ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i.
We investigated the relationship between the ATP-evoked rise of cytosolic Ca2+ concentration ([Ca2+]i) and barrier function in porcine aortic endothelial monolayers. ATP (0.01-100 microM) induced a transient rise of [Ca2+]i and reduced permeability in a concentration-dependent manner. In contrast, the Ca2+ ionophore ionomycin (1 microM) elicited a rise in [Ca2+]i comparable to that induced by ATP (10 microM), but it increased permeability. For the reduction of permeability, nucleotides were found to be in the following order of potency: ATP = ATPgammaS > ADP = UTP. Blockade of adenosine receptors by 8-phenyltheophylline (10 microM) did not affect ATP (10 microM)-induced reduction of permeability. ATP reduced permeability even in endothelial monolayers that had been loaded with the Ca2+ chelator BAPTA to prevent the rise in [Ca2+]i. U-73122 (1 microM), an inhibitor of phospholipase C (PLC), completely abolished the effect of ATP (10 microM) on permeability. It also abolished the translocation of protein kinase C (PKC) in response to ATP, which could also be achieved by the PKC inhibitors Go-6976 (100 nM) or bisindolylmaleimide I (1 microM). In the presence of PKC inhibitors, however, the permeability effect of ATP was not affected. The presence of inhibitors of adenylate or guanylate cyclase (50 microM SQ-22536 or 20 microM ODQ) prevented changes in cyclic nucleotides but did not affect the permeability effects of ATP. The study shows that ATP reduces macromolecule permeability via a PLC-mediated mechanism that is independent of the concomitant effects of ATP on cytosolic Ca2+, cyclic nucleotides, or PKC. (+info
NO causes perinatal pulmonary vasodilation through K+-channel activation and intracellular Ca2+ release.
Evidence suggests that nitric oxide (NO) causes perinatal pulmonary vasodilation through K+-channel activation. We hypothesized that this effect worked through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channel that requires release of intracellular Ca2+ from a ryanodine-sensitive store. We studied the effects of 1) K+-channel blockade with tetraethylammonium, 4-aminopyridine, a voltage-dependent K+-channel blocker, or glibenclamide, an ATP-sensitive K+-channel blocker; 2) cyclic nucleotide-sensitive kinase blockade with either KT-5823, a guanylate-sensitive kinase blocker, or H-89, an adenylate-sensitive kinase blocker; and 3) blockade of intracellular Ca2+ release with ryanodine on NO-induced pulmonary vasodilation in acutely prepared late-gestation fetal lambs. N-nitro-L-arginine, a competitive inhibitor of endothelium-derived NO synthase, was infused into the left pulmonary artery, and tracheotomy was placed. The animals were ventilated with 100% oxygen for 20 min, followed by ventilation with 100% oxygen and inhaled NO at 20 parts/million (ppm) for 20 min. This represents the control period. In separate protocols, the animals received an intrapulmonary infusion of the different blockers and were ventilated as above. Tetraethylammonium (n = 6 animals) and KT-5823 (n = 4 animals) attenuated the response, whereas ryanodine (n = 5 animals) blocked NO-induced perinatal pulmonary vasodilation. 4-Aminopyridine (n = 5 animals), glibenclamide (n = 5 animals), and H-89 (n = 4 animals) did not affect NO-induced pulmonary vasodilation. We conclude that NO causes perinatal pulmonary vasodilation through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channels and release of Ca2+ from ryanodine-sensitive stores. (+info
Isolation of a single carboxyl-carboxylate proton binding site in the pore of a cyclic nucleotide-gated channel.
The pore of the catfish olfactory cyclic nucleotide-gated (CNG) channel contains four conserved glutamate residues, one from each subunit, that form a high-affinity binding site for extracellular divalent cations. Previous work showed that these residues form two independent and equivalent high-pKa (approximately 7.6) proton binding sites, giving rise to three pH-dependent conductance states, and it was suggested that the sites were formed by pairing of the glutamates into two independent carboxyl-carboxylates. To test further this physical picture, wild-type CNG subunits were coexpressed in Xenopus oocytes with subunits lacking the critical glutamate residue, and single channel currents through hybrid CNG channels containing one to three wild-type (WT) subunits were recorded. One of these hybrid channels had two pH-dependent conductance states whose occupancy was controlled by a single high-pKa protonation site. Expression of dimers of concatenated CNG channel subunits confirmed that this hybrid contained two WT and two mutant subunits, supporting the idea that a single protonation site is made from two glutamates (dimer expression also implied the subunit makeup of the other hybrid channels). Thus, the proton binding sites in the WT channel occur as a result of the pairing of two glutamate residues. This conclusion places these residues in close proximity to one another in the pore and implies that at any instant in time detailed fourfold symmetry is disrupted. (+info
Levosimendan: effects of a calcium sensitizer on function and arrhythmias and cyclic nucleotide levels during ischemia/reperfusion in the Langendorff-perfused guinea pig heart.
The majority of clinically used inotropes act by increasing cytosolic calcium levels, which may hypothetically worsen reperfusion stunning and provoke arrhythmias. We tested the hypothesis that the calcium sensitizer levosimendan (levo) given during ischemia alone or ischemia and reperfusion would improve reperfusion function without promoting arrhythmias. The Langendorff-perfused guinea pig heart, subjected to 40-min low-flow ischemia (0.4 ml/min) with or without levo (10-300 nM) given during ischemia or ischemia/reperfusion was used. Left ventricular developed pressure (LVDP) was used as an index of mechanical function. The effect of levo (300 nM) or dobutamine (0.1 microM) on the incidence of ischemia/reperfusion arrhythmias was also investigated. Control hearts (vehicle-perfused) had LVDPs of 69.4 +/- 2.1 mm Hg whereas hearts treated with levo during ischemia and reperfusion (300 nM) had LVDPs of 104.5 +/- 2.7 mm Hg (p <.05). Hearts treated with levo during ischemia alone (10 nM) had reperfusion LVDPs of 95.8 +/- 4.2 mm Hg (p <.05) after 30-min reperfusion. Hearts treated with both levo and 10 microM glibenclamide (K(ATP) channel blocker) during ischemia had reperfusion LVDPs of 73.4 +/- 4.3 mm Hg after 30-min reperfusion. Of control hearts, 25% developed reperfusion ventricular tachycardia but not ventricular fibrillation. Levo-treated hearts had no ischemia/reperfusion arrhythmias whereas 83% (p <.05 versus control) of dobutamine-treated hearts developed ventricular tachycardia and 33% (p <.05 versus levo) developed reperfusion ventricular fibrillation. Levo improved reperfusion function without promoting arrhythmias in this model. This was possibly achieved by opening the K(ATP) channels during ischemia and sensitizing myocardial contractile apparatus instead of elevating cytosolic calcium levels in reperfused hearts. (+info
Temporal and spatial pattern of expression of cyclic nucleotide-gated channels in developing rat visual cortex.
Cyclic nucleotide-gated channels, ligand-gated and highly permeable to calcium, are good candidates for transducing signals received by migrating cells, growth cones and developing synapses. The level of calcium in growth cones is important for axon guidance. Further, cyclic nucleotides, whose levels can be altered by nitric oxide and other transmitters, are known to alter growth cone motility. We use rat visual cortex as a model in our semi-quantitative RT-PCR and in situ hybridization studies to determine the developmental time course and localization of all three CNG family members (rod, olfactory and cone/testis). We demonstrate that in the cortex, the three channel subtypes are each expressed in a distinct temporal and spatial pattern in only sensorimotor and occipital regions of the cortex. Specifically, the rod and olfactory subtypes are present at the time of migration and rapid dendritic outgrowth, and the cone/testis subtype is highly expressed after eye opening. These results suggest CNG channels may play a role in both early and late events in visual cortical development. (+info
The calcium/calmodulin-dependent phosphodiesterase PDE1C down-regulates glucose-induced insulin secretion.
To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic beta-cell line and used family and isozyme-specific PDE inhibitors to identify the PDEs that counteract glucose-stimulated insulin secretion. We demonstrate the presence of soluble PDE1C, PDE4A and 4D, a cGMP-specific PDE, and of particulate PDE3, activities in betaTC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmented glucose-stimulated insulin secretion in a dose-dependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from betaTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of betaTC3 cells is a novel isozyme possessing a K(m) of 0.47 microM for cAMP and 0.25 microM for cGMP. The PDE1C isozyme of betaTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC(50) = 7.5 and 4.5 microM, respectively) and resistant to vinpocetine (IC(50) > 100 microM). Increased responsiveness of PDE1C activity to calcium/calmodulin is evident upon exposure of cells to glucose. Enhanced cAMP degradation by PDE1C, due to increases in its responsiveness to calcium/calmodulin and in intracellular calcium, constitutes a glucose-dependent feedback mechanism for the control of insulin secretion. (+info
Active site characterization of RNase Rs from Rhizopus stolonifer: involvement of histidine and lysine in catalysis and carboxylate in substrate binding.
Chemical modification studies on purified RNase Rs revealed the involvement of a single histidine, lysine and carboxylate residue in the catalytic activity of the enzyme. RNA could not protect the enzyme against DEP- and TNBS-mediated inactivation whereas, substrate protection was observed in case of EDAC-mediated inactivation of the enzyme. K(m) and k(cat) values of the partially inactivated enzyme samples suggested that while histidine and lysine are involved in catalysis, carboxylate is involved in substrate binding. Active site nature of RNase Rs suggests that the inability of the enzyme to readily convert 2',3'-cyclic nucleotides to 3'-mononucleotides is probably due to the absence of catalytically active second histidine residue. (+info
Fluid secretion by isolated Malpighian tubules of Drosophila melanogaster Meig.: effects of organic anions, quinacrine and a diuretic factor found in the secreted fluid.
Para-aminohippuric acid (PAH, 0.2 and 1 mmol l(-)(1)) had no effect on the basal fluid secretion rate (FSR) of isolated Malpighian tubules of Drosophila melanogaster Meig. and did not affect stimulation of the FSR induced by adenosine 3',5'-monophosphate (cAMP). Phenol Red (phenolsulphonphthalein, PSP; 0.5 and 1 mmol l(-)(1)) slowed the FSR and abolished stimulation of the FSR by cAMP. Diodrast (1 mmol l(-)(1)) slightly, but significantly, reduced the FSR and greatly reduced the stimulation of the FSR normally provoked by cAMP and by the 3',5'-monophosphates of guanosine (cGMP), inosine (cIMP) and uridine (cUMP). However, stimulation of the FSR by the 3', 5'-monophosphate of cytidine (cCMP) was little affected by diodrast. Probenecid (0.2 or 1 mmol l(-)(1)) consistently stimulated the FSR, on average by approximately 25 %, but did not markedly inhibit the subsequent stimulation of the FSR by cAMP, cGMP or cIMP. However, the FSR of tubules stimulated by cGMP was temporarily lowered by probenecid. Quinacrine (0.1 mmol l(-)(1)) slowed basal FSR by an average of approximately 30 %, but subsequent stimulation of the FSR by cAMP was not noticeably affected. Both 0.1 mmol l(-)(1) cAMP and 1 mmol l(-)(1) probenecid stimulated adenylate cyclase activity in extracts of Malpighian tubules, but cIMP, cGMP, cUMP and diodrast were without effect in this regard. Uptake of radioactivity from a solution containing 500 nmol l(-)(1) [(3)H]cAMP and 9.5 micromol l(-)(1) cAMP was reduced by more than 90 % by 1 mmol l(-)(1) PSP, by approximately 40 % by 0.2 mmol l(-)(1) probenecid, by 36 % by 1 mmol l(-)(1) diodrast and by 30 % by 1 mmol l(-)(1) PAH. Neither 0.01 mmol l(-)(1) ouabain nor 0.1 mmol l(-)(1) quinacrine affected the uptake of [(3)H]cAMP by the Malpighian tubules. Fluid secreted by isolated Malpighian tubules of Drosophila melanogaster contains a factor that stimulated the FSR on average by approximately 50 %. The presence in the secreted fluid of cGMP at a concentration of 8.3 micromol l(-)(1) did not explain the stimulatory effect on FSR. These results support the existence of a carrier-mediated uptake of cyclic nucleotides into the Malpighian tubules of Drosophila melanogaster, possibly involving a multispecific transporter. (+info