Bt cAMP Research Articles

Cross-talk between protein kinase C and protein kinase a down-regulates the respiratory burst in polymorphonuclear leukocytes

In this paper it has been shown that increase in intracellular cAMP by epinephrine or its analogue dibutyryl cAMP (Bt 2cAMP) abolishes in a dose-dependent manner the protein kinase C (PKC)-mediated respiratory burst in polymorphonuclear leukocytes. The mechanism of inhibition has been shown to involve induction of cytosolic phosphoprotein phosphatase activity specific to cells receiving dual signals (PKC, PKA), as minimum respiratory burst was associated with cells with maximum phosphatase activity. Inclusion of specific PKA inhibitor completely restricted the development of dual signal-induced phosphatase activity in vitro, demonstrating the requirement of multisite phosphorylation of the phosphatase for the development of its activity. Purified phosphatase had a molecular weight of 78,000 and could exert its inhibitory effect on PKC-triggered respiratory burst in permeabilized cells, clearly showing that down-regulation of oxidase activity involved dephosphorylation by the phosphatase. Interaction of the purified phosphatase with eightfold purified NADPH oxidase as revealed by fluorescence studies further confirmed the role of the phosphatase in the respiratory burst event. Taken together, we have been able to establish that cross-talk between protein kinase C and protein kinase A is essential to ‘turn off’ generation of reactive oxygen species.

Supression by [D-pen 2, D-pen 5] enkephalin on cyclic amp dependent protein kinase-induced, but not protein kinase C-induced increment of intracellular free calcium in Ng 108-15 cells

In neuroblastoma X glioma NG108-15 cell lines, KCl 50 mM produced a significant increase in [Ca 2+]i which was blocked completely by voltage-dependent Ca 2+ channel antagonist verapamil. High K +-induced increase in [Ca 2+i can be suppressed by selective delta opioid agonist [D-Pen 2, D-Pen 5] enkephalin (DPDPE) (an effect completely reversed by opioid antagonist naloxone), but not by the mu agonist ohmefentanyl (OMF) or kappa agonist 66A-078. Aside from high K + stimulation, a number of chemicals can produce an increase in [Ca 2+]i, i.e., selective adenylate cyclase activator forskolin, the membrane permeable cyclic AMP (cAMP) analogue dibutyryl-cAMP (Bt 2cAMP) and the activator of protein kinase C (PKC) 12-O-tetradecanoylphorbol 13-acetate (TPA). All these effects can be readily blocked by verapamil. DPDPE blocks the increase in [Ca 2+]i induced by forskolin and Bt 2cAMP, but not that by TPA. The results suggest that cAMP dependent protein kinase-, but not PKC-induced Ca 2+ influx mechanism seems to be involved in the delta receptor mediated opioid effect.

Cyclic AMP mediates mu and delta, but not kappa opioid analgesia in the spinal cord of the rat

Intrathecal (i.t.) injection of the membrane-permeable cyclic AMP (cAMP) analogue dibutyryl-cyclic AMP(Bt 2cAMP) in 3 successive doses of 12.5, 12.5 and 25 ug in rats was very effective in reversing the antinociceptive effects produced by mu agonist morphine or delta agonist DPDPE, but not that by kappa agonist dynorphin A-(1–13). cAMP content of the spinal cord was significantly decreased by morphine, but not by dynorphin A-(1–13). The results imply that a decrease in spinal cAMP content may be important for the antinociceptive effect elicited by mu and delta, but not kappa opioid receptor agonists.

Phosphorylation of smg p21B in rat peritoneal mast cells in association with histamine release inhibition by dibutyryl-cAMP

IP 3 formation and histamine release from rat peritoneal mast cells stimulated by compound 48/80 were dose-dependently inhibited by Bt 2cAMP. These inhibitions were restored to the control level in the presence of H-8, a protein kinase A inhibitor. The 22 kDa protein in mast cells was revealed as a markedly phosphorylated protein by incubating with Bt 2cAMP, and this phosphorylation was also diminished by H-8. The 22 kDa phosphoprotein of rat mast cells comigrated with phosphorylated smg p21B, purified from human platelets and phosphorylated by protein kinase A in cell-free system, in both one- and two-dimensional PAGE analysis. Moreover, 22 kDa protein in mast cells was identified as smg p21B by immunoblot analysis using an antibody against smg p21B. From the present study, it became clear that smg p21B is phosphorylated by means or protein kinase A system in rat peritoneal mast cells, and it was assumed that phosphorylated smg p21B plays some important role in the suppression of IP 3 formation and histamine release from rat peritoneal mast cells.

Enhanced effectiveness of pulsatile 3',5'-cyclic adenosine monophosphate in stimulating prolactin and alpha-subunit gene expression.

cAMP is involved in the regulation of secretory activity in lactotrope, thyrotrope, and gonadotrope cells. The present study examined whether pulsatile or intermittent changes in cAMP are more effective than a continuous stimulation in increasing pituitary hormone gene expression. Pituitaries from adult female rats were dissociated, plated for 48 h (7-8 x 10(6) cells per well) to allow attachment to Matrigel-coated plastic coverslips, then inserted into perifusion chambers (five to eight chambers per group). After 24 h of treatment, the cells were recovered, RNA extracted, and messenger RNAs (mRNAs) determined by dot blot hybridization. Perfused cells were exposed to either hourly pulses of monobutyryl cAMP (Bt cAMP, 0.01, 0.1, or 1 mM; 1 mM butyrate pulses to controls), or continuously to forskolin (10 microM). Bt cAMP pulses increased both PRL and alpha-subunit mRNAs, maximal after the 0.1 mM dose for PRL (51% increase vs. butyrate controls) and after the 1 mM dose for alpha (60% increase). However, forskolin was ineffective in increasing PRL or alpha mRNA concentrations. TSH, LH, and FSH beta-subunit mRNAs were not altered by Bt cAMP pulses or forskolin. To confirm the different effects of pulsatile vs. continuous cAMP on PRL and alpha-subunit mRNAs, the response to pulsatile 8-bromo cAMP (1 mM) or Bt cAMP (0.5 mM) was compared to continuous Bt cAMP (0.5 mM). PRL and alpha-subunit mRNAs were increased by both cAMP analogs given in a pulsatile manner but not by continuous Bt cAMP. PRL and LH secretory responses (determined in perifusate samples after 2 h and 22 h of treatment) revealed that both PRL and LH release was increased by cAMP stimulation, given either in a pulsatile or continuous manner. These results show that PRL and alpha-subunit gene expression were sensitive to changes in cAMP stimulation, whereas that of TSH, LH, and FSH beta were unaltered. Only intermittent cAMP stimuli were effective in increasing PRL and alpha mRNAs. These data suggest that pulsatile fluctuations in intracellular cAMP may be essential for maximal expression of the PRL and alpha genes. Thus, intermittent changes in intracellular second messengers may be a necessary part of the pathway involved in the transduction of signals from the plasma membrane to the nucleus.

Enhanced effectiveness of pulsatile 3',5'-cyclic adenosine monophosphate in stimulating prolactin and alpha-subunit gene expression

cAMP is involved in the regulation of secretory activity in lactotrope, thyrotrope, and gonadotrope cells. The present study examined whether pulsatile or intermittent changes in cAMP are more effective than a continuous stimulation in increasing pituitary hormone gene expression. Pituitaries from adult female rats were dissociated, plated for 48 h (7-8 x 10(6) cells per well) to allow attachment to Matrigel-coated plastic coverslips, then inserted into perifusion chambers (five to eight chambers per group). After 24 h of treatment, the cells were recovered, RNA extracted, and messenger RNAs (mRNAs) determined by dot blot hybridization. Perfused cells were exposed to either hourly pulses of monobutyryl cAMP (Bt cAMP, 0.01, 0.1, or 1 mM; 1 mM butyrate pulses to controls), or continuously to forskolin (10 microM). Bt cAMP pulses increased both PRL and alpha-subunit mRNAs, maximal after the 0.1 mM dose for PRL (51% increase vs. butyrate controls) and after the 1 mM dose for alpha (60% increase). However, forskolin was ineffective in increasing PRL or alpha mRNA concentrations. TSH, LH, and FSH beta-subunit mRNAs were not altered by Bt cAMP pulses or forskolin. To confirm the different effects of pulsatile vs. continuous cAMP on PRL and alpha-subunit mRNAs, the response to pulsatile 8-bromo cAMP (1 mM) or Bt cAMP (0.5 mM) was compared to continuous Bt cAMP (0.5 mM). PRL and alpha-subunit mRNAs were increased by both cAMP analogs given in a pulsatile manner but not by continuous Bt cAMP. PRL and LH secretory responses (determined in perifusate samples after 2 h and 22 h of treatment) revealed that both PRL and LH release was increased by cAMP stimulation, given either in a pulsatile or continuous manner. These results show that PRL and alpha-subunit gene expression were sensitive to changes in cAMP stimulation, whereas that of TSH, LH, and FSH beta were unaltered. Only intermittent cAMP stimuli were effective in increasing PRL and alpha mRNAs. These data suggest that pulsatile fluctuations in intracellular cAMP may be essential for maximal expression of the PRL and alpha genes. Thus, intermittent changes in intracellular second messengers may be a necessary part of the pathway involved in the transduction of signals from the plasma membrane to the nucleus.

Effects of glucagon and an analogue of cAMP on tyrosine aminotransferase in isolated chick embryo hepatocytes

Hepatocytes were isolated from 17-day-old chick embryos by the use of collagenase. Glucagon and dibutyryl cAMP (bt 2cAMP), individually or in combination, stimulated tyrosine aminotransferase (TAT) activity and synthesis in the isolated hepatocytes; maximal stimulation occurred 4 h after exposure of hepatocytes to the inducers. The stimulatory effects produced by glucagon and bt 2cAMP were abolished by treatment of hepatocytes with cordycepin or cycloheximide. The effects of the hormone and the cyclic nucleotide were not additive. The induction of the enzyme by glucagon suggests a physiological role for the hormone in the expression of TAT activity during chick embryonic development.

Regulation of 1,25-dihydroxyvitamin D 3 receptor gene expression by parathyroid hormone and cAMP-agonists

We studied the effect of parathyroid hormone (PTH) and activation of the cAMP signal pathway on vitamin D receptor (VDR) mRNA levels in the phenotypically osteoblast cell line UMR 106. PTH caused a time- and dose-dependent increase of the VDR mRNA content with a maximum after 2 h. After 24 h the VDR mRNA level in PTH-treated cells returned to control level. In contrast, the 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3)-induced increase in VDR mRNA did not decline after 24 h. Inhibition of transcription with actinomycin D (10 μg/ml) completely abolished the PTH-induced increase of VDR mRNA and inhibition of translation with cycloheximide (1 μg/ml) resulted in superinduction of VDR mRNA. The role of cAMP in the induction of VDR mRNA was studied with several agents acting via the cAMP pathway. Incubation for 2 and 4 h with forskolin, Bt 2cAMP, PTHrP or prostaglandin E 2 caused an increase in the level of VDR mRNA comparable to that caused by PTH. The calcium ionophore A23187 did not affect VDR mRNA level. The present study demonstrates that PTH and activation of the cAMP signal pathway cause up-regulation of VDR via induction of VDR gene expression. The effect of cAMP on the VDR gene is suggestive for a cAMP responsive element in the VDR gene.

Stimulation of dense tubular Ca 2+ uptake in human platelets by cAMP

Elevation of intracellular cAMP is shown to increase the rate ( V) and maximal extent of Ca 2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt 2-cAMP). The free concentration of Ca 2+ in the dense tubular lumen ([Ca 2+] dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca 2+ concentration ([Ca 2+] cyt) was monitored in parallel experiments with quin2. Both [Ca 2+] cyt and [Ca 2+] dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 μM external Ca 2+, [Ca 2+] cyt is approx. 50 nM and [Ca 2+] dt is very low. When 2 mM external Ca 2+ is added, [Ca 2+] cyt rises to approx. 100 nM and the process of dense tubular Ca 2+ uptake can be resolved. Forskolin (10 μM) and Bt 2-cAMP increase the rate of dense tubular Ca 2+ uptake ( V) to 2.1 ± 0.60 and 1.70 ± 40 times control values (respectively). The agents also increase the final [Ca 2+] dt tto 1.72 ± 0.21 and 1.72 ± 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the V m of the dense tubular Ca 2+ pump. With [Iono] = 500 nM, [Ca 2+] cyt was raised to ⩾ 1.0 μM and V m of the dense tubular pump was elicited. (At[Iono]= 1.0 μM, the final [Ca 2+] dt values were degraded 15% due to shunting of Ca 2+ uptake.) Analysis showed that forskolin (10 μM) and Bt 2-cAMP (1 mM) increase the V m by a factors of 1.56 ± 40 and 1.56 ± 40, respectively. Analysis showed that neither agent changed the K m of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca 2+ across the DT membrane (1.7 min −1).

Cyclic AMP stimulates Ca 2+-ATPase-mediated Ca 2+ extrusion from human platelets

The effect of cAMP on active Ca 2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca 2+ in the cytoplasmic compartment ([Ca 2+] cy1). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca 2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca 2+] cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147–163). Forskolin, at a maximally effective concentration of 10 μM, was shown to stimulate Ca 2+ extrusion by increasing by a factor of 1.6 ± 0.5 the V m of a saturable component, previously identified with a Ca 2+ Mg 2+-ATPase located in the plasma membrane. Neither the K m (80 nM) or Hill coefficient (1.7 ± 0.3) of the Ca 2+-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na +/Ca 2+ exchanger) over the [Ca 2+] cyt range examined (50–1500 nM), Dibutyryl-cAMP (Bt 2-cAMP, 1 mM) stimulated the Ca 2+ Mg 2+-ATPase component of Ca 2+ extrusion by a factor of 2.0 ± 0.6. Separate experiments showed that 10 μM forskolin reduces the resting [Ca 2+] cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in V m of the pump, countered by a 37–74% increase in the rate constant for passive Ca 2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca 2+] cyt and (b) increasing the rate of Ca 2+ extrusion after the initial influx or triggered release event.

Cyclic GMP increases the rate of the calcium extrusion pump in intact human platelets but has no direct effect on the dense tubular calcium accumulation system

Sodium nitroprusside (SNP) and other agents that elevate cGMP levels are known to inhibit the aggregation of human platelets. Published data suggest that cGMP attenuation of agonist-induced Ca 2+ transients is involved in this effect. The present study shows that elevation of cGMP increases the rate of the Ca 2+ extrusion pump located in the plasma membrane (PM) but does not have a direct effect on the Ca 2+ accumulating pump of the dense tubules (DT). The study verifies that SNP can specifically elevate the cGMP level in the platelet. The kinetics of the Ca 2+ extrusion system were studied in situ in platelets overloaded with the cytoplasmic Ca 2+ indicator quin2 according to a published protocol developed in this laboratory. Elevation of cGMP by means of (10 μM) SNP increased the V m of the Ca 2+-ATPase pump by 63%, without affecting its K m (66–80 nM) or Hill coefficient (1.6–1.8). Dibutyryl-cGMP (Bt 2-cGMP), preincubated for 45 min at 1 mM, increased the V m by a factor of 2.2 ± 0.4. The experiments did not give any indication that SNP or Bt 2-cGMP change the rate of the Na +/Ca 2+ exchanger which makes a minor contribution to Ca 2+ extrusion in the studied [Ca 2+] cyt range. The rate constant for passive leakage of Ca 2+ across the PM was increased by 32 ± 4% by SNP and 90 ± 34% by Bt 2-cGMP. The net result is that the free Ca 2+ in the cytoplasm ([Ca 2+] cyt) at ‘rest’ is lowered from control values of 112 nM to 89 nM or 80 nM, respectively. The kinetics of Ca 2+ uptake by the dense tubules were determined in situ using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. Analysis showed that SNP and Bt 2-cGMP had no effect on the V m or K m of the dense tubular pump, and did not affect the rate constant for passive leakage. The agents did decrease resting [Ca 2+] dt by 25% or 30%, respectively, but this result can be explained purely in terms of the reduced [Ca 2+] cyt. The effects of cGMP (vs. cAMP) on the PM and DT pumps are closely correlated with reported effects of cGMP/cAMP induced phosphorylation of a protein of the molecular weight of the PM pump and a 22 kDa activator of the DT pump. Cyclic AMP increases the rate of both the PM and the DT pumps, whereas cGMP increases the rate of the PM pump only. In combination, treatment with maximally-effective doses of Bt 2-cGMP and Bt 2-cAMP had no greater effect on the PM pump than did either agent alone.

Mitogenic growth factors regulate differentially early gene mRNA expression: A study on two clones of 3T3 fibroblasts

The relationship between cell proliferation and mRNA levels of the immediate early genes c- fos, c- jun, and jun B has been investigated in two clones of 3T3 flbroblasts (D1-3T3 and N2-3T3) upon treatment with basic fibroblast growth factor (bFGF), thrombin, phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (Bt 2cAMP). The 3T3-derived clone D1-3T3 almost stops dividing upon serum deprivation, while the N2-3T3 clone does not. The proliferation of the two clones was stimulated by thrombin and PMA and inhibited by Bt 2cAMP. Basic FGF stimulated the growth of D1-3T3 but partly inhibited that of N2-3T3 cells. In spite of variable mitogenic response, immediate early genes, c- fos, c- jun, jun B, and c- myc, were induced by the growth factors and by PMA in both cell clones. In our experimental conditions the early gene mRNAs were expressed independently; i.e., the expression of one protooncogene had no bearing on the expression of the other. The cell growth was not directly related to the expression of a particular protooncogene mRNA. Data are presented showing that early gene mRNA expression induced by bFGF or thrombin was not mediated by protein kinase C activation while thrombin-induced mitosis was. Basic FGF induced a part of c- jun mRNA expression, but not mitosis, through a pertussis toxin-sensitive mechanism.

Identification, characterization, and hormonal regulation of 3', 5'-cyclic adenosine monophosphate-dependent protein kinases in rat Sertoli cells.

Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (approximately 9.2 +/- 0.8 pmol/mg protein) and C (approximately 7.3 +/- 0.7 pmol/mg protein). Stimulation with (Bt)2cAMP (0.1 mM) for 24 and 48 h revealed a time-dependent increase in [3H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N3-[32P]cAMP photoaffinity labeling, autophosphorylation by gamma-[32P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI alpha, 25% RII alpha, and very low levels of RII beta. Stimulation of Sertoli cells with (Bt)2cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI alpha (6.6-14 pmol/mg) and a 10- to 20-fold increase in RII beta (less than 0.1-1.1 pmol/mg), with little or no change in RII alpha (1.9-2.3 pmol/mg). Treatment with cAMP was associated with a slight increase in RI/RII ratio (3.3-4.1). mRNA levels for RII beta increased 30- to 50-fold after (Bt)2cAMP stimulation, whereas only minor changes in mRNA levels for RI alpha, RII alpha, and C alpha were observed (1.5- to 2.0-fold). mRNA levels for RI beta, C beta, and C gamma were not detected in either unstimulated or in cAMP-stimulated Sertoli cells. It is concluded that chronic treatment with cAMP changes the relative proportion of R subunits of PKA in a manner reflecting the changing levels in respective mRNAs. Furthermore, such treatment is associated with the appearance of a new PKA R subunit (RII beta), which is absent in untreated Sertoli cells.

Acute action of luteinizing hormone on mouse Leydig cells: Accumulation of mitochondrial phosphoproteins and stimulation of testosterone synthesis

Upon stimulation of Leydig cells with luteinizing hormone (LH) or dibutyryl-3',5'-cyclic AMP (Bt 2cAMP) at 37°C, two mitochondrial phosphoproteins accumulate with the same stimulant dose response as the increased rate of testosterone synthesis. The proteins pp32 and pp30 have apparent isoelectric points of 6.6 and 6.5 and molecular weights of approximately 32 and 30 kDa respectively, as determined by two-dimensional polyacrylamide gel electrophoresis. These two phosphoproteins are not detected in mouse adipose or liver cells nor in the total testicular cell population, of which Leydig cells constitute a small percentage. However, both proteins are also observed in mouse adrenal cells stimulated by ACTH or Bt 2cAMP. The appearance of pp32 and pp30 is prevented by inhibitors of cytosolic protein translation, indicating that only newly synthesized protein is available as a substrate for phosphorylation. Proteolytic peptide mapping indicates that both of these mouse Leydig and adrenal proteins have structural similarity to pp30 (formerly denoted as i b), the 30 kDa mitochondrial phosphoprotein that we have observed previously in peptide hormone or Bt 2cAMP-stimulated rat adrenal cortex (Pon, L.A, Hartigan, J.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 13309–13316; Alberta, J.A., Epstein, L.F., Pon, L.A. and Orme-Johnson, N.R. (1989) J. Biol. Chem. 264, 2368–2372) and rat corpus luteum cells (Pon, L.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6694–6599). Since pp32 is a larger mitochondrial protein of similar primary structure to pp30, it is a potential precursor of this protein. Finally, the detection of the mitochondrial phosphoprotein pp30 in a third steroidogenic tissue type and a third species provides further correlative evidence that the production of pp30 may be an integral part of the subcellular mechanism by which peptide hormones stimulate steroid hormone biosynthesis.

Cloning and expression of a cDNA encoding mouse indoleamine 2,3-dioxygenase

The depletion of an essential animo acid (aa), tryptophan, caused by interferon-γ (IFN-γ)-mediated induction of indoleamine 2,3-dioxygenase (IDO) in mouse allografted tumor cells, has been suggested as a reason for the allograft rejection, To elucidate the mechanism of this IDO induction, attempts were made to isolate cDNA clones encoding mouse IDO. In seven of 25 mouse cell lines, IDO was induced by IFN-γ, and the highest IDO induction was observed in the case of rectal cancer (CMT-93) cells, which were further stimulated two- to threefold by the simultaneous addition of dibutyryl cyclic AMP (Bt 2cAMP). A cDNA library was prepared from poly(A) + RNA isolated from CMT-93 cells treated with IFN-γ/Bt 2cAMP. The cDNA clones were isolated using the cDNA encoding human IDO as a probe. The mouse IDO cDNA encodes a 407-aa protein with an M r of 45639. The deduced aa sequence agreed with partial aa sequences derived from endopeptidase digestion of purified mouse IDO and revealed 61% homology with that of human IDO. Transient expression of the mouse IDO cDNA in COS-7 cells yielded a high level of IDO activity in the cells. Northern hybridization analysis of RNA in CMT-93 cells indicated that IFN-γ induced the IDO mRNA, and that the level of RNA was increased by simultaneous addition of Bt 2cAMP, while Bt 2cAMP itself had no effect on mRNA induction.

Insulin-like growth factor-1 and dibutyryl CAMP induce differentiation and decrease opioid receptor binding activity in N4TG1 neuroblastoma cells

The effects of fibroblast growth factor (FGF), insulin, insulin-like growth factor-1 (IGF-1), and dibutyryl cAMP (Bt 2cAMP) on cell proliferation and differentiation, opioid receptor binding activity, and expression of fos, myc, and ras proto-oncogenes were studied in N4TG1 neuroblastoma cells. IGF-1 and insulin increased thymidine incorporation and cell number, but Bt 2cAMP inhibited incorporation and FGF did not change thymidine incorporation after 24 h of treatment. Bt 2cAMP profoundly extended and IGF-1 slightly extended neurite-like processes, a morphologic marker of neuronal differentiation. A combination of IGF-1 and Bt 2cAMP potentiated neurite process extension. Thus, IGF-1 could induce both proliferation and differentiation of N4TG1 neuroblastoma cells. FGF did not cause neurite extension. N4TG1 neuroblastoma cells have a high density of α-opioid receptors. IGF-1 and Bt 2cAMP, but not FGF and other growth factors, decreased δ-opioid receptor binding activity with a reduced B max value and a normal apparent K d value. Using Northern blot and hybridization, both acidic and basic FGF elicited stimulatory effects on fos expression. Cycloheximide (CHX), a protein synthesis inhibitor, potentiated and prolonged the effect of FGF on fos expression. However, FGF had no effect on myc or ras expression. Neither IGF-1 nor Bt 2cAMP significantly changed fos, myc, or ras expression. The results suggest that fos, myc, or ras proto-oncogene expression is independent from or insufficient for IGF-1-induced and Bt 2cAMP-induced differentiation and regulation of opioid receptor synthesis in N4TG 1 neuroblastoma cells.

Effects of glucose, insulin, and cAMP on transcription of the serine dehydratase gene in rat liver

Starvation and diabetes both caused a dramatic induction of hepatic l-serine dehydratase (SDH) (EC 4.2.1.13) in rats. Increases in the activity of the enzyme which had been demonstrated in several previous studies were found to be associated with increases in the amount of SDH protein and its mRNA in our studies reported herein. Nuclear run-on experiments with isolated liver nuclei demonstrated that the increases in SDH activity were mainly the result of increases in the rate of SDH gene transcription. Refeeding of glucose to starved rats or the administration of insulin to diabetic rats caused a marked reduction in the amount of SDH mRNA. The rates of transcription as measured in isolated nuclei were reduced to uninduced levels within 30 min of either treatment. Following the administration of Bt 2-cAMP, the transcription rates of the SDH gene returned to the original induced rates within 40 min both in glucose-refed rats and in diabetic rats administered insulin. The results of these experiments indicate that the induction of SDH in rat liver in vivo is controlled predominantly at the level of gene transcription by the reciprocal action of cAMP and insulin.

Regulation of Fcγ receptor subtype expression on a human eosinophilic leukemia cell line EoL-3: Participation of cAMP and protein kinase C in the effects of interferon-γ and phorbol ester

We investigated the effects of interferon-γ (IFN-γ), phorbol myristate acetate (PMA), and dibutyryl cAMP (Bt 2cAMP) on FcγR subtype expression on a human eosinophilic leukemia cell line, EoL-3. Unstimulated EoL-3 cells expressed FcγRII as determined by monoclonal antibody (mAb) IV-3, whereas there was little or no FcγRI and FcγRIII expression as determined by mAbs 32.2 and 3G8, respectively. IFN-γ induced FcγRI expression, and Bt 2 cAMP, which did not induce FcγRI expression by itself, showed an additive effect on IFN-γ-induced FcγRI expression. FcγRII expression was augmented by IFN-γ, PMA, and Bt 2 cAMP. Bt 2 cAMP also showed an additive effect on IFN-γ-augmented FcγRII expression. FcγRIII expression could be induced only by IFN-γ plus Bt 2 cAMP. H-7, a protein kinase C (PK-C) inhibitor, suppressed the enhancement of FcγR subtype expression induced by these reagents. These results show that FCγR subtype expression on EoL-3 cells is regulated differently in each subtype and that cAMP and PK-C play important roles in the regulation.

Nucleotide-, Chemotactic Peptide- and Phorbol Ester-Induced Exocytosis in HL-60 Leukemic Cells

Undifferentiated and differentiated HL-60 leukemic cells possess nucleotide receptors which functionally couple to phospholipase C via pertussis toxin-sensitive guanine nucleotidebinding proteins (G-proteins).We investigated the role of extracellular nucleotides in the regulation of ϟ-glucuronidase release in HL-60 cells.In dibutyryl cyclic AMP (Bt 2cAMP)differentiated HL-60 cells, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), the phosphorothioate analogue of ATP, adenosine 5’-O-[3thio]triphosphate (ATP[γS]), and UTP increased cytosolic Ca 2+ from 100 nM up to 1.2 μM with EC 50 values of 4 nM, 1 μM and 100 nM, respectively.In these cells, ATP[γS] induced exocytosis with an EC 50 of 4 μM and an effectiveness amounting to 50–70 % of that of fMetLeu-Phe.ATP, ITP, UTP, CTP, and uridine 5’-O-[2-thio]diphosphate activated exocytosis as well.Phorbol myristate acetate (PMA) induced exocytosis with an EC 50 of 115 ng/ml and an effectiveness similar to that of ATP[γS].Cytochalasin B (CB) differently potentiated exocytosis induced by ATP[γS], fMet-Leu-Phe and PMA.Treatment of Bt 2cAMP-differentiated HL-60 cells with pertussis toxin (500 ng/ml) for 24 h resulted in ADP-ribosylation of more than 97.5 % of the G-proteins.Under these conditions, pertussis toxin almost completely inhibited the increase in cytosolic Ca 2+ and (β-glucuronidase release induced by fMet-Leu-Phe but only partially inhibited the effects of ATP[γS] and UTP.fMet-Leu-Phe at a nonstimulatory concentration (1 nM) potentiated ATP[γS]-induced β-glucuronidase release in the presence but not in the absence of CB.In contrast, ATP[γS] and fMet-Leu-Phe synergistically activated superoxide formation in the absence of CB.PMA potentiated superoxide formation induced by ATP[γS] or fMet-Leu-Phe and did not affect exocytosis induced by ATP[γS] or fMet-Leu-Phe.In undifferentiated HL-60 cells, fMet-Leu-Phe, ATP[γS], UTP and PMA did not induce β-glucuronidase release.fMet-Leu-Phe did not increase cytosolic Ca 2+ in undifferentiated HL-60 cells, whereas ATP[γS] and UTP were similarly potent and effective as in Bt 2cAMP-differentiated cells.In differentiated HL-60 cells, fMet-Leu-Phe induced aggregation, and ATP[γS] induced a transient shape change.Our results show (I) that exocytosis in HL-60 cells does not obligatorily depend on CB.(II) Purine and pyrimidine nucleotides activate exocytosis via pertussis toxin-sensitive and -insensitive signal transduction pathways.

Alterations in insulin receptor kinase activity during differentiation of HL-60 cells

Differentiation of the human promyelocytic leukemia cell line HL-60 into monocytes or macrophages is associated with increased expression of cell surface insulin receptors, while differentiation of these cells into granulocytes is associated with receptor loss. Here we demonstrate that differentiation of HL-60 cells into monocytes or granulocytes induced by 1;25(OH) 2vitD 3 or Bt 2cAMP, respectively, has no major effect on the specific activity of the insulin receptor kinase (IRK). By contrast, when HL-60 cells are incubated with a combination of 1;25(Oh) 2vitD 3 and Bt 2cAMP, their differentiation into adherent macrophages-like cells is accompanied by a 505 reduction in the specific activity of IRK. These findings suggest that acquisition or loss of insulin receptors during differentiation of HL-60 involves selective alterations in the functional aspects of these receptors. Our results also implicate the generation of specific regulatory signals that inhibit IRK activity when HL-60 cells are stimulated with a combination of 1;25(OH) 2vitD 3 and Bt 2cAMP.