Identification and characterization of a calmodulin-dependent nitric oxide synthase from GH3 pituitary cells

Abstract

A nitric oxide synthase activity stimulated more than 30-fold by the concurrent presence of Ca2+ and calmodulin (CaM), and inhibited by trifluoperazine (50 microM), has been identified in extracts of GH3 pituitary cells. The CaM-dependent nitric oxide synthase of the crude extract was stimulated more than 9-fold by (6R)-5,6,7,8-tetrahydro-L-biopterin with half-maximal stimulation occurring at a concentration of 300 nM. Fractionation of the extract on DEAE-cellulose enhanced nitric oxide synthase specific activity up to 300-fold and provided a preparation which on Western blot analysis possessed a 152 kDa protein which cross-reacted with antibodies to homogeneous bovine brain nitric oxide synthase. The DEAE-cellulose-purified enzyme exhibited apparent Km values of 4.3 microM, 0.4 microM, 0.3 microM and 4 nM for L-arginine, NADPH, Ca2+ and CaM respectively. The CaM-dependent nitric oxide synthase of GH3 extract bound to 2',5'-ADP-agarose and was eluted by NADPH with a 500-fold increased specific activity. Citrulline formation by the ADP-agarose-purified enzyme was inhibited by NG-nitro-L-arginine, NG-methyl-L-arginine and Nitro Blue Tetrazolium with apparent Ki values of 0.2, 1.8 and 7 microM respectively. The ADP-agarose-purified enzyme displayed cytochrome c reductase activity which was stimulated more than 18-fold by the concurrent presence of Ca2+ and CaM and inhibited by trifluoperazine. NG-Nitro-L-arginine and NG-methyl-L-arginine did not inhibit the cytochrome c reductase activity.

Inducible Nitric Oxide Synthase in the Anterior Pituitary Gland: Induction by Interferon-γ in a Subpopulation of Folliculostellate Cells and in an Unidentifiable Population of Non-hormone-secreting Cells

“…This discrepancy may be accounted for by the use of different stimulatory cytokines or may reflect the neoplastic state of the AtT20 cells. The latter would be in line with the presence of high levels of bc-NOS in pituitary adenomas (Lloyd et al 1995) and in the pituitary cell line GH 3 (Wolff and Datto 1992).…”

“…cNOS immunoreactivity is upregulated in pituitary adenomas (Lloyd et al 1995). High levels of bcNOS have been detected in the pituitary GH 3 cell line (Wolff and Datto 1992) and of iNOS in the corticotroph cell line AtT20 after induction with interleukin-1 ␤ (IL-1 ␤) (Ohta et al 1993).…”

Nitric oxide prevents phosphorylation of neuronal nitric oxide synthase at Serine1412 by inhibiting the Akt/PKB and CaM-K�II signaling pathways

“…5). It was shown that phosphorylation at Ser1412 by Akt/PKB is necessary for activation of nNOS by the NMDA receptor in cortical neurons (14). However, it is not clear which signaling is more susceptible to NO-mediated signaling pathways between the Akt/PKB and the CaM-K II under physiological and pathological conditions.…”

“…Here, we examined whether protein kinases Akt/PKB, CaM-K II and protein kinase A (PKA) mediate the phosphorylation of nNOS at Ser1412 and the regulatory role of NO derived from nNOS in rat pituitary tumor GH3 cells, which has been shown to express constitutive nNOS and produce NO (14).…”

Calcium(II)3 (3,5‐Diisopropylsalicylate)6(H2O)6 Activates Nitric Oxide Synthase: An Accounting for its Action in Decreasing Platelet Aggregation

“…Since a more lipophilic form of Ca may be able to undergo translocation across platelet membranes in a passive process, Calcium(II)a(3,5-diisopropylsalicylate)6(HzO) 6 [Ca(II)3(3,5-DIPS)6], a lipophilic Ca complex, was examined for its effects on platelet aggregation. Platelet aggregation was studied in an activated-platelet system containing mM ionically bonded Ca, in the form of CaCI_, and Thrombin, both being required for llatelet aggregation [4].…”