b receptor cDNA was cloned and stably expressed in CHO-AA8 Tet-Off cells under the control of tetracycline. When transfected cells were treated with arginine vasopressin (AVP), inositol phosphate production increased in a dose-dependent manner, indicating that the V3/V1b receptor couples to phospholipase C. Moreover, AVP did not stimulate cAMP production. Binding studies with [3H]AVP indicated that the affinity of the mouse V3/V1b receptor (Kd=0.5 nM) is similar to that reported for rat and human receptors. The rank order of potency established in competition binding experiments with different analogues was representative of a V3/V1b profile, distinct from V1a and V2. However, significant differences were found between human and mouse receptors tested in parallel. Thus the pharmacology of V3/V1b receptors can not be transposed among different species.

Parathyroid Ca2+-conducting currents are modulated by muscarinic receptor agonists and antagonists

1997 ◽  
Vol 273 (5) ◽  
pp. E880-E890 ◽  
Author(s):  
Wenhan Chang ◽  
Tsui-Hua Chen ◽  
Stacy A. Pratt ◽  
Benedict Yen ◽  
Michael Fu ◽  
...  
Keyword(s):  
Muscarinic Receptors ◽  
Inositol Phosphate ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Pipette Solution ◽  
Pcr Products ◽  
Inositol Phosphate Accumulation ◽  
Receptor Cdna ◽  
Dose Dependent

Parathyroid cells express Ca2+-conducting cation currents, which are activated by raising the extracellular Ca2+ concentration ([Ca2+]o) and blocked by dihydropyridines. We found that acetylcholine (ACh) inhibited these currents in a reversible, dose-dependent manner (50% inhibitory concentration ≈10−8 M). The inhibitory effects could be mimicked by the agonist (+)-muscarine. The effects of ACh were blunted by the antagonist atropine and reversed by removing ATP from the pipette solution. (+)-Muscarine enhanced the adenosine 3′,5′-cyclic monophosphate (cAMP) production by 30% but had no effect on inositol phosphate accumulation in parathyroid cells. Oligonucleotide primers, based on sequences of known muscarinic receptors (M1-M5), were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify receptor cDNA from parathyroid poly (A)+ RNA. RT-PCR products displayed >90% nucleotide sequence identity to human M2- and M4-receptor cDNAs. Expression of M2-receptor protein was further confirmed by immunoblotting and immunocytochemistry. Thus parathyroid cells express muscarinic receptors of M2 and possibly M4 subtypes. These receptors may couple to dihydropyridine-sensitive, cation-selective currents through the activation of adenylate cyclase and ATP-dependent pathways in these cells.

Characterization of cholecystokinin receptors on human gastric smooth muscle tumors

1984 ◽  
Vol 247 (4) ◽  
pp. G402-G410
Author(s):  
L. J. Miller
Keyword(s):  
Smooth Muscle ◽  
Dependent Manner ◽  
Binding Studies ◽  
Concentration Dependent Manner ◽  
Gastric Smooth Muscle ◽  
Smooth Muscle Tumors ◽  
Competition Binding ◽  
Specificity Of Binding ◽  
Enzyme Markers

Gastric smooth muscle cells are a physiological target for the polypeptide hormone cholecystokinin (CCK). Human tumors arising from this type of cell, leiomyosarcomas, can retain their ability to express a receptor for CCK. To begin to characterize the human CCK receptor, we established a scheme for fractionation of these tumors to yield a membrane preparation enriched in enzyme markers of plasmalemma that saturably binds CCK. In competition-binding studies using 125I-CCK-8, only peptides structurally related to CCK competed for binding, with 50% of binding inhibited by 0.075 nM CCK-8, 0.9 nM CCK-8-desulfate, 0.9 nM gastrin-17, and 2.5 nM CCK tetrapeptide. Specificity of binding was demonstrated by showing that structurally unrelated peptides did not compete for binding. Association and dissociation of binding were temperature dependent. We have also performed affinity labeling studies to define the molecular properties of the CCK binding site. In these, the membranes were incubated with 125I-CCK-33, washed, cross-linked with disuccinimidyl suberate, solubilized, and electrophoretically separated on a polyacrylamide gel. Autoradiography of the dried gel revealed labeling of a major component with Mr 75,000 and minor components with Mr 53,000, Mr 100,000, Mr 120,000, and Mr greater than 200,000. Labeling was inhibited by CCK-8 in a concentration-dependent manner. This was also specific for CCK and structurally related peptides. These results demonstrate that gastric leiomyosarcomas are a very good source of a human CCK receptor and suggest that they may provide an easily cultured tissue with which this receptor can be fully characterized.

Characterization of the gonadotrophin-releasing hormone receptor in αT3-1 pituitary gonadotroph cells

1993 ◽  
Vol 136 (1) ◽  
pp. 51-NP ◽  
Author(s):  
L. Anderson ◽  
G. Milligan ◽  
K. A. Eidne
Keyword(s):  
G Protein ◽  
Inositol Phosphate ◽  
Rank Order ◽  
Second Messenger ◽  
Time Dependent ◽  
Binding Studies ◽  
Gnrh Analogues ◽  
Α Subunit ◽  
Releasing Hormone ◽  
Gonadotrophin Releasing Hormone

ABSTRACT The present study has characterized the gonadotrophin-releasing hormone (GnRH) receptor in immortalized αT3-1 pituitary gonadotroph cells. GnRH and GnRH analogues produced both a dose- and time-dependent increase in total inositol phosphate (IP) accumulation. The rank order of potency of these analogues was the same as that obtained in parallel receptor-binding studies in αT3-1 cells. These responses were abolished following pretreatment with a GnRH antagonist. The use of a specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) assay demonstrated a rapid but short-lived rise in Ins(1,4,5)P3 production. Intracellular calcium ([Ca2+]i) was subsequently measured in αT3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. GnRH produced a biphasic rise in [Ca2+]i. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. G-protein involvement in the IP response to GnRH in αT3-1 cells was investigated using sodium fluoride (NaF) and pertussis toxin (PTx) which activate and inactivate G-proteins respectively. Like GnRH, NaF produced a dose- and time-dependent increase in IP accumulation. Activation of phospholipase C in these cells by either GnRH or NaF was PTx-insensitive, suggesting that the G-protein involved was neither Gi nor Go but more probably Gq. Immunoblot analysis of αT3-1 cell membranes using antisera raised against the predicted C-terminal decapeptide of the α subunit of Gq demonstrated the presence of Gq in αT3-1 cells. Collectively these results show that the GnRH receptors expressed in αT3-1 cells are coupled to the phosphatidylinositol second messenger pathway via a specific G-protein. αT3-1 therefore represents a convenient model in which to study GnRH-related second messenger pathways. Journal of Endocrinology (1993) 136, 51–58

Modulation of Ca2+ transients in neonatal and adult rat cardiomyocytes by angiotensin II and endothelin-1

1996 ◽  
Vol 270 (3) ◽  
pp. H857-H868 ◽  
Author(s):  
R. M. Touyz ◽  
J. Fareh ◽  
G. Thibault ◽  
B. Tolloczko ◽  
R. Lariviere ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Receptor Agonist ◽  
Dependent Manner ◽  
Induced Responses ◽  
Ang Ii ◽  
Binding Studies ◽  
Vasoactive Peptides ◽  
Adult Cardiomyocytes ◽  
Etb Receptor ◽  
Dose Dependent

Vasoactive peptides may exert inotropic and chronotropic effects in cardiac muscle by modulating intracellular calcium. This study assesses effects of angiotensin II (ANG II) and endothelin-1 (ET-1) on intracellular free calcium concentration ([Ca2+]i) in cultured cardiomyocytes from neonatal and adult rats. [Ca2+]i was measured microphotometrically and by digital imaging using fura 2 methodology. Receptor subtypes through which these agonists induce responses were determined pharmacologically and by radioligand binding studies. ANG II and ET-1 increased neonatal atrial and ventricular cell [Ca2+]i transients in a dose-dependent manner. ANG II (10(-11) to 10(-7) M) failed to elicit [Ca2+]i responses in adult cardiomyocytes, whereas ET-1 increased [Ca2+]i in a dose-dependent manner. The ETA receptor antagonist BQ-123 significantly reduced (P 7< 0.05) ET-1 induced responses, and the ETB receptor agonist IRL-1620 (10(-7) to 10(-5) M) significantly increased (P < 0.05) [Ca2+]i in neonatal and adult cardiomyocytes. ET-1 binding studies demonstrated 85% displacement by BQ-123 and approximately 15% by the ETB receptor agonist sarafotoxin S6c, suggesting a predominance of ETA receptors. Competition binding studies for ANG II failed to demonstrate significant binding on adult ventricular myocytes, indicating the absence or presence of very few ANG II receptors. These data demonstrate that ANG II and ET-1 have stimulatory [Ca2+]i effects on neonatal cardiomyocytes, whereas in adult cardiomyocytes, ANG II-induced effects are insignificant, and only ET-1-induced responses, which are mediated predominantly via ETA receptors, are preserved. Cardiomyocyte responses to vasoactive peptides may thus vary with cardiac development.

Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

1990 ◽  
Vol 5 (2) ◽  
pp. 159-166 ◽  
Author(s):  
N. G. N. Milton ◽  
E. W. Hillhouse ◽  
S. A. Nicholson ◽  
C. H. Self ◽  
A. M. McGregor
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Tissue Extracts ◽  
Rat Pituitary ◽  
Hypothalamic Tissue ◽  
Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

Adenosine Inhibits Tissue Factor Expression by LPS-stimulated Human Monocytes: Involvement of the A3 Adenosine Receptor

2002 ◽  
Vol 88 (07) ◽  
pp. 123-130 ◽  
Author(s):  
Matthieu Broussas ◽  
Pascale Cornillet-Lefèbvre ◽  
Gérard Potron ◽  
Philippe Nguyên
Keyword(s):  
Tissue Factor ◽  
Rank Order ◽  
Mrna Level ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Extrinsic Pathway ◽  
A3 Adenosine Receptor ◽  
Factor Expression ◽  
Cardioprotective Effects ◽  
Dose Dependent

SummaryTissue Factor (TF), an integral membrane glycoprotein that initiates the extrinsic pathway of blood coagulation, is thought to play a major part in coronary acute events. Adenosine, an endogenous nucleoside produced by the degradation of intracellular adenosine triphosphate, has been shown to exert many cardioprotective effects via an inhibition of platelets and neutrophils. This study was conducted to determine whether adenosine (ADO) could modulate the expression of TF by human monocytes. We found that ADO inhibited TF antigen and activity on endotoxin-stimulated monocytes in a dose-dependent manner. The mechanism was at least pre-translational since ADO caused a change in the TF mRNA level. Using ADO receptor-specific analogs, we showed that highly selective A3 agonist N6-(3-iodobenzyl)-adenosine-5’-N’-methyluronamide (IB-MECA) inhibited LPSinduced TF activity expression more potently than A1 agonist R-phenylisopropyladenosine (R-PIA) and A2 agonist CGS 2180. Furthermore, A1/A3 antagonist, xanthine amine congener (XAC) blocked the effect of ADO whereas A2a, A2b and A1 antagonists were ineffective. In addition, we observed that ADO agonists inhibited monocyte TF expression in LPS-stimulated whole blood. The rank order of agonist potency suggested that A2 and A3 receptors might be involved (2-Cado > CGS = IB-MECA > R-PIA). This was supported by the fact that A2 and A3 antagonists reversed the action of 2-Cado. We conclude that TF inhibition by ADO on human purified monocytes involved A3 receptors.

LUMI-AGGREGOMETER STUDIES OF THE INITIAL ATP-SECRETION FROM COLLAGEN-ADHERENT PLATELETS

1987 ◽  
Author(s):  
R Malmgren
Keyword(s):  
Platelet Adhesion ◽  
Rank Order ◽  
Shape Change ◽  
Dense Granule ◽  
Dependent Manner ◽  
Equal Degree ◽  
Atp Secretion ◽  
Ic50 Values ◽  
Dose Dependent ◽  
Platelet Shape

We have earlier, with the use of a lumi-aggregometer and sub-aggregating doses of collagen (0.2-0.8 ug/ml PRP), been able to detect the initial, aspirin-insensitive secretion of ATP from the collagen-adherent platelets, and to correlate this secretion to the doses of collagen, and onset and degree of subsequent shape change of non-adherent platelets (Malmgren, Thromb Res 4:445, 1986). The present study shows, that 200 ATU of hirudin,which reduced near-maximal aggregation and ATP-secretion induced by high collagen doses (2.5 ug/ml PRP) from 3.35 ± 0.2 uM to 2.85 ± 0.1 uM, did neither reduce the secreted amount of ATP that were 82.5 ± 15 nM in control samples and 90 ± 27.5 nM in hirudin-treated samples, nor reduce platelet shape change when platelets were challenged with 0.31 ug collagen /ml PRP. (200 ATU hirudin completely abolished an equal degree of platelet shape change induced by 0.01 U thrombin). Assuming that 3 % of the platelets in PRP were actually adhering to the collagen fibrils, the secreted amount corresponds to 14.6 ±0.04 pmoles ATP/106adheringplatelets, amounts which closely represented 100 % of their dense granule content. The finding confirms that hirudin does not inhibit platelet adhesion and also indicates, that thrombin-mediated activation of secretory pathways appears not to be involved during the initial phase of platelet-collagen interactions.Dipyridamole (DPA) and dibutyryl cAMP (DBcAMP) inhibited ATP-secretion and platelet aggregation in a dose-dependent manner at high collagen concentrations, but only DBcAMP caused a dose-dependent reduction of ATP secretion (IC50 =10-4 M) induced by sub-aggregating doses of collagen. DPA was devoid of effect in this respect and thus did not inhibit platelet adhesion.Yohimbine, dihydroergotamine and phentolamine reduced ATP-secretion induced by sub-aggregating collagen doses in the mentioned rank order of potency, and with IC50 values in the micromolar range. Ketanserin, ritanserin and propranolol were devoid of effect. The findings suggest that the initial collagen-plate-let interaction involve alfareceptor-mediated mechanisms that may encompass adhesion, while DBcAMP probably interacts with secretory mechanisms connected to phosphatidylinositol turnover.

scholarly journals A Mechano-Activated Cell Reporter System as a Proxy for Flow-Dependent Endothelial Atheroprotection

2018 ◽  
Vol 23 (8) ◽  
pp. 869-876
Author(s):  
Bendix R. Slegtenhorst ◽  
Oscar R. Fajardo Ramirez ◽  
Yuzhi Zhang ◽  
Zahra Dhanerawala ◽  
Stefan G. Tullius ◽  
...  
Keyword(s):  
Vascular Endothelium ◽  
Critical Role ◽  
Green Fluorescence Protein ◽  
Dependent Manner ◽  
Reporter System ◽  
Health And Disease ◽  
Fluorescence Protein ◽  
Biomechanical Stimuli ◽  
Dose Dependent

The vascular endothelium plays a critical role in the health and disease of the cardiovascular system. Importantly, biomechanical stimuli generated by blood flow and sensed by the endothelium constitute important local inputs that are translated into transcriptional programs and functional endothelial phenotypes. Pulsatile, laminar flow, characteristic of regions in the vasculature that are resistant to atherosclerosis, evokes an atheroprotective endothelial phenotype. This atheroprotective phenotype is integrated by the transcription factor Kruppel-like factor-2 (KLF2), and therefore the expression of KLF2 can be used as a proxy for endothelial atheroprotection. Here, we report the generation and characterization of a cellular KLF2 reporter system, based on green fluorescence protein (GFP) expression driven by the human KLF2 promoter. This reporter is induced selectively by an atheroprotective shear stress waveform in human endothelial cells, is regulated by endogenous signaling events, and is activated by the pharmacological inducer of KLF2, simvastatin, in a dose-dependent manner. This reporter system can now be used to probe KLF2 signaling and for the discovery of a novel chemical-biological space capable of acting as the “pharmacomimetics of atheroprotective flow” on the vascular endothelium.

scholarly journals Isolation and Characterization of a Lectin-like Protein (SBLP) from the Dried Roots of Scutellaria baicalensis (Lamiaceae)

2018 ◽  
Vol 13 (12) ◽  
pp. 1934578X1801301
Author(s):  
Huiqin Wang ◽  
Guanzhen Gao ◽  
Lijing Ke ◽  
Jianwu Zhou ◽  
Pingfan Rao
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Scutellaria Baicalensis ◽  
Dependent Manner ◽  
Scutellaria Baicalensis Georgi ◽  
Isolation And Characterization ◽  
Pas Staining ◽  
Ph Range ◽  
Dose Dependent

A novel lectin-like protein with MW 63.2 kDa, designated as SBLP, has been isolated and characterized from the dried roots of Scutellaria baicalensis Georgi (Lamiaceae). SBLP was purified by ammonium sulfate precipitation and anion exchange chromatography. It is a glycoprotein according to a PAS staining assay and consisting of protein (86.0%) and sugar (14.0%). Its N-terminal amino acid sequence was determined as GSAVGFLY by Edman degradation. SBLP showed hemagglutinating activity against human and rooster erythrocytes, which were stable below 60°C and in the pH range of 4 −10. Furthermore, SBLP was found to be stimulated by Ca2+, Na+, Ba2+, Zn2+ ions, which suggested it was a metal-dependent lectin. SBLP inhibited the growth of Fusarium oxysporum f.sp. lycopersici and Alternaria eichhorniae in the a dose-dependent manner, and suppressed the proliferation of HepG2 tumor cells with an IC50 of 1.00 μM. This is the first report of a lectin from Radix Scutellariae.

Benzodiazepines Block α2-Containing Inhibitory Glycine Receptors in Embryonic Mouse Hippocampal Neurons

2003 ◽  
Vol 90 (1) ◽  
pp. 89-99 ◽  
Author(s):  
Liu Lin Thio ◽  
Ananth Shanmugam ◽  
Keith Isenberg ◽  
Kelvin Yamada
Keyword(s):  
Hippocampal Neurons ◽  
Clinical Effects ◽  
Dependent Manner ◽  
Glycine Receptors ◽  
Binding Studies ◽  
Chloride Currents ◽  
Embryonic Mouse ◽  
Voltage Dependent ◽  
Polymerase Chain ◽  
Dose Dependent

Inhibitory glycine receptors (GlyRs) in the mammalian cortex probably contribute to brain development and to maintaining tonic inhibition. Given their presence throughout the cortex, their modulation likely has important physiological consequences. Although benzodiazepines potentiate γ-aminobutyric acidA receptors (GABAARs), they may also modulate GlyRs because binding studies initially suggested that they act at GlyRs. Furthermore, their diminished ability to potentiate neonatal GABAARs suggests that they may exert their beneficial clinical effects at another site in the developing brain. Therefore we examined the effect of benzodiazepines on whole cell currents mediated by GlyRs in cultured embryonic mouse hippocampal neurons. First, we determined the GlyR subunit composition in this preparation. Glycine, β-alanine, and taurine activate strychnine-sensitive chloride currents in a dose-dependent manner. Maximal concentrations of the three agonists produce equal, nonadditive responses as expected of full agonists. The pharmacological properties of the GlyR currents including their pattern of modulation by picrotoxinin, picrotin, and tropisetron indicate that GlyRs consist of α2β heteromers and α2 homomers. Reverse transcriptase polymerase chain reaction (RTPCR) studies confirmed the presence of α2 and β subunits. Second, we found that micromolar concentrations of some benzodiazepines, including chlordiazepoxide and nitrazepam, inhibit GlyR currents. Nitrazepam inhibition of GlyRs is noncompetitive, is not voltage dependent, and does not reflect enhanced desensitization. Thus benzodiazepines allosterically inhibit α2-containing GlyRs in embryonic mouse hippocampal neurons via a “low”-affinity site.

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