zaprinast and 6-anilino-5-8-quinolinedione

Dendroaspis natriuretic peptide (DNP), a newly-described natriuretic peptide, relaxes gastrointestinal smooth muscle. L-type calcium channel currents play an important role in regulating smooth muscle contraction. The effect of DNP on L-type calcium channel currents in gastrointestinal tract is still unclear. This study was designed to investigate the effect of DNP on barium current (I(Ba)) through the L-type calcium channel in gastric antral myocytes of guinea pigs and cGMP-pathway mechanism. The whole-cell patch-clamp technique was used to record L-type calcium channel currents. The content of cGMP in guinea pig gastric antral smooth muscle and perfusion solution was measured using radioimmunoassay. DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in perfusion medium. DNP concentration-dependently inhibited I(Ba) in freshly isolated guinea pig gastric antral circular smooth muscle cells (SMCs) of guinea pigs. DNP-induced inhibition of I(Ba) was partially blocked by LY83583, an inhibitor of guanylate cyclase. KT5823, a cGMP-dependent protein kinase (PKG) inhibitor, almost completely blocked DNP-induced inhibition of I(Ba). However, DNP-induced inhibition of I(Ba) was potentiated by zaprinast, an inhibitor of cGMP-sensitive phosphodiesterase. Taken together, DNP inhibits L-type calcium channel currents via pGC-cGMP-PKG-dependent signal pathway in gastric antral myocytes of guinea pigs.

Topics: Aminoquinolines; Animals; Calcium Channels, L-Type; Carbazoles; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Elapid Venoms; Electrophysiology; Enzyme Inhibitors; Guanylate Cyclase; Guinea Pigs; Intercellular Signaling Peptides and Proteins; Myocytes, Smooth Muscle; Peptides; Purinones; Pyloric Antrum; Radioimmunoassay

In this study, we sought to investigate the effect of dendroaspis natriuretic peptide (DNP) on calcium-activated potassium current (I K(Ca)) and its mechanism in gastric antral circular smooth muscle cells (SMCs) using the whole-cell patch-clamp technique. DNP concentration-dependently increased macroscopic I K(Ca) and spontaneous transient outward currents (STOCs) in freshly isolated guinea pig gastric antral circular SMCs. The effects of DNP on I K(Ca) and/or STOCs were not blocked by applying calcium-free bath solution or the ryanodine receptor (RyR) antagonist ryanodine (10 microM), but they were inhibited by the inositol triphosphate receptor (IP3R) inhibitor heparin or the guanylate cyclase inhibitor LY83583. Moreover, a DNP-induced increase in STOCs was potentiated by the cyclic guanosine monophosphate (cGMP)-sensitive phosphoesterase inhibitor zaprinast. In conclusion, our results suggest that DNP increases I K(Ca) in gastric antral circular SMCs by increasing cGMP production and activating IP3Rs.

Topics: Aminoquinolines; Animals; Calcium; Calcium Channel Blockers; Dose-Response Relationship, Drug; Drug Interactions; Elapid Venoms; Enzyme Inhibitors; Female; Guinea Pigs; Inositol 1,4,5-Trisphosphate Receptors; Intercellular Signaling Peptides and Proteins; Male; Membrane Potentials; Muscle Contraction; Muscle, Smooth; Myocytes, Smooth Muscle; Nicardipine; Peptides; Phosphodiesterase Inhibitors; Potassium Channels, Calcium-Activated; Purinones; Pyloric Antrum; Ryanodine; Ryanodine Receptor Calcium Release Channel

Isolated rat retina was preloaded with [(14)C]glutamate and subsequently superfused to follow release of glutamate (Glu). After 20 min of superfusion in the dark, exposure of the [(14)C]Glu preloaded rat retina to a single train of white light pulses reduced Glu efflux significantly in the absence as well as in the presence of low (4 microM) and high (0.5 mM) concentrations of the Glu uptake inhibitor trans-L-pyrrolidine-2,4-dicarboxylate (t-PDC). The dark-light response was the highest in the presence of 4 microM t-PDC by establishing a plateau at 75% +/- 7% of the tonic Glu release in the dark (100%). Displaying transient to saturating responses with increasing relative luminance, time series of four trains of white light pulses arrived at a plateau of 85% +/- 10%. The cyclic guanosine monophosphate (cGMP) phosphodiesterase inhibitor Zaprinast (200 microM) antagonized the effect of the light series, leading to a plateau of 115% +/- 9%. Exposure of the retina to the guanylyl cyclase inhibitor LY83583 (30 and 100 microM) showed fast, transient responses characterized by peaks at 90% +/- 1% and 80% +/- 3%, respectively.

Topics: Aminoquinolines; Animals; Carbon Radioisotopes; Cyclic GMP; Dark Adaptation; Dicarboxylic Acids; Enzyme Inhibitors; Glutamic Acid; Male; Neurons; Neurotransmitter Uptake Inhibitors; Organ Culture Techniques; Phosphodiesterase Inhibitors; Photic Stimulation; Purinones; Pyrrolidines; Rats; Rats, Long-Evans; Retina; Synaptic Transmission; Vision, Ocular

Hypoxic pulmonary vasoconstriction (HPV) is essential for matching lung perfusion with ventilation, thus optimizing pulmonary gas exchange. Preceding studies provided evidence for a role of both nitric oxide (NO) and superoxide/ H(2)O(2) formation in this vasoregulatory mechanism. Both agents might be operative via stimulation of guanylate cyclase with formation of the vasodilatory cyclic guanosine monophosphate (cGMP), the loss of which under conditions of hypoxia contributes to HPV. This view is challenged by the recent suggestion of increased rather than decreased superoxide/H(2)O(2) formation in hypoxia. We addressed the role of NO-dependent versus NO-independent guanylate cyclase activity in hypoxic and pharmacologically evoked vasoconstriction in perfused rabbit lungs. Two inhibitors of soluble guanylate cyclase, LY83583 (2 to 16 microM) and methylene blue (20 to 60 microM), increased baseline pulmonary artery pressure under normoxic conditions and markedly amplified the vasoconstrictor response to both hypoxia and the stable thromboxane analogue U46619. Under conditions of preblocked lung NO synthesis (N(G)-mono-methyl-L-arginine), however, additional guanylate cyclase inhibition further enhanced the vasoconstrictor response to U46619 but did not influence the strength of HPV. The selective phosphodiesterase V inhibitor Zaprinast (1 to 10 microM), used for prolongation of the cGMP half-life, reduced the hypoxia-induced pressor response to a larger extent than the pressor response to U46619. This difference was lost under conditions of preblocked NO synthesis. Equilibration of the lung perfusate with molecular NO suppressed the HPV more potently than the U46619-induced vasoconstrictor response. We conclude that NO-dependent guanylate cyclase activity has an important role in attenuating the vasoconstrictor response to alveolar hypoxia in rabbit lungs. In contrast, no evidence was obtained for a role of NO-independent cGMP formation in HPV. In this feature, HPV differs from that elicited by the thromboxane analogue U46619.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aminoquinolines; Animals; Aspirin; Blood Pressure; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Female; Guanylate Cyclase; Hypoxia; In Vitro Techniques; Lung; Male; Methylene Blue; Nitric Oxide; omega-N-Methylarginine; Pulmonary Artery; Purinones; Rabbits; Vasoconstriction; Vasoconstrictor Agents

The phototransduction mechanism of the extra-ocular photoreceptor cells Ip-2 and Ip-1 in the mollusc Onchidium ganglion was examined. Previous work showed that the depolarizing receptor potential of another extra-ocular photoreceptor cell, A-P-1 is produced by a decrease of the light-sensitive K+ conductance activated by a second messenger, cGMP and is inactivated by the hydrolysis of cGMP. Here, a hyperpolarizing receptor potential of Ip-2 or Ip-1 was associated with an increase in membrane conductance. When Ip-2 or Ip-1 was voltage-clamped near the resting membrane potential, light induced an outward photocurrent corresponding to the above hyperpolarization. The spectral sensitivity had a peak at 510 nm. The shift of reversal potentials of the photocurrent depended on the Nernst equation of K(+)-selective conductance. The photocurrent was blocked by 4-AP and L-DIL, which are effective blockers of the A-P-1 light-sensitive K+ conductance. These results suggested that the hyperpolarization is mediated by increasing a similar light-sensitive K+ conductance to that of A-P-1. The injection of cGMP or Ca2+ into a cell produced a K+ current that mimicked the photocurrent. 4-AP and L-DIL both abolished the cGMP-activated K+ current, while TEA suppressed only the Ca(2+)-activated K+ current. These results indicated that cGMP is also a second messenger that regulates the light-sensitive K+ conductance. The photocurrent was blocked by LY-83583, a guanylate cyclase (GC) inhibitor, but was unaltered by zaprinast, a phosphodiesterase (PDE) inhibitor. Together, the present results suggest that increasing the internal cGMP in Ip-2 or Ip-1 cells light-activates GC rather than inhibits PDE, thereby leading to an increase of the light-sensitive K+ conductance and the hyperpolarization.

Topics: 1-Methyl-3-isobutylxanthine; 4-Aminopyridine; 8-Bromo Cyclic Adenosine Monophosphate; Abdomen; Aminoquinolines; Animals; Cyclic GMP; Electric Conductivity; Electrophysiology; Enzyme Inhibitors; Membrane Potentials; Mollusca; Phosphodiesterase Inhibitors; Photic Stimulation; Photoreceptor Cells, Invertebrate; Potassium; Purinones; Second Messenger Systems; Tetraethylammonium; Vision, Ocular

1. Photoactivated mechanical responses that resulted from exposure to 3-NO2-1,4-dihydropyridines (3-NO2-DHP5) or NO-donors were examined in rat isolated oesophageal smooth muscle with a view to determining the role of calcium and cyclic GMP. 2. Isometric contractile force was recorded in preparations bathed in normal Tyrode or 110 mM K(+)-depolarizing solution. Exposure to (+)-PN 202791, (+/-)-Bay K 8644 and (-)-PN 2020791 or the photodegradable NO-donors, sodium nitroprusside (SNP), streptozotocin (STZ) and sodium nitrite photosensitized precontracted tunica muscularis mucosae preparations in a concentration-dependent fashion. Photosensitizing potency followed the order: (+/-)-PN 202791 > (+/-)-Bay K 8644 > (-)-PN 202791 > SNP > STZ > NaNO2. 3. A low amplitude, slow photorelaxation (slope: 1 mg s-1) was obtained with the L-channel antagonists (-)-PN 202791 and (+)-Bay K 4407. Photosensitization by the agonist enantiomers (+)-PN 202 791 and (-)-Bay K 5407, as well as racemic Bay K 8644, was mimicked by NO donors and showed at least three different components, consisting of (i) a fast relaxation (slope: 140 mg s-1), (ii) a fast "off-contraction', and (iii) a delayed slow relaxation. The fast components, but not the delayed slow relaxation, were abolished by blockade of L-type voltage-operated calcium channels, chelation of extracellular calcium and skinning of the plasmalemma, suggesting their mediation by a process linked to calcium entry through L-channels. 4. Both cyclopiazonic acid (3-30 microM) and ryanodine (30 microM) inhibited the fast response. This inhibition was accelerated in the presence of extracellular calcium and resembled that seen in tissues exposed to the calcium ionophore A 23187 (1 microM). In calcium depleted tissues, cyclopiazonic acid (3 microM) prevented restoration of the cis-dioxolane-induced contraction following re-exposure to a calcium containing high K+ buffer, but failed to inhibit the photoresponse. 5. Both the fast and slow relaxations were potentiated by zaprinast (10 microM) and inhibited by LY B3583 (10 microM). However, in calcium-depleted, calyculin A-precontracted preparations only the slow relaxation was evident. 6. The present results support the conclusion that: (i) functional L-channels are required for the expression of the fast components of the 3-NO2-DHP- or NO-donor-induced photoresponse, (ii) NO photorelease followed by activation of soluble guanylyl cyclase is responsible for the photosensitizing activity o

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; 3',5'-Cyclic-GMP Phosphodiesterases; Aminoquinolines; Animals; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium-Transporting ATPases; Chelating Agents; Egtazic Acid; Esophagus; Guanylate Cyclase; In Vitro Techniques; Indoles; Marine Toxins; Muscle, Smooth; Nicotinic Acids; Nitric Oxide; Oxadiazoles; Oxazoles; Phosphoprotein Phosphatases; Photosensitizing Agents; Purinones; Rats; Rats, Sprague-Dawley; Ryanodine

The role of cGMP as a second messenger for renin secretion is contentious. This was investigated using a superfused collagenase-dispersed rat kidney cortex cell preparation devoid of indirect influences on renin secretion. Nitroprusside, atriopeptin II and 8-Br-cGMP all increased renin release but the dose-response relationships were biphasic. At low dose ranges there was a positive correlation between increasing drug concentration and renin secretion, but at high drug concentrations, a negative correlation was apparent. Methylene blue, a guanylate cyclase inhibitor, also suppressed baseline renin release at 10(-5) and 10(-6) M, but stimulated release at 10(-3) M. Using mid-range drug concentrations, the cGMP specific phosphodiesterase inhibitor MB22948 potentiated renin release in response to nitroprusside and 8-Br-cGMP. Inhibition of guanylate cyclase with either methylene blue or LY83583 attenuated renin release in response to nitroprusside, but, as expected, had no effect on 8-Br-cGMP induced release. We conclude that, under physiological conditions, cGMP is a stimulatory second messenger for renin release. This activity is mimicked at low dose ranges by 8-Br-cGMP, nitroprusside and atriopeptin II. In response to high doses of these drugs an unknown inhibitory pathway is activated and this opposes, in a dose-related manner, the stimulatory actions of cGMP for renin release.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aminoquinolines; Animals; Atrial Natriuretic Factor; Cyclic GMP; Guanylate Cyclase; In Vitro Techniques; Kidney Cortex; Methylene Blue; Nitroprusside; Peptide Fragments; Purinones; Rats; Renin; Second Messenger Systems

We have demonstrated previously that in response to hypoxia, isolated rat pulmonary arteries show an initial endothelium-dependent relaxation followed by an endothelium-independent transient contraction. In the presence of increased extracellular Ca2+, both of these responses were enhanced in endothelium-intact arteries. Nitro-L-arginine, a blocker of the biosynthesis of endothelium-derived relaxing factor (EDRF), abolished the initial endothelium-dependent relaxation and Ca(2+)-induced enhancement of hypoxic contraction in endothelium-intact arteries but did not alter responses in endothelium-denuded vessels. Inhibition of prostaglandin formation with indomethacin had no effect on the hypoxia-elicited responses. Preincubation with LY 83583, an inhibitor of guanylate cyclase activation, abolished the initial hypoxia-elicited relaxation and subsequent contraction. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor, decreased tone under O2 but not under N2, causing an apparent enhancement of the contraction to hypoxia. Thus the modulation of hypoxic responses by the endothelium is dependent on changes in EDRF production, and a decrease in smooth muscle cGMP not involving an EDRF mechanism appears to mediate the endothelium-independent hypoxic contraction observed in the isolated rat pulmonary artery.

Topics: Aminoquinolines; Animals; Arginine; Cyclic GMP; Hypoxia; In Vitro Techniques; Indomethacin; Male; Nitroarginine; Pulmonary Artery; Purinones; Rats; Rats, Inbred Strains

Previously we showed that atrial natriuretic factor (ANF) decreases cardiac cell volume by inhibiting ion uptake by Na+/K+/2Cl- cotransport. Digital video microscopy was used to study the role of guanosine 3',5'-monophosphate (cGMP) in this process in rabbit ventricular myocytes. Each cell served as its own control, and relative cell volumes (volume(test)/volume(control)) were determined. Exposure to 10 microM 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 +/- 0.007; the ED50 was 0.77 +/- 0.33 microM. Activating guanylate cyclase with 100 microM sodium nitroprusside also decreased cell volume to 0.889 +/- 0.009. In contrast, 8-bromo-adenosine 3',5'-monophosphate (8-Br-AMP; 0.01-100 microM) neither altered cell volume directly nor modified the response to 8-Br-cGMP. The idea that cGMP decreases cell volume by inhibiting Na+/K+/2Cl- cotransport was tested by blocking the cotransporter with 10 microM bumetanide (BUM) and removing the transported ions. After BUM treatment, 10 microM 8-Br-cGMP failed to decrease cell volume. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methanesulfonate also prevented 8-Br-cGMP from shrinking cells. The data suggest that 8-Br-cGMP, like ANF, decreases ventricular cell volume by inhibiting Na+/K+/2Cl-cotransport. Evidence that ANF modulates cell volume via cGMP was also obtained. Pretreatment with 10 microM 8-Br-cGMP prevented the effect of 1 microM ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10 microM) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprinast; 100 microM) amplified the volume decrease caused by a low dose of ANF (0.01 microM) approximately fivefold. In contrast, neither 100 microM 8-Br-cAMP nor 50 microM forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by 125I-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2Cl- cotransport. Basal cGMP levels also appear to modulate cell volume.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aminoquinolines; Animals; Atrial Natriuretic Factor; Chlorides; Cyclic GMP; Guanylate Cyclase; In Vitro Techniques; Myocardium; Potassium; Purinones; Rabbits; Second Messenger Systems; Sodium; SRS-A

Atrial natriuretic factor (ANF) has been shown to bind to specific receptors on vascular smooth muscle cells (VSMC) and to cause an increase in intracellular cyclic GMP (cGMP) content. We have recently demonstrated that a prominent Na+,K+,Cl- cotransport system is present in VSMC and that a permeable cGMP analog (8-bromo-cGMP) stimulates activity of the cotransporter. We have also shown that the ANF peptide, rat atriopeptin III, stimulates Na+,K+,Cl- cotransport and elevates intracellular cGMP levels in VSMC. In the present study, we tested the hypothesis that ANF stimulation of Na+,K+,Cl- cotransport occurs via an increase in cGMP levels. When the quinolinedione, 6-anilo-5,8-quinolinedione (LY83583) (10 microM), was used to block formation of cGMP in VSMC from primary cultures of rat thoracic aorta, it was found that both basal and rat atriopeptin III (100 nM)-stimulated Na+,K+,Cl- cotransport were significantly inhibited. The effect of LY83583 was dose-dependent and the half-maximal inhibitory concentration was 0.5 microM. LY83583 also inhibited cotransport in the presence of a maximal concentration of 8-bromo-cGMP. However, this inhibition was not seen in cells also treated with 2-O-propoxyphenyl-8-azapurin-6-one (M&B 22,948), an inhibitor of cGMP phosphodiesterase. M&B 22,948 alone also increased levels of cotransport. Since inhibition of cGMP formation blocks ANF-stimulated Na+,K+,Cl- cotransport and inhibition of cGMP breakdown enhances Na+, K+, Cl- cotransport, we conclude that ANF stimulation of Na+,K+,Cl- cotransport in VSMC is mediated via increase in intracellular cGMP levels.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Aminoquinolines; Animals; Aorta, Thoracic; Atrial Natriuretic Factor; Carrier Proteins; Cyclic GMP; Muscle, Smooth, Vascular; Purinones; Rats; Sodium-Potassium-Chloride Symporters