Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1–9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover,Bacillus anthracisandBacillus pertussisproduce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2′(3′)-O-(N-methylanthraniloyl)-guanosine 5′-[γ-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl2.N-Methylanthraniloyl (MANT)-GTP inhibited C1·C2 with aKiof 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5′-[γ-thio]triphosphate potently inhibited C1·C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5′-[γ-thio]triphosphate and uridine 5′-[γ-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2′,5′-dideoxyadenosine 3′-triphosphate was the most potent compound (IC50, 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC withKivalues in the 200–400 nmrange. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl2against MgCl2reduced inhibitor potencies at ACs and sGC 1.5–250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.

Download Full-text

Molecular Analysis of the Interaction of Anthrax Adenylyl Cyclase Toxin, Edema Factor, with 2′(3′)-O-(N-(methyl)anthraniloyl)-Substituted Purine and Pyrimidine Nucleotides

Molecular Pharmacology ◽

10.1124/mol.108.052340 ◽

2008 ◽

Vol 75 (3) ◽

pp. 693-703 ◽

Cited By ~ 28

Author(s):

Hesham M. Taha ◽

Jennifer Schmidt ◽

Martin Göttle ◽

Srividya Suryanarayana ◽

Yuequan Shen ◽

...

Keyword(s):

Adenylyl Cyclase ◽

Molecular Analysis ◽

Pyrimidine Nucleotides ◽

Edema Factor ◽

Purine And Pyrimidine Nucleotides

Download Full-text

Spontaneous and Receptor-Controlled Soluble Guanylyl Cyclase Activity in Anterior Pituitary Cells

Molecular Endocrinology ◽

10.1210/mend.15.6.0648 ◽

2001 ◽

Vol 15 (6) ◽

pp. 1010-1022 ◽

Cited By ~ 26

Author(s):

Tatjana S. Kostic ◽

Silvana A. Andric ◽

Stanko S. Stojilkovic

Keyword(s):

Adenylyl Cyclase ◽

Guanylyl Cyclase ◽

Anterior Pituitary ◽

Soluble Guanylyl Cyclase ◽

Gh3 Cells ◽

Pituitary Cells ◽

Cgmp Production ◽

Pituitary Tissue ◽

Anterior Pituitary Cells ◽

Inducible Nos

Abstract Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH3 immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH3 cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH3 cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-γ further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC50s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.

Download Full-text

Dependence of Electrical Activity and Calcium Influx-Controlled Prolactin Release on Adenylyl Cyclase Signaling Pathway in Pituitary Lactotrophs

Molecular Endocrinology ◽

10.1210/me.2005-0363 ◽

2006 ◽

Vol 20 (9) ◽

pp. 2231-2246 ◽

Cited By ~ 28

Author(s):

Arturo E. Gonzalez-Iglesias ◽

Yonghua Jiang ◽

Melanija Tomić ◽

Karla Kretschmannova ◽

Silvana A. Andric ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Adenylyl Cyclase ◽

Guanylyl Cyclase ◽

Cyclic Nucleotide ◽

Action Potentials ◽

Soluble Guanylyl Cyclase ◽

Cgmp Production ◽

Camp And Cgmp ◽

Kinase A

Abstract Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+ influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenyltio)-2′-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.

Download Full-text