Channel modulators affect PGE(2) binding to bovine aortic endothelial cells.
ABSTRACT PGE(2), PGF(2alpha) and the thromboxane agonist U-46619 bind to bovine aortic endothelial cells and compete on the same binding site with similar affinity. In addition, binding remains unaffected by prolonged exposure to the ligand. These characteristics differ significantly from those of any known G-coupled prostaglandin receptor. Binding of PGE(2) to the cells is reduced in the presence of the cyclic nucleotides cGMP and cAMP, and is unaffected by protein kinase inhibitors. Removal of permeable cyclic nucleotides from the cell medium results in a fast and complete restoration of PGE(2) binding to the cells, suggesting that both cyclic nucleotides reduce PGE(2) binding by a reversible interaction with the prostaglandin-binding site, without the involvement of second messenger-activated protein kinases. Our data further show that binding of prostaglandins to bovine aortic endothelial cells is sensitive to heavy metals and to activators and blockers of calcium, ATP-sensitive K(+) and chloride channels. Nickel, a specific cyclic nucleotide-gated (CNG) channel activator, decreases PGE(2) binding and so do the CNG channel activators Rp-8-Br-PET-cGMPS and Sp-8-Br-PET-cGMPS. On the other hand, the calcium channel blockers pimozide, diltiazem as well as LY-83,583, a guanylate cyclase inhibitor, which were reported to block CNG channels, enhance PGE(2) binding. The sensitivity of PGE(2) binding to selective CNG channel modifying agents, as well as the rapid and reversible interaction with cyclic nucleotides, may suggest that the common low-affinity prostanoid-binding site on bovine aortic endothelial cells is associated with a molecular entity, which possess several properties of a CNG channel.
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ABSTRACT: Sequences specific for cyclic nucleotide-gated channels (CNG channels) have been amplified by PCR from cDNA of heart, aorta, sino-atrial node, cerebellum, C-cells and kidney. The complete amino acid sequence of a CNG channel from rabbit aorta has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CNG channel in Xenopus oocytes.FEBS Letters 09/1993; 329(1-2):134-8. · 3.58 Impact Factor
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ABSTRACT: The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.The Journal of General Physiology 06/1999; 113(5):601-20. · 4.73 Impact Factor
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ABSTRACT: Calcium agonists induce membrane depolarization in endothelial cells through an unknown mechanism. Present studies tested the hypothesis that pulmonary artery endothelial cells express a cyclic nucleotide-gated (CNG) cation channel activated by store-operated calcium entry to produce membrane depolarization. In the whole-cell configuration, voltage-clamped cells revealed a large non-inactivating, outwardly rectifying cationic current in the absence of extra- or intracellular Ca(2+) that was reduced upon replenishment of Ca(2+). The inward current was non-selective for K(+), Na(+), Cs(+), and Rb(+) and was not inhibited by high tetraethylammonium concentrations. cAMP and cGMP stimulated the current and changed the cation permeability to favor Na(+). Moreover, 8-bromo-cAMP stimulated the current in voltage-clamped cells in the perforated patch mode. The cationic current was inhibited by the CNG channel blocker LY83,583, and reverse transcriptase-polymerase chain reaction cloning identified expression of a CNG channel resembling that seen in olfactory neurons. Activation of store-operated calcium entry using thapsigargin increased a current through the CNG channel. Stimulation of the current paralleled pulmonary artery endothelial cell membrane depolarization, and both the current and membrane depolarization were abolished using LY83,583. Taken together, these data demonstrate activation of store-operated calcium entry stimulates a CNG channel producing membrane depolarization. Such membrane depolarization may contribute to slow feedback inhibition of store-operated calcium entry.Journal of Biological Chemistry 07/2000; 275(25):18887-96. · 4.65 Impact Factor