Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells.

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The 'receptorless' mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.     

The effect of bromodeoxyuridine and dibutyryl cyclic AMP on the induction of placental alkaline phosphate in human choriocarcinoma cells.

The effect of 5-bromo-2'-deoxyuridine (BrdUrd) and dibutyryl cyclic AMP (Bt2cAMP) on the expression of the placental isoenzyme of human alkaline phosphatase was examined in BeWo choriocarcinoma cells. By using a combination of specific immunoprecipitation and polyacrylamide-gel electrophoresis of cells labelled either metabolically with [35S]methionine or cell-surface-labelled with 125I, both BrdUrd (5 micrograms/ml) and 1 mM-Bt2cAMP were shown to result in the enhanced accumulation of a specific protein. This protein has immunochemical identity and co-electrophoreses with placental alkaline phosphatase in two-dimensional gels. These results clearly demonstrate that the induction of placental alkaline phosphatase activity in choriocarcinoma cells treated with these agents is a consequence of the accumulation of specific enzyme protein rather than of altered catalytic activity.     

Effect of hormones on cyclic AMP levels in cultured human cells.

Cultured cells derived from human adipose tissue grew more slowly and had significantly higher basal levels of cyclic AMP than cultured fibroblasts. Cyclic AMP levels in cultured adipose tissue cells were unaffected by epinephrine and were elevated 15-fold by prostaglandin E₁ while fibroblast cyclic AMP levels were elevated 27-fold by epinephrine and 95-fold by prostaglandin E₁. These results support the postulate that the cultured adipose tissue cell is a distinct cell type which may represent an adipocyte or preadipocyte in culture.     

Carbachol enhances forskolin-stimulated cyclic AMP accumulation via activation of calmodulin system in human neuroblastoma SH-SY5Y cells.

We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin-stimulated cAMP levels in a dose-dependent manner (EC50 = 3 microM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC50 = 3 microM)-, adenosine- and prostaglandin E1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line.     

Differential role for cyclic AMP response element binding protein-1 in multiple stages of B cell development, differentiation, and survival

CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser(119-ala) phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed approximately 70% increase in pre-B1 (CD43(+)B220(+)CD24(+(int))) and approximately 60% decreased pre-BII (CD43(+)B220(+)CD24(++(high))) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed approximately 4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2(-/-) mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43(+)B220(+)CD24(high) population that is normally absent in RAG-2(-/-) mice. In the periphery, the Tg mice exhibited decreased CD21(dim)CD23(high)IgM(+) follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.     

Immunohistochemical detection of c-fos proteins in cultured human glial cells — induction by cyclic AMP and phorbol ester

Summary The expression of c-fos protein in cultured human glial cells derived from the brain and spinal cord was investigated immunocytochemically. Primary cultures of fetal glial cells were maintained in culture for three weeks and deprived of animal sera for 22 h. The glial cell nature of the cells was ascertained by GFAP-immunoreactivity. Incubations with phorbol dibutyrate, 8-Br-cAMP and sodium nitroprusside representing signal transduction pathways of PKC, PKA and cyclic GMP kinase, respectively, were carried out for 60 and 120 min. The control serum-deprived cultures did not display c-fos protein immunoreactivity (c-fos-IR), whereas phobol dibutyrate incubation for 120 min induced strong c-fos-IR in the nuclei of both brain and spinal cord derived glial cells. Semiquantitative intensity measurements revealed a slight c-fos-IR induction after 8-Br-cAMP as well, but not after sodium nitroprusside. The observations suggest that c-fos protein is involved in PKC and PKA signal transduction in cultured human glial cells.     

Effects of dibutyryl cyclic AMP and prostaglandin E1 on cultured human glioma cells

Dibutyryl cyclic AMP (db-cAMP) and prostaglandin E₁ (PGE₁) induced morphological alterations in cultured human glioma cells (138 MG). Cells in serum-free medium, treated with db-cAMP (1 mM) or PGE₁ (10μg/ml), within 1–3 h showed multiple thin processes resembling those of normal glial cells. These processes increased in size during a 24 h incubation. In serum-containing medium the appearance of cells with multiple processes was delayed. The induced morphological alterations were reversible upon exchange with fresh serum-containing but not with serum-deprived medium. Actinomycin D (5 μg/ml) did not prevent the changes induced by PGE₁ or db-cAMP. Inhibition of protein synthesis with cycloheximide (10 μg/ml) did not arrest the initial (1–3 h) changes in morphology but blocked further growth of the processes on prolonged incubation. Vinblastine sulphate (0.1 μg/ml) completely inhibited the alterations induced by PGE₁ or db-cAMP.     

Identification of the cyclic AMP responsive element (CRE) that mediates transcriptional regulation of the pyruvate carboxylase gene in HepG2 cells

Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis. Here we investigated the effect of various hormones including cAMP, dexamethasone and insulin on the abundance of PC mRNA in the human hepatocyte cell line, HepG2. Treatment of HepG2 cells with 1 μM of glucagon increased the expression of PC mRNA threefold within 72 h. Treatment with 1 mM 8-Br-cAMP caused the abundance of PC mRNA to increase by 2-3-fold by 48 h, peak at fourfold at 72 h, and remain unchanged to 96 h. This is in contrast to phosphoenolpyruvate carboxykinase (PEPCK) for which expression was decreased after 72 h, suggesting a distinct difference in the control of these two enzymes in the long term. Dexamethasone or insulin alone did not affect the abundance of PC mRNA whereas treatment of HepG2 cells with the combination of 1 mM 8-Br-cAMP and 0.5 μM dexamethasone further increased the abundance of PC mRNA, suggesting the predominant role of 8-Br-cAMP over dexamethasone. Transient transfection of the luciferase reporter construct driven by a 1.95 kbp 5′-flanking sequence of the mouse PC gene and a plasmid encoding the human cAMP-responsive element binding protein increased luciferase reporter activity to 7-fold similar to that observed with a PEPCK promoter-luciferase reporter construct. Deletion of the 5′-flanking region of the PC gene to 781 bp resulted in the complete loss of CREB-mediated induction of reporter gene, suggesting the presence of the cAMP-responsive unit is located between 1.95 kbp and 781 bp upstream of the mouse PC gene. Electrophoretic mobility shifted and chromatin immunoprecipitation assays demonstrated that CREB bind to −1639/−1631 CRE of mouse PC gene in vitro and in vivo, respectively.     

ACTH receptor-mediated induction of leukocyte cyclic AMP

Studies were conducted to determine whether lymphocyte ACTH receptors behave as their structurally similar adrenal cell counterparts, in terms of adenylate cyclase activation and cyclic AMP (cAMP) production in the presence of ACTH. Treatment of mouse mononuclear splenocytes with ACTH (10(-5) to 10(-10) M) induced a consistent rise in cAMP. ACTH treatment of more homogenous cell populations, represented by Molt 4 T lymphoblast and S49A T cell lymphoma lines, yielded a dramatic, dose-related increase in cAMP levels for S49A cells but not for Molt 4 cells. Immunofluorescence assays, employing an antiserum to the adrenal cell ACTH receptor, indicated that 45% of splenocytes, 69% of S49A cells, and less than 1% of Molt 4 cells possess ACTH receptors. Radioligand binding studies confirmed that Molt 4 cells possess many fewer receptors than S49A cells, and probably fail to respond to ACTH because they lack the appropriate receptor. This is the first report of ACTH induction of leukocyte cAMP, evidence important to understanding the mechanisms by which this neuroendocrine hormone influences immune responses.