atrial-natriuretic-factor and iberiotoxin

The aim of this study was to test the hypothesis that vasorelaxing action of vasonatrin peptide (VNP) is due to activation of the large-conductance Ca(2+)-activated potassium channel (BK(Ca)) via guanylyl cyclase (GC)-coupled natriuretic peptide receptors (NPRs) in vascular smooth muscle cells (VSMCs). Contraction experiments were performed using human radial artery, whereas BK(Ca) current by patch clamp was recorded in cells from rat mesenteric artery. Contractility of rings cut from human radial artery was detected in vitro. As a result, VNP induced a dose-dependent vasorelaxation of human radial artery, which could be mimicked by 8-Br-cGMP, and suppressed by TEA, a blocker of BK(Ca), HS-142-1, a blocker of GC-coupled NPRs, or methylene blue (MB), a selective inhibitor of guanylyl cyclase. Sequentially, whole-cell K(+) currents were recorded using patch clamp techniques. BK(Ca) current of VSMCs isolated from rat mesentery artery was obtained by subtracting the whole cell currents after applications of 10(-7) mol/l iberiotoxin (IBX) from before its applications. In accordance with the results of arterial tension detection, BK(Ca) current was significantly magnified by VNP, which could also be mimicked by 8-Br-cGMP, whereas suppressed by HS-142-1, or MB. Taken together, VNP acts as a potent vasodilator, and NPRA/B-cGMP-BK(Ca) is one possible signaling system involved in VNP induced relaxation.

Topics: Animals; Atrial Natriuretic Factor; Cyclic GMP; Dose-Response Relationship, Drug; Guanylate Cyclase; Humans; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Mesenteric Arteries; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Peptides; Radial Artery; Rats; Receptors, Atrial Natriuretic Factor; Tetraethylammonium; Vasodilation

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides were investigated in epithelium-denuded guinea pig tracheal rings, treated with indomethacin (5 microM) and phosphoramidon (1 microM) and contracted with histamine (3 microM). Atrial natriuretic peptide (ANP) was a more potent relaxant than C-type natriuretic peptide whereas ANP-(4-23) was inactive suggesting the involvement of ANP(A) receptors in the relaxant effect of ANP. ODQ (1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one, 10 microM), a selective inhibitor of soluble guanylyl cyclase, markedly inhibited the relaxant response to sodium nitroprusside. The relaxant response to ANP was not altered by ODQ demonstrating the involvement of particulate guanylyl cyclase. ANP-induced relaxations, as well as sodium nitroprusside-induced relaxations, were similarly potentiated by rolipram (4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one, 3 microM), a type IV phosphodiesterase inhibitor, and by zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one, 10 microM), a type V phosphodiesterase inhibitor. ANP-mediated response was unaffected by glibenclamide (10 microM), a selective blocker of ATP-sensitive K(+) channels, and by apamin (1 microM), a selective blocker of small-conductance Ca(2+)-activated K(+) channels. Iberiotoxin (100 nM) extensively prevented the relaxant effect of ANP suggesting the activation of large-conductance Ca(2+)-activated K(+) channels. In addition, ANP (10 nM) and ANP-(4-23) (100 nM) significantly reduced forskolin (1 microM)-stimulated cAMP accumulation suggesting, for the first time, the presence of functional ANP(C) receptors in guinea pig airway smooth muscle. However, relaxations to forskolin and to isoproterenol were not altered in the presence of ANP-(4-23) or ANP demonstrating that the inhibitory effect of ANP-(4-23) and ANP on adenylyl cyclase was not sufficient to alter the functional response induced by these two activators of adenylyl cyclase.

Topics: Adenylyl Cyclases; Animals; Apamin; Atrial Natriuretic Factor; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Glyburide; Guanylate Cyclase; Guinea Pigs; Histamine; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Natriuretic Peptide, C-Type; Nitroprusside; Oxadiazoles; Peptide Fragments; Peptides; Phosphodiesterase Inhibitors; Potassium Channel Blockers; Quinoxalines; Rolipram; Trachea

We coexpressed the human large-conductance, calcium-activated K (K(Ca)) channel (alpha- and beta-subunits) and rat atrial natriuretic peptide (ANP) receptor genes in Xenopus oocytes to examine the mechanism of guanylyl cyclase stimulatory coupling to the channel. Exposure of oocytes to ANP stimulated whole cell K(Ca) currents by 21 +/- 3% (at 60 mV), without altering current kinetics. Similarly, spermine NONOate, a nitric oxide donor, increased K(Ca) currents (20 +/- 4% at 60 mV) in oocytes expressing the channel subunits alone. Stimulation of K(Ca) currents by ANP was inhibited in a concentration-dependent manner by a peptide inhibitor of cGMP-dependent protein kinase (PKG). Receptor/channel stimulatory coupling was not completely abolished by mutating the cAMP-dependent protein kinase phosphorylation site on the alpha-subunit (S869; Nars M, Dhulipals PD, Wang YX, and Kotlikoff MI. J Biol Chem 273: 14920-14924, 1998) or by mutating a neighboring consensus PKG site (S855), but mutation of both residues virtually abolished coupling. Spermine NONOate also failed to stimulate channels expressed from the double mutant cRNAs. These data indicate that nitric oxide donors stimulate K(Ca) channels through cGMP-dependent phosphorylation and that two serine residues (855 and 869) underlie this stimulatory coupling.

Topics: Animals; Atrial Natriuretic Factor; Catalytic Domain; Cyclic GMP-Dependent Protein Kinases; Gene Expression; Guanylate Cyclase; Humans; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Molecular Sequence Data; Muscle, Smooth; Mutagenesis; Nitric Oxide Donors; Nitrogen Oxides; Oocytes; Patch-Clamp Techniques; Peptides; Phosphorylation; Potassium Channels; Potassium Channels, Calcium-Activated; Rats; Receptors, Atrial Natriuretic Factor; Spermine; Xenopus laevis

The relaxant effects of intracellular concentration of cyclic guanosine monophosphate (cGMP) on spontaneous tone in human tracheal smooth muscle were investigated in comparison with guinea pig, using isometric tension records. In both human and guinea pig tracheas, application of atrial natriuretic peptide (ANP) and 8-brom cGMP (a membrane permeable analogue of cGMP) caused an inhibition of spontaneous tone in a concentration-dependent fashion. However, ANP was less potent in relaxation of tracheal smooth muscle in human than guinea pigs, and values of % relaxation induced by 1 mmol/l ANP in human and guinea pigs were 37.1 +/- 5.3 and 82.7 +/- 10.5%, respectively (n = 6). In the presence of 30 nmol/l iberiotoxin (IbTX), a potent and selective large conductance Ca(2+)-activated K+ (BKCa) channel inhibitor, relaxant actions of ANP on human tracheal smooth muscle were markedly suppressed, and values of % relaxation by 1 mmol/l ANP decreased to 8.4 +/- 1.2% (n = 6). On the other hand, 8-brom cGMP was roughly equipotent in relaxating tracheal smooth muscle in these two species, different from ANP, and inhibitory effects of 8-brom cGMP on both human and guinea pig tracheal smooth muscle were also markedly suppressed in the presence of 30 nmol/l IbTX, similar to ANP. These results demonstrate that augmentation of BKCa channel activity may play a functionally important role in the cGMP-induced relaxation in human airway smooth muscle. However, ANP may have modest potency as a bronchodilator.

Topics: Aged; Animals; Atrial Natriuretic Factor; Cyclic GMP; Guinea Pigs; Humans; In Vitro Techniques; Isometric Contraction; Large-Conductance Calcium-Activated Potassium Channels; Male; Muscle Relaxation; Muscle Tonus; Muscle, Smooth; Peptides; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated; Trachea

1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca2+ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips. 2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca2(+)-activated K+ (BKCa) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC70 values for ANP and EC50 values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 mumol/L nifedipine, an antagonist of voltage-operated Ca2+ channels (VOCC). 3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 mumol/L glibenclamide, an inhibitor of ATP-sensitive K+ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca2(+)-activated K+ channels. When these agents were applied to tissues precontracted by high (40 mmol/L) K+, the relaxant effects of these agents markedly diminished. 4. The extracellular Ca2(+)-dependent contraction was inhibited in the presence of 0.3 mumol/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration-response curves to extracellular Ca2+ (0.03-2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca2+ was affected by these agents. 5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca2+ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca2+ by ANP was suppressed. 6. We conclude that ANP and 8-brom cGMP activate BKCa channels and that the inhibition of Ca2+ influx through VOCC, mediated by BKCa channel activation, may be involved in cGMP-dependent bronchodilation.

Topics: Animals; Apamin; Atrial Natriuretic Factor; Calcium; Cyclic GMP; Cytosol; Extracellular Space; Glyburide; Guinea Pigs; Intracellular Fluid; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Nifedipine; Peptides; Potassium; Potassium Channel Blockers; Potassium Channels; Scorpion Venoms; Trachea

The functional importance of Ca++ activated K+ (K(Ca)) channels in cGMP mediated relaxation of pressurized septal arteries (internal basal diameter 213 +/- 4 microm) was investigated. Vascular tone was increased by the thromboxane A2 analogue, U-46619 and internal pressure was maintained at 60 mmHg. Vessels were tested with an endothelium independent agonist (nitroprusside) and endothelium dependent agonist (acetylcholine) of nitric oxide which activates soluble guanylate cyclase. Receptor activation of particulate guanylate cyclase was tested by atrial natriuretic peptide. Direct changes in intracellular cGMP concentration were done with the cell permeable analog, 8-Bromo-cGMP. Tetraethylammonium ion (TEA+), 1 mM, significantly inhibited relaxation to nitroprusside from 10(-7) to 10(-3) M with a maximal inhibition of 53 +/- 8% at 10(-3) M. Relaxation to acetylcholine from 10(-9) M to 10(-5) M was significantly inhibited by TEA+ with a maximal inhibition of 52 +/- 13% at 10(-7) M. TEA+ significantly inhibited relaxation to 8-Bromo-cGMP from 10(-6) M to 10(-3) M with a maximal inhibition of 59 +/- 14% at 10(-4) M. The relaxation response to atrial natriuretic peptide from 10(-12) M to 10(-7) M was significantly inhibited by TEA+ with a maximal inhibition of 84 +/- 5% at 10(-11) M. The large conductance K(Ca) channel blocker, iberiotoxin, eliminated the relaxation response to 8-Bromo-cGMP (10(-3) M). The results suggest that a large portion of the dilator action of cGMP is mediated by effects on K+ membrane channels.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Animals; Atrial Natriuretic Factor; Calcium; Coronary Vessels; Cyclic GMP; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroprusside; Peptides; Potassium Channels; Prostaglandin Endoperoxides, Synthetic; Rats; Scorpion Venoms; Tetraethylammonium; Tetraethylammonium Compounds; Thromboxane A2; Vasoconstrictor Agents