cyclic-gmp and sphingosine-1-phosphate

In preovulatory ovarian follicles, the oocyte is maintained in meiotic prophase arrest by natriuretic peptide precursor C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2). LH treatment results in the decrease of NPR2 guanylyl cyclase activity that promotes resumption of meiosis. We investigated the regulatory mechanism of LH-activated epidermal growth factor (EGF) receptor signaling on NPR2 function. Cumulus cell-oocyte complex is cultured in the medium with 30 nM NPPC to prevent oocyte spontaneous maturation. In this system, EGF could stimulate oocyte meiotic resumption after 4 hours of incubation. Further study showed that EGF elevated intracellular calcium concentrations of cumulus cells and decreased cGMP levels in cumulus cells and oocytes, and calcium-elevating reagents ionomycin and sphingosine-1-phosphate mimicked the effects of EGF on oocyte maturation and cGMP levels. EGF-mediated cGMP levels and meiotic resumption could be reversed by EGF receptor inhibitor AG1478 and the calcium chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester. EGF also decreased the expression of Npr2 mRNA in cumulus cells, which may not be involved in meiotic resumption, because the block of NPR2 protein de novo synthesis by cycloheximide had no effect on NPPC and EGF-mediated oocyte maturation. However, EGF had no effect on oocyte maturation when meiotic arrest was maintained in the present of cGMP analog 8-bromoadenosine-cGMP. These results suggest that EGF receptor signaling induces meiotic resumption by elevating calcium concentrations of cumulus cells to decrease NPR2 guanylyl cyclase activity.

Topics: Animals; Animals, Outbred Strains; Calcium Ionophores; Calcium Signaling; Chelating Agents; Cumulus Cells; Cyclic GMP; Epidermal Growth Factor; ErbB Receptors; Female; Luteinizing Hormone; Lysophospholipids; Mice; Natriuretic Peptide, C-Type; Oocytes; Oogenesis; Protein Kinase Inhibitors; Protein Precursors; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Sphingosine; Tissue Culture Techniques

NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway.. The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+]i in depolarized RA (120 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of 10 micromol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 micromol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA-activating phospholipid sphingosine-1-phosphate (S1P, 12 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+]i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP-induced dilations.. NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+-desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.

Topics: ADP Ribose Transferases; Animals; Arteries; Botulinum Toxins; Calcium; Cricetinae; Cyclic GMP; Enzyme Inhibitors; Female; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Muscles; Mutagenesis, Site-Directed; Myosin-Light-Chain Phosphatase; Nitric Oxide; Phosphoprotein Phosphatases; Protein Serine-Threonine Kinases; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Transfection; Vascular Patency; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents