NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival.

Transcription factor NRF2 is an important regulator of oxidative stress. It is involved in cancer progression, and has abnormal constitutive expression in acute myeloid leukaemia (AML). Posttranscriptional regulation by microRNAs (miRNAs) can affect the malignant phenotype of AML cells. In this study, we identified and characterised NRF2-regulated miRNAs in AML. An miRNA array identified miRNA expression level changes in response to NRF2 knockdown in AML cells. Further analysis of miRNAs concomitantly regulated by knockdown of the NRF2 inhibitor KEAP1 revealed the major candidate NRF2-mediated miRNAs in AML. We identified miR-125B to be upregulated and miR-29B to be downregulated by NRF2 in AML. Subsequent bioinformatic analysis identified putative NRF2 binding sites upstream of the miR-125B1 coding region and downstream of the mir-29B1 coding region. Chromatin immunoprecipitation analyses showed that NRF2 binds to these antioxidant response elements (AREs) located in the 5' untranslated regions of miR-125B and miR-29B. Finally, primary AML samples transfected with anti-miR-125B antagomiR or miR-29B mimic showed increased cell death responsiveness either alone or co-treated with standard AML chemotherapy. In summary, we find that NRF2 regulation of miR-125B and miR-29B acts to promote leukaemic cell survival, and their manipulation enhances AML responsiveness towards cytotoxic chemotherapeutics.

Granulocyte macrophage-colony stimulating factor and interleukin-3 increase expression of type II tumour necrosis factor receptor, increasing susceptibility to tumour necrosis factor-induced apoptosis. Control of leukaemia cell life/death switching.

Tumour necrosis factor (TNF) induces apoptosis in a range of cell types via its two receptors, TNFR1 and TNFR2. Here, we demonstrate that proliferation and TNFR2 expression was increased in human leukaemic TF-1 cells by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), with TNFR1 expression unaffected. Consequently, they switch from a proliferative to a TNF-induced apoptotic phenotype. Raised TNFR2 expression and susceptibility to TNF-induced apoptosis was not a general effect of proliferation as IL-1beta and IFN-gamma both proliferated TF-1 cells with no effect on TNFR expression or apoptosis. Although raised TNFR2 expression correlated with the apoptotic phenotype, stimulation of apoptosis in GM-CSF-pretreated cells was mediated by TNFR1, with stimulation of TNFR2 alone insufficient to initiate cell death. However, TNFR2 did play a role in apoptotic and proliferative responses as they were blocked by the presence of an antagonistic TNFR2 antibody. Additionally, coincubation with cycloheximide blocked the mitotic effects of GM-CSF or IL-3, allowing only the apoptotic responses of TNF to persist. TNF life/death was also observed in K562, but not MOLT-4 and HL-60 human leukaemic cell types. These findings show a cooperative role of TNFR2 in the TNF life/death switching phenomenon.

TNF-alpha receptors simultaneously activate Ca2+ mobilisation and stress kinases in cultured sensory neurones.

The cytokine tumour necrosis factor-alpha (TNF) has been implicated in autoimmune diseases and may play an indirect role in activation of pain pathways. In this study we have investigated the possibility that TNF directly activates cultured neonatal rat dorsal root ganglion (DRG) neurones and provides a signalling pathway from cells in the immune system such as macrophages to sensory neurones. Expression of TNF receptor subtypes (TNFR1 and TNFR2) on sensory neurones was identified using immunohistochemistry, fluorescence-activated cell sorting analysis and RT-PCR. Biochemical and immunocytochemical analysis showed that TNF activated p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) but not p42/p44 MAPK. TNF treatment evoked transient Ca2+-dependent inward currents in 70% of DRG neurones. These TNF-evoked currents were significantly attenuated by ryanodine or thapsigargin or by inclusion of BAPTA in the patch pipette solution. Responses were also evoked in subpopulations of cultured DRG neurones by human mutant TNFs that cross-reacted with rat receptors and selectively activated TNFR1 or TNFR2 subtypes. TNF-evoked transient increases in [Ca2+]i were also detected in 34% of fura-2-loaded DRG neurones. The link between TNF receptor activation and Ca2+ release from stores remains to be elucidated. However, responses to TNF were mimicked by sphingolipids, including sphingosine-1-phosphate, which evoked a transient rises in [Ca2+]i in a pertussis toxin-insensitive manner in fura-2-loaded DRG neurones. We conclude that distinct receptors TNFR1 and TNFR2 are expressed on cultured DRG neurones and that they are functionally linked to intracellular Ca2+ mobilisation, a response that may involve sphingolipid signalling.

Type II tumour necrosis factor-alpha receptor (TNFR2) activates c-Jun N-terminal kinase (JNK) but not mitogen-activated protein kinase (MAPK) or p38 MAPK pathways.

The pleitropic actions of tumour necrosis factor-alpha (TNF) are transmitted by the type I 55 kDa TNF receptor (TNFR1) and type II 75 kDa TNF receptor (TNFR2), but the signalling mechanisms elicited by these two receptors are not fully understood. In the present study, we report for the first time subtype-specific differential kinase activation in cell models that respond to TNF by undergoing apoptotic cell death. KYM-1 human rhabdomyosarcoma cells and HeLa human cervical epithelial cells, engineered to overexpress TNFR2, displayed c-Jun N-terminal kinase (JNK) activation by wild-type TNF, a TNFR1-specific TNF mutant and a TNFR2-specific mutant TNF in combination with an agonistic TNFR2-specific monoclonal antiserum. A combination of the TNFR2-specific mutant and agonistic antiserum elicited maximal endogenous or exogenous TNFR2 responsiveness. Moreover, alternative expression of a TNFR2 deletion mutant lacking its cytoplasmic domain rendered the cells unable to activate JNK activity through this receptor subtype. The profile of JNK activation by TNFR1 was more transient than that of TNFR2, with TNFR2-induced JNK activity also being more sensitive to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone. Conversely, only activation of the TNFR1 could stimulate mitogen-activated protein kinase (MAPK) or p38 MAPK activities in a time-dependent manner. The role of TNFR2 activation in enhanced apoptotic cell death was confirmed with agonistic monoclonal antisera in cells expressing high levels of TNFR2. Activation of TNFR2 alone elicited cell death, but full TNF-induced death required stimulation of both receptor types. These findings indicate that efficient activation of TNFR2 by soluble TNFs is achievable with co-stimulation by antisera, and that both receptors differentially modulate extracellular signal-regulated kinases contributing to the cytokine's cytotoxic response.

Modulated kinase activities in cells undergoing tumour necrosis factor-induced apoptotic cell death.

Tumour necrosis factor-alpha (TNF) has a variety of cellular effects including apoptotic and necrotic cytotoxicity. TNF activates a range of kinases, but their role in cytotoxic mechanisms is unclear. HeLa cells expressing elevated type II 75 kDa TNF receptor (TNFR2) protein, analysed by flow cytometry and Western analysis, showed altered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK; but not MAPK) protein content and activation. There was greater JNK activation, but reduced p38MAPK activation in dying cells compared to those still to enter TNF-induced apoptosis. Moreover, cells displaying more rapid apoptosis possess higher levels of type I 55 kDa TNFR1 receptor isoform, but less TNFR2. These findings reveal differential kinase activation in TNF-induced apoptotic death.

Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons.

Agonist-induced internalization of G-protein-coupled receptors is an important mechanism for regulating receptor abundance and availability at the plasma membrane. In this study we have used immunolabeling techniques and confocal microscopy to investigate agonist-induced internalization and trafficking of CB(1) receptors in rat cultured hippocampal neurons. The levels of cell surface CB(1) receptor immunoreactivity associated with presynaptic GABAergic terminals decreased markedly (by up to 84%) after exposure to the cannabinoid agonist (+)-WIN55212, in a concentration-dependent (0.1-1 microm) and stereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB(1) receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surface labeling (by 43% at 1 microm). Differential labeling of cell surface and intracellular pools of receptor demonstrated that the reduction in cell surface immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB(1) receptors are translocated toward the soma. The internalization process did not require activated G-protein alpha(i) or alpha(o) subunits. A different pattern of cell surface CB(1) receptor expression was observed using an undifferentiated F-11 cell line, which had pronounced somatic labeling. In these cells substantial CB(1) receptor internalization was also observed after exposure to (+)-WIN55212 (1 microm) for relatively short periods (30 min) of agonist exposure. In summary, this dynamic modulation of CB(1) receptor expression may play an important role in the development of cannabinoid tolerance in the CNS. Agonist-induced internalization at presynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.

Stimulation of stress-activated but not mitogen-activated protein kinases by tumour necrosis factor receptor subtypes in airway smooth muscle.

The multifunctional cytokine tumour necrosis factor-alpha (TNF) displays many physiological effects in a variety of tissues, especially proliferative and cytotoxic actions in immunological cells. Recently, we uncovered an important new mechanism by which TNF can sensitise airway smooth muscle (ASM) to a fixed intracellular Ca2+ concentration which in vivo would produce a marked hypercontractility of the airways. Here, we report that both 50-60 kDa type I TNFR (TNFR1) and 70-80 kDa type II TNFR (TNFR2) receptor subtypes were expressed in ASM cells and selectively activated the stress kinases, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (p38 MAPK). However, TNF caused no activation of p42/p44 MAPK or cytosolic phospholipase A(2) activity. In contrast, TNF stimulation of the TNFR1, but not the TNFR2, elicited nuclear factor-kappaB transcription factor function, a species known to be important in mediation of certain inflammatory cellular responses. This is the first report of TNF receptor subtypes in ASM cells causing selective kinase activation, which may prove important in therapeutic strategies for treating immune airway disorders such as chronic obstructive pulmonary disease and asthma.

Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception.

Cannabinoids modulate nociceptive processing in models of acute, inflammatory and neuropathic pain. We have investigated the location and function of cannabinoid receptors on cultured neonatal dorsal root ganglion (DRG) neurones and F-11 cells, a dorsal root ganglionxneuroblastoma hybridoma which displays several of the features of authentic DRG neurones. CB(1) receptor immunolabelling was observed on the cell bodies and as fine puncta on processes of both cultured DRG neurones and F-11 cells. Additionally, fluorescence-activated cell sorting (FACS) analysis provided evidence that both CB(1) and CB(2) receptors are expressed on populations of cells within the cultured DRG and F-11 cells. The cannabinoid receptor agonist (+)-WIN55212 (10 and 100 nM) inhibited the mean voltage-activated Ca(2+) current in DRG neurones by 21% and 30%, respectively. The isomer, (-)-WIN55212 (10 and 100 nM) produced significantly less inhibition of 6% and 10% respectively. The CB(1) selective receptor antagonist SR141716A (100 nM) enhanced the peak high voltage-activated Ca(2+) current by 24% and simultaneous application of SR141716A (100 nM) and (+)-WIN55212 (100 nM) resulted in a significant attenuation of the inhibition obtained with (+)-WIN55212 alone. These data give functional evidence for the hypothesis that the analgesic actions of cannabinoids may be mediated by presynaptic inhibition of transmitter release in sensory neurones.

Unmodified calcium concentrations in tumour necrosis factor receptor subtype-mediated apoptotic cell death.

Tumour necrosis factor-alpha (TNF) receptors mediate a variety of effects dependent on cell type. A role for Ca2+ in TNF-induced death remains uncertain. Here we investigated restricting intracellular/extracellular Ca2+ in HeLa epithelial carcinoma cells expressing low and high levels of p75TNFR receptor subtype and KYM-1 rhabdomyosarcoma cells, models of rapid TNF-induced apoptosis. Ca2+ -chelators EGTA and BAPTA-AM as well as microsomal Ca2+ -ATPase inhibitor thapsigargin, did not alter TNF-induced death. TNF was also unable to alter resting [Ca2+]i levels which remained < 200 nM even during times when these cells were undergoing apoptotic cell death. These findings indicate no role for modulated Ca2+ concentrations in TNF-induced apoptotic cell death.

Selective down-regulation of the G(q)alpha/G11alpha G-protein family in tumour necrosis factor-alpha induced cell death.

Investigations into the regulation of heterotrimeric GTP-binding protein alpha-subunits in models of tumour necrosis factor-alpha (TNF)-induced cell death, revealed the selective down-regulation of the G(q)alpha/G11alpha family of G-proteins. The human HeLa and murine L929 cells treated with recombinant human TNF for up to 24 h displayed down-regulated G(q)alpha/G11alpha family protein levels, but not G(s)alpha, G(i)alpha and G(o)alpha protein levels as determined by Western analyses. This effect of TNF was observed in a concentration--and time-dependent manner, consistent with the profiles of TNF-induced cell death observed. Moreover, the functioning of G(q)alpha/G11alpha family proteins were found to be impaired in TNF-treated cells, as measured by agonist-induced [Ca2+]i release. In contrast, G(s)alpha activity was unaltered by TNF-treatment, determined by measurement of agonist-induced intracellular cyclic AMP generation. These findings in TNF-induced cytotoxic models, indicate a novel 'cross-talk' mechanism by which TNF alters Ca2+-signalling mechanisms, which may contribute towards the apoptotic and necrotic cell death.

Tumour necrosis factor-alpha activates a calcium sensitization pathway in guinea-pig bronchial smooth muscle.

1. The effects of tumour necrosis factor-alpha (TNF) on guinea-pig bronchial smooth muscle contractility were investigated. 2. The Ca2+-activated contractile response of permeabilized bronchial smooth muscle strips was significantly increased after incubation with 1 microgram ml-1 TNF for 45 min. This TNF-induced effect was not due to a further increase in intracellular Ca2+. 3. The TNF-induced Ca2+ sensitization was, at least partly, the result of an increase in myosin light chain20 phosphorylation. 4. The intracellular signalling pathway involved in this effect of TNF was further investigated. Sphingomyelinase, a potential mediator of TNF, had no effect on Ca2+ sensitivity of permeabilized bronchial smooth muscle. Also, p42/p44 mitogen-activated protein kinase (p42/p44mapk), activated by TNF in some cell types, did not show an increased activation in bronchial smooth muscle after TNF treatment. 5. In conclusion, TNF may activate a novel signalling pathway in guinea-pig bronchial smooth muscle leading to an increase in myosin light chain20 phosphorylation and a subsequent increase in Ca2+ sensitivity of the myofilaments. This pathway does not appear to involve sphingomyelinase-liberated ceramides or activation of p42/p44mapk. Given the importance of TNF in asthma, this TNF-induced Ca2+ sensitization of the myofilaments may represent a mechanism responsible for airway hyper-responsiveness.

Tumor necrosis factor-alpha mediates both apoptotic cell death and cell proliferation in a human hematopoietic cell line dependent on mitotic activity and receptor subtype expression.

The TF-1 human erythroleukemic cell line exhibits opposing physiological responses toward tumor necrosis factor-alpha (TNF) treatment, dependent upon the mitotic state of the cells. Mitotically active cells in log growth respond to TNF by rapidly undergoing apoptosis whereas TNF exposure stimulates cellular proliferation in mitotically quiescent cells. The concentration-dependent TNF-induced apoptosis was monitored by cellular metabolic activity and confirmed by both DNA epifluorescence and DNA fragmentation. Moreover, these responses could be detected by measuring extracellular acidification activity, enabling rapid prediction (within approximately 1.5 h of TNF treatment) of the fate of the cell in response to TNF. Growth factor resupplementation of quiescent cells, resulting in reactivation of cell cycling, altered TNF action from a proliferative stimulus to an apoptotic signal. Expression levels of the type II TNF receptor subtype (p75TNFR) were found to correlate with sensitivity to TNF-induced apoptosis. Pretreatment of log growth TF-1 cells with a neutralizing anti-p75TNFR monoclonal antibody inhibited TNF-induced apoptosis by greater than 80%. Studies utilizing TNF receptor subtype-specific TNF mutants and neutralizing antisera implicated p75TNFR in TNF-dependent apoptotic signaling. These data show a bifunctional physiological role for TNF in TF-1 cells that is dependent on mitotic activity and controlled by the p75TNFR.

Protein kinase C isoforms in pituitary cells displaying differential sensitivity to phorbol ester.

Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the alpha, beta, delta, epsilon, zeta and theta isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH3 somatomammotrophic cell line. AtT-20/D16-V corticotrophic and alphaT3-1 gonadotrophic murine cell lines contained no PKC-delta. The gamma or eta isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca2+-independent PKCs in alphaT3-1 and GH3 cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca2+-dependent PKC activities were of similar sensitivity to PDBu in GH5, alphaT3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca2+-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca2+-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells.

Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors.

Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively activated mutants of receptors should provide a convenient means to detect inverse agonists. Incubation of cells either transiently or stably transfected with a constitutively activated mutant of the human beta 2-adrenoceptor with the beta 2-inverse agonists betaxolol or sotalol, which are both able to inhibit CAM beta 2-adrenoceptor-mediated basal adenylyl cyclase activity, resulted in a strong upregulation of levels of the receptor. In the stable cells lines this effect was prevented by co-incubation with neutral antagonists but could not be reproduced by an adenylyl cyclase P-site ligand which also inhibited basal adenylyl cyclase levels.

Up-regulation of a constitutively active form of the beta2-adrenoceptor by sustained treatment with inverse agonists but not antagonists.

In neuroblastoma X glioma hybrid, NG1O8-15, cells transfected to stably express a constitutively active mutant (CAM) form of the human beta2-adrenoceptor, the beta-adrenoceptor ligands sotalol and betaxolol functioned as inverse agonists as they reduced basal adenylyl cyclase activity whereas the antagonists dihydroalprenolol and propranolol did not. Maintained presence of the CAMbeta2-adrenoceptor inverse agonists but not the antagonists in the culture medium of the cells resulted in a substantial, concentration-dependent, up-regulation of the CAMbeta2-adrenoceptor. Up-regulation of the CAMbeta2-adrenoceptor by the inverse agonists was prevented by co-incubation of the cells with either propranolol or dihydroalprenolol. Neither maintained elevation of cAMP levels nor the inhibition of adenylyl cyclase activity altered the ability of the inverse agonist ligands to cause receptor up-regulation.

Inverse agonist-induced up-regulation of the human beta2-adrenoceptor in transfected neuroblastoma X glioma hybrid cells.

Neuroblastoma X glioma hybrid NG108-15 cells were transfected to express stably either the wild-type human beta2-adrenoceptor or a constitutively active mutant (CAM) version of this receptor. Basal adenylyl cyclase activity in cells expressing the CAM beta2-adrenoceptor correlated well with the level of expression of the receptor and was substantially greater than that in cells expressing the wild-type beta2-adrenoceptor. The CAM beta2-adrenoceptor displayed higher affinity for the agonist isoprenaline than the wild-type receptor but not for the antagonist alprenolol or the inverse agonist betaxolol. Pretreatment of cells harboring the CAM beta2-adrenoceptor with betaxolol resulted in a large (4-7-fold within 24 hr) up-regulation in levels of this receptor. This was not observed after exposure of the CAM beta2-adrenoceptor-expressing cells to alprenolol, and a much smaller effect of betaxolol was produced in cells expressing the wild-type receptor. Betaxolol-mediated up-regulation of the CAM beta2-adrenoceptor was both time and concentration dependent. However, this up-regulation did not result in a substantial alteration in the cellular distribution profile of the receptor. Half-maximal up-regulation of the CAM beta2-adrenoceptor required concentrations of betaxolol similar to those needed to cause half-maximal inhibition of basal adenylyl cyclase activity, indicating the receptor up-regulation is associated with the inverse agonist properties of this compound. Despite the large up-regulation of CAM beta2-adrenoceptor levels, treatment with betaxolol did not significantly alter levels of the G protein that couples to this receptor (G(Salpha)). After sustained treatment with betaxolol, Northern analyses did not demonstrate up-regulation of either CAM beta2-adrenoceptor or G(Salpha) mRNA, and up-regulation of the receptor was prevented by cotreatment of the cells with cycloheximide. These data indicate that the up-regulation of the receptor by betaxolol is likely to reflect an increase in translational efficiency of existing mRNA and/or stabilization of the receptor polypeptide from proteolytic degradation and indicate that such effects can be produced by inverse agonists but not by neutral antagonists.

Agonist regulation of adenylate cyclase activity in neuroblastoma x glioma hybrid NG108-15 cells transfected to co-express adenylate cyclase type II and the beta 2-adrenoceptor. Evidence that adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity.

Stable cell lines, derived from NG108-15 cells and transfected to express both the beta 2-adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [3H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the beta 2-adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5'-[beta, gamma-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced beta 2-adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration-effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones over-expressing adenylate cyclase type II. A maximally effective concentration of the beta-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5'-N-ethylcarbox-amidoadenosine (acting at an endogenous A2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.

Elevated cPLA2 levels as a mechanism by which the p70 TNF and p75 NGF receptors enhance apoptosis.

The 70-kDa TNF cell surface receptor (p70TNFR) and the related 75-kDa nerve growth factor (NGF) receptor (p75NGFR) can enhance cell death. Expression of p70TNFR or p75NGFR in HeLa cells resulted in enhanced TNF-induced apoptotic cell death with a corresponding elevation of cytosolic phospholipase A2 (cPLA2) levels. This response was apparent by 24 h, did not occur with NGF treatment or when vector or TrkA NGFR were expressed and was reversed by dexamethasone pretreatment. These findings reveal a novel mechanism by which the p70TNFR and p75NGFR achieve enhanced TNFR-mediated programmed cell death by elevating cPLA2 levels.

Analysis of the role of receptor number in defining the intrinsic activity and potency of partial agonists in neuroblastoma x glioma hybrid NG108-15 cells transfected to express differing levels of the human beta 2-adrenoceptor.

Many agonist ligands are known experimentally to display a range of efficacies and potencies in different tissues and preparations. To analyze the role of the levels of receptor expression and availability in the intrinsic activities and potencies of agonists, the function of a number of beta-adrenoceptor ligands was examined in clones of neuroblastoma x glioma hybrid NG108-15 cells transfected to express differing levels of the human beta 2-adrenoceptor, as well as after treatment of these cell lines with the irreversible beta-adrenoceptor antagonist bromoacetyl alprenolol menthane (BAAM). Clone beta N22 expressed approximately 10-fold higher levels of the receptor than did clone beta N17. In measurements of agonist stimulation of adenylyl cyclase activity in membranes of these cells or agonist stimulation of the formation of the complex of Gs alpha and adenylyl cyclase, which acts as the high affinity binding site for [3H]forskolin in whole cells, a series of beta-adrenoceptor agonists, including dichloroisoprenaline, ephedrine, dobutamine, and salbutamol, displayed higher intrinsic activity and showed concentration-response curves that were substantially to the left (lower EC50 values) in clone beta N22, compared with clone beta N17. Treatment of clone beta N22 cells with varying concentrations of BAAM reduced the intrinsic activity of these ligands and shifted the concentration-response curves for these agents to the right. In clone beta N22 cells and membranes, reduction in the observed intrinsic activity for ephedrine required elimination of a smaller fraction of the beta 2-adrenoceptor reserve than for salbutamol and reduction in the effect of the full agonists isoprenaline and epinephrine was noted only with high fractional elimination of the receptor pool. The effect of isoprenaline was substantially reduced, however, by BAAM treatment of clone beta N17 cells, where the beta 2-adrenoceptor number approached extremely low levels. Analysis of the data using the formalisms of Whaley et al. [Mol. Pharmacol. 45:481-489 (1994)] showed that prediction of alterations in agonist potency with receptor number for full agonists can be adequately extended to partial agonists.