h-89 and dibutyryl-cyclic-3--5--cytidine-monophosphate

Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 μM free Ca2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca2 + increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 μM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 μM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca2 + stimulation of oxidative phosphorylation

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Diphosphate; Animals; Calcium; Catalytic Domain; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic CMP; Cytochrome b Group; Electron Transport; Electron Transport Complex I; Electron Transport Complex III; Electron Transport Complex IV; Isoquinolines; Mitochondria, Heart; NAD; Oxidative Phosphorylation; Oxygen Consumption; Protein Kinase Inhibitors; Spectrometry, Fluorescence; Spectrophotometry; Sulfonamides; Swine

The lacrimal gland is primarily responsible for the aqueous portion of the tear film. Simultaneous addition of cholinergic agonists or growth factors with cAMP-dependent agonists potentiates secretion. Recent investigations revealed that cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity stimulated by cholinergic agonists and growth factors that could account for this potentiation. In this study the authors identify the signal transduction pathway used by cAMP to inhibit MAPK activity.. Rat lacrimal gland acini were incubated with H89, an inhibitor of protein kinase A, before the addition of dibutyryl cAMP (dbcAMP, 10(-3) M) for 30 minutes. Basal MAPK and CREB activity and MAPK activity after stimulation with the cholinergic agonist carbachol (Cch) or epidermal growth factor (EGF) for 5 minutes was determined. The effect of dbcAMP on EGF receptor activity and basal and stimulated Ras, Raf-1, mitogen-activated protein kinase kinase (MEK), and MAPK activity was determined. The effect of a Rap-1 inhibitor, GGTI-298, on MAPK activity after the addition of dbcAMP was also determined.. H89 relieved the inhibition of cAMP on MAPK activity and inhibited CREB activity. Incubation with dbcAMP did not have any effect either on the EGF receptor or on Ras but significantly inhibited both basal and Raf-1 and MEK activity stimulated with Cch or EGF. GGTI-298 did not have any effect on cAMP-dependent decrease in MAPK activity.. The authors conclude that cAMP mediates the inhibition of MAPK by PKA in a Raf-1-dependent manner.

Topics: Animals; Blotting, Western; Carbachol; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclic CMP; Epidermal Growth Factor; ErbB Receptors; Isoquinolines; Lacrimal Apparatus; Male; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-raf; Rats; Rats, Sprague-Dawley; Sulfonamides

Bradykinin (BK) is an endogenous peptide with diverse biological actions and is considered to be an important mediator of the inflammatory response in both the peripheral and the central nervous systems. BK has attracted recent interest as a potential mediator of K(+) conductance, Cl(-) channels, and Ca(2+)-activated K(+) channels. However, few reports have associated BK with the voltage-gated K(+) current. In this study, we demonstrated that BK suppressed the transient outward potassium current (I(A)) in mouse Schwann cells using whole cell recording techniques. At a concentration of 0.1 muM to 5 muM, BK reversibly inhibited I(A) in a dose-dependent manner with the modulation of steady-state activation and inactivation properties. The effect of BK on I(A) current was abolished after preincubation with a B(2) receptor antagonist but could not be eliminated by B(1) receptor antagonist. Intracellular application of GTP-gammaS induced an irreversible decrease in I(A), and the inhibition of G(s) using NF449 provoked a gradual augmentation in I(A) and eliminated the BK-induced effect on I(A,) while the G(i)/(o) antagonist NF023 did not. The application of forskolin or dibutyryl-cAMP mimicked the inhibitory effect of BK on I(A) and abolished the BK-induced effect on I(A). H-89, an inhibitor of PKA, augmented I(A) amplitude and completely eliminated the BK-induced inhibitory effect on I(A). In contrast, activation of PKC by PMA augmented I(A) amplitude. A cAMP assay revealed that BK significantly increased intracellular cAMP level. It is therefore concluded that BK inhibits the I(A) current in Schwann cells by cAMP/PKA-dependent pathways via activation of the B(2) receptor.

Topics: Adamantane; Animals; Benzenesulfonates; Bradykinin; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic CMP; Enzyme Activators; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Guanosine 5'-O-(3-Thiotriphosphate); Isoquinolines; Membrane Potentials; Mice; Mice, Inbred ICR; Potassium; Potassium Channels, Voltage-Gated; Protein Kinase C; Protein Kinase Inhibitors; Receptor, Bradykinin B1; Receptor, Bradykinin B2; Schwann Cells; Sciatic Nerve; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Time Factors

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.

Topics: Animals; Apoptosis Regulatory Proteins; Astrocytes; Cell Differentiation; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Cyclic CMP; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioma; Humans; Isoquinolines; Membrane Proteins; Rats; S100 Proteins; Sulfonamides