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.

Inhibition of depolarisation-induced calcium influx into GH3 cells by arachidonic acid: the involvement of protein kinase C.

The influx of 45Ca2+ induced in GH3 cells by exposure to 60 mM K+ medium was inhibited by arachidonic acid (AA) in a concentration-dependent manner. This action of AA was not prevented by inhibitors of its metabolism but was reversed by the inhibitors of protein kinase C (PKC), H7 and staurosporine but not their less active congeners HA 1004 and K252a, respectively. Presumed down-regulation of PKC by pretreatment with phorbol 12,13-dibutyrate (PDBu) also greatly diminished the effect of AA. Experiments to assess effects of AA on 45Ca2+ efflux and on cytosolic Ca2+ concentrations indicated that an additional PKC-independent action of AA involving the release of intracellularly stored calcium was present. Both direct activation of certain PKC isoform(s) by AA and the synergistic influence on PKC activity by its concomitant raising of intracellular Ca2+ concentrations, may be physiologically important in the regulation of depolarisation-induced Ca2+ entry.

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.

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.

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.

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.

Calcium influx through 'L'-type channels into rat anterior pituitary cells can be modulated in two ways by protein kinase C (PKC-isoform selectivity of 1,2-dioctanoyl sn-glycerol?).

The depolarisation-induced influx of 45Ca2+ into anterior pituitary tissue and GH3 cells through 'L'-type, nimodipine-sensitive channels was investigated. In anterior pituitary prisms, phorbol esters, activators of protein kinase C, caused an enhancement of K(+)-induced 45Ca2+ influx. However, in the GH3 anterior pituitary cell line, phorbol esters inhibited K(+)-induced 45Ca2+ influx. The modulation by phorbol esters in both tissues was stereo-specific and time- and concentration-dependent. The diacylglycerol analogue, 1,2-dioctanoyl sn-glycerol was able to mimic the phorbol ester-induced enhancement of calcium influx into anterior pituitary pieces, but was ineffective in GH3 cells. 1,2-Dioctanoyl sn-glycerol may selectively activate an isoform of protein kinase C which is responsible for enhanced 'L'-type Ca(2+)-channel activity.

Characterisation of protein kinase C isoforms and enzymic activity from the alpha T3-1 gonadotroph-derived cell line.

Western blots of alpha T3-1 cell extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. These revealed the presence of PKC types alpha, epsilon and zeta, but beta, gamma, delta and eta were not detected. The potency with which partially-purified cytosolic PKC from alpha T3-1 cells was activated by phorbol 12,13-dibutyrate (PDBu), mezerein and 1,2-dioctanoyl-sn-glycerol was assessed in the presence and absence of Ca2+. The inhibitors staurosporine, K252a, H7, GF109203X and Ro 31-8220 were tested on basal activity, PDBu-induced activity and Ca(2+) + PDBu-induced kinase activity. Each inhibitor showed distinct differences in their IC50 values under the three conditions, suggesting that these inhibitors may exhibit different potencies on the PKC isoforms present in alpha T3-1 cells. Although histone IIIs was used as the phosphate acceptor for most of these experiments, the efficiency of alpha, epsilon and zeta peptide and GS peptide substrates were also determined, with epsilon peptide giving the greatest activity in the presence of PDBu or Ca2+. Each substrate displayed a different pattern of activation under the conditions tested. Overall, the findings suggest that 3 or more PKC isoforms with varying specificities are present in gonadotroph-derived alpha T3-1 cells and that the contribution of each isoform should be considered when these cells are used in models of anterior pituitary cell function where PKC is involved.

Functional expression of 5-HT1c receptor cDNA in COS 7 cells and its influence on protein kinase C.

Two subtypes of receptors for serotonin (5-hydroxytryptamine; 5-HT) are known to stimulate inositol (1,4,5)-trisphosphate production, the 5-HT1c and 5-HT2 receptors. In this study we investigated the ability of 5-HT1c receptors, transiently expressed in COS 7 cells, to functionally interact with protein kinase C-alpha, the indigenous (phorbol ester-responsive) isoform of the enzyme in those cells. Serotonin caused translocation of the [3H]phorbol 12,13-dibutyrate (PDBu) binding site of PKC-alpha from the cytosolic to the membrane fraction in a Ca(2+)-dependent manner which was prevented by the 5-HT1c receptor antagonist mianserin. The lipid activators of PKC, PDBu and 1,2-dioctanoyl-sn-glycerol (DOG) also caused translocation, but through a mechanism apparently quite independent of Ca2+.

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.

Evidence for a distinct H7-resistant form of protein kinase C in rat anterior pituitary gland.

Inhibition of phorbol 12,13-dibutyrate-induced protein kinase C (PKC) activity from rat midbrain, anterior pituitary and a number of other tissues, as well as COS 7 cells, was studied in vitro. In anterior pituitary, Ca(2+)-independent activity was notably resistant to H7 but sensitive to staurosporine and Ro 31-8220. All Ca(2+)-dependent activity was sensitive to these three inhibitors. Mezerein and 1,2-dioctanoyl-sn-glycerol also activated this H7-insensitive PKC from anterior pituitary. The distribution of this activity, prominently expressed in pituitary and perhaps also lung, and its characteristic resistance to H7 but not other inhibitors, does not obviously correlate with that of any of the well-characterised PKCs, and may reflect either a novel or a modified isoform.

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.

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.

Involvement of multiple protein kinase C isozymes in the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein kinase C (PKC)-mediated enhancement of calcium- and guanine nucleotide-evoked adrenocorticotrophin (ACTH) secretion. 2. A profile of the PKC isozymes present in AtT-20 cells was obtained by Western blotting analysis and it was found that AtT-20 cells express the alpha, beta, epsilon and zeta isoforms of PKC. 3. PKC isozymes were activated by the use of substances reported to activate particular isoforms of the enzyme. The effects of these substances were investigated in both intact and electrically-permeabilized cells. Phorbol 12-myristate 13-acetate (PMA, EC50 = 1 +/- 0.05 nM, which activates all isozymes of PKC, except the zeta isozyme), thymeleatoxin (TMX, EC50 = 10 +/- 0.5 nM, which activates the alpha, beta and gamma isozymes) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA, EC50 = 3 +/- 0.5 nM, a beta 1-selective isozyme activator) all stimulated ACTH secretion from intact cells in a concentration-dependent manner. Maximal TMX stimulated ACTH secretion was of a similar degree to that obtained in response to PMA but maximal dPPA-stimulated ACTH secretion was only 60-70% of that obtained in response to PMA or TMX. 4. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 100 nM to 10 microM. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) enhanced the amount of ACTH secreted at every concentration of calcium investigated. PMA (100 nM) and TMX (100 nM)significantly enhanced ACTH secretion in the effective absence of calcium (i.e. where the free calcium concentration is nM).5. GTP-gamma-S stimulated ACTH secretion from permeabilized cells in a concentration-dependent manner with a threshold of 1 micro M. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) increased the amount of ACTH secretion evoked by every concentration of GTP-gamma-S investigated.6. The PKC inhibitor, chelerythrine chloride (10 micro M), blocked the PMA (100 nM)-evoked enhancement of calcium- and GTP-micro-S-stimulated ACTH secretion but did not significantly alter calcium- or GTP-micro-S-evoked secretion itself.7. The present paper indicates that AtT-20 cells express multiple isoforms of PKC and that these act at different sites in the secretory pathway for ACTH secretion. The alpha and epsilon isozymes of PKC can act distal to calcium entry to modulate the ability of increased cytosolic calcium concentrations to stimulate ACTH secretion. This site of action is either at the level of, or at some stage distal to, a GTP-binding protein which mediates the effects of calcium upon ACTH secretion. The beta isozyme of PKC may act ata stage early in the secretory pathway to regulate the cytosolic calcium concentration.

Oestradiol-17 beta modulates the actions of pharmacologically distinct forms of protein kinase C in rat anterior pituitary cells.

Phorbol ester-induced release of LH and GH from rat anterior pituitary tissue in vitro is differentially inhibited by some, but not other, inhibitors of protein kinase C (PKC), suggesting that pharmacologically distinct species of PKC may have different functional roles in these cells. Since stimulus-induced anterior pituitary hormone release can be enhanced by oestradiol-17 beta (OE2) pretreatment, we investigated the effect of OE2 treatment of long-term (4 weeks) ovariectomized rats on the amount, activity and cellular actions of pharmacologically distinct PKC species in rat anterior pituitary tissue. Here we report that OE2 treatment enhanced phorbol 12,13-dibutyrate (PDBu)-induced LH but not GH release measured in vitro. This effect of OE2 on LH release may involve synthesis of additional PKCs that are not targeted by the synthetic diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG). Measurements of anterior pituitary PKC activity and [3H]phorbol ester-binding studies suggested that the facilitatory action of OE2 on LH release may occur, at least in part, by altering the quantity and activity of PKC(s). Our results also demonstrate that the OE2-induced PKC(s) which facilitate LH release may be of the type that are not dependent upon raised intracellular Ca2+ for their activation and display distinct pharmacological properties (being readily activated by PDBu, but not by DOG, and are staurosporine-sensitive but H7-insensitive). This facilitatory action of OE2 on PKC-induced LH release does not appear to involve OE2-induced changes in the affinity of existing PKC(s) for PDBu, or changes in the amount of releasable LH in the pituitary prior to the stimulus.

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.

The involvement of dihydropyridine-sensitive calcium channels in phorbol ester-induced luteinizing hormone and growth hormone release.

We examined the role of voltage-activated, L-type, Ca2+ channels in phorbol ester-induced luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue. The L-type Ca2+ channel inhibitor, nimodipine (NMD), inhibited phorbol 12,13-dibutyrate (PDBu)-induced GH release but had no significant effect on LH release. The L-type Ca2+ channel activator BAY K 8644 had no effect on PDBu-induced GH release but potentiated PDBu-induced LH release. In contrast, 60 mM K(+)-induced LH and GH release were inhibited by NMD, whereas BAY K 8644 had no effect. When PDBu and either K+ or BAY K 8644 were used together, they acted synergistically to evoke levels of LH release greater than addition of release caused by each secretagogue alone. However, the release of GH was additive with PDBu and either K+, BAY K 8644. The protein kinase C (PKC) inhibitor staurosporine inhibited both PDBu-induced LH release and GH release. A structurally different PKC inhibitor, H7, significantly inhibited PDBu-induced LH release but had no effect on PDBu-induced GH release. Both staurosporine and H7 inhibited LH release induced by PDBu and BAY K 8644 together. In contrast, although staurosporine inhibited GH release induced by PDBu and BAY K 8644, H7 significantly potentiated this response. A difference in the action of these two inhibitors was also apparent on K(+)-induced hormone release where staurosporine partially blocked K(+)-induced LH and GH release but H7 had no effect on the release of either hormone. Data obtained in 45Ca2+ influx experiments further suggested that a staurosporine-sensitive, but H7-resistant, PKC-like kinase may tonically maintain L-channels in a voltage-sensitive state, as down-regulation of PKC in dispersed anterior pituitary cells by long term PDBu treatment caused a significant reduction in K(+)-induced 45Ca2+ influx. We conclude that phorbol ester-induced GH release, but not LH release, is a result of L-type Ca2+ channel activation which may occur by means of alterations in the channel itself to increase its responsiveness to a given depolarisation.

The differential effects of protein kinase C activators and inhibitors on rat anterior pituitary hormone release.

We investigated the possibility that various protein kinase C (PKC) activators and inhibitors may differentially affect luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue, incubated in vitro. Activators of PKC induced LH release with the following order of potency: mezerein > phorbol 12,13-dibutyrate (PDBu). Mezerein and PDBu were equipotent on GH release. A range of PKC inhibitors (including compounds highly selective for PKC) potently and completely inhibited PKC activator-induced LH and GH release. Chelerythrine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) were less potent inhibitors of PDBu-induced GH release than of LH release. A component of PDBu- and mezerein-induced LH release was inhibited by H7 with high potency, but a second H7-insensitive component was detected. Mezerein- and PDBu-induced GH release consisted of an H7-resistant component only. When the regulatory domain of PKCs from different sources was investigated by displacement of [3H]PDBu binding, the affinity for mezerein was 3-5-fold greater than that for PDBu at PKCs from cerebral cortex, lung and alpha and beta isoforms extensively purified from brain. Anterior pituitary PKCs were unusual in showing closely matched affinity for mezerein and PDBu, reminiscent of their equivalent potency on GH release. In order to investigate the potency of the catalytic domain inhibitor H7 on PKCs from different sources, enzyme activity assays were carried out on partially purified cytosolic PKCs from midbrain and anterior pituitary and on extensively purified PKC alpha and PKC beta. The Ca(2+)-independent component of PDBu-induced (phosphatidylserine-dependent) activity from anterior pituitary alone showed unusually low potency of inhibition by H7 but was potently inhibited by staurosporine and Ro 31-8220. In contrast, the Ca(2+)-dependent PKC activity in anterior pituitary was inhibited by H7, staurosporine and Ro-31-8220 with high potency as in all other preparations. These results are consistent with the presence and active role in secretion of pharmacologically distinct forms of PKC (or PKC-like kinases) in rat anterior pituitary cells.

Evidence that protein kinase C alpha has reduced affinity towards 1,2-dioctanoyl-sn-glycerol: the effects of lipid activators on phorbol ester binding and kinase activity.

The effect of 1,2-diacylglycerols on specific binding of [3H]phorbol 12,13-dibutyrate to cytosolic protein kinase C (PKC) was investigated in tissues reported to contain different proportions of PKC isoforms. In lung, frontal cerebral cortex and cerebellum cytosols (enriched in PKC alpha, beta and gamma, respectively) displacement of specific binding by phorbol 12,13-dibutyrate or diacylglycerols containing unsaturated acyl chains was of similar potency for each tissue. A range of 1,2-diacylglycerols containing saturated acyl chains exhibited varying affinities for [3H]phorbol 12,13-dibutyrate binding sites in each tissue; defining an optimal acyl chain length of around 14 carbons in each case. However, the affinities of saturated diglycerides were consistently lower in lung cytosol than in frontal cerebral cortex and cerebellum cytosols, with the greatest differences occurring at lower acyl chain lengths, especially with 1,2-dioctanoyl-sn-glycerol. Furthermore, a mixed micelle assay of PKC activity showed that 1,2-dioctanoyl-sn-glycerol displayed reduced potency at PKC alpha partially-purified from COS 7 cell cytosol compared to the mixture of PKC isoforms present in rat midbrain cytosol. Both low potency of 1,2-dioctanoyl-sn-glycerol as a displacer of [3H]phorbol 12,13 dibutyrate binding and the ability of arachidonic acid to act as an allosteric enhancer of binding, correlated with the proportional PKC alpha content of a range of tissues reported in the literature. In PKC enzyme activity assays, 1,2-dioctanoyl-sn-glycerol, but not phorbol 12,13-dibutyrate, was correspondingly a much poorer activator of PKC alpha from COS 7 cells than of the broad consensus of isoforms in rat midbrain. When alpha and beta isoforms were extensively-purified on DEAE-cellulose then hydroxyapatite, both the low affinity of 1,2-dioctanoyl-sn-glycerol for [3H]phorbol 12,13-dibutyrate binding sites and their allosteric regulation by arachidonic acid were confirmed to be characteristic of the alpha rather than the beta isoforms.

Influence of magnetic resonance imaging on diagnosis and therapeutic intention.

RATIONALE AND OBJECTIVES: We assessed the contribution of magnetic resonance (MR) imaging to diagnostic and therapeutic decision making. METHODS: In a before-after observational study, we collected information from clinicians before and after patients were given MR examinations. We studied 406 cases selected from consecutive referrals to a single MR imaging facility in Manitoba between November 1, 1991, and October 30, 1992, for diagnosis of suspected brain, spinal column, or large-joint disorder. We examined changes in diagnoses, changes in clinician diagnostic confidence, and changes in therapeutic intentions after MR examinations. RESULTS: Overall, MR imaging findings contributed to a change in referring physicians' diagnoses or diagnostic confidence in 76% of the cases. Referring physicians reported a change in provisional diagnosis in 42% of the cases. In 67% of these cases, the referring physician's provisional diagnosis was ruled out by normal examination findings; in the remaining 33% of the cases, an alternate diagnosis was offered by the consulting radiologist. In the 58% of the cases in which the provisional diagnosis was not altered by MR imaging findings, clinical confidence in the provisional diagnosis increased in 46% of the cases and decreased in 12% of the cases. Management plans were reported to be altered in 54% of the cases; in 24% of the cases, therapeutic intentions changed from lower to higher levels of intervention. CONCLUSION: Although MR imaging had a substantial influence on clinicians' decisions concerning diagnoses, the influence of MR imaging findings on therapeutic decision making, and therefore on patients' health status, was more moderate.