8-bromoguanosino-3--5--cyclic-monophosphorothioate and zaprinast

This study investigates the effect of protein kinase G (PKG) activation upon proliferation of human cultured prostatic stromal cells. The PKG II activator (8-pCPT-cGMP; IC50 of 113+/-42 nM) and the phosphodiesterase inhibitor, zaprinast (up to 50 microM), but not the PKG I isoform activators (APT-cGMP and PET-cGMP), reduced foetal calf serum-stimulated proliferation. The effect of 8-pCPT-cGMP (30 microM) was blocked by Rp-8-Br-cGMPS (5 microM) and Rp-8-pCPT-cGMP (5 microM), but not Rp-cAMPS (5 microM). 8-pCPT-cGMP (30 microM) and zaprinast (50 microM), but not PET-cGMP (30 microM), caused a significant increase in atypical nuclei and an increase in annexin-V staining. These data indicate that activation of PKG II induces apoptosis of human cultured prostatic stromal cells.

Topics: Aged; Apoptosis; Azides; Cell Division; Cell Nucleus; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Humans; Isoenzymes; Male; Phosphoric Diester Hydrolases; Prostate; Prostatic Hyperplasia; Purinones; Serum; Stromal Cells; Thionucleotides

Adenosine is an important regulator of neuronal excitability. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to suppress excitation. This action can be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by exposure to zaprinast. To explore the mechanism of this phenomenon further, we examined the effect of zaprinast on adenosine release itself in cultured rat forebrain neurons. Zaprinast significantly stimulated extracellular adenosine accumulation. The effect of zaprinast on adenosine appeared to be mediated by increasing intracellular cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA): (i) zaprinast stimulated intracellular cAMP accumulation; (ii) a cAMP antagonist (Rp-8-Br-cAMP) significantly reduced the zaprinast effect on adenosine; (iii) an inhibitor of phosphodiesterase (PDE)1 (vinpocetine) and an activator of adenylate cyclase (forskolin) mimicked the effect of zaprinast on adenosine. We also found that zaprinast had no effect on adenosine in astrocyte cultures, and tetrodotoxin completely blocked zaprinast-evoked adenosine accumulation in neuronal cultures, suggesting that neuronal activity was likely to be involved. Consistent with a dependence on neuronal activity, NMDA receptor antagonists (MK-801 and D-APV) and removal of extracellular glutamate by glutamate-pyruvate transaminase blocked the effect of zaprinast. In addition, zaprinast was shown to stimulate glutamate release. Thus, our data suggest that zaprinast-evoked adenosine accumulation is likely to be mediated by stimulation of glutamate release by a cAMP- and PKA-dependent mechanism, most likely by inhibition of PDE1 in neurons. Furthermore, regulation of cAMP, either by inhibiting cAMP-PDE activity or by stimulating adenylate cyclase activity, may play an important role in modulating neuronal excitability. These data suggest the existence of a homeostatic negative feedback loop in which increases in neuronal activity are damped by release of adenosine following activation of glutamate receptors.

Topics: Adenosine; Animals; Animals, Newborn; Astrocytes; Cells, Cultured; Cyclic AMP; Cyclic GMP; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Activation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Extracellular Space; Female; Glutamic Acid; Intracellular Space; Male; Membrane Potentials; Models, Neurological; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Pregnancy; Prosencephalon; Purinones; Rats; Rats, Sprague-Dawley; Thionucleotides

1. The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10(-1) M), NO (10(-10) - 10(-6) M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura-2 fluorimetry. 2. Zaprinast (10(-4) M), increased NO-stimulated levels of cyclic GMP by 3-20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. 3. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10(-5) M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10(-10) - 10(-8) M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10(-7) - 10(-6) M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10(-7) - 10(-5) M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. 4. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Angiotensin II; Animals; Aorta; Calcium; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Manganese; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Oxadiazoles; Phosphodiesterase Inhibitors; Protein Kinase C; Purinones; Quinoxalines; Rabbits; Thionucleotides; Vasoconstrictor Agents; Vasodilator Agents