ucn-1028-c and 8-bromoadenosine-3--5--cyclic-monophosphorothioate

Lactoferrin (LF) is a multifunctional protein. While its functions and mechanism of actions are actively being investigated, the cellular signals that regulate LF expression have not been as explored. We have previously demonstrated that LF is upregulated by estrogen in the reproductive system. In this study, we show that the expression of LF was stimulated by bacterial lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) in normal mouse mammalian HC-11 cells. When cells were exposed to either LPS or dsRNA, the mRNA and protein of LF were increased in a dose- and time-dependent manner, yet the kinetics of LF induction by dsRNA or LPS were different. The LPS and dsRNA-induced LF was mainly released into the culture medium where it blocked TNF-alpha production in exposed cells. We explored the mechanisms of LF induction by LPS and dsRNA using specific inhibitors and found that the induction could be attenuated by inhibitors to PKC, NF-kappaB, p38 and JNK, but not by an inhibitor to PKA. Interestingly, ERK inhibitor was effective against dsRNA but not against LPS induction of LF. These data suggest that LF was induced by LPS and dsRNA through PKC, NF-kappaB and MAPK pathways which in turn play an inhibitory role in the continuation of innate inflammation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Blotting, Western; Cell Line; Cyclic AMP-Dependent Protein Kinases; Cytokines; Epithelial Cells; Gene Expression; Lactoferrin; Lipopolysaccharides; Mammary Glands, Animal; Mice; Microscopy, Confocal; Naphthalenes; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; RNA, Double-Stranded; Signal Transduction; Thionucleotides; Tumor Necrosis Factor-alpha

We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Acetylcholine; Analysis of Variance; Animals; Cyclic AMP-Dependent Protein Kinases; Isoquinolines; Membrane Potentials; Naphthalenes; Neuromuscular Junction; Protein Kinase C; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Thionucleotides

1. The effect of purified G protein subunits alphas and betagamma on L-type Ca2+ channels in vascular smooth muscle and the possible pathways involved were investigated using freshly isolated smooth muscle cells from rabbit portal vein and the whole-cell patch clamp technique. 2. Cells dialysed with either Galphas or Gbetagamma exhibited significant increases in peak Ba2+ current (IBa) density (148 % and 131 %, respectively) compared with control cells. The combination of Galphas and Gbetagamma further increased peak IBa density (181 %). Inactive Galphas and Gbetagamma did not have any effect on Ca2+ channels. 3. The stimulatory effect of Galphas on peak IBa was entirely abolished by the protein kinase A inhibitor Rp-8-Br-cAMPS, or the adenylyl cyclase inhibitor SQ 22536. On the other hand, the stimulatory response of Ca2+ channels to Gbetagamma was not affected by the protein kinase A inhibitors Rp-8-Br-cAMPS and KT 5720, or by the Ca2+-dependent protein kinase C inhibitor bisindolylmaleimide 1, but was completely blocked by the protein kinase C inhibitor calphostin C. Pretreatment of cells with phorbol 12-myristate 13-acetate for over 18 h prevented the stimulatory effect of Gbetagamma on peak IBa. In addition, acute application of phorbol 12,13-dibutyrate enhanced peak IBa density in control cells, which could be entirely blocked by calphostin C. 4. These data indicate that enhancement of Ba2+ currents by Galphas and Gbetagamma can be attributed to increased activity of protein kinase A and protein kinase C, respectively. No direct membrane-delimited pathway for Ca2+ channel regulation by activated Gs proteins could be detected in vascular smooth muscle cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcium Channels; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; GTP-Binding Proteins; In Vitro Techniques; Indoles; Male; Maleimides; Models, Biological; Muscle, Smooth, Vascular; Naphthalenes; Patch-Clamp Techniques; Phorbol 12,13-Dibutyrate; Portal Vein; Protein Conformation; Protein Kinase C; Pyrroles; Rabbits; Signal Transduction; Tetradecanoylphorbol Acetate; Thionucleotides