Inhibitor Of Cyclic Nucleotide-dependent Protein Kinases Research Articles

Flagellar Adhesion between Mating Type Plus and Mating Type Minus Gametes Activates a Flagellar Protein-tyrosine Kinase during Fertilization in Chlamydomonas

When Chlamydomonas gametes of opposite mating type are mixed together, flagellar adhesion through sex-specific adhesion molecules triggers a transient elevation of intracellular cAMP, leading to gamete activation in preparation for cell-cell fusion and zygote formation. Here, we have identified a protein-tyrosine kinase (PTK) activity that is stimulated by flagellar adhesion. We determined that the protein-tyrosine kinase inhibitor genistein inhibited fertilization, and that fertilization was rescued by dibutyryl cAMP, indicating that the genistein-sensitive step was upstream of the increase in cAMP. Incubation with ATP of flagella isolated from non-adhering and adhering gametes followed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies showed that adhesion activated a flagellar PTK that phosphorylated a 105-kDa flagellar protein. Assays using an exogenous protein-tyrosine kinase substrate confirmed that the activated PTK could be detected only in flagella isolated from adhering gametes. Our results indicate that stimulation of the PTK is a very early event during fertilization. Activation of the PTK was blocked when gametes underwent flagellar adhesion in the presence of the protein kinase inhibitor staurosporine, but not in the presence of the cyclic nucleotide-dependent protein kinase inhibitor, H8, which (unlike staurosporine) does not block the increases in cAMP. In addition, incubation of gametes of a single mating type in dibutyryl cAMP failed to activate the PTK. Finally, flagella adhesion between plus and minus fla10-1 gametes, which have a temperature-sensitive lesion in the microtubule motor protein kinesin-II, failed to activate the PTK at elevated temperatures. Our results show that kinesin-II is essential for coupling flagellar adhesion to activation of a flagellar PTK and cAMP generation during fertilization in Chlamydomonas.

The urokinase-plasminogen activator system in ovine macrophages and neutrophils

The urokinase-plasminogen activator (u-PA) system in resting and activated ovine macrophages and neutrophils was examined. Macrophages and neutrophils were isolated from a total of 28 lactating sheep of the Chios breed. Low amounts of u-PA were found intracellularly or membrane-bound in resting macrophages and neutrophils. However, incubation of resting macrophages or neutrophils with purified u-PA (10 IU/ml) revealed extensive binding of u-PA to cell membranes. Excess amino terminal fragment of the u-PA molecule, a proteolytically inactive fragment of u-PA (amino acids 1–135) blocked binding of u-PA to macrophage or neutrophil cell membrane. These results indicate that the binding of u-PA is specific and that resting neutrophils and macrophages have unoccupied u-PA receptors on their cell membrane. Addition of phorbol myristate acetate (PMA) led to an increase ( P<0.01) in total cell-associated and membrane-bound u-PA activity and a decrease ( P<0.01) in free, unoccupied u-PA binding sites of macrophages or neutrophils. No significant effects on total cell-associated or membrane-bound u-PA were found when macrophages or neutrophils were treated with 4-phorbol-12,13-didecanoate, a phorbol ester that does not activate protein kinase C (PKC). Furthermore, the PMA-induced increase in total cell-associated u-PA activity of sheep macrophages or neutrophils was completely ablated by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 100 μM) but was unaffected by the cyclic nucleotide-dependent protein kinase inhibitor N-(2-quanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA-1004, 100 μM). Thus, PKC plays a role in the modulation of u-PA system by PMA in ovine macrophages and neutrophils.

Stimulation of cardiac glucose transport by inhibitors of oxidative phosphorylation

Previously we showed that hypoxia in heart stimulates glucose transport via translocation of glucose transporters from intracellular membranes to the plasma membrane. We later showed that rotenone, an inhibitor of oxidative phosphorylation, also decreased intracellular transporters. Here, using another membrane fractionation technique, we show that rotenone increases plasma membrane transporters, and that another respiratory chain inhibitor, azide, acts similarly. Thus, they likely activate the same signaling pathway as hypoxia. Genistein, a tyrosine kinase inhibitor, inhibited insulin- and azide-stimulated 3-O-methylglucose transport similarly in cardiac myocytes. It also increased glucose transporters in the plasma membranes of perfused hearts even though it inhibited glucose uptake, suggesting effects on membrane trafficking. Another tyrosine kinase inhibitor, lavendustin A, and the cyclic nucleotide-dependent protein kinase inhibitors H-8 and H-7 had little effect on basal or azide-stimulated transport. Polymyxin B was a weak inhibitor of basal, insulin-stimulated, and azide-stimulated transport. A nitric oxide donor and a nitric oxide synthase inhibitor had no effect on basal and azide-stimulated transport. The results indicate that tyrosine kinases; protein kinases A, G, and C; and nitric oxide are not involved in the hypoxic activation of cardiac glucose transport.

Protein kinase C signalling pathway is involved in the regulation of vascular endothelial growth factor expression in human bladder transitional carcinoma cells

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the growth and metastasis of various cancers and plays a prominent role in vesical angiogenesis regulation. In this study, we investigated the effect of the phorbol 12-myristate 13-acetate (PMA) on the expression of VEGF in human bladder transitional carcinoma cells (RT4). RT4 cells expressed three VEGF isoforms (VEGF 189, VEGF 165, VEGF 121). PMA increased VEGF mRNA expression time-dependently with a peak at 4 h. PMA increased the half-life of VEGF mRNA. The amount of VEGF protein in conditioned media was increased by PMA in a dose-dependent manner with a maximal effect at 10 −7 M. Staurosporine and calphostin C (PKC inhibitors) decreased PMA-induced VEGF mRNA expression as opposed to protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. Thus, in RT4 cells, VEGF expression is up-regulated by PMA via the PKC signalling pathway and according to a posttranscriptional mechanism.

Calmodulin and a cyclic nucleotide-dependent protein kinase facilitate the prolactin-induced increase in tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons.

Many aspects of tuberoinfundibular dopaminergic neuronal function are increased by elevated prolactin (PRL) levels, including the activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. This study evaluated the roles of calmodulin, cyclic nucleotide-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II in the PRL-induced increase in tyrosine hydroxylase activity. Ovariectomized rats were treated with haloperidol or ovine PRL (oPRL) for 20-30 h before the experiment, respectively. Treatment with haloperidol increased circulating PRL levels 8-fold and tyrosine hydroxylase activity in the stalk-median eminence 1.8-fold. Treatment with oPRL increased tyrosine hydroxylase activity 1.9-fold. W-7, a calmodulin antagonist, reversed both the haloperidol- and oPRL-induced increase in tyrosine hydroxylase activity to control levels. H-8, a cyclic nucleotide-dependent protein kinase inhibitor, also reversed the haloperidol induced increase in tyrosine hydroxylase activity. KN62, a selective calcium/calmodulin-dependent protein kinase II inhibitor, attenuated the haloperidol-induced increase in tyrosine hydroxylase activity, but KNO4, a structurally related control compound, had no effect. By contrast, the oPRL- and haloperidol-induced increases in tyrosine hydroxylase activity were not altered by KN93, a selective calcium/calmodulin-dependent protein kinase II inhibitor. These data indicate that calmodulin and a cyclic nucleotide-dependent protein kinase contribute to the PRL-induced increase in tyrosine hydroxylase activity, but the role of calcium/calmodulin-dependent protein kinase II is still unclear.

Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors.

Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway. Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs.

Flagellar Adhesion between mt+ and mt−Chlamydomonas Gametes Regulates Phosphorylation of the mt+-specific Homeodomain Protein GSP1

During fertilization in Chlamydomonas, flagellar adhesion between mt(+) and mt(-) gametes induces a cAMP-dependent signal transduction pathway that prepares the gametes for cell fusion and zygote formation. Previously, our laboratory identified a homeodomain protein (GSP1) whose expression was restricted to the cell bodies of mt(+) gametes and whose transcript level was up-regulated during flagellar adhesion. In this report, we describe a new form of GSP1 that appears early during gamete interactions. Immunoblot analysis showed that in addition to the 120-kDa form of GSP1 normally present in mt(+) gametes, a 122-kDa form was detected when the cells were mixed with mt(-) gametes. The more slowly migrating form of GSP1 was detectable within minutes after gametes were mixed together, and its appearance did not require new protein synthesis. Thus, the 122-kDa form represents a post-translational modification of the pre-existing 120-kDa form of GSP1. Moreover, conversion to the 122-kDa form did not require cell fusion. Although the 120-kDa form was expressed 10 h after vegetative cells were transferred to gametic induction medium, the 122-kDa form was detected only after mt(+) gametes were induced to undergo the sexual signaling that accompanies fertilization. Incubation of mt(+) gametes with dibutyryl cAMP led to the appearance of the 122-kDa form of GSP1, and the cyclic nucleotide-dependent protein kinase inhibitor H-8 inhibited the adhesion-induced conversion. Incubation of GSP1 immunoprecipitated from signaling mt(+) gametes with alkaline phosphatase showed that the conversion was due to phosphorylation. The results indicate that flagellar adhesion induces a rapid, cAMP-dependent phosphorylation of the homeodomain protein GSP1 early during fertilization in Chlamydomonas.

Role of Protein Kinase C Isoforms in Phorbol Ester-induced Vascular Endothelial Growth Factor Expression in Human Glioblastoma Cells

Aberrant expression of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), has been demonstrated to be associated with most human solid tumors. Both transcriptional and post-transcriptional mechanisms have been shown to modulate VEGF expression in a multitude of cell types. Here we report that when protein kinase C (PKC) pathways were activated in human glioblastoma U373 cells by phorbol 12-myristate 13-acetate (PMA), VEGF mRNA expression was up-regulated via a post-transcriptional mRNA stabilization mechanism. PMA treatment exhibited no increase in VEGF-specific transcriptional activation as determined by run-off transcription assays and VEGF promoter-luciferase reporter assays. However, PMA increased VEGF mRNA half-life from 0.8 to 3.6 h which was blocked by PKC inhibitors but not by protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. When U373 cells were transfected with antisense oligonucleotide sequences to the translation start sites of PKC-alpha, -beta, -gamma, -delta, -epsilon, or -zeta isoforms, both PKC-alpha and -zeta antisense oligonucleotides showed substantial inhibition of PMA-induced VEGF mRNA. In addition, overexpression of PKC-zeta resulted in a strong constitutive up-regulation of VEGF mRNA expression. This study demonstrates for the first time that specific PKC isoforms regulate VEGF mRNA expression through post-transcriptional mechanisms.

ATP-induced inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells: lack of involvement of known purinoceptor-coupled signaling pathways.

P2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca2+]i, activation of phospholipases C (PLC) and A2 (PLA2), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na+,K+,Cl- cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier ( approximately 50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca2+]i that was abolished by a chelator of Ca2+i, BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+, Cl- cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4, 5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na+,K+, Cl- cotransport activity. Inhibitors of PLA2 (AACOCF3), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na+,K+,Cl- cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na+ channels (amiloride), Cl- channels (NPPB) and Na+/H+ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na+,K+,Cl- cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na+,K+,Cl- cotransport and suggest the presence of a novel P2-receptor-coupled signaling mechanism.

Cyclic GMP inhibits cytoplasmic Ca 2+ oscillation by increasing Ca 2+-ATPase activity in rat megakaryocytes

The regulatory effects of cyclic GMP on purinoceptor-operated cytoplasmic Ca 2+ oscillation of rat megakaryocytes were investigated by using whole-cell patch-clamp technique. ATP-induced oscillatory K + currents though Ca 2+-activated K + channels ( I KCas) were depressed by pretreatment with the guanylate cyclase activator, sodium nitroprusside, and a stable membrane-permeable cGMP analogue, 8-bromo-cGMP. The inhibition by sodium nitroprusside was blocked by treatment with a cyclic nucleotide-dependent protein kinase inhibitor, N-[2-(methylamino)]-5-isoquinolinesulfonamide·HCl (H-8) (10 μM), but not by a selective cAMP-dependent-protein kinase inhibitor, Rp-cAMPS (100 μM). The oscillatory I KCa directly evoked by intracellular d-myo-inositol-trisphosphate (IP 3) perfusion was also inhibited by the application of sodium nitroprusside. The inhibitory effect of sodium nitroprusside disappeared when the ATP-induced oscillatory I KCa was changed to a monophasic sustained I KCa current by inhibition of Ca 2+-ATPase. These results suggested that cGMP depressed Ca 2+ mobilization by improving Ca 2+-ATPase activity by phosphorylation.

Effect of protein kinase modulators on the regulation of cathepsin B activity in THP-1 human monocytic leukemia cells.

Cathepsin B (CB), a lysosomal cysteine proteinase, is implicated in cancer metastasis and inflammatory tissue injury. We examined the effects of the protein kinase agonists and inhibitors on the regulation of CB activity in THP-1 human monocytic cells by two macrophage activators, lipopolysaccharide (LPS) and interferon- (IFN- ). CB elevation induced by LPS alone or LPS followed by IFN- was blocked by protein kinase C (PKC) inhibitors staurosporine, H-7, phloretin and bisindolylmaleimide, and by cyclic nucleotide-dependent protein kinase inhibitors HA 1004, H-8, H-89 and cAMP-dependent protein kinase (PKA) inhibitor. The CB activity by LPS and IFN- were augmented by diacylglycerol kinase inhibitor. PKC activator, phorbol 12-myristate 13-acetate (PMA) and PKA activator, dibutyryl cAMP could replace LPS in priming the cells for IFN- stimulation but 8-bromo-cGMP did not. These findings suggest that the activation of PKC and PKA appears to be involved at least in part in the induction of CB activity in THP-1 cells.

Direct blockade of both cloned rat rod photoreceptor cyclic nucleotide-gated non-selective cation (CNG) channel α-subunit and native CNG channels from Xenopus rod outer segments by H-8, a non-specific cyclic nucleotide-dependent protein kinase inhibitor

Using excised inside-out patch techniques, a non-specific cyclic nucleotide-dependent protein kinase inhibitor, H-8 ( N-2-(methylamino)ethyl-5-isoquinolinesulfonamide), has been shown to suppress directly the activities of both rat rod photoreceptor cyclic GMP-gated channel α-subunits expressed in Xenopus oocytes and native cGMP-gated channels from Xenopus rod outer segments. When co-applied with 100 μM cGMP on the cytoplasmic side of the patches, current suppression by H-8 increased with membrane depolarization. Blockade by H-8 was not relieved by supersaturating concentrations of cGMP (1 mM). In addition, blockade by H-8 showed significant inhibition of channel activity at negative holding potentials when acting from the extracellular side of the channel. The results were consistent with the conclusion that H-8 can act as an open channel blocker from either side of the channel. Even though H-8 is a much more effective kinase inhibitor, it is frequently used at concentrations that cause inhibition of cGMP-gated channels and this effect needs to be taken into account when evaluating results obtained with this compound.

Effects of spinal versus supraspinal administration of cyclic nucleotide-dependent protein kinase inhibitors on morphine tolerance in mice

The consequences of becoming tolerant to the analgesic effects of morphine include increased risk of unwanted side effects, such as respiratory depression, because the patient is required to take larger doses of the opioid to get the same relief from pain. Many studies suggest that phosphorylation plays a role in the neuroplasticity associated with opioid tolerance. This study examines the effect of inhibiting cyclic nucleotide-dependent protein kinase activity in the brain or spinal cord of morphine-tolerant mice. KT5720, a cyclic adenosine monophosphate (cAMP)-dependent protein kinase inhibitor, or KT5823, a cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, was centrally administered in morphine-tolerant and placebo-treated mice prior to a systemically administered challenge dose of morphine. KT5720 completely reversed morphine tolerance in the tail-flick assay when the pretreatment was administered intracerebroventricularly (i.c.v.); KT5823 had no effect on morphine via this route. When either of these drugs was administered intrathecally (i.t.), the activity of morphine was greatly diminished in the tolerant animals, with no effect on morphine antinociception in the placebo group. These data suggest that cAMP-dependent protein kinase activity may be upregulated in the brain with morphine tolerance, and that this upregulation is critical to the expression of tolerance to the antinociceptive effects of morphine. In the spinal cord, however, the activity of cyclic nucleotide-dependent protein kinases, and possibly their substrate proteins, may be affected by chronic morphine exposure such that inhibition of these kinases produces hyperalgesia.

Regulation of Ca(2+)-activated K+ channels in pulmonary vascular smooth muscle cells: role of nitric oxide.

Nitric oxide (NO.) is believed to mediate nitrovasodilators and acetylcholine-induced vasodilatation via increasing intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. The cellular mechanisms involved in No.-mediated pulmonary vasodilatation are complex and include membrane hyperpolarization. Using the patch-clamp technique in cell-attached and inside-out configurations, we examined the effect of NO. gas, 3-morpholinosydnomimine hydrochloride (SIN-1), and perfusate from ACh-stimulated human pulmonary arterial endothelial cells, or endothelium-derived relaxing factors (EDRF), on the Ca(2+)-dependent K+ (KCa) channels in isolated cultured human pulmonary arterial smooth muscle cells (HPSMC). NO., SIN-1, and EDRF caused similar increases in KCa channel activity. Inhibiting cGMP generation with methylene blue or inhibiting the effect(s) of cGMP with the cGMP antagonist 8-bromoguanosine 3',5'-cyclic monophosphorothioate Rp isomer Rp-cGMPS prevented the NO.- and SIN-1-mediated activation of KCa channels, respectively. Treating the human pulmonary arterial endothelial cells with methylene blue blocked the EDRF-mediated activation of KCa channels in HPSMC. The cGMP analogue 8-bromo-cGMP increased KCa channel activity in intact cells and in excised inside-out HPSMC membrane patches. In the presence of cGMP and ATP, the alpha-isozyme of the cGMP-dependent protein kinase (I alpha-cGMP-PK) significantly increased KCa channel activity, and the channel activation was further increased on addition of the protein phosphatase inhibitors okadaic acid and calyculin A. Furthermore, the cGMP-mediated KCa channel activation was reduced by the cyclic nucleotide-dependent protein kinase inhibitor N-[2-methylamino)ethyl]-5-isoquinlinesulfonamide (H-8). Thus, in HPSMC, the mechanism of NO.- and native EDRF-induced KCa channel activation appears to be mediated via cGMP-I alpha-cGMP-PK phosphorylation of KCa channels.

Caffeine-induced chloride current in dissociated rat hepatocytes.

Current responses to caffeine in single hepatocytes dissociated from adult rat liver were investigated with the conventional whole cell patch-recording configuration. Caffeine produced a sustained inward current (Icaf) with increasing conductance at a holding potential of -40 mV. The reversal potential of Icaf was close to the Cl- equilibrium potential. Icaf was not affected by the internal perfusion of 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or Cs+, whereas the Ca(2+)-activated K+ outward current elicited by A-23187 was inhibited by intracellular BAPTA or Cs+. A 1 mM 3-isobutyl-1-methylxanthine (IBMX) was about equipotent to 1 mM caffeine in inducing the current. Icaf was not modulated by the external application of N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), a cyclic nucleotide-dependent protein kinase inhibitor, or intracellular perfusion with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). It was concluded that caffeine induced an increase in membrane Cl- conductance without utilizing the rise of intracellular free Ca2+ or adenosine 3',5'-cyclic monophosphate (cAMP) and without mediating G protein, suggesting the possible existence of caffeine receptor-Cl- channel complexes on liver plasma membrane.

Gamma interferon induced increases in intracellular cathepsin B activity in PMA primed THP-1 cells are blocked by inhibitors of protein kinase C.

Macrophage proteinases including cathepsin B (CB) are implicated in the tissue injury of inflammatory lesions. We have previously shown that interferon-gamma (IFN-gamma) increases intracellular levels of the lysosomal proteinase, CB, in THP-1 cell primed with phorbol 12-myristate 13-acetate (PMA). We have now examined the role of protein kinase C (PKC) in this effect. Following activation with PMA, the intracellular CB activity was significantly increased in the presence of 500 U/ml IFN-gamma. With the addition of protein kinase C (PKC) inhibitors bisindolylmaleimide, staurosporine, H-7, or phloretin a reversal of the effect of IFN-gamma was noted whereas the addition of the cyclic nucleotide-dependent protein kinase inhibitors HA 1004, H-8, H-89, or cAMP-Dependent Protein Kinase (PKA) Inhibitor did not block the effect. Although diacylglycerol (DAG) did not replace PMA in the study. Diacylglycerol Kinase Inhibitor induced a more pronounced augmentation and PKC depletion inhibited the effect. This suggests that a PKC-dependent pathway is involved in the response of CB in PMA primed THP-1 cells to IFN-gamma.

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.

Mechanical strain-enhanced fetal lung cell proliferation is mediated by phospholipase C and D and protein kinase C.

The signaling pathways by which intermittent strain (60 cycles/min, 15 min/h) regulates proliferation of mixed fetal rat lung cell in vitro have been investigated. Adenosine 3',5'-cyclic monophosphate (cAMP) content and cAMP-dependent protein kinase (PKA) activity were not affected by strain. The stimulatory effect of strain on DNA synthesis was also not influenced by the cyclic nucleotide-dependent protein kinase inhibitors H-8 or HA-1004, the adenylate cyclase inhibitor SQ-22536, or a PKA inhibitor and cAMP antagonist, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). In contrast, intracellular concentrations of two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), were dramatically increased after a short period of strain. This increase in second messengers was accompanied by an increased tyrosine phosphorylation of phospholipase C-gamma 1. Phospholipase D activity was also increased by strain. Mechanical strain elicited a shift in the subcellular distribution of PKC activity from cytosol to membranes shortly after the onset of strain. The specific activity of PKC in the membranes increased 6- to 10-fold within 5-15 min and remained increased throughout a 48-h period of intermittent strain. Strain-induced PKC activation and DNA synthesis were blocked by the PKC inhibitors H-7, staurosporine, and calphostin C, as well as by the phospholipase C inhibitor U-73,122. We conclude that mechanical strain of mixed fetal rat lung cells activates phospholipid turnover via phospholipases, followed by PKC activation, which then triggers the downstream events that lead to cell proliferation.

Histamine augments cytokine-stimulated IL-11 production by human lung fibroblasts.

Histamine mediates its effects via histamine receptors and by participating in a multicellular cytokine cascade. IL-11 is a stromal cell-derived cytokine with biologic activities that overlap with IL-6. To further understand the biology of histamine and IL-11, we determined whether histamine regulates the production of IL-11 by human lung fibroblasts. Histamine was a weak stimulator of IL-11 production. Importantly, it also interacted in a synergistic fashion with TGF-beta 1 to further augment IL-11 protein production and mRNA accumulation. This synergistic interaction was not altered by the H2 receptor antagonist cimetidine and could not be reproduced with the H2 receptor agonist 4-methylhistamine. In addition, it was not abrogated by the cyclic nucleotide-dependent protein kinase inhibitor N-(2-1-guanidinoethyl)-5 isoquinolinesulfonamide hydrochloride), and histamine and TGF-beta 1 did not stimulate intracellular cAMP. In contrast, the synergy was abrogated by the H1 histamine receptor antagonists diphenhydramine and pyrilamine, could be reproduced when histamine was replaced with the H1 agonist 2-methylhistamine, and was abrogated by the calmodulin antagonists N-(6-aminohexyl)-1-napthalenesulfonamide), N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide), and trifluoperazine dichloride and by the intracellular calcium chelator 1,2-bis-(2-amino-5-bromo-phenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester. In addition, although TGF-beta 1 did not alter cytosolic Ca2+, histamine caused a biphasic increase in cytosolic Ca2+, and the majority of cells incubated with TGF-beta 1 plus histamine exhibited sustained Ca2+ oscillations. These studies demonstrate that histamine is an important regulator of fibroblast IL-11 production, that histamine interacts with TGF-beta 1 in the induction of this cytokine, and that this interaction is mediated, to a great extent, by a pretranslational mechanism that is dependent on H1 receptors and a calcium/calmodulin-dependent activation pathway.

Involvement of protein kinase C and protein tyrosine kinase in lipopolysaccharide-induced TNF-alpha and IL-1 beta production by human monocytes.

Bacterial LPS stimulates human monocytes to secrete inflammatory cytokines, which are involved in several disease processes. However, the mechanism of LPS activation of cytokine expression and secretion is not completely understood. In this study, we investigated the signal transduction pathways involved in LPS-stimulated TNF-alpha and IL-1 beta secretion. TNF-alpha and IL-1 beta secretion were completely blocked by protein kinase C (PKC) and cyclic nucleotide-dependent protein kinase inhibitor, H-7, but were not affected by H-89, a specific cyclic nucleotide-dependent protein kinase inhibitor. In addition, LPS was found to induce activation of PKC, reaching maximal activity at 30 min and returning to unstimulated levels after 60 min. LPS stimulation only slightly increased intracellular levels of diacylglycerol, the natural activator of PKC, and pretreatment of monocytes with the diacylglycerol-kinase inhibitor, R59022, did not affect LPS-stimulated TNF-alpha secretion. LPS-induced PKC activation was found not to be affected by blocking of the LPS receptor, CD14, with mAb or by inhibition of protein tyrosine kinase with herbimycin A. However, these agents suppressed LPS-induced TNF-alpha secretion and TNF-alpha mRNA accumulation. The results suggest that TNF-alpha and IL-1 beta secretion after LPS stimulation of human monocytes requires the activation of protein tyrosine kinase and PKC, upstream to the activation of gene transcription. The activation of PKC by LPS is probably mediated by a diacylglycerol-independent pathway.