Overexpression of protein kinase C isoform epsilon but not delta in human interleukin-3-dependent cells suppresses apoptosis and induces bcl-2 expression.

Hematopoietic progenitor cells die by apoptosis after removal of the appropriate colony-stimulating factor (CSF). Recent pharmacologic data have implicated protein kinase C (PKC) in the suppression of apoptosis in interleukin-3 (IL-3) and granulocyte-macrophage (GM)-CSF-dependent human myeloid cells. Because IL-3 and GM-CSF induce increases in diacylglycerol without mobilizing intracellular Ca++, it seemed that one of the novel Ca++ independent isoforms of PKC was involved. We report here that overexpression of PKC epsilon in factor-dependent human TF-1 cells extends cell survival in the absence of cytokine. Overexpression of PKC delta does not have this effect. By 72 to 96 hours after cytokine withdrawal, the PKC epsilon transfectants remain distributed in all phases of the cell cycle, as shown by fluorescence-activated cell sorting (FACS) analysis, while little intact cellular DNA is detectable in vector or PKC delta transfectants. PKC epsilon induces bcl-2 protein expression fivefold to sixfold over the levels in empty vector transfectants, whereas the levels in PKC delta transfectants are similar to those in vector controls.

Human interleukin-3 receptor modulates bcl-2 mRNA and protein levels through protein kinase C in TF-1 cells.

Upon withdrawal of interleukin-3 (IL-3) from human factor-dependent erythroleukemic cell line TF-1, bcl-2 mRNA and protein levels decrease within 8 to 24 hours. Accompanying this decrease is the onset of apoptosis as determined by flow cytometric analysis of DNA degradation. By 8 to 18 hours of deprivation approximately 70% to 80% of the cells have entered apoptosis. Downregulation of protein kinase (PK) by a 24-hour incubation in 100 nmol/L 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the presence of IL-3 dramatically reduced bcl-2 mRNA levels, and induced apoptosis in the presence of IL-3. We have also found that even in the presence of IL-3, two inhibitors of PKC, light-activated calphostin and H-7, substantially reduced the levels of bcl-2 mRNA between 8 and 24 hours as measured by a semi-quantitative reverse transcriptase/polymerase chain reaction assay method; however, the cyclic nucleotide-dependent PK inhibitor HA 1004, that is a structural analog of H-7 but a poor inhibitor of PKC, did not reduce bcl-2 levels in the presence of IL-3. This decrease in bcl-2 mRNA was accompanied by a decline in bcl-2 protein levels by 8 to 24 hours after addition of light-activated calphostin. In addition to interfering with the maintenance of bcl-2 mRNA levels, inhibition of PKC with H-7 inhibited the induction of bcl-2 mRNA in factor-deprived TF-1 cells restimulated with IL-3. The cyclic nucleotide-dependent PK inhibitor HA 1004 did not inhibit IL-3-induced bcl-2 mRNA. Studies with actinomycin D showed that transcription plays a major role in maintaining bcl-2 levels in TF-1 cells, and it is therefore likely that IL-3 plays a role in maintaining bcl-2 transcription through activation of PKC in these cells.

Cloning and sequencing of a chick embryo cDNA encoding the 20-kDa hypusine-containing protein, eIF-5A.

Chick embryo contains 18- and 20-kDa isoforms of eukaryotic translation initiation factor 5A (eIF-5A). cDNA clones corresponding to the 20-kDa eIF-5A were isolated and sequenced. A full-length cDNA clone encodes a 153-amino-acid (aa) protein. The deduced aa sequence exactly matches with the partial aa sequence determined for this protein and shows high identity to that of human or rabbit eIF-5A. The results of Southern and Northern hybridization provide evidence for multiple transcripts for chick embryo eIF-5A or an eIF-5A-like protein that presumably derive from more than one gene.

Multiple fluorescent ligands for dopamine receptors. I. Pharmacological characterization and receptor selectivity.

We report the synthesis and pharmacological characterization of novel fluorescently labeled ligands with high affinity and specificity for D1 and D2 dopamine receptors. D1-selective antagonist probes have been synthesized using (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1H]-3- benzazepin-7-ol, the 4'-amino derivative of the high affinity D1-selective antagonist, SCH-23390, while D2-selective antagonist probes were synthesized using the high affinity, D2-selective agonist, N-(p-aminophenethyl)spiperone (NAPS). In addition, we have synthesized fluorescent probes using an amino-derivative of the high affinity, D2-selective agonist, 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT or N-0434). These ligands were coupled to the fluorescent moieties, fluorescein, rhodamine, coumarin, Texas red, Cascade blue, or Bodipy. This resulted in a wide variety of dopaminergic ligands which fluoresce at different wavelengths: Cascade blue and coumarin are blue fluorophores, fluorescein and Bodipy, are yellow-green, and Texas red and rhodamine are red. The interaction of these fluorescent ligands with dopamine and serotonin receptors was evaluated by examining their ability to compete for radioligand binding to D1 and D2 dopamine receptors and 5-HT1A, 5-HT1C and 5-HT2 serotonin receptors. We report here that these novel fluorescent ligands exhibit high affinity and, in general, selectivity for either D1 or D2 dopamine receptors. In addition, we demonstrate that the fluorescent derivatives of PPHT retain the full agonist efficacy exhibited by the parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)

A putative AP-2 binding site in the 5' flanking region of the mouse POMC gene.

Using extracts of AtT-20 cell nuclei, protein binding sites on the POMC gene 5'-flanking region were examined with an exonuclease protection approach. One such binding site, located from -119 to -106 bp upstream from the mouse POMC gene transcription initiation site, which exhibited a close homology to the activator protein-2 (AP-2) site [1]. A double-stranded oligonucleotide containing this site was subsequently used in gel shift assays to demonstrate AP-2 consensus sequence binding activity in extracts of AtT-20 cell nuclei. Gel shift competition experiments using both homologous and heterologous competitor DNA sequences revealed that the AP-2 like factor(s) exhibited specific binding to the mouse AP-2 consensus sequence. Furthermore, AP-2 factor binding was also modulated by a CTF/NF1-like factor. Pretreatment of AtT-20 cell nuclear extracts with alkaline phosphatase prior to inclusion in gel shift assays led to a reduction in the intensities of AP-2 factor-specific bands, indicating a potential involvement of protein phosphorylation in AP-2 factor binding in AtT-20 cells.

Interleukin 1 induces early protein phosphorylation and requires only a short exposure for late induced secretion of beta-endorphin in a mouse pituitary cell line.

Previous work has shown that prolonged pretreatment of a mouse anterior pituitary cell line, AtT-20 cells, with the cytokine interleukin 1 (IL-1) stimulates beta-endorphin release and potentiates the secretion induced by many secretagogues. Desensitization of protein kinase C (PKC) by pretreatment with phorbol ester [phorbol 12-tetradecanoate 13-acetate (TPA)] for 8 hr abolished the secretion induced by TPA as well as the enhancement of TPA-induced beta-endorphin release produced by IL-1. Desensitization of PKC only partly abolished the potentiating effects of IL-1 on corticotropin-releasing factor-induced beta-endorphin secretion. In contrast, IL-1-induced beta-endorphin release was independent of PKC. We observed that treatment of AtT-20 cells with IL-1 markedly phosphorylated 19-, 20-, and 60-kDa proteins within minutes, presumably by early activation of protein kinases. Prolonged treatment with TPA, which was shown to desensitize an 87-kDa protein (a substrate for PKC), had no effect on IL-1-induced phosphorylation of 20-, 60-, and 87-kDa proteins, indicating that the phosphorylation of these proteins does not involve PKC. IL-1 does not generate cAMP in AtT-20 cells, suggesting that a cAMP-dependent protein kinase is also not involved. Prolonged treatment with IL-1 abolishes the capacity of cytokine to induce the phosphorylation of 20- and 60-kDa proteins. The presence of IL-1 was required initially only for a short time to induce late secretion in AtT-20 cells. These observations indicate that once IL-1 generates an early signal, its presence is no longer necessary for the subsequent secretion of beta-endorphin.

Effect of 6-iodoamiloride in various models of experimental hypertension.

6-Iodoamiloride, an analogue of the sodium channel blocker amiloride, is a vasodilator-depressor, diuretic-natriuretic, and antikaliuretic agent. In these experiments we intravenously infused 6-iodamiloride (0.38 mg/100 g body weight) over a 10- to 11-minute period into rats with reduced renal mass-saline hypertension or one-kidney, one clip hypertension. The infusion produced a prompt but transient fall in blood pressure. These findings are in contrast to those in spontaneously hypertensive rats (SHR), in which the same infusion of 6-iodoamiloride produced a prompt, pronounced, and sustained fall in blood pressure. Studies from a number of laboratories suggest that vascular smooth muscle cells from the SHR have increased permeability to sodium whereas vascular smooth muscle cells from the other two models do not. Thus, 6-iodoamiloride may have potential both as a diagnostic probe and a therapeutic agent for hypertension characterized by increased vascular smooth muscle cell permeability to sodium.