8-bromo cGMP Research Articles

Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal

Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

Sildenafil Effect on the Human Bladder Involves the L-cysteine/Hydrogen Sulfide Pathway: A Novel Mechanism of Action of Phosphodiesterase Type 5 Inhibitors

BackgroundPhosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the treatment of lower urinary tract symptom (LUTS), although their mechanism of action is still unclear. PDE5-Is cause bladder detrusor relaxation, and this effect is partially independent of nitric oxide. Hydrogen sulfide (H2S) is a newly discovered transmitter with myorelaxant properties. It is predominantly formed from L-cysteine by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). ObjectiveTo evaluate whether the L-cysteine/H2S pathway contributes to the relaxing effect of sildenafil on the human detrusor dome. Design, setting, and participantsSamples of bladders obtained from men undergoing open prostatectomy for benign prostatic hyperplasia (BPH) were used. The presence of CBS and CSE enzymes was assessed by western blot. H2S production was measured by a colorimetric assay in basal and stimulated conditions with L-cysteine and in response to sildenafil (1, 3, 10, and 30μM), 8-bromo–cyclic guanosine monophosphate (8-bromo–cGMP; 100μM) or dibutyryl–cyclic adenosine monophosphate (dibutyryl-cAMP; 100μM). A curve concentration effect of sodium hydrosulfide (NaHS), H2S donor (0.1μM to 10mM), L-cysteine (0.1μM to 10mM), and sildenafil (0.1–10μM) was performed on precontracted detrusor dome strips. To investigate H2S signaling in a sildenafil effect, CBS and CSE inhibitors were used. Outcome measurements and statistical analysisAnalysis of variance was used, followed by the Bonferroni post hoc test. Results and limitationsCBS and CSE are present in the human bladder dome and efficiently convert L-cysteine into H2S. Both NaHS and L-cysteine relaxed human strips. Sildenafil caused (1) a relaxation of bladder dome strips and (2) a concentration-dependent increase in H2S production. Both effects were significantly reduced by CBS and CSE inhibitors. Similar to sildenafil, both 8-bromo-cGMP and dibutyryl-cAMP caused an increase in H2S production. ConclusionsThe sildenafil relaxant effect on the human bladder involves the H2S signaling pathway. This effect may account in part for the efficacy of PDE5-Is in LUTS. A better definition of the pathophysiologic role of the H2S pathway in the human bladder may open new therapeutic approaches.

Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors

BackgroundLinaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out.ResultsIn ischemia-damaged pig jejunum, using measurements of transepithelial resistance, 3H-mannitol fluxes, short-circuit current (Cl− secretion) and occludin localization, active linaclotide failed to effectively promote repair of the epithelial barrier or recovery of short-circuit current, whereas lubiprostone promoted barrier repair and increased short-circuit current. In control pig jejunum, 1 μM linaclotide and 1 μM lubiprostone both caused similar increases in short-circuit current (Cl− secretion). In T84 cells, using measurements of transepithelial resistance, fluxes of fluorescent macromolecules, occludin and mitochondrial membrane potential, active linaclotide was virtually ineffective against damage caused by interferon-γ and tumor necrosis factor-α, while lubiprostone protected or promoted repair of epithelial barrier and cell function. Barrier protection/repair by lubiprostone was inhibited by methadone, a ClC-2 inhibitor. Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca2+]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide. In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin. However the physiological implications of these small but statistically significant changes remain unclear.ConclusionsConsidering the physiological importance of epithelial barrier function and cell integrity and the known impact of stressors, the finding that lubiprostone, but not active linaclotide, exhibits the additional distinct property of effective protection or repair of the epithelial barrier and cell function after stress suggests potential clinical importance for patients with impaired or compromised barrier function such as might occur in IBS.

Inhibition of calcifying nodule formation in cultured porcine aortic valve cells by nitric oxide donors

Calcific aortic stenosis displays some similarities to atherosclerosis including evidence of endothelial dysfunction. Whether nitric oxide (NO), which is produced by valvular endothelium, has direct protective effects extending to calcification processes in aortic valve cells has not previously been examined. In vitro calcifying nodules in porcine aortic valve interstitial cell cultures, formed in response to transforming growth factor-beta1 (TGF-β1) 5 ng/ml, were inhibited by NO donors DETA-NONOate 5–100 µM, and sodium nitroprusside (SNP) 3 µM. Raising intracellular cGMP concentrations, via 8-bromo cGMP 1 mM or via brain natiuretic peptide and C-type natiuretic peptide 0.1 µM, inhibited TGF-β1-induced nodule formation, potentially implicating the cGMP pathway in the NO effect. Stimulation of interstitial cells with substance P or calcium ionophone (A23187) caused NO release and increased intracellular cGMP respectively. However in the presence of TGF-β1 basal levels of NO production via nitric oxide synthase (NOS) were insufficient to affect nodule formation. Increased dihydroethidium (DHE) fluorescence in response to TGF-β1, which was inhibited by DETA-NONOate and TEMPOL, suggested a role for intracellular superoxide in TGF-β1 signalling. Moreover, nodule formation was suppressed by superoxide scavengers TEMPOL, hydralazine and polyethylene glycol-superoxide dismutase (PEG-SOD), but not SOD. In conclusion, NO donors, or agents raising intracellular cGMP levels, may protect aortic valve interstitial cells from early events leading to calcification.

Investigation of the regulation of transcriptional changes in Ancylostoma caninum larvae following serum activation, with a focus on the insulin-like signalling pathway

The exit from dauer in the free-living nematode Caenorhabditis elegans is under the control of a single amphidial neuron (ASJ) of the insulin-like signalling pathway. Mutations of this pathway have the ability to suppress entry into the dauer stage. It has been postulated that insulin-like signalling plays a significant role in the response to serum stimulation in vitro of the third-stage larvae (L3s) of the canine hookworm Ancylostoma caninum. To test for the possible involvement of the insulin-like signalling cascade in the response to serum stimulation, the effects of two signalling stimulants (8-bromo cGMP and arecoline) and four inhibitors, namely 4,7-phenanthroline, phosphoinositide-3 kinase (PI3K), Akt inhibitor IV and rapamycin on feeding and on levels of selected activation-associated mRNAs in serum-stimulated L3s were explored. L3s of A. caninum were pre-incubated with or without the appropriate inhibitor/agonist. Following serum-stimulation, the feeding activity was assessed. The transcription levels of a number of activation-associated mRNAs linked to particular expressed sequence tags (ESTs) were investigated by reverse transcription, real-time PCR (rtPCR). The treatment of worms with 4,7-phenanthroline completely suppressed feeding and significantly reduced the differential levels of most activation-associated mRNAs, whereas the treatment with cGMP resulted in the resumption of feeding in almost 85% of the L3s and yielded a specific transcriptional profile consistent with that following serum stimulation. The treatment of L3s with arecoline resulted in the resumption of feeding in ∼85% of L3s, but did not result in a transcriptomic profile consistent with activation. A complete reduction in feeding was recorded in the presence of the PI3K inhibitor LY294002 (1 mM) and resulted in a pronounced dampening of differential transcription in response to serum stimulation for the molecules examined. Akt inhibitor IV resulted in a ∼70% reduction in feeding but had almost no effect on the level of any of the activation-associated mRNAs studied. Rapamycin was shown to have a weak effect on feeding, and several of the mRNAs studied exhibited greater than expected transcription following treatment. The complexities of activation-associated transcription could not be addressed using the current approach. A larger number of mRNAs needs to be investigated in order to predict or identify regulatory mechanisms proposed to function in the insulin-like signalling pathway in A. caninum.

Cyclic GMP/Protein Kinase G Phosphorylation of Smad3 Blocks Transforming Growth Factor-β–Induced Nuclear Smad Translocation

See related article, pages 185–192 In response to hemodynamic overload, the heart undergoes a complex adaptive remodeling process that involves cardiac myocyte hypertrophy, transformation of fibroblast into myofibroblast, high-level expression of extracellular matrix (ECM) proteins, interstitial fibrosis, and cell death.1 Differentiation of cardiac fibroblasts into myofibroblasts is critical to the production and deposition of collagens and plays a decisive role in myocardial fibrosis and morphological alterations during the progression of adaptive myocardial hypertrophy to decompensation and heart failure.1,2 Among the plethora of identified fibrogenic factors, transforming growth factor (TGF)-β3 plays a fundamental role in hypertrophic and fibrotic remodeling of the heart, where it regulates cardiomyocyte growth, fibroblast activation, and ECM deposition.4,5 The expression of ventricular TGF-β mRNA and protein is increased in numerous models of pathological cardiac hypertrophy and in cardiac cells in response to putative hypertrophic stimuli.6,7 In vitro, TGF-β activates myofibroblast transformation and increases ECM production.7 Blockade of TGF-β signaling is predicted to blunt fibrosis.5 Recent studies from Chen et al8 convincingly demonstrated that disruption of TGF-β signaling by inducible dominant-negative mutation of the TGF-β receptor type II (TβRII) gene significantly reduced the pressure overload–induced myofibroblast transformation and interstitial fibrosis in mouse heart. Thus, there is considerable interest in understanding how signaling by TGF-β receptors is transduced and how the inevitable damage produced could be mitigated. Of the 3 isoforms of TGF-β expressed in mammals,9,10 TGF-β1 is expressed in the adult heart, where it is secreted by cardiomyocytes and myofibroblasts and retained in significant amounts in ECM as a latent cytokine. Recent advances have led to clear description of downstream of TGF-β signal transduction pathways initiated by binding of TGF-β to membrane-bound heteromeric receptor kinases (TβRI and TβRII) that transduce intracellular signals via both Smad and non-Smad pathways …

Human Atrial Natriuretic Peptide Suppresses Torsades de Pointes in Rabbits

The increase in inward current, primarily L-type Ca2+ current, facilitates torsades de pointes (TdP). Because human atrial natriuretic peptide (ANP) moderates the L-type Ca2+ current, in our study it was hypothesized that ANP counteracts TdP. We tested the effect of ANP, guanosine 3', 5'-cyclic monophosphate analogue (8-bromo cGMP) and hydralazine on the occurrence of TdP in a rabbit model. In control rabbits, administration of methoxamine and nifekalant almost invariably caused TdP (14/15). In contrast, ANP (10 microg . kg(-1) . min(-1)) markedly abolished TdP (2/15), whereas hydralazine failed to show a comparable anti-arrhythmic action (10/15). TdP occurred only in 1 of 15 rabbits treated with 8-bromo cGMP. Presence of early afterdepolarization-like hump in the ventricular monophasic action potential was associated with the occurrence of TdP. Results suggest that ANP affects TdP in the rabbit model, and that this anti-arrhythmic effect of ANP is not necessarily shared by other vasodilating agents.

Regulation of Ion Transport in Eel Intestine by the Homologous Guanylin Family of Peptides

Since the gene expression of guanylin peptides and their receptors, guanylyl cyclase Cs, is enhanced in the intestine of seawater (SW)-adapted eels compared with fresh water (FW)-adapted fish, the guanylin family may play an important role in SW adaptation in eels. The present study analyzed the effect of three homologous guanylin peptides, guanylin, uroguanylin and renoguanylin, on ion movement through the eel intestine, and examined the target of guanylin action using Ussing chambers. The middle and posterior parts of the intestine, where water and ion absorption occurs actively in SW eels, exhibited serosa-negative transepithelial potential, while the anterior intestine was serosa-positive. Mucosal application of each guanylin in the middle or posterior intestine reduced the short-circuit current (Isc) dose dependently and reversed it at high doses, and reduced electric tissue resistance. The effects were greater in the middle intestine than in the posterior intestine. All three guanylins showed similar potency in the middle segment, but guanylin was more potent in the posterior segment. 8-bromo cGMP mimicked the effect of guanylins. The intestinal response to guanylin was smaller in FW eels. The mucosal presence of NPPB utilized as a CFTR blocker, but not of other inhibitors of the channels/transporters localized on the luminal surface in SW fish intestine, inhibited the guanylin-induced decrease in Isc. In eels, therefore, the guanylin family may be involved in osmoregulation by the intestine by binding to the receptors and activating CFTR-like channels on the mucosal side through cGMP production, perhaps resulting in Cl(-) and HCO3(-) secretion into the lumen.

Abstract 537: Sildenafil Suppresses Cardiac Hypertrophy through Inhibition of Canonical Transient Receptor Potential Cation Channel 6 Expression in Cardiac Myocytes

Background: We have shown that a phosphodiesterase 5A (PDE5A) inhibitor, sildenafil blocks cardiac pathological hypertrophy through inhibition of Gαq mediated signaling and calcineurin (Cn) signaling. However, the precise molecular mechanism is still unknown. Recently canonical transient receptor potential cation channel 6 (TRPC6) and its up-regulation have been shown to play a critical role for Gαq-mediated Cn activation in pathogenesis of cardiac hypertrophy. Therefore, we hypothesized that PDE5A inhibition blocks TRPC6 gene induction in cardiac myocytes and thus prevents cardiac hypertrophy. Methods and Results: We examined TRPC6 expression levels using real-time RT-PCR in hypertrophied mouse heart created by transverse aortic constriction (TAC). TRPC6 mRNA expression was significantly increased after TAC. Sildenafil effectively attenuated TAC-induced hypertrophy, Cn protein level and upregulation of TRPC6 mRNA. To investigate role of TRPC6 in sildenafil’s anti-hypertrophic effects, we studied cultured adult mouse cardiac myocytes (AMCM) and neonatal rat cardiac myocytes (NRCM). Stimulation with Endothelin 1 (ET1) or angiotensin II (AngII) increased cell surface area, leucine uptake, Cn protein expression and TRPC6 gene expression. Sildenafil dose-dependently blocked these increases. 8-bromo cGMP also blocked TRPC6 induction by ET1 and AngII. In addition, 8-bromo cGMP stimulation following adenovirus-mediated overexpression of cGMP-dependent protein kinase I-α (PKG) showed a marked decrease in TRPC6 expression. These results suggest that PKG activation regulates TRPC6 gene in cardiac myocytes. Using adenovirus-based expression of artificial PDE5A-gene silencing miRNA, PDE5 protein was effectively knocked down both in NRCM and AMCM. Importantly, PDE5-miRNA completely blocked ET1-mediated increase of Cn and TRPC6 same as sildenafil. Conclusion: Sildenafil effectively blocks TRPC6 induction both in vivo and in vitro . TRPC6 gene regulation is PKG and PDE5A dependent in cardiac myocytes. Given that TRPC6 induction triggers Gαq-mediated maladaptive signaling, especially Cn, these data show that the inhibition of TRPC6 gene expression contributes to the Gαq-coupled anti-hypertrophic mechanism of sildenafil.

Asymmetrical Dimethylarginine Inhibits Shear Stress–Induced Nitric Oxide Release and Dilation and Elicits Superoxide-Mediated Increase in Arteriolar Tone

l-arginine is the substrate used by NO synthase to produce the vasodilator NO. However, in several human diseases, such as hyperhomocysteinemia, diabetes mellitus, and hypertension, there is an increase in serum levels of methylated l-arginines, such as asymmetrical dimethylarginine (ADMA), which cannot be used by NO synthase to produce NO. Yet, the functional consequence of increased levels of ADMA on the vasomotor function of resistance vessels has not been delineated. We hypothesized that elevated levels of exogenous ADMA inhibit NO mediation of flow/shear stress-dependent dilation of isolated arterioles. In the presence of indomethacin, isolated arterioles from rat gracilis muscle (approximately 165 microm at 80 mm Hg) were incubated with ADMA (10(-4) mol/L), which eliminated the dilations to increases in intraluminal flow (control: from 164+/-5.4 to 188+/-3.8 microm versus ADMA: from 171+/-6.1 to 173+/-6.3 microm at 20 microL/min). ADMA did not affect dilations to nifedipine (10(-6) mol/L; control: 63.4+/-2%, ADMA: 65.8+/-3%) or 8-bromo cGMP (10(-4) mol/L; control: 51.2+/-2.1%, ADMA: 49.3+/-3.4%). In addition, ADMA elicited significant constriction of arterioles (from 173+/-17 microm to 138+/-16 microm at 80 mm Hg), which was prevented by previous incubation of arterioles with polyethylene-glycol (PEG) superoxide dismutase (SOD; 120 U/mL, control: 155+/-11 microm versus ADMA: 150+/-14 microm). Correspondingly, ADMA increased PEG-SOD reversible manner the production of vascular superoxide assessed by lucigenin-enhanced chemiluminescence and ethidium bromide fluorescence. Thus, increased levels of ADMA in various diseases could inhibit the regulation of arteriolar resistance by shear stress-induced release of NO and elicit superoxide-mediated increase in basal tone, both of which favor the development of hypertension.

The arginine‐NO pathway modulates lipolysis in adipose tissues of obese human subjects

Previous studies involving high extracellular concentrations of various NO donors (20 μM to 2 mM) showed that they strongly inhibited lipolysis in adipose tissue. However, the physiological significance of these findings remains obscure. On the basis of our recent report that dietary supplementation with L-arginine (the physiological nitrogenous precursor of NO) reduced fat mass in Zucker diabetic fatty rats, the present study was conducted to test the hypothesis that the arginine-NO pathway modulates lipolysis in adipose tissues of obese human subjects. Subcutaneous and omental fat tissues (~100 mg) were obtained from 8 adult women with BMI between 30 and 40 kg/m2, minced into small fragments, and incubated at 37 oC for 3 h in 1 ml of Krebs bicarbonate buffer (pH 7.4) containing 5 mM glucose, 4% BSA, and one of the following: 0, 0.5, 1, 5, 10, 25, 50, 100, or 500 μM sodium nitroprusside (SNP; an NO donor); 0, 0.5 or 2 mM L-arginine; or 5 μM 8-bromo cGMP (a mediator of NO effect). At the end of the incubation, the medium was analyzed for glycerol as an indicator of lipolysis. SNP at 0 to 5 μM dose-dependently increased (P < 0.05) lipolysis by up to 50–54% in both subcutaneous and omental fat tissues, whereas SNP at 25 to 500 μM dose-dependently decreased (P < 0.05) lipolysis by up to 90% in the tissues. L-Arginine at 0.5 and 2 mM increased (P < 0.05) lipolysis by ~ 24% and 50%, respectively, in both fat tissues, whereas 5 μM 8-bromo cGMP stimulated their lipolysis by 47–55%. Our results indicate that physiologic levels of NO increase, while excess levels inhibit, lipolysis in human adipose tissue. The dose-dependent lipolytic effects of NO clarify the discrepancies in the literature regarding a role for NO in fat metabolism. Supported by GRECC-BVAMC, NIH, and Texas A&M.

Molt-inhibiting hormone-mediated regulation of ecdysteroid synthesis in Y-organs of the crayfish ( Procambarus clarkii): Involvement of cyclic GMP and cyclic nucleotide phosphodiesterase

Crustacean molt-inhibiting hormone (MIH), a polypeptide secreted by the X-organ/sinus gland complex of the eyestalks, regulates molting by inhibiting the synthesis of ecdysteroids by Y-organs. Previous results indicate the biosynthetic activity of Y-organs is likely controlled not only by the level of hemolymphatic MIH, but also by the responsiveness of Y-organs to MIH. The present studies were conducted to (a) identify the second messenger that mediates MIH-induced suppression of ecdysteroidogenesis, and (b) assess the possible involvement of cyclic nucleotide phosphodiesterase (PDE) in determining the responsiveness of Y-organs to MIH. Adding 8-bromo cAMP or 8-bromo cGMP to incubation medium significantly suppressed ecdysteroid production by Y-organs of the crayfish ( Procambarus clarkii). Incubating Y-organs with MIH produced a significant increase in glandular cGMP, but MIH had no effect on glandular cAMP. The composite data indicate that MIH-induced suppression of ecdysteroidogenesis in Y-organs of P. clarkii is mediated by cGMP . Subsequently, Y-organs from various stages of the molt cycle were incubated with MIH, 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of PDE), or both. Y-Organs from middle and late premolt stages were poorly responsive to MIH alone. Including IBMX in the incubation medium enhanced the responsiveness of the Y-organs to MIH at these stages. Moreover, glandular PDE activity in the Y-organs at these stages was significantly higher than other stages. The combined results suggest that molt cycle-associated changes in PDE activity affect the ability of MIH to stimulate cGMP accumulation and suppress ecdysteroidogenesis in Y-organs of P. clarkii.

Differential effects of melatonin and its downstream effector PKCα on subcellular localization of RGS proteins

Regulators of G protein signaling (RGS) are proteins that bind specifically to activated Galpha subunits of heterotrimeric G proteins to terminate signaling by both Galpha and Gbetagamma subunits. Signal-induced RGS redistribution may affect their activity in G protein-mediated signaling. We have previously shown that melatonin and the cell permeable cGMP analog 8-bromo cGMP, which lead to protein kinase C (PKC) activation, enhanced cytoplasmic distribution of RGS10 and RGS2 in prostate carcinoma PC3-AR cells. In the present study, we transfected PC3-AR cells with myc-tagged Galphai/Galphaq specific RGS proteins RGS2, RGS4 and RGS10 and examined the effects of melatonin, 8-bromo cGMP and PKC inhibitors on their nuclear-cytoplasmic partitioning. RGS10 and RGS2 were predominantly localized in the nucleus and perinuclear regions whereas RGS4 was mostly cytoplasmic in the PC3-AR cells. Melatonin and the cell permeable cGMP analog 8-bromo cGMP, previously found to activate PKCalpha in the PC3-AR cells, enhanced cytoplasmic localization of RGS10 and RGS2 but induced nuclear accumulation of RGS4. The isozyme specific PKC inhibitor GO6976 (PKCalpha and PKCbeta1) but not hispidin (PKCbeta) negated the effects of melatonin on RGS10, RGS2 and RGS4 localization. These findings indicate that PKCalpha, a downstream effector of the melatonin receptor, differentially affects nuclear/cytoplasmic localization of both Galphai and Galphaq specific RGS proteins. These observations provide further insight into melatonin's ability to fine-tune multiple membrane G-proteins signaling in cells.

Prednisolone augments superoxide formation in porcine pulmonary artery endothelial cells through differential effects on the expression of nitric oxide synthase and NADPH oxidase.

1. Prednisolone, a potent anti-inflammatory drug, has proved ineffective in treating acute respiratory distress syndrome (ARDS). ARDS is associated with superoxide (O(2)(*-)) generation, which negates nitric oxide (NO). NO also downregulates NADPH oxidase and inhibits O(2)(*-) formation. A possible reason for the lack of effect of prednisolone may due to an inhibition of eNOS expression. In order to test this proposal, the effect of prednisolone on O(2)(*-) formation and the expression of gp91(phox) (catalytic subunit of NADPH oxidase) and eNOS in pig pulmonary artery (PA) segments and PA endothelial cells (PAECs) and PA vascular smooth muscle cells (PAVSMCs) was investigated. 2. PA segments and cells were incubated with prednisolone and tumour necrosis factor-alpha (TNF-alpha) for 16 h. O(2)(*-) formation was measured spectrophometrically and gp91(phox) and eNOS expression by Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride, the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ) and the stable cGMP analogues, 8-bromo cGMP and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP). NO release was studied using a fluorescence assay and O(2)(*-)-NO interactions with a nitrite/nitrate assay. 3. Prednisolone elicited significant increase in O(2)(*-) formation in intact PA segments and PAECs, but not PAVSMCs, in a concentration-dependent manner. In endothelium-denuded segments, prednisolone slightly enhanced O(2)(*-) release. TNF-alpha further increased prednisolone-enhanced O(2)(*-) formation in intact PA segments and PAECs. NADPH oxidase inhibitor, apocynin, inhibited O(2)(*-) formation. Increased O(2)(*-) release and gp91(phox) expression in PAECs elicited by prednisolone was blocked by SIN-1 (3-morpholinosydnonimine hydrochloride), DETA-NONOate, 8-pCPT-cGMP and 8-bromo cGMP. The effects of SIN-1 on gp91(phox) expression were reversed by ODQ. Finally, eNOS protein expression was significantly reduced by prednisolone. 4. Prednisolone increases O(2)(*-) in porcine PAECs through a downregulation of endogenous eNOS expression. Since the NO-cGMP axis inhibits gp91(phox) expression, the resultant decrease in endogenous NO formation then augments NADPH oxidase activity, which in turn results in increased O(2)(*-) formation. Since O(2)(*-) promotes inflammation, this mechanism may explain why prednisolone is ineffective in treating ARDS. Therapeutically, the coadministration of an NO donor may render prednisolone more effective in treating ARDS.

Regulation of spontaneous activity in rabbit corpus cavernosum myocytes by nitric oxide

The aim of the present study is two fold. Firstly, to determine if the nitric oxide (NO)/cGMP pathway can modulate spontaneous transient inward and outward currents (STICs and STOCs), in rabbit corpus cavernosum smooth muscle. Secondly, to see if nitric oxide can effect intracellular calcium oscillations observed in these cells. Male New Zealand white rabbits were humanely killed with an injection of pentobarbitone (I.V.), and the corpus cavernosum was dissected from the penis and cut into 1 mm3 pieces. From this, single smooth muscle cells were isolated in a medium containing collagenase as described previously [1]. The cells were then studied at 37°C using the perforated patch technique. When cells were held under voltage clamp at -60 mV, using caesium containing pipettes, over 70% fired STICs. These events were shown to be carried by a Ca2+-activated Clconductance, and were sensitive to inhibition of IP3-mediated Ca2+-release [1]. Modulation by the NO/cGMP pathway was investigated by applying nitrosocysteine, 3-(5-hydroxymethyl-2furyl)-1-benzyl indazole (YC-1), and 8-bromo cGMP, all three of which abolished STIC activity. In addition, the protein kinase G 1 (GK1) specific activator SP-8-Br-PETcGMPS (25 and 100 μM), significantly reduced STICs. This provides one possible mechanism whereby the NO/ cGMP pathway could inhibit tone in corpus cavernosum smooth muscle.

Calcium-Independent, cGMP-Mediated Light Adaptation in Invertebrate Ciliary Photoreceptors

Calcium is thought to be essential for adaptation of sensory receptor cells. However, the transduction cascade of hyperpolarizing, ciliary photoreceptors of the scallop does not use IP3-mediated Ca release, and the light-sensitive conductance is not measurably permeable to Ca2+. Therefore, two typical mechanisms that couple the light response to [Ca]i changes seem to be lacking in these photoreceptors. Using fluorescent indicators, we determined that, unlike in their microvillar counterparts, photostimulation of ciliary cells under voltage clamp indeed evokes no detectable change in cytosolic Ca. Notwithstanding, these cells exhibit all of the hallmarks of light adaptation, including response range compression, sensitivity shift, and photoresponse acceleration. A possible mediator of Ca-independent sensory adaptation is cGMP, the second messenger that regulates the light-sensitive conductance; cGMP and 8-bromo cGMP not only activate light-dependent K channels but also reduce the amplitude of the light response to an extent greatly in excess of that expected from simple occlusion between light and chemical stimulation. In addition, these substances accelerate the time course of the photocurrent. Tests with pharmacological antagonists suggest that protein kinase G may be a downstream effector that controls, in part, the cGMP-triggered changes in photoresponse properties during light adaptation. However, additional messengers are likely to be implicated, especially in the regulation of response kinetics. These observations suggest a novel feedback inhibition pathway for signaling sensory adaptation.

Modulation of spontaneous Ca2+-activated Cl- currents in the rabbit corpus cavernosum by the nitric oxide-cGMP pathway.

The whole-cell perforated patch clamp technique was used to study membrane currents in isolated rabbit corpus cavernosum smooth muscle cells. Depolarization from -80 mV to the range -40 to -10 mV evoked a nifedipine-sensitive Ca(2+) current that was followed by a slower inward current that activated over several hundred milliseconds. The slow current reversed near the Cl(-) equilibrium potential (E(Cl)) and was reduced by anthracene-9-carboxylic acid (A9C; 1 mm) and niflumic acid (100 microm), suggesting that it was a Ca(2+)-activated Cl(-) current. When held constantly at -60 mV, over 70% of cells fired spontaneous transient inward currents (STICs), the amplitudes of which were reduced by A9C and niflumic acid. STICs reversed near E(Cl) in a symmetrical Cl(-) gradient and when [Cl(-)](o) was substituted with glutamate or I(-), the reversal potential shifted to more positive or more negative values, respectively, confirming that STICs were mediated by Cl(-) channels. STICS were also blocked by cyclopiazonic acid, 2-aminoethoxydiphenyl borate (2-APB) and 2-nitro-4-carboxyl-N,N-diphenylcarbamate (NCDC), suggesting that they depended on IP(3)-mediated Ca(2+)-release from the sarcoplasmic reticulum. Modulation by the NO-cGMP pathway was investigated by applying nitrosocysteine, 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1), and 8-bromo cGMP, all three of which abolished STIC activity. YC-1 also reduced noradrenaline-evoked inward currents, but had no effect on similar currents evoked by caffeine, suggesting that cGMP selectively inhibited IP(3)-mediated Ca(2+) release. We propose that Ca(2+)-activated Cl(-) currents underlie detumescent tone in the corpus cavernosum, and that modulation of this mechanism by the NO-cGMP pathway is important during penile erection.

Cyclic GMP-mediated activation of a glibenclamide-sensitive mechanism in the rabbit sphincter of Oddi.

We investigated whether glibenclamide-sensitive potassium channels are involved in cyclic GMP (cGMP)-mediated relaxation of the rabbit Oddi's sphincter. Changes in isometric tension were measured in the presence of atropine (1 microM) and guanethidine (4 microM). Concentration-response curves for nitroglycerin, vasoactive intestinal polypeptide (VIP), and sodium nitroprusside (SNP) were shifted to the right in the presence of (p-chloro-D-Phe6, Leu17)-VIP (VIPa), a VIP receptor antagonist. Glibenclamide (1 microM) attenuated the relaxations to VIP, nitroglycerin, or 8-bromo cGMP. In the presence of tetrodotoxin (TTX), glibenclamide attenuated relaxations to VIP without effect on those to nitroglycerin. Furthermore, nitroglycerin increased both cAMP and cGMP concentrations, however, it failed to increase the tissue cAMP concentration in the presence of TTX. VIPa also blocked the increase in content of either cyclic nucleotide. VIP increased cAMP with a TTX-sensitive increase in cGMP content. 8-Bromo cGMP (1 microM) significantly increased the tissue cAMP content. This was blocked by either TTX or VIPa (both 1 microM). We conclude that ATP-sensitive potassium channel (KATP) activation contributes to cGMP-mediated relaxation of the Oddi's sphincter of the rabbit. Activation of KATP results from a cyclic AMP-mediated process due to cGMP-dependent VIP release from neurons.

Inhibitory effect of C-type natriuretic peptide (CNP) on cultured cardiac myocyte hypertrophy: interference between CNP and endothelin-1 signaling pathways.

C-type natriuretic peptide (CNP) is known to play a role in the local regulation of vascular tone. We recently found that CNP is also produced by cardiac ventricular cells. However, its local effect on myocyte hypertrophy remains to be elucidated. The present study investigated the effects of CNP on cultured cardiac myocyte hypertrophy and the interaction between CNP and endothelin-1 (ET-1) signaling pathways. CNP attenuated basal and ET-1-augumented protein synthesis, atrial natriuretic peptide secretion, hypertrophy-related gene expression, GATA-4 and MEF-2 DNA binding activities, Ca(2+)/calmodulin-dependent kinase II activity, and ERK phosphorylation. CNP also inhibited ET-1-induced increase in intracellular Ca(2+) concentration. These effects of CNP were mimicked by a cGMP analog, 8-bromo cGMP. However, the inhibitory effects of CNP on the hypertrophic response of myocytes were significantly diminished at high concentrations of ET-1. Although CNP increased intracellular cGMP levels in myocytes, ET-1 suppressed CNP-induced cellular cGMP accumulation. A protein kinase C activator and Ca(2+) ionophore mimicked this suppressive effect of ET-1. We further examined the effect of CNP on the paracrine action of ET-1 secreted from cardiac nonmyocytes. CNP and 8-bromo cGMP significantly inhibited ET-1 secretion from nonmyocytes. Although nonmyocyte-conditioned medium increased the protein synthesis in myocytes through endogenous ET-1 action, this increase was significantly attenuated by pretreatment of nonmyocytes with CNP and 8-bromo cGMP. These findings demonstrate that CNP inhibits ET-1-induced cardiac myocyte hypertrophy via a cGMP-dependent mechanism, and conversely, ET-1 inhibits CNP signaling by a protein kinase C- and Ca(2+)-dependent mechanism, suggesting mutual interference between CNP and ET-1 signaling pathways.

Nitric Oxide-Induced Intracellular Ca2+ Modulation in Macrovascular Endothelial Cells

Background and Objectives:Nitric oxide (NO) reduces the intracellular Ca concentration ([Ca]i) in smooth muscle cells, whereas the effect of NO on [Ca]i in endothelial cells is still controversial. Therefore, the effect of NO on the [Ca]i, and its mechanism in mouse aortic endothelial cells (MAEC) and human umbilical vein endothelial cells (HUVEC) were examined. Materials and Methods:In primary cultured MAEC and HUVEC, cells were loaded with fura 2-AM and [Ca]i and measured using a microfluorometer. Results:The NO donor, sodium nitroprusside (SNP), reduced the [Ca]i in 72% of the cells tested (n=100). In the remaining cells, the effect of SNP was biphasic, or the [Ca]i was increased. In addition, the membrane-permeable cGMP, 8-bromo cGMP, decreased the [Ca]i. The effects of SNP and 8-bromo cGMP were inhibited by the soluble guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one (ODQ), and the cGMP-dependent protein kinase inhibitor, KT5823, respectively. In contrast, in the presence of 8-bromo cGMP or ODQ, SNP increased the [Ca]i. Conclusion:These results suggest that NO inhibits the [Ca ]i through a cGMPdependent mechanism and increases the [Ca]i through a cGMP-independent mechanism. (Korean Circulation J 2004;34(6):600-609)