Agonist-like activity of antibodies directed against the second extracellular loop of the human cardiac serotonin 5-HT4(e) receptor in transfected COS-7 cells

We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.     

Functional domains of delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition

Opioid receptors (delta, mu) belong to the large superfamily of G protein coupled receptors that inhibit adenylyl cyclase. We have studied the effects of seven synthetic peptides representing selected cytoplasmic regions of the murine delta-opioid receptor on forskolin-mediated adenylyl cyclase activity in membranes of D2 and Neuro(2A) cells stably expressing the delta- and mu-opioid receptors respectively. The entire third intracellular loop (i3), its amino-terminal portion (i3.1) and the carboxyl-terminal region of the second cytoplasmic loop (i2.2) enhanced dose-dependently the agonist-mediated inhibition of cAMP accumulation. The peptide-mediated effects are blocked by pertussis toxin treatment and are not observed in parental cells that lack these receptors. The inhibitory effects of the peptides on adenylyl cyclase were markedly attenuated when membranes from D2 and Neuro(2A) cells were preincubated with antisera against Gi(2) alpha and G beta subunits of G proteins. Our results provide evidence on domains of the delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition.     

Extracellular Ca2+ sensitivity of mGluR1alpha associated with persistent glutamate response in transfected CHO cells

We previously reported that the metabotropic glutamate receptor 1alpha (mGluR1alpha) can be activated not only by glutamate but also by extracellular Ca2+ (Ca2+o), and that Ser 166 in the extracellular domain determines the sensitivity to Ca2+o. In the present study, we investigated by intracellular Ca2+ (Ca2+i) imaging, the effect of Ca2+o on the glutamate responses of Chinese Hamster Ovary (CHO) cells stably expressing mGluR1alpha wild-type (CHO-wt). As a negative control, we carried out similar experiments using CHO cells expressing Ser166Asp mutant of mGluR1alpha (CHO-S166D) or the substance P receptor (CHO-SPR), which were not activated by Ca2+o application. We observed a remarkable prolongation of the duration of the glutamate response in CHO-wt cells in a Ca2+o concentration dependent manner. In CHO-S166D cells and CHO-SPR cells, only a small sustained component of the glutamate response was observed in the presence of Ca2+o. These sustained components were blocked by SKF-96365, a blocker of receptor-operated Ca2+-influx. Thus, it was concluded that the Ca2+o-sensing function of mGluR1alpha-wt induced the persistent opening of the receptor-operated Ca2+-permeable channels, probably by persistent activation of the receptor by glutamate. We additionally observed that the dose-response relationship of CHO-S166D and CHO-SPR shifted significantly by changing Ca2+o concentration, i.e. Ca2+o was required to maintain the normal ligand responses of these receptors.     

Characterization of adenosine A1 receptors in human proximal tubule epithelial (HK-2) cells

BACKGROUND: Adenosine A1 receptors (A1ARs) modulate various aspects of renal functions, such as hormone release, hemodynamics and tubular absorption. Here we set up to demonstrate the expression of A1ARs in an immortalized cell line (HK-2) derived from normal adult human proximal tubule. We also examined the mechanism whereby A1ARs signal in HK-2 cells and their potential role in renal physiology such as sodium-dependent phosphate transport. METHODS: Ligand binding assay of A1ARs was performed using plasma membranes of HK-2 cells and a selective high-affinity A1AR radioligand [3H]DPCPX. HK-2 cells in 96-well plates were treated with various agents (forskolin, adenosine receptor agonists, and antagonists) to activate or inhibit adenylate cyclase. Intracellular cyclic AMP accumulation was measured using cAMP flashplates. mRNA levels of adenosine receptors in HK-2 cells was determined by real-time PCR technique. Sodium-dependent phosphate transport across cell membrane was measured after 15-minute incubation of phosphorus-33 in transport buffer with HK-2 cells at room temperature. RESULTS: In HK-2 cells, A1ARs were expressed at a density of 211 +/- 74 fmol/mg membrane proteins. [3H]DPCPX bound to A1ARs on HK-2 cell membranes with Kd of 8.3 +/- 2.2 nM. Activation of A1ARs inhibited isoproterenol-stimulated adenylate cyclase activity through pertussis toxin-sensitive Gi protein in HK-2 cells. Coexpression of adenosine A2a receptors at a seemingly lower level than A1ARs was revealed by synergistically activating adenylate cyclase with forskolin. Real-time RT-PCR further demonstrated the expression of both A1AR and A2aAR in HK-2 cells. Sodium-dependent phosphate transport was augmented by activation of A1ARs in HK-2 cells. CONCLUSION: A1ARs are expressed in human proximal tubule epithelial (HK-2) cells and modulate sodium-dependent phosphate transport.     

Quantification of human caudate d2 dopamine receptor density with positron emission tomography: a review of the model

The radioligand [11C] N-methylspiprone (NMSP) has been used to assess relative and absolute D2 dopamine receptor densities in the living human brain with positron emission tomography (PET). In those studies, a three-compartment model was used to analyze the NMSP accumulation in the caudate nucleus. Absolute receptor densities were determined from studies in both the presence and absence of the inhibitor haloperidol; haloperidol was used to reduce the number of available receptors. We show that the mathematical analysis of the model requires that occupancy of D2 dopamine receptors by NMSP be negligible. The published parameter values (K1, k2, k3 k4(t), and BM'max) for studies in the presence of haloperidol are shown to be inconsistent with this requirement. Potential sources of the inconsistency include a high mass dose of NMSP, as well as the need to assign values for the association and dissociation rate constants for haloperidol, and for the association rate constant for NMSP. For these reasons, the method for absolute receptor density determination may be in error. If sufficiently low masses of NMSP are injected, however, the value of BM'max might provide a measure of relative receptor density.     

Unaltered agonist potency upon inducible 5-HT7(a) but not 5-HT4(b) receptor expression indicates agonist-independent association of 5-HT7(a) receptor and Gs

We compared adenylyl cyclase (AC) activation by the G protein-coupled human serotonin (5-HT) receptors 5-HT4(b) and 5-HT7(a) using an ecdysone-inducible expression system, which allowed for reproducible expression of increasing receptor densities in clonal HEK293 (EcR293) cell lines. Low constitutive expression of receptors (2-70 fmol/mg protein) was observed and could be titrated up to 50-200-fold (approximately 400-7000 fmol/mg protein) by the ecdysone analogue ponasterone A. Although 5-HT-stimulated AC activity increased with receptor density, interclonal variation precluded comparisons of coupling efficiency. Interestingly, the potency of 5-HT to stimulate AC increased with increasing receptor density only in clones expressing 5-HT4(b) receptors. The potency for 5-HT did not change in clones expressing 5-HT7(a) receptors, even though 5-HT-stimulated AC activity approached asymptotic levels. This indicates that potency of 5-HT for stimulation of AC through the 5-HT7(a) receptor is independent of receptor-Gs stoichiometry and is consistent with a model where the 5-HT7(a) receptors are tightly associated with G protein, independent of agonist binding. This supports the existence of a complex between inactive receptor and G protein, as predicted by the cubic ternary complex model. In such a system, spare receptors do not lead to increased potency of an agonist with increased receptor density.     

Differential expression of MHC class II antigens and cathepsin L by subtypes of cortical epithelial cells in the rat thymus: an immunoelectron microscopic study

To better comprehend the thymic microenvironment, it is necessary to identify the antigenic profile of cortical thymic epithelial cells (cTECs) that are involved in the development of major histocompatibility complex (MHC)-restricted T cells. Ultrastructurally, cTECs can be classified into four morphologically distinct subtypes: subcapsular/perivascular (EC1), pale (EC2), intermediate (EC3) and dark (EC4) cells. Several immunohistochemical studies were done on cTECs at the light and electron microscopic levels, but not with reference to the above subtypes. In the present paper, we analysed the expression of MHC class II antigen and cathepsin L by individual cTEC subtypes at the electron microscopic level. We show that (1) MHC class II antigens are expressed on the cell surfaces except on the basal surface of EC1, both on the cell surface and in intracytoplasmic vacuoles of EC2, and only in the intracytoplasmic vacuoles of EC3 and EC4, and (2) that cathepsin L is expressed strongly and uniformly throughout the cytoplasm of EC2, but weakly and non-uniformly in the cytoplasm of EC1, EC3 and EC4. These results show that MHC class II antigen expression and cathepsin L expression is heterogeneous in cTEC subtypes and suggest that EC2 might play a significant role in the development of CD4+ T cells.     

Stable expression of human homomeric and heteromeric AMPA receptor subunits in HEK293 cells

Human homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors (GluRs) were stably expressed in HEK293 cells with cDNAs encoding the flip splice variant of GluR1, GluR2, GluR3, GluR4 subunit, and the GluR1/GluR2, GluR3/GluR2, and GluR4/GluR2 combination. The lethal combination of GluR2 and GluR4 subunits was found in high expression levels of both receptors. The AMPA-evoked current voltage relationships demonstrated the functional channel properties, such as a double rectification in GluR1, GluR3, and GluR4 receptors, and a linear relation in receptors assembled from GluR2 alone and coexpression of GluR2 with the other subunits. All the transfectants exhibited higher selectivity for AMPA than glutamate in dose-dependent current responses. [3H]AMPA binding revealed that the homomeric and heteromeric receptors displayed a single binding site in Scatchard analysis, with dissociation constant (Kd) values in the range of 14.5-49.3 nM. The Bmax values were in the range of 0.57-7.66 pmol/mg protein. The ligand displacement potency for [3H]AMPA binding was CNQX > glutamate > NS257 in all of the transfectants. These results suggest that stable transformants expressing human homomeric and heteromeric AMPA receptors will be useful tools to define selectivity and potential site of action for AMPA receptor modulators.     

NKG2D配体在视网膜母细胞瘤RB细胞的表达及意义

目的：探讨视网膜母细胞瘤RB细胞株上NKG2D配体的表达情况及其对同种异体NK细胞杀伤活性的影响。方法：采用半定量RT-PCR法检测RB细胞及人红白血病细胞K562细胞上NKG2D配体MICA、MICB、ULBP1、ULBP2、ULBP3的mRNA表达情况。应用MTT法检测人NK细胞对肿瘤细胞的杀伤活性。结果：K562细胞表面表达MICA、MICB、ULBP1、ULBP2、ULBP3,RB细胞表面表达MICA、MICB、ULBP2、ULBP3不表达ULBP1。K562细胞表面NKG2D的配体mRNA表达强度明显强于RB细胞（P〈0．05）。当效靶比分别为5：1、10：1、20：1、40：1时NK细胞对K562、RB细胞的杀伤活性分别为28．32％±2．06％、9．11％±0．89％;45．62％±1．17％、22．87％±1．38％;60．79％±1．78％、32．69％±0．37％;73．83％±3．05％、50．29％±2．49％,在各效靶比时NK细胞对K562细胞的杀伤活性较RB细胞明显增强（P〈0．05）。结论：NKG2D配体影响NK细胞对靶细胞的杀伤活性,NKG2D配体表达水平可能决定着NK细胞抗肿瘤免疫应答的强弱,提高NKG2D配体的表达有可能提高NK细胞的抗肿瘤作用。     

Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was examined as host for heterologous expression of the G protein-coupled VPAC1 receptor. Rat VPAC1 receptor cDNA and two chimeric constructs encoding the yeast mating factor pre-pro alpha-leader peptide fused in-frame to rat VPAC1 receptor were expressed in yeast cells under control of a galactose inducible promoter. The rat VPAC1 receptor was fused to the HSV tag epitope to ensure proper immunological detection of the receptor. Crucial conditions for high-level expression of active rat VPAC1 receptor included growth in amino acid supplemented minimal medium, fusion to the yeast alpha-leader peptide and a temperature shift from 30 degrees C to 15 degrees C before promoter induction. Western blotting showed that the expressed receptor was highly glycosylated and a band of 47 kDa was obtained upon endoglycosidase H treatment. Binding with radioiodinated vasoactive intestinal polypeptide revealed a KD of 2.5 nM and an IC50 of 15 nM when displacing with unlabeled vasoactive intestinal polypeptide. VPAC1 receptor density quantified by Western blotting was 510 pmol/mg membrane protein of which only 66 pmol/mg were able to bind vasoactive intestinal polypeptide.     

Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors

GABA(A) receptors composed of alpha1beta3 gamma2 and alpha1beta3 subunits were expressed in insect Sf9 cells and solubilized in 1% Triton X100. In sucrose density gradients, [3H]-Ro15-1788 binding activity, in the case of alpha1beta3 gamma2, and [3H]-muscimol binding activity, in the case of alpha1beta3 containing receptors sedimented as a single sharp peak suggesting the formation of receptors containing a defined number of subunits. When alpha1beta3gamma2 -containing receptors were incubated with an alpha-subunit specific antibody (bd24), a single class of antibody receptor complex was formed irrespective of the receptor-antibody ratio. This is consistent with two alpha subunits cross-linked within the receptor by the antibody. Similar results were obtained using a beta-subunit specific antibody (bd17). Several classes of antibody-receptor complex were formed when receptors were pre-incubated with a gamma specific antibody (anti gamma(2) 1-15 Cys). This profile is consistent with the presence of a single gamma subunit in each complex. Experiments with alpha1beta3 subunit containing receptors and antibody bd24 produced a profile similar to that seen with alpha1beta3 gamma2 receptors, consistent with two alpha subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with three beta subunits per receptor. This method permits the rapid determination of subunit stoichiometries of homogeneous receptor populations     

Unmasking different constitutive activity of four chemoattractant receptors using Na+ as universal stabilizer of the inactive (R) state.

Neutrophils express receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) complement C5a, leukotriene B4 (LTB4) and platelet-activating factor (PAF). The aim of this study was to analyze the constitutive activity of chemoattractant receptors by studying binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to the G-protein Gi alpha 2 beta 1 gamma 2 expressed in Sf9 cells. We used Na+ as modulator of constitutive activity because there are no known inverse agonists for the C5a receptor (C5aR), LTB4 receptor (BLTR) and PAF receptor (PAFR). In the absence of NaCl, PAF and LTB4 exhibited larger relative stimulatory effects on GTP gamma S binding than fMLP and C5a. NaCl showed larger inhibitory effects on basal GTP gamma S binding in membranes expressing the formyl peptide receptor (FPR) and C5aR than in membranes expressing BLTR and PAFR. The order of potency of NaCl at inhibiting basal GTP gamma S binding was FPR > C5aR approximately BLTR > PAFR. As a result of the inhibitory effect of NaCl on basal GTP gamma S binding, the relative stimulatory effects of agonists were increased. By quantitatively analyzing the expression levels of chemoattractant receptors and Gi alpha 2 and the stoichiometry of receptor/G-protein coupling we obtained no evidence for structural instability of constitutively active receptors and catalytical G-protein activation. Taken together, the FPR and C5aR exhibit higher constitutive activity than the BLTR and PAFR. Na+ acts as a universal stabilizer of the inactive (R) state in chemoattractant receptors. The different potencies of NaCl at suppressing basal G-protein activity with different receptors indicate that chemoattractant receptors differ from each other in their Na(+)-affinity.     

Polyclonal antibody effects on the human cardiac 5-HT4(e) receptors depend upon the expression system

The initial objective of this work was to examine the effects of an antibody (Anti-G21V) directed against the second extracellular loop of human heart 5-HT4 receptors expressed in Chinese hamster ovary (CHO) cells. The antibody anti-G21V had no effect upon either basal cAMP-or 5-HT-evoked increases in cAMP in CHO cells, whereas it had shown an agonist-like effect in COS-7 cells. Analysis of agonist fractions of h5-HT4(e) receptors in CHO and COS-7 cells revealed that equilibrium constant could underlie the different responses of the receptor toward the anti-G21V antibody. Therefore, different expression systems could give rise to functional differences in 5-HT4 receptor behavior.     

Performance Tradeoff Between Energy Conservation and User Fairness for D2D Communication Underlaying Cellular Networks

Device-to-Device (D2D) communication is viewed as an emerging technology in the fifth generation systems to fully explore the proximate gain residing in lo-cal communicating pairs. But the interference imposed to the existing Cellular links (C-link) should be addressed in the optimization for underlaid D2D links (D-link). There is a performance tradeoffbetween Energy conservation (EC) and the number of links with satisfied Quality-of-service (QoS) requirement or System capacity (SC). We aim to weight the above tradeofffor D-links by specifying it into optimizing EC on the condition that SC is maximized and QoS requirements of C-links are fully guaranteed. A three-stage joint power control and channel assignment mecha-nism is proposed including feasibility check, SC maximiza-tion and greedy optimization for EC. Thanks to numeri-cal results, we illustrate the necessity to conduct our per-formance tradeoffand observe the gain when multiple re-source variables are in elaborate cooperation.     

Extracellular Ca2+ sensitivity of mGluR1alpha induces an increase in the basal cAMP level by direct coupling with Gs protein in transfected CHO cells

We previously reported that the metabotropic glutamate receptor R1alpha (mGluR1alpha) can be activated not only by applying glutamate but also by raising extracellular Ca2+ (Ca2+o) concentration, and that the constant stimulation by Ca2+o causes morphological change of transfected Chinese Hamster Ovary (CHO) cells (Kubo Y Miyashita T and Murata Y (1998) Science 279, 1722-1725). The physiological role of the Ca2+o-sensing function of mGluR1alpha, however, is not fully clear yet, especially because Ca2+ is constitutively present in the extracellular space unlike other neurotransmitters. In this work, we aimed to elucidate the physiological significance of the Ca2+o-sensing function of mGluR1alpha. The effect of mGluR1alpha activation by Ca2+o on the morphological change of CHO cells was mimicked by forskolin. The effect of mGluR1alpha activation on the morphological change was suppressed by the inhibitors of adenylate cyclase, protein kinase A (PKA) and MAP kinase kinase (MAPKK), and the effect of forskolin was also decreased by the inhibitors of PKA and MAPKK. These results demonstrate the involvement of cAMP, PKA, MAPKK, MAPK pathway in the morphological change. We actually confirmed that the Ca2+o stimulation of mGluR1alpha increased the basal cAMP level of transfected CHO cells. This increase in cAMP was observed even when only the membrane fraction of mGluR1alpha transfected CHO cells were used, and the increase was inhibited by anti-Gs alpha antibody. Taken together, we concluded that the Ca2+o-sensing function of mGluR1alpha and the continuous stimulation by Ca2+o caused the increase in the basal cAMP level by direct coupling with Gs, and triggered the subsequent activation of PKA, MAPKK, and MAPK cascade which resulted in the morphological change of transfected CHO cells.     

Quantification of the dopamine D2 receptor in the living human caudate nucleus by PET: comparison of in vivo and in vitro kinetic parameters

It is demonstrated that the set of kinetic parameters (including first-order rate constants for the dissociation of N-methylspiperone (NMSP) from, and second-order rate constants for the association of NMSP to, the dopamine D2 and serotonin S2 receptors of the caudate nucleus) which can be derived from previously reported human caudate PET (positron emission tomographic) data is not uniquely determined, but that multiple sets generate approximately equivalent curve fits. In particular, the set consisting of the in vitro values can generate the PET data. Thus, the in vitro rate constants may apply in vivo.     

The parathyroid hormone 2 (PTH2) receptor

The human PTH2 receptor binds and is activated at high potency by PTH and by the recently discovered peptide tuberoinfundibular peptide of 39 residues (TIP39). Rat and zebrafish PTH2 receptors are more weakly activated by PTH, suggesting that TIP39 may be the natural ligand for the PTH2 receptor. Unlike the PTH1 receptor, the PTH2 receptor interacts extremely weakly with parathyroid hormone-related peptide (PTHrP). The PTH2 receptor is strongly coupled to stimulation of cAMP accumulation, and more weakly, in a cell-specific manner to increases in intracellular calcium concentration. A variety of evidence supports the general model of receptor amino terminal sequences binding ligand carboxyl terminal sequences with high affinity, and ligand amino terminal sequences activating the receptor through interaction with the "juxtamembrane" portion of the receptor. The receptor is present at greatest levels in the nervous system. It is expressed in scattered cells in the cerebral cortex and basal ganglia and at relatively high abundance in the septum, midline thalamic nuclei, several hypothalamic nuclei, and the dorsal horn of the spinal cord. Peripherally, expression in pancreatic islet somatostatin cells is most dramatic. Functional hypotheses based on the receptor's distribution are being tested. Recent data support involvement in hypothalamic releasing-factor secretion and pain.     

2D and 3D Hybrid Systems for Enhancement of Chondrogenic Differentiation of Tonsil‐Derived Mesenchymal Stem Cells

2D/3D hybrid cell culture systems are constructed by increasing the temperature of the thermogelling poly(ethylene glycol)‐poly(l ‐alanine) diblock copolymer (PEG‐l ‐PA) aqueous solution in which tonsil tissue‐derived mesenchymal stem cells and graphene oxide (GO) or reduced graphene oxide (rGO) are suspended, to 37 °C. The cells exhibit spherical cell morphologies in 2D/3D hybrid culture systems of GO/PEG‐l ‐PA and rGO/PEG‐l ‐PA by using the growth medium. The cell proliferations are 30%–50% higher in the rGO/PEG‐l ‐PA hybrid system than in the GO/PEG‐l ‐PA hybrid system. When chondrogenic culture media enriched with TGF‐β3 is used in the 2D/3D hybrid systems, cells extensively aggregate, and the expression of chondrogenic biomarkers of SOX 9, COL II A1, COL II, and COL X significantly increases in the GO/PEG‐l ‐PA 2D/3D hybrid system as compared with the PEG‐l ‐PA 3D systems and rGO/PEG‐l ‐PA 2D/3D hybrid system, suggesting that the GO/PEG‐l ‐PA 2D/3D hybrid system can be an excellent candidate as a chondrogenic differentiation platform of the stem cell. This paper also suggests that a 2D/3D hybrid system prepared by incorporating 2D materials with various surface biofunctionalities in the in situ forming 3D hydrogel matrix can be a new cell culture system.     

How the edge permeability of a 2D island influences the transition from 2D to 3D growth

In the context of the simple growth model of two level pyramids consisting of 2D islands, we examine the incipient transition from 2D to 3D growth. It is shown that the characteristic time of transition to 3D growth can depend nonmonotonically on temperature. Under high temperatures, the formation of 3D islands is caused by atoms detaching from the edge of a 2D island to its surface. Under low temperatures, 3D-island formation is caused by adsorbed atom transition from the initial surface over the edge of a 2D island, without visiting the position in the kink. The last mechanism takes place under the weak migration of the adsorbed atoms along an island edge, if the Schwoebel barrier and potential well for the adsorbed atom on the 2D-island surface relative to the initial surface are present.