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1.
The abilities of unfractionated mononuclear cells (MNC), monocytes (98-99% pure), and lymphocytes (98-99% pure) to carry out the lysis of target cells in the ADCC, NK, NOCC, and MICC assays were compared. Lymphocytes by themselves were able to lyse the CRBC (ADCC), K-562 (NK), and RRBC (MICC) target cells. The monocytes were very effective in the lysis of the CRBC (MICC) target cells. However, the lysis of two other target cells--RRBC (NOCC) and HRBC (ADCC)--required the simultaneous presence of both lymphocytes and monocytes in order to effect optimal lysis. Soluble factor(s) secreted by the cytotoxic cells capable of lysing the target cells were detected only in the NK assay. The activity of the soluble cytotoxic factor (NKCF) was only 25-40% of that exhibited by the cytotoxic NK cells and it was secreted by the cytotoxic cells after 48 hr of culture and not 24 hr of culture which is the usual assay condition. The NKCF was cytotoxic only to the NK target cells and not to the target cells used in the ADCC, NOCC, and MICC cytotoxic assays. Different classes of lymphocytes were cytotoxic in the monocyte-independent assays [ADCC (CRBC), NK (K-562), and MICC (RRBC)]. The null lymphocytes and the T lymphocytes were the primary cytotoxic cells in the ADCC and MICC assays, respectively, whereas the T, B, and null cells were almost equally cytotoxic in the NK assay. With respect to the monocyte-dependent assays [ADCC (HRBC), NOCC (RRBC), and MICC (CRBC)], the cytotoxic activity of any one class of lymphocytes failed to approach that of the unfractionated MNC. The T cells were the most cytotoxic; the B cells exhibited limited cytotoxic activity in only the ADCC assay and the null cells showed no cytotoxic activity. However, the combination of T and non-T cells and, to a lesser extent, T and B cells, exhibited much greater cytotoxic activity than the individual cells and together were as cytotoxic as the unfractionated MNC. It is concluded that, depending upon the selection of the target cells, lysis in the ADCC, NK, NOCC, and MICC assays may be effected by lymphocytes only, by monocytes only, by both monocytes and lymphocytes, or as a result of lymphocyte-monocyte collaboration. In the latter instance more than one class of lymphocytes must be present in order for maximum cytotoxic activity to be expressed. 相似文献
2.
Subpopulations of human peripheral blood lymphocytes were prepared by rosetting techniques employing neuraminidase-treated sheep erythrocytes (SRBCn), sheep erythrocytes coated with IgM and murine complement (EAC′), and bovine erythrocytes coated with IgG and IgM. The isolated subpopulations were tested in assays of natural cytotoxicity (NC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). B cells (SRBCn?, EAC′+) did not mediate cytotoxicity. L cells (SRBCn?, EAC′?) mediated NC and ADCC but not MICC. T cells (SRBCn+) mediated NC, ADCC, and MICC. Separation of T cells into Fc-IgG (Tγ) and Fc-IgM (Tμ) subsets revealed that Tγ cells mediated NC, ADCC, and MICC while Tμ cells mediated only MICC. Thus MICC but not NC or ADCC was solely T-cell mediated. Tγ and L cells were functionally distinguishable in that Tγ cells but not L cells mediated MICC. Tγ cells and Tμ cells differed with regard to NC and ADCC effector function while both subsets mediated MICC. 相似文献
3.
Studies of complement receptors on cytotoxic effector cells in human peripheral blood 总被引:1,自引:0,他引:1
J M Vierling C J Steer B M Bundy W Strober E A Jones N E Hague D L Nelson 《Cellular immunology》1978,35(2):403-413
The question of whether cells bearing complement receptors (CR) mediate cytotoxicity in vitro against allogeneic Chang liver cell targets was investigated by assessing peripheral blood mononuclear cells (PBMC) from normal humans for cell surface characteristics and cytotoxic capacity before and after depletion of CR+ cells capable of forming rosettes with sheep erythrocytes coated with 19S antibody and mouse complement (EAC) and depletion of Fc receptor-bearing cells capable of forming rosettes with human O+ erythrocytes coated with Ripley antibody (EA-Ripley). PBMC depleted of CR+ cells by density centrifugation contained markedly reduced proportions of phagocytes and sIg + cells and increased proportions of both sIg ?, FcR+ cells as well as cells forming rosettes with sheep erythrocytes (E). PBMC depleted of CR+ cells mediated cytotoxicity to an extent equal to or greater than that mediated by unfractionated PBMC in assays of spontaneous cell-mediated cytotoxicity (SCMC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). Cells harvested from the EAC-rosette enriched pellet mediated cytotoxicity 5- to 10-fold less than unfractionated PBMC; however, the cytotoxic activity of the pellet could not be attributed to CR + effector cells since similar cytotoxic activity was present in cell pellets obtained by density centrifugation of PBMC which had been incubated with E coated with 19S antibody or E alone. PBMC depleted of EA-Ripley rosette-forming cells contained decreased proportions of sIg?, FcR+ cells and increased proportions of CR+ cells; PBMC so depleted contained virtually no SCMC and ADCC effector cell activity. These findings indicate that at least the majority of effector cells which mediate SCMC, ADCC, and MICC do not bear CR. 相似文献
4.
The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed 51Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events. 相似文献
5.
Anthony S. Fauci Tohru Murakami David D. Brandon D.Lynn Loriaux Mortimer B. Lipsett 《Cellular immunology》1980,49(1):43-50
Glucocorticosteroid receptors were measured in subpopulations of human peripheral blood T cells identified by the presence of an Fc receptor for IgG (TG) or an Fc receptor for IgM (TM). TM cells are selectively depleted from the circulation by in vivo administration of glucocorticosteroids as opposed to TG cells which are relatively resistant to the lymphodepletive effects of these hormones. However, this selective lymphodepletive effect of glucocorticosteroids on TM cells could not be explained on the basis of detectable differences in intracytoplasmic glucocorticosteroid receptors in these T-cell subpopulations since TG and TM cells had quantitatively similar glucocorticosteroid receptors as well as remarkably similar dexamethasone binding affinities and dissociation constants. Hence, the strikingly different effects of glucocorticosteroids on the circulatory kinetics of TG and TM cells must be explained by mechanisms other than those at the level of the glucocorticosteroid receptor. 相似文献
6.
An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine-labelled cells, using FCM-derived data 总被引:1,自引:0,他引:1
M. C. Johansson B. Baldetorp P. O. Bendahl R. Johansson S. M. Oredsson† 《Cell proliferation》1994,27(8):475-488
Abstract. Chinese hamster ovary cells in vitro were pulse-labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd-labelled cells in relation to that of G1 and G2 cells, was calculated. RM was then used to calculate DNA synthesis time (TS), at all post-labelling times (t). Since labelled cells in G2 and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G2 and M (Tg2+M) phases is finite, a non-linear relationship exists between RM and post-labelling time. Because of this, the use of a linear formula in the calculation of TS yields results that are affected by t. We found that RM data can be corrected with regard to TG2+M resulting in the derivation of a non-linear TS formula. This non-linear TS formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G1, S and G2+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd-labelled cells in each window at every post-labelling time. Plots of the fraction of BrdUrd-labelled cells v. post-labelling time were then made for each window. TS obtained in this way was in agreement with TS obtained with the corrected RM method. In conclusion, we present a method to calculate Ts which theoretically first makes the determination of RM independent of TG2+M, and secondly compensates for the non-linear function of RM with post-labelling time caused by accumulation of BrdUrd-labelled cells in G2+ M. 相似文献
7.
The nature of the cell types capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cells from athymic nude and euthymic heterozygous control littermate mice as well as Sephadex anti-Fab immunoabsorbent column purified spleen cell populations from normal (CS7BL/6) mice. Chicken erythrocytes (CRBC) and the mouse lymphoma, EL-4, were used as target cells in both cytotoxicity assays. MICC utilizing CRBC targets was mediated by several effector cell types whereas MICC utilizing EL-4 lymphoma targets was T-cell dependent. ADCC against both CRBC and EL-4 lymphoma targets occurred independently of the presence of T-cells. In addition, effector cell populations incapable of mediating MICC against EL-4 lymphoma targets were capable of mediating ADCC against the same EL-4 targets. Thus, utilizing the appropriate target cells, EL-4 but not CRBC, a sharp distinction can be made between the effectors for ADCC and MICC: ADCC is T-cell independent while MICC is dependent on the presence of mature thymus-derived cells. Furthermore these studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions plays a critical role in defining the types of effector cells capable of mediating these cytotoxicity reactions. 相似文献
8.
We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets. 相似文献
9.
KATSUMA DAN SHOJI TANAKA KIMIE YAMAZAKI YOSHIHIRO KATO 《Development, growth & differentiation》1980,22(3):589-598
Cell cycles have been analyzed in 10 divisions up to the time of hatching in the embryos of the sea urchin, Hemicentrotus pulcherrimus. In the first 5 cleavages, division synchrony is very high. On the average, TGC= 55.4 min, TG1= 0 min, Ts= 12 min, TG2=±0 min, TM= 42 min. In the remaining 5 cleavages, TGC becomes longer: 70 min for the 7th to 246 min for the 10th cleavage. G1 and G2 become definitely recognizable and become longer along with Ts. TM stays more or less constant. Plots of the changing lengths of the four compartments (G1, S, G2, M) on the Y-axis against TGC (X-axis) can be fitted to the following 4 regression equations; TG1= 0.28TGC - 19.7, Ts= 0.609TGC - 15.2, TG2= 0.104TGC - 4.72 and TM= 0.007TGC+ 39.6. 相似文献
10.
A model for the action of vinblastine (VLB) on cells multiplying exponentially in vivo with a generation time, TG, has been derived. It is based on the assumption that cells attempting to pass through mitosis in the presence of VLB lose their proliferative capacity and that this lethal effect occurs only when the cells are exposed to a concentration of VLB which is above a critical value, Ck. The model leads to two predictions. First, that the percentage of cells surviving at any time after exposure to a dose, D, of VLB is 100% if D < Dk and decreases to 0% after a time, TG, following a dose D ≥ Dk·2T G/T1/2, where Dk represents the dose of VLB required to produce the concentration Ck, and T1/2 is the half-life of the VLB in vivo. Second, that the time, TG, at which the percentage of cells surviving an exposure to VLB, at doses greater than Dk·2U G/T1/2, decreases to zero should be equal to the generation time of the cells. Both of these predictions were confirmed experimentally which indicates that the model adequately explains the action of VLB in vivo. 相似文献
11.
Marek Rola-Pleszczynski 《Cellular immunology》1981,64(2):220-226
Following in vitro sensitization with HSV-infected cells, Tγ cells comprise most of the cytotoxic effector cell population. However, whereas freshly obtained Tγ cells exhibit theophylline sensitivity in the sheep erythrocyte rosette assay, presensitized Tγ cells are theophylline resistant. Similarly, when T cells are fractionated according to their theophylline sensitivity before the sensitization culture, theophylline-resistant Tμ cells appear as the precursors of Tγ cytotoxic effector cells, the Tμ-Tγ switch occurring with a transitory eclipse of Fc receptors, and maintenance of theophylline resistance. 相似文献
12.
Lymphocytes from the organized gut-associated lymphoid tissues (GALT) of adult guinea pigs were examined for surface markers characteristic of T and B lymphocytes and for their capacity to function as effector cells in mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) reactions. GALT lymphocytes formed rosettes with rabbit erythrocytes, a T-cell marker, and underwent proliferative responses in vitro in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). GALT lymphocytes were cytotoxic in vitro for erythrocyte and DBA mastocytoma targets in the presence of PHA. A population of GALT lymphocytes bound aggregated γ-globulin; however, they functioned poorly in ADCC reactions. Thus, organized GALT in the guinea pig contains lymphocytes capable of functioning in T-cell-dependent MICC reactions but either lacks the effector cell population which mediates ADCC or contains an effector cell which functions poorly in ADCC. 相似文献
13.
T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed lymphocyte culture. The sensitized cells were fractionated on the basis of the presence of Fc receptors for IgG (TG+) or IgM (TM+), or the absence of both IgG and IgM receptors (TG?,M?). The Cytotoxicity of the T cells was found to reside principally in the TG?,M? subset. The degree of target cell lysis by this subset was related to the effector-to-target cell ratio. The sensitized T cells were also separated into subsets by treatment with monoclonal OKT4 antibody and complement (yielding OKT8+ cells) or OKT8 antibody and complement (yielding OKT4+ cells). The OKT8+ subset was the more cytotoxic of the two subsets, and this Cytotoxicity was again related to the effector-to-target cell ratio. The T-cell subsets obtained by these methods were characterized by immunologie and morphologic means. The Cytotoxicity of the total sensitized T-cell population or the TG?,M? subset could be neutralized to a considerable extent by anti-human α-lymphotoxin (anti-α-LT) serum, and α-LT thus appears to have an important role in cytolysis in this system. 相似文献
14.
R. Allen White David J. Grdina† Marvin L. Meistrich† Raymond E. Meyn‡ Tod S. Johnson§ 《Cell proliferation》1984,17(3):237-245
Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times TG1, Ts and TG2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G1 state have a 14-hr cycle time with TG1, Ts and TG2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G2M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G1 population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time. 相似文献
15.
In the guinea pig, the killer cell in in vitro ADCC assays, is found to be the Fc receptor-bearing Kurloff cell. This killer Kurloff cell is under hormonal control, estrogen treatment significantly increasing the Kurloff cell numbers in blood, spleen, and thymus, and markedly augmenting the lytic capacity of these lymphoid compartments. The cytotoxic killer lymphocyte, if present, appears to be a minor effector cell in guinea pig ADCC. Selective lymphocyte subpopulation and Kurloff-cell depletion procedures reveal that the killer Kurloff cell may also possess a variety of other membrane markers (T+, C3+, Ig+). The particular membrane profile of a cytotoxic Kurloff cell is determined by its lymphoid site of residence and the hormonal status of the animal. 相似文献
16.
The influence of one DNA region on the stability of an adjoining region (telestability) was examined. Melting curves of three block DNA's, d(C15A15)·d(T15G15), d(C20A15)·d(T15G20), and d(C20A10)·d(T10G20) were analyzed in terms of the nearest neighbor Ising model. Comparisons of predicted and experimental curves were made in 0.01 M and 0.1 M sodium ion solutions. The nearest neighbor formalism was also employed to analyze block DNA transition in the presence of actinomycin, a G·C specific molecule. The results show that nearest neighbor base-pair interaction cannot predict the melting curves of the block DNA's. Adjustments in theoretical parameters to account for phosphate repulsion assuming a B conformation throughout the DNA's do not alter this conclusion. Changes in the theoretical parameters, which provide good overall agreement, are consistent with a substantial stabilization of the A·T region nearest the G·C block. The melting temperature T A·T for the average A·T pari in d(C20A10)·d(T10G20), with 10 A·T pairs, appears to be 4°C greater than TA·T for d(C15A15)·d(T15G15) and d(C20A15)·d(T15G20), both with 15 A·T pairs. Actinomycin bound to the G·C end effectively stabilizes the A·T end by 9°C. These results indicate a long-range contribution to the interactions governing DNA stability. A possible mechanism for these interactions will be discussed. 相似文献
17.
《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2020,1865(7):158704
Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that Gq-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed Gq regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gαq to phospholipase C (PLC)-β and the thromboxane A2 receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M3 and bradykinin B2 receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca2+]i increases. Surprisingly, WDR36 also augmented M3 receptor-induced [Ca2+]i increases, which was due to increased Ca2+ mobilization while the Ca2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by Gq-coupled M3, B2 and H1 receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B2 receptors. It is concluded that WDR36 reduces signalling of Gq-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gαq and PLCβ. Our results support a role of WDR36 in orchestration of Gq signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy. 相似文献
18.
Summary We describe the in vitro influence of 3,5,3′-triiodo-l-thyronine (T3),l-thyroxine (T4), a thyroid-stimulating hormone (TSH), and/or estradiol (E2: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S+G2+M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor,
analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction,
which is selective and quantitative (stoichiometric) with respect to DNA. We show that T3, T4, and TSH at 0.01 μM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three
hormones bring about a significant transient increase in the S+G2+M fraction as does E2. Furthermore, our data indicate that E2 and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics.
This work is supported by grants awarded by the IRSIA and the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium). 相似文献
19.
Previous studies in man have shown that T cells with suppressor activity were mainly found among a subset bearing Fc receptors for IgG (Tγ). Recently, we found that virus-induced cytotoxic effector cells were also found predominantly among Tγ cells. In the present studies, we present evidence that similar, possibly overlapping T-cell populations can mediate both suppressor and cytotoxic activities when sensitized in vitro with virus-infected cells. In fact, both activities are found within the positively selected Tγ subset, but not in the Tγ-depleted population; both activities are abolished by irradiation but not by treatment with mitomycin C; a 1-hr exposition to theophylline at the onset of sensitization enhances both cytotoxic and suppressor activities. The data suggest that development of antiviral cell-mediated immune responses in vivo may also be accompanied by a concurrent induction of nonspecific suppressor cells. Such suppressor activity may play a role in the depressed cellular immune responsiveness which is associated with several systemic virus infections. 相似文献
20.
Tianlai Tang Juliann G. Kiang Thomas Cote Brian M. Cox 《Journal of neurochemistry》1995,65(4):1612-1621
Abstract: δ-Receptor agonists induce a concentration-dependent increase in intracellular calcium concentration ([Ca2+]i) in ND8-47 cells by activating dihydropyridine-sensitive Ca2+ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [d -Ser2,Leu5]enkephalin-Thr (DSLET)-induced increase in [Ca2+]i. Cholera toxin (10 nM, 24 h) had no effect on DSLET-induced response. Pretreatment of the cells with 1 µM DSLET for 1 h resulted in a 30% inhibition of DSLET-induced increase in [Ca2+]i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 µM DSLET for 24 h demonstrate a nearly 90% decrease in [3H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits αi2, αi3, αs, and αq were detected in ND8-47 cell membranes by western blot; αo and αi1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the α-subunits. These results suggest that the DSLET-induced increase in [Ca2+]i is mediated through pertussis toxin-sensitive G proteins (probably Gi2 or Gi3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density. 相似文献