h-89 and 6-anilino-5-8-quinolinedione

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large amounts of BMP2 applied. Therefore, reduction of BMP2 amounts while maintaining efficacy is of clinical importance. As nitric oxide (NO) signaling plays a role in bone fracture healing and an association with the BMP2 pathway has been indicated, this study aimed to investigate the relationship of BMP2 and NO pathways and whether NO can enhance BMP2-induced signaling and osteogenic abilities in vitro. To achieve this, the stable BMP reporter cell line C2C12BRELuc was used to quantify BMP signaling, and alkaline phosphatase (ALP) activity and gene expression were used to quantify osteogenic potency. C2C12BRELuc cells were treated with recombinant BMP2 in combination with NO donors and substrate (Deta NONOate, SNAP & L-Arginine), NOS inhibitor (LNAME), soluble guanylyl cyclase (sGC) inhibitor (LY83583) and activator (YC-1), BMP type-I receptor inhibitor (LDN-193189), or protein kinase A (PKA) inhibitor (H89). It was found that the NOS enzyme, direct NO application, and sGC enhanced BMP2 signaling and improved BMP2 induced osteogenic activity. The application of a PKA inhibitor demonstrated that BMP2 signaling is enhanced by the NO pathway via PKA, underlining the capability of BMP2 in activating the NO pathway. Collectively, this study proves the ability of the NO pathway to enhance BMP2 signaling.

Topics: Alkaline Phosphatase; Aminoquinolines; Animals; Bone Morphogenetic Protein 2; Cell Line; Cyclic AMP-Dependent Protein Kinases; Enzyme-Linked Immunosorbent Assay; Isoquinolines; Mice; Nitric Oxide; Nitric Oxide Donors; Pyrazoles; Pyrimidines; Signal Transduction; Sulfonamides

The involvement of adenylate cyclase-cyclic adenosine monophosphate (AC-cAMP) in gonadotropin-stimulated testicular steroidogenesis is well known. Little is known about the role of guanylate cyclase-cyclic guanosine monophosphate (GC-cGMP) or early chloride conductance stimulated by gonadotropins in steroidogenesis. Human chorionic gonadotropin (hCG) 1 IU/L caused significant androgen secretion without a discernible effect on cAMP production. Despite negligible intracellular cAMP, the protein kinase A inhibitor H89 blocked basal and hCG-stimulated steroidogenesis. The GC inhibitors methylene blue (MB) and LY83583 decreased androgen secretion, but hCG did not stimulate cGMP production and there was not a steroidogenic response to exogenous cGMP. A chloride-channel inhibitor, diphenylamine-2-carboxylate (DPC), at concentrations up to 0.6 mmol/L stimulated basal steroid secretion and hCG 10 IU/L stimulated cAMP production, but higher concentrations had an inhibitory effect. Substitution of chloride by gluconate enhanced basal steroid secretion, but nitrate completely abolished the effect of 1 IU/L hCG on androgen secretion, which could be partially overcome by increasing the gonadotropin concentration. In conclusion, chloride, perhaps by activating AC-cAMP, mediates the steroidogenic action of gonadotropins in mouse Leydig tumor cells (MLTC-1). Inorganic nitrate probably inhibited steroidogenesis via conversion to nitric oxide (NO) without involving the GC-cGMP pathway. Nevertheless, the results obtained with GC inhibitors suggest a role for the GC-cGMP pathway in Leydig cell steroidogenesis.

Topics: Aminoquinolines; Androgens; Animals; Calcium Channel Blockers; Chlorides; Chorionic Gonadotropin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Enzyme Inhibitors; Gonadotropins; Guanylate Cyclase; Isoquinolines; Leydig Cell Tumor; Mice; Nitrates; ortho-Aminobenzoates; Sulfonamides; Testosterone

Pituitary adenylate cyclase-activating peptide (PACAP) receptors and their signaling pathways were characterized in dispersed rabbit gastric muscle cells. 125I-PACAP-27 and 125I-vasoactive intestinal peptide (VIP) binding to muscle cells were inhibited equally by PACAP and VIP (mean inhibitory concentration 0.8 to 1.3 nM) and desensitized to the same extent (70-80%) by exposure to either peptide. PACAP, like VIP, increased cytosolic free Ca2+ and the formation of L-[3H]citrulline, NO-3/NO-2, guanosine 3',5'-cyclic monophosphate (cGMP), and adenosine 3'5'-cyclic monophosphate (cAMP) and induced relaxation (mean effective concentration 1.8 +/- 0.1 nM) that was partly inhibited by NG-nitro-L-arginine (L-NNA), VIP-(10-28), and PACAP 6-38. L-[3H]citrulline and cGMP formation were blocked by nifedipine, L-NNA, and pertussis toxin (PTx), implying activation of a G protein-coupled, Ca(2+)-calmodulin-dependent nitric oxide (NO) synthase. PACAP-induced relaxation was inhibited to the same extent (46-49%) by nifedipine, L-NNA, PTx, and the protein kinase G inhibitor KT-5823; the inhibition reflected the component of relaxation mediated by the NO-cGMP pathway. The residual relaxation was abolished by the protein kinase A inhibitor H-89. The pattern of inhibition of all responses was identical to that observed with VIP. Desensitization with VIP or PACAP abolished cAMP formation but had no effect on L-[3H]citrulline and cGMP formation induced by either peptide. Receptor protection with VIP or PACAP preserved fully all responses (L-[3H]citrulline, cGMP, and cAMP formation and relaxation) to either peptide. The complete cross-competition, cross-desensitization, cross-antagonism, and cross-protection of receptors by either VIP or PACAP are consistent with interaction of both peptides with the same receptors; the receptors consist of two classes, each coupled to a distinct signaling pathway.

Topics: Adenylate Cyclase Toxin; Alkaloids; Aminoquinolines; Animals; Calcium; Carbazoles; Cells, Cultured; Citrulline; Cyclic AMP; Cyclic GMP; Cytosol; Enzyme Inhibitors; GTP-Binding Proteins; Guanylate Cyclase; Indoles; Isoquinolines; Kinetics; Muscle Relaxation; Muscle, Smooth; Neuropeptides; Neurotransmitter Agents; Nifedipine; Nitric Oxide Synthase; Nitroarginine; Pertussis Toxin; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Kinase Inhibitors; Rabbits; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Signal Transduction; Stomach; Sulfonamides; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

At birth, pulmonary vasodilation occurs as air-breathing life begins. The mechanism of O2-induced pulmonary vasodilation is unknown. We proposed that O2 causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel (KCa) via a cyclic nucleotide-dependent kinase. We tested this hypothesis in hemodynamic studies in acutely prepared fetal lambs and in patch-clamp studies on resistance fetal pulmonary artery smooth muscle cells. Fetal O2 tension (PaO2) was increased by ventilating the ewe with 100% O2, causing fetal total pulmonary resistance to decrease from 1.18 +/- 0.14 to 0.41 +/- 0.03 mmHg per ml per min. Tetraethylammonium and iberiotoxin, preferential KCa-channel inhibitors, attenuated O2-induced fetal pulmonary vasodilation, while glibenclamide, an ATP-sensitive K+-channel antagonist, had no effect. Treatment with either a guanylate cyclase antagonist (LY83583) or cyclic nucleotide-dependent kinase inhibitors (H-89 and KT 5823) significantly attenuated O2-induced fetal pulmonary vasodilation. Under hypoxic conditions (PaO2 = 25 mmHg), whole-cell K+-channel currents (Ik) were small and were inhibited by 1 mM tetraethylammonium or 100 nM charybdotoxin (CTX; a specific KCa-channel blocker). Normoxia (PaO2 = 120 mmHg) increased Ik by more than 300%, and this was reversed by 100 nM CTX. Nitric oxide also increased Ik. Resting membrane potential was -37.2 +/- 1.9 mV and cells depolarized on exposure to CTX, while hyperpolarizing in normoxia. We conclude that O2 causes fetal pulmonary vasodilation by stimulating a cyclic nucleotide-dependent kinase, resulting in KCa-channel activation, membrane hyperpolarization, and vasodilation.

Topics: Alkaloids; Aminoquinolines; Animals; Carbazoles; Charybdotoxin; Endothelium, Vascular; Enzyme Inhibitors; Female; Fetus; Glyburide; Guanylate Cyclase; Hypoxia; Indoles; Isoquinolines; Membrane Potentials; Models, Cardiovascular; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Potassium Channels; Pregnancy; Protein Kinase Inhibitors; Pulmonary Artery; Sheep; Sulfonamides; Tetraethylammonium; Tetraethylammonium Compounds; Vasodilation