cyclic-gmp and staurosporine-aglycone

Prolonged hyperoxic exposure contributes to neonatal lung injury, and airway hyperreactivity is characterized by enhanced contraction and impaired relaxation of airway smooth muscle. Our previous data demonstrate that hyperoxia in rat pups upregulates expression of brain-derived neurotrophic factor (BDNF) mRNA and protein, disrupts NO-cGMP signaling, and impairs cAMP production in airway smooth muscle. We hypothesized that BDNF-tyrosine kinase B (TrkB) signaling plays a functional role in airway hyperreactivity via upregulation of cholinergic mechanisms in hyperoxia-exposed lungs. Five-day-old rat pups were exposed to >or=95% oxygen or room air for 7 days and administered daily tyrosine kinase inhibitor K-252a (50 microg x kg(-1) x day(-1) i.p.) to block BDNF-TrkB signaling or vehicle. Lungs were removed for HPLC measurement of ACh or for in vitro force measurement of lung parenchymal strips. ACh content doubled in hyperoxic compared with room air-exposed lungs. K-252a treatment of hyperoxic pups restored ACh content to room air levels. Hyperoxia increased contraction and impaired relaxation of lung strips in response to incremental electrical field stimulation. K-252a administration to hyperoxic pups reversed this increase in contraction and decrease in relaxation. K-252a or TrkB-Fc was used to block the effect of exogenous BDNF in vitro. Both K-252a and TrkB-Fc blocked the effects of exogenous BDNF. Hyperoxia decreased cAMP and cGMP levels in lung strips, and blockade of BDNF-TrkB signaling restored cAMP but not cGMP to control levels. Therefore, hyperoxia-induced increase in activity of BDNF-TrkB receptor signaling appears to play a critical role in enhancing cholinergically mediated contractile responses of lung parenchyma.

Topics: Acetylcholine; Animals; Animals, Newborn; Brain-Derived Neurotrophic Factor; Carbazoles; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Hyperoxia; Indole Alkaloids; Lung; Lung Injury; Muscle Relaxation; Muscle, Smooth; Rats; Rats, Sprague-Dawley; Receptor, trkB; Signal Transduction

Our previous results showed that inhibition of protein tyrosine phosphatases (PTP) by orthovanadate is an appropriate strategy to mimic nerve growth factor (NGF) effects in neurons, including enhanced phosphorylation of TrkA, stimulation of downstream survival signaling pathways, and protection against apoptotic stress. In this study, we wanted to trigger such NGF-like survival signaling in primary hippocampal neurons with the more specific PTP inhibitors ethyl-3,4-dephostatin (DPN), 4-O-methyl-ethyl-3,4-dephostatin (Me-DPN), and methoxime-3,4-dephostatin. It was striking that only the nitric oxide (NO)-releasing dephostatin analogs DPN and Me-DPN, but not the nitrosamine-free methoxime derivative (which did not release NO), enhanced TrkA phosphorylation and protected the neurons against staurosporine (STS)-induced apoptosis. The established NO donor S-nitroso-N-acetylpenicillamine (SNAP) also enhanced TrkA phosphorylation and prevented apoptosis similarly to DPN and Me-DPN. Analysis of the major signaling pathways downstream of TrkA revealed that both SNAP and DPN enhanced phosphorylation of Akt and the mitogen-activated kinases (MAPK) Erk1/2. Blocking of these signaling pathways by the PI3-K inhibitor wortmannin or the MAPK kinase inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] equally abolished the neuroprotective effect of the NO donors. It was striking that inhibition of the soluble guanylyl cyclase (sGC) by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or protein kinase G (PKG) inhibition by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823) also blocked the neuroprotective effect of the NO donors, and ODQ clearly attenuated SNAP-induced phosphorylation of TrkA, Akt, and MAPK. In conclusion, NO release by the dephostatin derivatives and subsequent stimulation of sGC and PKG is essential for their neuroprotective effects. In primary neurons, such NO-activated survival signaling involves NGF-like effects, including enhanced phosphorylation of TrkA and activation of PI3-K/Akt and MAPK pathways.

Topics: Animals; Carbazoles; Cell Survival; Cyclic GMP; Hippocampus; Immunohistochemistry; In Vitro Techniques; Indole Alkaloids; MAP Kinase Signaling System; Nerve Growth Factor; Neurons; Nitric Oxide Donors; Penicillamine; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction

Binding of folic acid (an intrinsic agonist) to the cell surface receptors evokes transmembrane signals for activation and adaptation of guanylate cyclase in Dictyostelium discoideum. The activation signal activates this enzyme and then the adaptation signal terminates the activation. As a result, these two signals cooperatively induce a transient activation of guanylate cyclase. We investigated transmembrane signal transduction for guanylate cyclase using 2,3-dimercapto-1-propanol (BAL, a thiol-reducing reagent) since BAL induces or modifies the transmembrane signal(s). We found that BAL induced prolonged or continuous activation of guanylate cyclase. Thus, the mode of the activation is drastically different (transient versus continuous) between folic acid and BAL. We also found that the BAL-induced continuous activation was not observed when the cells were stimulated with BAL + folic acid, while folic acid + BAL transiently induced more cGMP accumulation than folic acid alone. We lastly showed that K252a, a protein kinase inhibitor, enhanced both the folic acid-induced and the BAL-induced activation of guanylate cyclase. Our results suggest that BAL induces or mimics the activation signal for guanylate cyclase. The lack of termination in the BAL-induced activation suggests that BAL does not induce the adaptation signal or that the adaptation does not inhibit the BAL-induced activation. The former possibility is more likely since folic acid suppresses the BAL-induced continuous activation. The effect of K252a suggests that protein phosphorylation plays a role in suppression of guanylate cyclase.

Topics: Animals; Carbazoles; Cyclic GMP; Dictyostelium; Dimercaprol; Enzyme Activation; Folic Acid; Guanylate Cyclase; Indicators and Reagents; Indole Alkaloids; Oxidation-Reduction; Protein Kinase C

K-252 compounds (K-252a and b isolated from Nocardiopsis sp. (1) and their synthetic derivatives) were found to inhibit cyclic nucleotide-dependent protein kinases and protein kinase C to various extents. The inhibitions were of the competitive type with respect to ATP. K-252a was a non-selective inhibitor for these three protein kinases with Ki values 18-25 nM. K-252b showed a comparable potency for protein kinase C (Ki, 20nM), whereas inhibitory potencies for cyclic nucleotide-dependent protein kinases were reduced. KT5720 and KT5822 selectively inhibited cAMP-dependent (Ki, 60nM) and cGMP-dependent (Ki, 2.4nM) protein kinases, respectively.

Topics: Adenosine Triphosphate; Carbazoles; Cyclic GMP; Indole Alkaloids; Protein Kinase C; Protein Kinase Inhibitors; Structure-Activity Relationship