Cyclic Nucleotide-dependent Protein Kinases Research Articles

Tyrosine kinase but not phospholipid/Ca2+ signaling pathway is involved in interferon-γ stimulation of I-a expression in macrophages

The specific signal transduction pathway(s) involved in the induction of the expression of the MHC class II molecule, la, on macrophages by interferon-γ (IFN-γ) is unclear. In this paper, we assessed the role of several signal transduction pathways including calcium mobilization, phospholipase C, protein kinase C and cyclic nucleotide-dependent protein kinase, and the tyrosine kinase pathways. IFN-γ was unable to mobilize intracellular calcium, unlike platelet-activating factor, which stimulated a threefold increase in cytosolic Ca2+ concentration in macrophages. Inhibition of the phospholipase C pathway by U73122 or ET-180CH3 and of phosphatidic acid phosphohydrolase by propranolol did not suppress IFN-γ-induced la expression. In addition, inhibition of protein kinase C by calphostin C or cyclic nucleotide-dependent protein kinase by HA1004 did not suppress la expression. However, IFN-γ-induced la expression was significantly suppressed when the tyrosine kinase pathway was inhibited with herbimycin A and genestein. In addition, those two inhibitors suppressed tyrosine phosphorylation of several proteins in macrophages that may or may not be involved in the induction of la expression. Thus, IFN-γ used only the tyrosine kinase signaling pathway, but not the phospholipid/Ca2+ signaling pathways, to induce la expression in macrophages. © 1996 Wiley-Liss, Inc.

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.

Inductive effect of Porphyromonas gingivalis fimbriae on differentiation of human monocytic tumor cell line U937.

This study used the human monocytic tumor cell line U937 to examine whether Porphyromonas gingivalis fimbriae induce differentiation of monocyte/macrophage progenitors. When the progenitor cells were incubated with P. gingivalis fimbriae, the incubation resulted in increased Fc rosette formation and increased CD11b production by the cells. The presence of a protein kinase C inhibitor, H7 or calphostin C, in the medium eliminated the stimulatory effects of P. gingivalis fimbriae on Fc rosette formation. Furthermore, CD11b expression was inhibited by calphostin C. In contrast, HA1004 , an inhibitor of cyclic nucleotide-dependent protein kinase, had no effect on P. gingivalis fimbriae-induced Fc rosette formation or CD11b expression. These results demonstrate that P. gingivalis fimbriae are a potent inducer of the differentiation of the monocyte/macrophage tumor cell line U937, most probably via cyclic nucleotide-independent protein kinase C.

Adenylate and guanylate cyclases in Tetrahymena.

Cyclic nucleotides, such as cyclic AMP (cAMP) and cyclic GMP (cGMP), are of fundamental importance in regulating the many physiologic processes in a wide variety of living cells, acting as messengers interacting between receptor sites on the cell membrane and intracellular activity. Both adenylate cyclase (A-cyclase) and guanylate cyclase (G-cyclase), which catalyze the formation of cyclic nucleotides, are known to exist in most organisms. A free-living unicellular eukaryote, Tetrahymena, represents a suitable cell system for studies of the regulation of intracellular events by cyclic nucleotides (Nozawa and Thompson 1971), since it contains high activities of cyclic nucleotide phosphodiesterase (PDE; Dickinson et al. 1977; Kudo et al. 1980), A-cyclase (Rozensweig and Kindler 1972; Voichick et al. 1973), G-cyclase Nagao et al. 1979; Nakazawa et al. 1979), cyclic nucleotide-dependent protein kinases (Murofushi 1974), and calmodulin (CaM; Kakiuchi et al. 1981; Yazawa et al. 1981), which are involved in the metabolism of cyclic nucleotides. This chapter will dwell on the regulatory mechanisms in the metabolism of Tetrahymena cyclic nucleotides involved in the two cyclases, particularly as they relate to the molecular mechanisms of regulation of those enzymes and CaM, and will discuss the structural features of Tetrahymena CaM which endow it with its special functional capabilities, and its intracellular distribution.KeywordsAdenylate CyclaseCyclic NucleotideGuanylate CyclaseMiddle Exponential PhaseCyclic Nucleotide MetabolismThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

14] Cyclic GMP-dependent protein kinase in nitric oxide signaling

Cyclic GMP-dependent protein kinase is now implicated in a number of important cellular signaling events. The role of PKG in processes as diverse as the regulation of intracellular Ca 2+ levels in smooth muscle tissues to its potential role in gene expression has been the subject of investigations over the past few years. Despite the importance of this enzyme in cellular regulation, few details of the molecular mechanisms of action of PKG are available. There are a number of important issues to consider, however, when studying the role of NO, cGMP, and PKG in cellular function. In the first case, it is important to acknowledge the diversity of effects of NO on cellular processes. At submicromolar concentrations of NO-generating drugs such as nitroprusside, NO is known to activate soluble guanylate cyclase. Predictably, this leads to the activation of PKG and the phosphorylation of proteins relevant to the signaling cascade under investigation. At higher concentrations of NO-generating drugs, however, other effects of NO occur that may be unrelated to PKG activation. These include cross-activation of PKA by cGMP, and the modification of proteins by the NO radical. Another important consideration when investigating the role of cGMP and PKG in cell regulation is the nonspecific actions of cyclic nucleotide analogs (e.g., 8-Br-cyclic nucleotides) and drugs used to inhibit protein kinase activity. For example, high concentrations of cyclic nucleotide analogs may cross-activate both cyclic nucleotide-dependent protein kinases when incubated with cultured cells at high concentrations for prolonged periods of time. And finally, the specificity of protein phosphorylation catalyzed by protein kinases must be considered. Both PKA and PKG, for example, catalyze the phosphorylation of identical residues in protein substrates in vitro . In the intact cell, the pattern of protein phosphorylation may be affected by the localization of the kinases or the presence of adaptor or anchoring proteins. Many of these experimental problems may be addressed with appropriate pharmacological protocols (dose-response curves and time courses), and there are now available specific cDNAs for expressing catalytic domains or subunits of protein kinases. In this way, the specific role of PKG may be addressed through transfection studies.

Cyclic nucleotide-induced termination of vitellogenin uptake by Hyalophora cecropia follicles

Endocytosis of vitellogenin by isolated follicles of Hyalophora cecropia terminated after membrane-permeable analogs of cAMP or cGMP were added to the culture medium. Depending on the concentration of the analog, a lag period of 30 min to 3 h preceded termination. Forskolin and IBMX both stimulated a rise in endogenous cAMP, and this also induced termination, as did pharmacological activation of the cyclic nucleotide-dependent protein kinases PKA and PKG. Inhibitors of PKA or PKG protected follicles from the corresponding cyclic nucleotide effect. When cAMP or cGMP was added to homogenates of vitellogenic follicles, a 32 kDa polypeptide was phosphorylated; inhibition of PKA, prevented phosphorylation of this protein. The rate of vitellogenin uptake did not accelerate significantly when PKA or PKG was inhibited in culture, which suggests that these kinases are normally inactive or operating below threshold during the several days of vitellogenesis. They seem thus not to be involved in the steady-state modulation of protein upake. A more likely function of this control pathway in follicle development would be to trigger the termination of vitellogenesis, which normally occurs spontaneously in follicles of this species as they reach a length of 2 mm.

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.

Endothelium-dependent hyperpolarization. Beyond nitric oxide and cyclic GMP.

Blood flow and blood pressure are determined by an integration of reflex, humoral, and local vascular control mechanisms. Knowledge of these mechanisms has mushroomed over the past 15 years, particularly in the area of local endothelium-dependent vasomotor control. This has stemmed from the pioneering report in 1980 by Robert Furchgott1 demonstrating that the endothelium releases a vasodilator substance in response to acetylcholine. This concept has been expanded with knowledge that the endothelium releases a variety of relaxing and contracting factors that regulate the underlying smooth muscle.2 The most widely known endothelium-derived relaxing factor, nitric oxide, is released from endothelial cells in response to shear stress or stimulation of different receptors for a variety of neurohumoral mediators on the endothelial cell surface.3 The increase in endothelial cell calcium initiated by these stimuli increases the activity of a constitutively expressed enzyme, nitric oxide synthase, which converts l-arginine to nitric oxide and citrulline.4 5 6 7 Nitric oxide thus formed diffuses to and inhibits contraction of the underlying vascular smooth muscle.3 The physiological significance of the production of endothelial nitric oxide is suggested by the vasoconstriction observed in most vascular beds8 9 10 11 12 13 and the increase in systemic arterial blood pressure,14 15 16 which occurs on infusion of inhibitors of nitric oxide synthase. This observation further implies that under normal conditions, endothelial cells are locally liberating nitric oxide which effectively inhibits vasoconstriction arising by other mechanisms. Thus, normal vascular homeostasis depends in the periphery on a balance between neurally and humorally mediated vasoconstriction in skeletal muscle, mesentery, and the kidney, and local endothelium-dependent vasodilatation. In the truly vital areas of the heart, brain, and genitalia, vasodilator neural mechanisms reinforce the vasodilator influence of the endothelium.17 All is not known regarding the …

Role of cyclic nucleotides in store-mediated external Ca2+ entry in human platelets

This study explores the role of cyclic nucleotides (i.e. cyclic AMP and cyclic GMP) in store-regulated external Ca2+ entry in human platelets. To stimulate store-regulated external Ca2+ entry, thapsigargin was used to deplete Ca2+ from the dense tubules, and sodium nitroprusside and iloprost respectively were used to stimulate endogenous cyclic GMP and cyclic AMP formation. Pretreatment with sodium nitroprusside and iloprost (a) attenuated the thapsigargin-evoked external Ca2+ entry and (b) reduced the rate of Ca2+ release from the dense tubules. The effects on external Ca2+ entry and Ca2+ release from the dense tubules were exerted independently and were apparently mediated through activation of the respective cyclic nucleotide-dependent protein kinases. Both sodium nitroprusside and iloprost reduced tyrosine kinase phosphorylation of a number of proteins, particularly a 72 kDa protein band. Both agents also attenuated the thapsigargin-evoked tyrosine kinase phosphorylation of the 72 kDa band. Intracellular Ca2+ depletion resulted in a reduction in tyrosine kinase-mediated phosphorylation of a number of protein bands, including the 72 kDa band and the further attenuation of thapsigargin-mediated tyrosine phosphorylation of this band. The effects of the cyclic nucleotides on cellular Ca2+ homoeostasis in thapsigargin-treated platelets were not exerted via acceleration of Ca2+ extrusion or Ca2+ sequestration into the mitochondria. We conclude that cyclic nucleotides participate in store-regulated control of external Ca2+ entry by slowing down the rate of external Ca2+ entry and Ca2+ release from intracellular Ca2+ stores. These effects are apparently mediated via cyclic nucleotide-dependent protein kinases and the attenuation of protein phosphorylation by tyrosine kinases.

Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper.

An iterative approach to the a priori determination of the substrate specificity of cAMP- and cGMP-dependent protein kinases (PKA and PKG) by the use of peptide libraries on cellulose paper is described. The starting point of the investigation was an octamer library with the general structure Ac-XXX12XXX, where X represents mixtures of all 20 natural amino acids and 1 and 2 represent individual amino acid residues. The library thus contained all possible 2.56 x 10(10) octamers, divided into 400 sublibraries with defined amino acids 1 and 2 each consisting of 6.4 x 10(7) sequences. After phosphorylation with the kinases in the presence of [gamma-32P]ATP, the sublibrarys Ac-XXXRRXXX and Ac-XXXRKXXX were identified as the best substrates for PKA and PKG, respectively. The second-generation libraries had the structures Ac-XXXRR12X and Ac-XXXRK12X for PKA and PKG and resulted in the most active sequence pools Ac-XXXRRASX and Ac-XXXRKKSX. After delineation of every position in the octameric sequence and extension of the investigation to decameric peptides, the best sequences, Ac-KRAERKASIY and Ac-TQKARKKSNA, were obtained for PKA and PKG, respectively. Promising octameric and decameric peptides were assembled 5 or 10 times each and assayed in order to determine the experimental scatter inherent in the approach. The kinetic data of several octameric and decameric sequences were determined in solution and compared to data for known substrates. The recognition motif of PKA was confirmed by this approach, and a novel substrate sequence for PKG was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Signal Requirements for Production of Luteinizing Hormone Releasing-Hormone by Human T Cells

We have previously demonstrated that both human CD4+ and CD8+ T lymphocytes produce enhanced levels of luteinizing hormone-releasing hormone (LHRH) mRNA and peptide upon stimulation with monoclonal antibody directed at the CD3 component of the T cell receptor for antigen (TCR) or mitogenic lectin. In the current study, we define the signaling pathways that control TCR-mediated LHRH production by using agents known to affect distinct signals, and compare the messenger systems required for LHRH response to other T-cell-associated activation responses, such as expression of CD69 and interleukin-2 receptor (IL-2R) molecules and production of interleukin-2 (IL-2). Results indicate that the activation of protein kinase C (PKC) is essential for LHRH production by previously nonstimulated T cells, not increased concentration of cytosolic-free calcium ([Ca]2+]1). Phorbol ester (PMA), a direct activator of PKC, provoked LHRH production and cell surface expression of CD69 and IL-2R molecules by T cells, but not IL-2 synthesis. The synthesis of IL-2 by T cells required costimulation with PMA and ionomycin, a Ca2+ ionophore. Consistent with these observations, H7, an inhibitor of PKC, prevented T cells from producing LHRH upon activation with mitogen. However, LHRH production was not suppressed by HA 1004, which inhibits all cyclic nucleotide-dependent protein kinases except for PKC. Genistein, a selective inhibitor of protein tyrosine kinase, blocked PMA-induced increase in LHRH production, but not CD69 and IL-2R expression, suggesting that protein tyrosine phosphorylation events distal from PKC activation may play a role in regulating LHRH response.

Disruption of the Cytokeratin Cytoskeleton and Inhibition of Hepatocytic Autophagy by Okadaic Acid

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 n M). Only small effects were seen with vinblastine (10 μ M) or cytochalasin D (10 μ M). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against α- and β-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 μ M), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 μ M) and prunin (100 μ M) as well as KN-62 (10 μ M), a specific inhibitor of Ca 2+/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 μ M), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 μ M), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.

Intracellular mechanisms involved in the regulation of vascular smooth muscle tone

Vascular smooth muscle contraction is thought to occur by a mechanism similar to that described for striated muscles, i.e., via a cross-bridge cycling--sliding filament mechanism. This symposium focused on Ca2+ signalling and the role of intracellular free Ca2+ concentration, [Ca2+]i, in regulating vascular tone: how contractile stimuli leading to an increase in [Ca2+]i trigger vasoconstriction and how relaxant signals reduce [Ca2+]i causing vasodilation. M.P. Walsh opened the symposium with an overview emphasizing the central role of myosin phosphorylation-dephosphorylation in the regulation of vascular tone and identifying recent developments concerning regulation of [Ca2+]i, Ca2+ sensitization and desensitization of the contractile response, Ca(2+)-independent protein kinase C induced contraction, and direct regulation of cross-bridge cycling by the thin filament associated proteins caldesmon and calponin. The remainder of the symposium focused on three specific areas related to the regulation of vascular tone: Ca2+ signalling in relation to smooth muscle structure, structure-function relations of myosin, and the role of cyclic GMP (cGMP) dependent protein kinase. G.J. Kargacin described how smooth muscle cells are structured and how second messenger signals such as Ca2+ might be modified or influenced by this structure. J. Kendrick-Jones then discussed the results of mutagenesis studies aimed at understanding how the myosin light chains, particularly the phosphorylatable (Ca(2+)-calmodulin dependent) regulatory light chains, control myosin. The vasorelaxant effects of signalling molecules such as beta-adrenergic agents and nitrovasodilators are mediated by cyclic nucleotide dependent protein kinases, leading principally to a reduction in [Ca2+]i. T.M. Lincoln described the roles of cyclic nucleotide dependent protein kinases, in particular cyclic GMP dependent protein kinase, in vasodilation.

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.

Inhibition of cyclic nucleotide-dependent protein kinases accounts for human umbilical artery vabospask

Inhibition of cyclic nucleotide-dependent protein kinases accounts for human umbilical artery vabospask

Impaired cyclic nucleotide-dependent vasorelaxation in human umbilical artery smooth muscle

Activation of either the adenylate cyclase pathway with forskolin or the guanylate cyclase pathway with sodium nitroprusside fails to induce active relaxation of serotonin-precontracted human umbilical artery smooth muscle (HUASM) but causes active relaxation of serotonin-precontracted bovine carotid artery smooth muscle (BCASM). This difference in response appears to be unique to HUASM in that all other vascular muscles exhibit relaxation in response to these substances. Forskolin and sodium nitroprusside stimulation leads to respective increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) concentrations in HUASM and BCASM. cAMP- and cGMP-dependent protein kinases are both present in HUASM and can be activated in homogenates of HUASM by the addition of exogenous cAMP and cGMP, respectively. When either forskolin or nitroprusside acts in BCASM, two low-molecular-weight proteins display an increase in the extent of phosphorylation. Neither protein shows such an increase when HUASM is treated with either agent. Thus the inability of HUASM to display active relaxation appears to be secondary to impaired activation of cyclic nucleotide-dependent protein kinases. The refractoriness to active relaxation may contribute to the vasospasm that occurs in the umbilical vasculature with parturition.

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.

Structure and function of cyclic nucleotide-dependent protein kinases.

Cyclic AMP-dependent protein kinase (cAK) was first described in 19631964 (43, 132) as a cAMP-dependent glycogen synthase kinase that, in the presence of Mg/ A TP, transferred the 'V-phosphate of A TP to serines or threonines on many cellular enzymes. It was found to phosphorylate a number of proteins and was permanently named in 1968 (168). Ensuing years produced vast numbers of reports regarding properties of cAK and the signal mechanisms that elicit its activation. Many of these have been reviewed recently (8, 33,47, 85, 141, 155, 181). In 1970 cyclic GMP-de­ pendent protein kinase (cGK) was discovered (86). cGK is homologous in structure and function to cAK (24, 49, 59, 66, 93, 141, 154). The role of cGMP in physiological processes is increasingly appreciated (15, 39, 92, 94, 113, 170), but cGK is only one of several intracellular receptors for cGMP. Studies of the heterogeneity, function, and regulation of cAK and cGK have provided insight into the enzymology of diverse protein kinases and into the roles of these kinases in cellular processes.

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.

Phosphofructokinase from the posterior gills of the euryhaline crab, Eriocheir sinensis: evidence for its regulation by phosphorylation

Phosphofructokinase from the posterior gills of the euryhaline crab Eriocheir sinensis acclimated to freshwater is likely regulated in part via phosphorylation induced by endogenous cyclic nucleotide-dependent protein kinases. Phosphofructokinase from gill extracts devoid of low molecular weight compounds by chromatography through a PD10 Sephadex column, incubated in the presence of cAMP or cGMP protein kinases activators (cAMP or cGMP, Mg-ATP and Mg2+), shows an increased catalytic activity. This treatment is accompanied by 32P incorporation into the proteins immunoprecipitated with anti-mammalian phosphofructokinase polyclonal antibodies cross-reacting with the analog crustacean enzyme. Our results indicate that the covalent modification induced by these nucleotide-dependent protein kinases activates the glycolytic enzyme by increasing its affinity for its substrate and, when the activation is specifically due to cAMP-dependent protein kinases, by also reducing the homotropic cooperativity between its multiple substrate binding sites.