okadaic-acid and alpha-beta-methyleneadenosine-5--triphosphate

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K(i) = 64 microM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P(2)) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-gamma-S, whereas alpha, beta-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P(2)Y(2) purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N(G)-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P(2)Y(2) receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.

Topics: Adenosine Triphosphate; Affinity Labels; Alkaloids; Animals; Carbazoles; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Extracellular Space; Indoles; Ion Channel Gating; Kidney Tubules, Collecting; Membrane Potentials; Mice; Mice, Inbred C57BL; Naphthalenes; NG-Nitroarginine Methyl Ester; Okadaic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Phosphorylation; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Protein Kinases; Rats; Rats, Sprague-Dawley; Receptors, Purinergic; Small-Conductance Calcium-Activated Potassium Channels; Thionucleotides; Uridine Triphosphate

The effects of okadaic acid (30 microM), a protein phosphatase inhibitor, on noradrenaline (NA) release evoked by 70 mM KCl and 100 microM ouabain were evaluated in guinea-pig vas deferens. Release of NA evoked by high KCl was inhibited by okadaic acid but this inhibition was antagonized by Bay K 8644. Furthermore, okadaic acid, like Ca(2+)-channel blockers, reduced NA release by ouabain. However, ATP-release induced by alpha,beta-methylene ATP was virtually unaffected by okadaic acid or Ca(2+)-free medium. These findings suggest that phosphatases may play an important role in Ca(2+)-channel activation and consequent NA exocytosis from adrenergic nerves.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine Triphosphate; Animals; Calcium; Calcium Channels; Ethers, Cyclic; Exocytosis; Guinea Pigs; In Vitro Techniques; Kinetics; Male; Norepinephrine; Okadaic Acid; Ouabain; Phosphoprotein Phosphatases; Potassium Chloride; Time Factors; Vas Deferens

We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATP. ATP gamma S (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinases, leading to irreversible protein thiophosphorylation (Gratecos, D., and E.H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP gamma S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATP gamma S requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATP gamma S irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATP gamma S. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATP gamma S, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calcium-stimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Calcium; Cell Membrane; Cytoplasmic Granules; Ethers, Cyclic; Exocytosis; Fertilization; Intracellular Membranes; Membrane Fusion; Okadaic Acid; Ovum; Phosphoproteins; Phosphorylation; Sea Urchins; Signal Transduction; Time Factors