Effect Of Cyclic Guanosine Monophosphate Research Articles

Cardioprotective effects of Prolame and SNAP are related with nitric oxide production and with diminution of caspases and calpain-1 activities in reperfused rat hearts.

Cardiac tissue undergoes changes during ischemia-reperfusion (I-R) that compromise its normal function. Cell death is one of the consequences of such damage, as well as diminution in nitric oxide (NO) content. This signaling molecule regulates the function of the cardiovascular system through dependent and independent effects of cyclic guanosine monophosphate (cGMP). The independent cGMP pathway involves post-translational modification of proteins by S-nitrosylation. Studies in vitro have shown that NO inhibits the activity of caspases and calpains through S-nitrosylation of a cysteine located in their catalytic site, so we propose to elucidate if the regulatory mechanisms of NO are related with changes in S-nitrosylation of cell death proteins in the ischemic-reperfused myocardium. We used two compounds that increase the levels of NO by different mechanisms: Prolame, an amino-estrogenic compound with antiplatelet and anticoagulant effects that induces the increase of NO levels in vivo by activating the endothelial nitric oxide synthase (eNOS) and that has not been tested as a potential inhibitor of apoptosis. On the other hand, S-Nitroso-N-acetylpenicillamine (SNAP), a synthetic NO donor that has been shown to decrease cell death after inducing hypoxia-reoxygenation in cell cultures. Main experimental groups were Control, I-R, I-R+Prolame and I-R+SNAP. Additional groups were used to evaluate the NO action pathways. Contractile function represented as heart rate and ventricular pressure was evaluated in a Langendorff system. Infarct size was measured with 2,3,5-triphenyltetrazolium chloride stain. NO content was determined indirectly by measuring nitrite levels with the Griess reaction and cGMP content was measured by Enzyme-Linked ImmunoSorbent Assay. DNA integrity was evaluated by DNA laddering visualized on an agarose gel and by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling assay. Activities of caspase-3, caspase-8, caspase-9 and calpain-1 were evaluated spectrophotometrically and the content of caspase-3 and calpain-1 by western blot. S-nitrosylation of caspase-3 and calpain-1 was evaluated by labeling S-nitrosylated cysteines. Our results show that both Prolame and SNAP increased NO content and improved functional recovery in post-ischemic hearts. cGMP-dependent and S-nitrosylation pathways were activated in both groups, but the cGMP-independent pathway was preferentially activated by SNAP, which induced higher levels of NO than Prolame. Although SNAP effectively diminished the activity of all the proteases, a correlative link between the activity of these proteases and S-nitrosylation was not fully established.

STUDY OF CARDIOVASCULAR EFFECTS OF CGMP PATHWAY ACTIVATION ON EXPERIMENTAL RAT MODEL OF METABOLIC SYNDROME

Objective: Metabolic syndrome is a fast growing problem associated with an increased risk of cardiovascular death. This association has been shown to be multifactorial where endothelial dysfunction acts as a major contributor. However, few studies have explored the effects of cyclic guanosine monophosphate (cGMP) pathway activation on cardiovascular dysfunction related to metabolic syndrome. The aim was to investigate the effects of a long-term administration of BAY 41–2272, a cGMP pathway activator on cardiovascular reactivity in spontaneously hypertensive rats (SHR) with an experimentally-induced metabolic syndrome. Design and method: Thirty-six 9-week-old rats were randomly divided into 3 groups: Standard diet-fed rats (control group), Cafeteria diet (CAF)-fed rats and CAF diet-fed rats treated with BAY 41–2272 (5 mg/kg) daily for 12 weeks. CAF consisted of allowing rats free access to energy dense human foods during 12 weeks. Body weight, abdominal circumference, arterial pressure and glucose tolerance test were monitored. Cumulative concentration-response curves to isoproterenol (0.1 nM – 1 μM), phenylephrine (1 nM – 10 μM), acetylcholine (1 nM – 10 μM), insulin (1 nM – 3 μM) and sodium nitroprusside (SNP) (0.1 nM – 0.1 μM) were performed on isolated persfused heart and thoracic aorta. A significance level of 0.05 was used for statistical comparison purposes. Results: 12 weeks of CAF feeding induced obesity, arterial hypertension and glucose intolerance. Compared to the control group, there was a significant decrease in the positive inotropic effect of isoproterenol and in the α1-adrenergic response in CAF fed-group. Long-term BAY 41–2272 treatment significantly improved the metabolic abnormalities, restored the cardiac inotropy and the α1-adrenergic response. CAF fed-rats showed also an altered insulin-induced vasorelaxation which was significantly improved after BQ-123 preincubation (1 μM), a selective endothelin 1 receptor antagonist. However, BAY 41–2272 did not improve the insulin-induced vasorelaxation. No difference in acetylcholine and SNP-induced vasorelaxations was observed between all groups. Conclusions: In this study, we demonstrated the positive effects of BAY 41–2272 on metabolic alterations and cardiovascular reactivity in SHR with metabolic syndrome. These results suggest that cGMP pathway modulation may be a promising therapeutic target in the management of the metabolic syndrome.

Morphology-Based Analysis of Myoblasts for Prediction of Myotube Formation

The development of new drugs depends on the efficiency of drug screening. Phenotype-based screening has attracted interest due to its considerable potency for the discovery of first-in-class drugs. In general, fluorescently labeled imagery is the leading technique for phenotype-based screening; however, there are growing requirements to understand total culture profiles, which are unclear after end-point assays. In this study, we demonstrate that morphology-based cellular evaluation of unlabeled cells is an efficient approach to evaluate myotube formation assays. One of our aims was to study the myogenic differentiation process in C2C12 cells to discern the differences between cellular responses to different medium conditions (serum concentrations and insulin dosages). Our results show that predictive morphological profiles that strongly correlate with myogenic differentiation can be generated from myotube images, even in the confluent stage. The differentiation rate after 14 days can be quantitatively predicted with the highest accuracy by means of images taken on days 0–11.5. In addition, for the application of our morphology-based cellular evaluation, the effect of cyclic guanosine monophosphate (cGMP) on myogenic differentiation was analyzed. Our results show that the quantitated morphological profile from these images can be an effective descriptor for analysis of the myotube-recovering effect of cGMP.

Cyclic Guanosine Monophosphate (cGMP)-Dependent Protein Kinase II Blocks Epidermal Growth Factor (EGF)/Epidermal Growth Factor Receptor (EGFR)-Induced Biological Effects on Osteosarcoma Cells.

BackgroundThe present work was performed to detect the potential inhibitory effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKG II) on epidermal growth factor (EGF) receptor-induced biological activity and related signal cascades in osteosarcoma cells.Material/MethodsWe transfected the osteosarcoma MG-63 cell line with an adenoviral vector encoding PKG II cDNA (Ad-PKGII) and incubated the transfected cells with 250 μM 8-pCPT-cGMP to activate the PKG II. We stimulated the MG-63 cells with100 ng/ml EGF, and then detected their proliferation using a CCK-8 assay. Transwell assay was used to examine MG-63 cell migration; and Western blot analysis was used to detect expression of matrix metalloproteinase 9 (MMP-9) and activation of ERK and Akt.ResultsStimulating cells by 100 ng/ml EGF promoted MG-63 cell proliferation and migration, ERK and Akt phosphorylation, and MMP-9 expression. These effects of EGF were inhibited in MG-63 cells infected with Ad-PKGII and incubated with 8-pCPT-cGMP.ConclusionsOur results demonstrate that Ad-PKGII infection significantly inhibited EGF-induced proliferation and migration, as well as the associated-signal cascades; which indicates that PKG II might be a potential anti-cancer factor.

Nitric oxide (NO) enhances pemetrexed cytotoxicity via NO‑cGMP signaling in lung adenocarcinoma cells in�vitro and in�vivo

Pemetrexed (PEM) is a novel, multitargeted, antifolate, antineoplastic agent for the treatment of non-small cell lung cancer and malignant pleural mesothelioma. Additional effects of nitric oxide (NO) donors on the chemosensitivity of cancers have been reported. However, the effects of an NO donor on PEM-induced cytotoxicity remain unknown. In this study, we investigated the effects of the NO donors, NOC-18 on the cytotoxicity in A549 cells in vitro and of nitroglycerin (GTN), on the tumor growth of Lewis lung carcinoma cells in a murine syngraft model treated with PEM. The effects of NO donors on the expression of proteins associated with PEM metabolism, including thymidylate synthase (TS), reduced folate carrier 1 (RFC1), folylpolyglutamate synthase (FPGS), γ-glutamyl hydrolase (GGH) and multidrug resistance-related protein (MRP)5, and the effects of cyclic guanosine mono-phosphate (cGMP) signaling on these proteins were examined in A549 cells. Treatment with 100 nM NOC-18 for 3 days significantly enhanced PEM-induced cytotoxicity and increased the expression of RFC1 and FPGS in A549 cells. Treatment with 10 nM 8-bromo-cGMP (8-Br-cGMP) for 3 days also increased the expression of RFC1 and FPGS in A549 cells. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µm) significantly reversed the increase in RFC1 and FPGS expression induced by 100 nM NOC-18 in A549 cells. Combination therapy with GTN and PEM significantly reduced tumor growth compared with PEM alone in the syngraft model. The enhanced antitumor effect of GTN plus PEM was significantly reversed by the concomitant addition of ODQ. These findings suggest that NO donors, such as NOC-18 and GTN, enhance the anticancer effects of PEM by increasing the RFC1 and FPGS expression and stimulating cGMP signaling pathways in cancer cells.

Present and Future Treatment Strategies for Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare progressive disorder historically associated with mortality in <3 years post-diagnosis. The etiology of PAH is complex, multifactorial, and likely involves the interplay between genetic and environmental factors. These are reviewed with emphasis on the nitric oxide pathway. Use of treatment modalities including vasodilator therapy have resulted in improved symptoms, hemodynamics, and survival in these patients. Vasodilators, including the calcium channel antagonists, prostanoids, and endothelin receptor antagonists, have been used to counteract potential imbalances in vasoactive mediators in PAH patients; all have produced improved long-term symptomatology and hemodynamics. Only the prostanoid epoprostenol has improved survival in IPAH patients. Although these medications have worked well in many patients with PAH, each of them has limitations. The phosphodiesterase-5 (PDE-5) inhibitors are a relatively new form of treatment for PAH. They are designed to potentiate the effects of cyclic guanosine monophosphate, thereby mimicking endogenous nitric oxide within the vasculature. PDE-5 inhibitors are selective pulmonary vasodilators effective in animal models of pulmonary hypertension. The published clinical studies evaluating their use have been small in size to date but appear to demonstrate benefit. The recently completed 12-week randomized placebo-controlled Sildenafil Use in Pulmonary Hypertension (SUPER-1) trial demonstrated improvement in 6-minute walk distance and hemodynamics in patients receiving sildenafil. These data suggest that the PDE-5 inhibitors are effective in treating PAH and that it is likely that their usage will increase over time. The purpose of this review is to present a current view of the pathogenesis and treatment of PAH, with an emphasis on the use of PDE-5 inhibitors in these patients.

Activation of the PI3-K/Akt Pathway Mediates cGMP Enhanced-Neurogenesis in the Adult Progenitor Cells Derived from the Subventricular Zone

The intracellular mechanisms that regulate neurogenesis remain unclear. Using neurospheres isolated from the subventricular zone (SVZ) of the adult rat, we investigated the effect of cyclic guanosine monophosphate (cGMP) and its signaling pathway on the induction of neurogenesis. Neurospheres expressed phosphodiesterase 5 (PDE5) and treatment of neurospheres with Sildenafil, a specific inhibitor of PDE5, significantly increased cGMP levels and neurogenesis. In addition, incubation of neurospheres with Sildenafil significantly phosphorylated Akt, which was associated with an increase of phosphorylation of glycogen synthase kinase 3 (GSK-3), a downstream target of Akt. Coincubation of neurospheres with Sildenafil and LY 294002, a pharmacological inhibitor of PI3-K/Akt, abolished Sildenafil-induced phosphorylated Akt and GSK-3. Furthermore, LY 294002 blocked Sildenafil-increased SVZ cell proliferation. These data suggest that Sildenafil-enhanced neurogenesis likely occurs through activation of the PI3-K/Akt/GSK-3 pathway.

Experimental Evaluation of the Effects of the Intraportal Administration of Cyclic Guanosine Monophosphate on Ischemia/Reperfusion in the Porcine Liver

This study was done to examine the protective effects of cyclic guanosine monophosphate (cGMP), a second messenger of nitric oxide, for ischemia/reperfusion injury of the liver, since it is known to induce vasodilatation and to inhibit platelet aggregation. Using an experimental model of porcine liver ischemia, 8-bromoguanosine 3′,5′ monophosphate, a cGMP analog, was continuously administered into the portal vein before ischemia and after reperfusion 30 min for each in the cGMP group (n=6). Saline water was administered in the same way in the control group (n=6). The cardiac output (CO), mean arterial blood pressure (MAP), portal venous flow (PVF), hepatic arterial flow (HAF), hepatic tissue blood flow (HTBF), and hepatic tissue cGMP level were determined. Hepatic enzymes and the bile discharge were also assessed as indicators of hepatic function. The hepatic tissue cGMP level was significantly higher, and PVF, HTBF, and the bile discharge were significantly greater in the cGMP group, while there were no remarkable differences between the groups with CO, MAP, HAF, and hepatic enzymes. In conclusion, the continuous supplementation of cGMP into the portal vein was found to be beneficial for preserving both the hepatic circulation and, consequently, the hepatic function of after warm ischemia of porcine liver.

Cyclic GMP protein kinase mediates negative metabolic and functional effects of cyclic GMP in control and hypertrophied rabbit cardiac myocytes.

We tested the hypothesis that in isolated cardiac myocytes, the negative metabolic and functional effects of cyclic guanosine monophosphate (GMP) are mediated by cyclic GMP protein kinase activity, and that these effects are altered in renal hypertensive (one-kidney, one-clip, 1K1C) cardiac hypertrophic rabbits. By using isolated cardiac myocytes from control and 1K1C rabbits, oxygen consumption (Mvo2; O2 nl/ min/10(5) cells), cyclic GMP (fmol/10(5) cells), and cell shortening (percentage) data were collected (a) at baseline; (b) with cyclic GMP protein kinase inhibitors KT5823 (10(-6) M) or Rp8-pCPT-cGMP (5 x 10(-6) M); (c) with the cyclic GMP phosphodiesterase inhibitor zaprinast (10(-6), 10(-4) M); and (d) with zaprinast (10(-6), 10(-4) M) and protein kinase inhibitors. Basal levels of cyclic GMP were similar in control versus 1K1C myocytes (62 +/- 10 vs. 66 +/- 17 pmol/10(5) myocytes). Zaprinast produced a dose-dependent increase in cyclic GMP in both control and 1K1C myocytes. The addition of KT5823 did not significantly affect cyclic GMP levels. Zaprinast significantly and dose dependently decreased Mvo2, and KT5823 partially restored it in control and 1K1C. Zaprinast also significantly decreased percentage shortening, and KT5823 partially restored it in control. Similar results were obtained with Rp-8pCPT-cGMP, although neither inhibitor was effective without zaprinast. The hypertrophied myocytes demonstrated comparable responses to all agents. These data suggest that the cyclic GMP protein kinase activity was not significant under basal conditions; however, the importance of cyclic GMP protein kinase in control and 1K1C myocytes was significant under conditions of increased intracellular cyclic GMP.

Society of Neurosurgical Anesthesia and Critical Care. Orlando, Florida, October 16, 1998.

Society of Neurosurgical Anesthesia and Critical Care. Orlando, Florida, October 16, 1998.

Atrial Natriuretic Peptide-Induced Release of Cyclic Guanosine Monophosphate by Coronary Bypass Grafts

Background. Superior long-term patency rates of the internal mammary artery (IMA) versus saphenous vein (SV) after coronary artery bypass grafting are well documented. Higher production rates of vasodilating and platelet-inhibiting mediators (prostacyclin and nitric oxide) by the IMA seem to have a major impact on its long-term durability and resistance to coronary artery graft disease. For the right gastroepiploic artery (RGEA) marked release of protective mediators is reported as well. The vasodilating effect of cyclic guanosine monophosphate (cGMP) released after stimulation by atrial natriuretic peptide might serve as another graft protective system. The aim of the present study was to determine cGMP release by IMA, RGEA, and SV after atrial natriuretic peptide challenge. Methods. Samples of human IMA (n = 19), RGEA (n = 7), and SV (n = 18) discarded during coronary artery bypass grafting were stimulated with 10 −6 mol/L atrial natriuretic peptide after a resting phase in nutrient me-dium. Release of cGMP was determined by 125-iodide radioimmunoassay. Results. Basal cGMP production rates of the IMA (759.9 ± 277.0 fmol/cm 2) and RGEA (739.9 ± 186.0 fmol/cm 2) were higher than production rates of SV (281.2 ± 64.0 fmol/cm 2). Application of atrial natriuretic peptide led to a statistically significant increase of cGMP release in IMA grafts (1,939.3 ± 778.0 fmol/cm 2), whereas RGEA (618.4 ± 141.3 fmol/cm 2) and SV (221.7 ± 64.5 fmol/cm 2) remained at basal levels ( p < 0.05). Conclusions. From these data we conclude that the IMA in comparison with the RGEA and SV produces more extracellular cGMP when stimulated by atrial natriuretic peptide. This effect might support the cGMP-mediated protective properties of nitric oxide and could underline the extraordinary suitability of the IMA as a bypass conduit.

Different effects of cGMP and cAMP in the intestine of the European eel, Anguilla anguilla.

The regulation of salt absorption in the sea water eel intestine was studied by evaluating the effects of theophylline, 8 Br cyclic adenosine monophosphate, 8 Br cyclic guanosine monophosphate, atriopeptin III, porcine vasoactive intestinal peptide and prostaglandin E1 on the short-circuit current, the transepithelial voltage difference and conductance and on the dilution potentials. It was shown that theophylline increased the transepithelial conductance and reduced the magnitude of the dilution potentials, indicating that the drug increases the anion conductance of the tight junctions. In addition its inhibitory effect on short-circuit current and transepithelial voltage difference suggests that theophylline also affects the transcellular transport mechanisms. It was shown that 8 Br cyclic guanosine monophosphate and 8 Br cyclic adenosine monophosphate affect transcellular mechanisms underlying C1- transport since both compounds reduced short-circuit current and transepithelial voltage difference; however, cyclic adenosine monophosphate is less effective since unlike cyclic guanosine monophosphate, even at maximal concentration, it was not able to completely abolish transepithelial voltage difference and short-circuit current. The effects of cyclic guanosine monophosphate and cyclic adenosine monophosphate were not additive even if cyclic guanosine monophosphate may produce further inhibition of ion transport in 8 Br cyclic adenosine monophosphate-treated tissues. In addition, cyclic guanosine monophosphate but not cyclic adenosine monophosphate reduced the magnitude of the dilution potentials, suggesting that cyclic guanosine monophosphate acts also on the paracellular pathway. Rat atriopeptin III, a peptide known to increase cyclic guanosine monophosphate cellular levels, behaved like 8 Br cyclic guanosine monophosphate since it lowered the dilution potentials and reduced short-circuit current and transepithelial voltage difference to near zero values, suggesting that the hormone modulates both paracellular and transcellular transport mechanisms, probably acting on the Na-K-2Cl cotransport. Agents acting via cyclic adenosine monophosphate, like porcine Basoactive intenstinal peptide and prostaglandin, behaved like 8 Br cyclic adenosine monophosphate. They were less effective in inhibiting ion transport and did not interfere with the paracellular pathway.

Maxi K+ channels are stimulated by cyclic guanosine monophosphate-dependent protein kinase in canine coronary artery smooth muscle cells

By using a patch clamp technique, we examined the effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G kinase) on Ca(2+)-activated maxi K+ channels in canine coronary artery smooth muscle cells. Maxi K+ channels (274 +/- 4 pS in symmetrical 140 mM KCl at 24-26 degrees C) were activated by cytoplasmic Ca2+ and were completely blocked by 100 nM charybdotoxin (CTX). G kinase (300 U/ml) added to the cytoplasmic face of the membrane patch shifted the voltage dependence of these channels by about 25 mV in the negative direction in the presence of 1 microM Ca2+, 50 microM cGMP and 1 mM magnesium adenosine triphosphate. At -50 mV and 1 microM Ca2+, G kinase treatment increased the mean number of open channels 4.5-fold compared with the control. alpha-Human atrial natriuretic peptide (ANP, 100 nM) reduced the isometric tension of coronary arterial rings elicited by 14 or 24 mM KCl, but failed to relax the artery contracted by 34 mM KCl. Addition of 100 nM CTX augmented tension development elicited by 24 mM KCl and totally prevented ANP from relaxing the arterial rings. These results indicate that G kinase-dependent protein phosphorylation activates maxi K+ channels in canine coronary smooth muscle, and further suggest that the G kinase-induced activation of maxi K+ channels may cause hyperpolarization and relaxation of coronary artery.

Effect of cyclic guanosine monophosphate on hypoxic and angiotensin-II-induced pulmonary vasoconstriction

We examined, in isolated blood perfused rat lungs, the effect of the cell permeable 8-bromo derivative of cGMP on pulmonary vasoconstriction induced by either alveolar hypoxia or angiotensin II. 8-Bromo cGMP dose-dependently reduced both hypoxia-(IC50 = 2.2 X 10(-5) M) and angiotensin-II-induced pulmonary vasoconstriction (IC50 = 5.0 X 10(-5) M). This effect of 8-bromo cGMP on pulmonary vasoconstriction was not affected by cyclooxygenase blockade. M & B 22948 (0.1 mM), an inhibitor of cGMP-phosphodiesterase, reduced synergistically with 8-bromo cGMP the hypoxia or angiotensin-II-induced vasoconstriction. The cGMP-phosphodiesterase inhibitor M & B 22948, by itself, selectively reduced hypoxia-induced vasoconstriction, suggesting a modulating effect of endogenous cGMP during hypoxic vasoconstriction.

Responses of morphologically identified cortical neurons to intracellularly injected cyclic AMP

Intracellular injections of cyclic adenosine monophosphate (cAMP) and horseradish peroxidase (HRP) were made in neurons of the motor cortex of awake cats. Eighty-six percent of injected cells responded to cAMP and HRP with a rapid decrease in input resistance. The decreases in input resistance occurred immediately after injection and began to return toward baseline 2 to 3 min later. The decreases were significantly greater than the small decreases in input resistance normally seen in uninjected cells held for 2 min or more after penetration and exceeded comparably small decreases in input resistance seen after control injections of 5′-AMP plus HRP. Pyramidal cells of layer V were identified as responding to cAMP with a decreased input resistance. A spiny stellate cell of layer III and a pyramidal cell of layer VI were also identified that showed similar responses. Increased rates of discharge were also observed after penetration with electrodes containing cAMP, but significant changes in input resistance were not found in association with the increased rates of discharge. After pressure injection of cAMP, the rates of discharge decreased toward more normative values. Our findings indicate that cAMP has an effect on cortical neurons similar to that found in some types of invertebrate (molluscan) neurons and dissimilar to the effect of cyclic guanosine monophosphate.

Effect of cyclic guanosine monophosphate on some indices of carbohydrate metabolism of muscle tissue during wound healing

Effect of cyclic guanosine monophosphate on some indices of carbohydrate metabolism of muscle tissue during wound healing