Cyclic Monophosphate Analogue Research Articles

Unraveling the Binding Mode of Cyclic Adenosine-Inosine Monophosphate (cAIMP) to STING through Molecular Dynamics Simulations.

The stimulator of interferon genes (STING) plays a significant role in immune defense and protection against tumor proliferation. Many cyclic dinucleotide (CDN) analogues have been reported to regulate its activity, but the dynamic process involved when the ligands activate STING remains unclear. In this work, all-atom molecular dynamics simulations were performed to explore the binding mode between human STING (hSTING) and four cyclic adenosine-inosine monophosphate analogs (cAIMPs), as well as 2',3'-cGMP-AMP (2',3'-cGAMP). The results indicate that these cAIMPs adopt a U-shaped configuration within the binding pocket, forming extensive non-covalent interaction networks with hSTING. These interactions play a significant role in augmenting the binding, particularly in interactions with Tyr167, Arg238, Thr263, and Thr267. Additionally, the presence of hydrophobic interactions between the ligand and the receptor further contributes to the overall stability of the binding. In this work, the conformational changes in hSTING upon binding these cAIMPs were also studied and a significant tendency for hSTING to shift from open to closed state was observed after binding some of the cAIMP ligands.

Theophylline-induced endothelium-dependent vasodilation is mediated by increased nitric oxide release and phosphodiesterase inhibition in rat aorta.

This study aimed to examine the endothelial dependence of vasodilation induced by the phosphodiesterase inhibitor theophylline in isolated rat thoracic aortas and elucidate the underlying mechanism, with emphasis on endothelial nitric oxide (NO). The effects of various inhibitors and endothelial denudation on theophylline-induced vasodilation, and the effect of theophylline on vasodilation induced by NO donor sodium nitroprusside, cyclic guanosine monophosphate (cGMP) analog bromo-cGMP, and β-agonist isoproterenol in endothelium-denuded aorta were examined. The effects of theophylline and sodium nitroprusside on cGMP formation were also examined. We examined the effect of theophylline on endothelial nitric oxide synthase (eNOS) phosphorylation and intracellular calcium levels. Theophylline-induced vasodilation was greater in endothelium-intact aortas than that in endothelium-denuded aortas. The NOS inhibitor, NW-nitro-L-arginine methyl ester; non-specific guanylate cyclase (GC) inhibitor, methylene blue; and NO-sensitive GC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one inhibited theophylline-induced vasodilation in endothelium-intact aortas. Theophylline increased the vasodilation induced by sodium nitroprusside, bromo-cGMP, and isoproterenol. Theophylline increased cGMP formation in endothelium-intact aortas, and sodium nitroprusside-induced cGMP formation in endothelium-denuded aortas. Moreover, theophylline increased stimulatory eNOS (Ser1177) phosphorylation and endothelial calcium levels, but decreased the phosphorylation of inhibitory eNOS (Thr495). These results suggested that theophylline-induced endothelium-dependent vasodilation was mediated by increased endothelial NO release and phosphodiesterase inhibition.

This Month in Anesthesiology

This Month in Anesthesiology

Isoflurane Disrupts Postsynaptic Density-95 Protein Interactions Causing Neuronal Synapse Loss and Cognitive Impairment in Juvenile Mice via Canonical NO-mediated Protein Kinase-G Signaling.

Inhalational anesthetics are known to disrupt PDZ2 domain-mediated protein-protein interactions of the postsynaptic density (PSD)-95 protein. The aim of this study is to investigate the underlying mechanisms in response to early isoflurane exposure on synaptic PSD-95 PDZ2 domain disruption that altered spine densities and cognitive function. The authors hypothesized that activation of protein kinase-G by the components of nitric oxide (NO) signaling pathway constitutes a mechanism that prevents loss of early dendritic spines and synapse in neurons and cognitive impairment in mice in response to disruption of PDZ2 domain of the PSD-95 protein. Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 wild-type PDZ2 peptide or soluble guanylyl cyclase activator YC-1 along with their respective controls. Primary neurons at 7 days in vitro were exposed to isoflurane or PSD-95 wild-type PDZ2 peptide for 4 h. Coimmunoprecipitation, spine density, synapses, cyclic guanosine monophosphate-dependent protein kinase activity, and novel object recognition memory were assessed. Exposure of isoflurane or PSD-95 wild-type PDZ2 peptide relative to controls causes the following. First, there is a decrease in PSD-95 coimmunoprecipitate relative to N-methyl-d-aspartate receptor subunits NR2A and NR2B precipitate (mean ± SD [in percentage of control]: isoflurane, 54.73 ± 16.52, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 51.32 ± 12.93, P = 0.001). Second, there is a loss in spine density (mean ± SD [spine density per 10 µm]: control, 5.28 ± 0.56 vs. isoflurane, 2.23 ± 0.67, P < 0.0001; and PSD-95 mutant PDZ2 peptide, 4.74 ± 0.94 vs. PSD-95 wild-type PDZ2 peptide, 1.47 ± 0.87, P < 0.001) and a decrease in synaptic puncta (mean ± SD [in percentage of control]: isoflurane, 41.1 ± 14.38, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 50.49 ± 14.31, P < 0.001). NO donor or cyclic guanosine monophosphate analog prevents the spines and synapse loss and decline in the cyclic guanosine monophosphate-dependent protein kinase activity, but this prevention was blocked by soluble guanylyl cyclase or protein kinase-G inhibitors in primary neurons. Third, there were deficits in object recognition at 5 weeks (mean ± SD [recognition index]: male, control, 64.08 ± 10.57 vs. isoflurane, 48.49 ± 13.41, P = 0.001, n = 60; and female, control, 67.13 ± 11.17 vs. isoflurane, 53.76 ± 6.64, P = 0.003, n = 58). Isoflurane-induced impairment in recognition memory was preventable by the introduction of YC-1. Activation of soluble guanylyl cyclase or protein kinase-G prevents isoflurane or PSD-95 wild-type PDZ2 peptide-induced loss of dendritic spines and synapse. Prevention of recognition memory with YC-1, a NO-independent activator of guanylyl cyclase, supports a role for the soluble guanylyl cyclase mediated protein kinase-G signaling in countering the effects of isoflurane-induced cognitive impairment.

Efficient Delivery of Hydrophilic Small Molecules to Retinal Cell Lines Using Gel Core-Containing Solid Lipid Nanoparticles.

In this study, we developed a novel solid lipid nanoparticle (SLN) formulation for drug delivery of small hydrophilic cargos to the retina. The new formulation, based on a gel core and composite shell, allowed up to two-fold increase in the encapsulation efficiency. The type of hydrophobic polyester used in the composite shell mixture affected the particle surface charge, colloidal stability, and cell internalization profile. We validated SLNs as a drug delivery system by performing the encapsulation of a hydrophilic neuroprotective cyclic guanosine monophosphate analog, previously demonstrated to hold retinoprotective properties, and the best formulation resulted in particles with a size of ±250 nm, anionic charge > −20 mV, and an encapsulation efficiency of ±60%, criteria that are suitable for retinal delivery. In vitro studies using the ARPE-19 and 661W retinal cell lines revealed the relatively low toxicity of SLNs, even when a high particle concentration was used. More importantly, SLN could be taken up by the cells and the release of the hydrophilic cargo in the cytoplasm was visually demonstrated. These findings suggest that the newly developed SLN with a gel core and composite polymer/lipid shell holds all the characteristics suitable for the drug delivery of small hydrophilic active molecules into retinal cells.

The Synthesis of Nucleoside 2',3'-Cyclic Monophosphate Analogs with Regio- and Stereospecificity.

This article presents a new method for the rapid, stereoselective and regioselective synthesis of nucleoside 2',3'-O,O-phosphorothioate and 2',3'-O,O-phosphoroselenoate molecules. The method avoids the use of protection groups, chiral reagents, and chiral metal catalysts, as well as complicated chiral separations. This synthetic method has been applied successfully to all of the natural nucleosides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 2-chloro-1,3,2-dithiophospholane (6) Basic Protocol 2: Preparation of 2-cyanoethoxy-thio-dithiophospholane (8) Basic Protocol 3: Preparation of 2-cyanoethoxy-seleno-dithiophospholane (9) Basic Protocol 4: Preparation of uridine-2',3'-O,O-phosphorothioate (Sp, exo; 1A) Basic Protocol 5: Preparation of uridine-2',3'-O,O-phosphoroselenoate (Sp, exo; 11) Basic Protocol 6: Preparation of adenosine-2',3'-O,O-phosphorothioate (Sp, exo; 12) Basic Protocol 7: Preparation of thymidine-2',3'-O,O-phosphorothioate (Sp, exo; 13) Basic Protocol 8: Preparation of cytosine-2',3'-O,O-phosphorothioate (Sp, exo; 14) Basic Protocol 9: Preparation of guanosine-2',3'-O,O-phosphorothioate (Sp, exo; 15).

C-Type Natriuretic Peptide Plays an Anti-Inflammatory Role in Rat Epididymitis Induced by UPEC.

Human epididymitis is mainly caused by retrograde urinary tract infection with uropathogenic Escherichia coli (UPEC). This disease is an important factor (accounting for 20–30%) causing male infertility. C-type natriuretic peptide (CNP), a protein composed of 22 amino acids, is proved to play an immunoregulatory role in respiratory and cardiovascular systems. CNP is expressed extremely high in the epididymis, but whether CNP plays the same role in acute epididymitis is unclear. At first, we established an acute caput epididymitis model in rats with UPEC and treated them with CNP to measure inflammatory damage. Then RNA-seq transcriptome technology was used to reveal potential signal pathways. Secondly, the turbidity and activity of UPEC were assessed using a microplate reader and the amount of UPEC by agar plates after incubation with CNP. Thirdly, macrophages in caput epididymis were tested by immunohistochemistry (IHC). Meanwhile, lipopolysaccharide (LPS) with or without CNP was used to stimulate the macrophage (RAW264.7) in vitro and to detect the expression level of pro-inflammatory factors. Finally, the macrophage (RAW264.7) was treated with CNP, 8-Br-cGMP [cyclic guanosinc monophosphate (cGMP) analog] and KT5823 [protein kinase G (PKG) inhibitor], and the expression level of nuclear factor-k-gene binding (NF-kB) signal pathway was examined. The results showed that the damage of epididymis induced by UPEC as well as the pro-inflammatory factors could be alleviated significantly with CNP treatment. CNP could inhibit the activity and numbers of bacteria in both in vivo and in vitro experiments. Moreover, CNP repressed the invasion, and the expression of pro-inflammatory factors (such as NF-kB, IL-1β, IL-6, TNF-α) in macrophages and its effect could be inhibited by KT5823. Therefore, we drew a conclusion from the above experiments that CNP alleviates the acute epididymitis injury induced by UPEC. On one hand, CNP could inhibit the growth of UPEC. On the other hand, CNP could decrease invasion and inflammatory reaction of macrophages; the mechanism was involved in inhibiting NF-kB signal pathway through the cGMP/PKG in macrophages. This research would open up the possibility of using CNP as a potential treatment for epididymitis.

Regulation of stanniocalcin-1 secretion by BeWo cells and first trimester human placental tissue from normal pregnancies and those at increased risk of developing preeclampsia.

Stanniocalcin‐1 (STC‐1) is a multi‐functional glycosylated peptide present in the plasma of healthy women postpartum and increased further in pregnancies complicated by preeclampsia. Although the STC‐1 gene is expressed by the placenta what regulates its secretion and from which cells at the feto‐maternal interface is unknown. Here, we demonstrate for the first time that the syncytiotrophoblast and cytotrophoblast are a major site of STC‐1 protein expression in first trimester placental tissue. Further, in response to low oxygen, first trimester chorionic villous tissue from pregnancies at increased risk of developing preeclampsia secreted significantly more STC‐1 than normal tissue under the same conditions. Using the human trophoblast cell line BeWo we have shown that low oxygen increased the secretion of STC‐1 but it required co‐stimulation with the Adenosine‐3', 5'‐cyclic monophosphate (cAMP) analogue, 8‐Bromo adenosine‐3', 5'‐cyclic monophosphate cAMP (8 Br‐cAMP) to reach significance. Inhibition of Hypoxia inducible factor 2α (HIF‐2α) and the Phosphatidylinositol‐3 kinase (PI3‐Kinase)/AKT/Serum and glucocorticoid‐induced kinase‐1(SGK‐1) pathway resulted in significant inhibition of STC‐1 secretion. As both low oxygen and cAMP are known to play a central role in placental function, their regulation of STC‐1 points to a potentially important role in the maintenance of a normal healthy pregnancy and we would hypothesize that it may act to protect against prolonged placental hypoxia seen in preeclampsia.

Addressing regio- and stereo-specificity challenges in the synthesis of nucleoside 2',3'-cyclic monophosphate analogs - a rapid and facile synthesis of nucleoside-2',3'-O,O-phosphoro-thioate or -selenoate, and elucidation of the origin of the rare specificity.

A new facile, rapid, stereo- and regio-selective one-pot synthesis of nucleoside-2',3'-O,O-phosphorothioate and selenoate analogs has been developed. This method avoids the need for protection strategies and chiral reagents, chiral metal catalysts, or chiral separations. This synthetic method has been applied to all natural nucleosides (U/A/G/C/T). Furthermore, we have deciphered the origin of the stereo- and regio-selectivity of the reaction.

Activation of Protein Kinase G After Repeated Cocaine Administration Is Necessary for the Phosphorylation of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor GluA1 at Serine 831 in the Rat Nucleus Accumbens.

Phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the striatum plays a crucial role in regulating the receptor-coupled signaling cascades leading to behavioral changes associated with psychostimulant exposure. The present study determined if activation of protein kinase G (PKG) contributes to the phosphorylation of AMPA receptor GluA1 subunit at the position of serine 831 (GluA1-S831) in the rat nucleus accumbens (NAc) after repeated cocaine administration. The results demonstrated that repeated intraperitoneal (i.p.) injections of cocaine (20 mg/kg) once a day for seven consecutive days significantly increased the level of phosphorylated (p)GluA1-S831. This increase was decreased by the intra-NAc infusion of either the cyclic guanosine monophosphate (cGMP) analog, Rp-8-Br-PET-cGMPS (5 nmol/1 μL), or the PKG inhibitor, KT5823 (2 nmol/1 μL). Repeated cocaine administration increased PKG binding activity to GluA1. This increase in GluA1-S831 phosphorylation after repeated cocaine administration was decreased by the intra-NAc infusion of the synthetic peptide (Tat-tagged interfering peptide (Tat-GluA1-i)), that interferes with the binding of PKG to GluA1. Intra-NAc infusion of the interfering peptide also reduced the repeated cocaine-induced increase in locomotor activity. These findings suggest that activated PKG, after repeated exposure to cocaine, binds to AMPA receptor GluA1 and is required for the phosphorylation of S831, contributing to behavioral changes.

Atrial Natriuretic Peptide Affects Stimulus-Secretion Coupling of Pancreatic β-Cells.

Atrial natriuretic peptide (ANP) influences glucose homeostasis and possibly acts as a link between the cardiovascular system and metabolism, especially in metabolic disorders like diabetes. The current study evaluated effects of ANP on β-cell function by the use of a β-cell-specific knockout of the ANP receptor with guanylate cyclase activity (βGC-A-KO). ANP augmented insulin secretion at the threshold glucose concentration of 6 mmol/L and decreased KATP single-channel activity in β-cells of control mice but not of βGC-A-KO mice. In wild-type β-cells but not β-cells lacking functional KATP channels (SUR1-KO), ANP increased electrical activity, suggesting no involvement of other ion channels. At 6 mmol/L glucose, ANP readily elicited Ca2+ influx in control β-cells. This effect was blunted in β-cells of βGC-A-KO mice, and the maximal cytosolic Ca2+ concentration was lower. Experiments with inhibitors of protein kinase G (PKG), protein kinase A (PKA), phosphodiesterase 3B (PDE3B), and a membrane-permeable cyclic guanosine monophosphate (cGMP) analog on KATP channel activity and insulin secretion point to participation of the cGMP/PKG and cAMP/PKA/Epac (exchange protein directly activated by cAMP) directly activated by cAMP Epac pathways in the effects of ANP on β-cell function; the latter seems to prevail. Moreover, ANP potentiated the effect of glucagon-like peptide 1 (GLP-1) on glucose-induced insulin secretion, which could be caused by a cGMP-mediated inhibition of PDE3B, which in turn reduces cAMP degradation.

The cAMP analogs have potent anti-proliferative effects on medullary thyroid cancer cell lines.

The oncogenic activation of the rearranged during transfection (RET) proto-oncogene has a main role in the pathogenesis of medullary thyroid cancer (MTC). Several lines of evidence suggest that RET function could be influenced by cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity. We evaluated the in vitro anti-tumor activity of 8-chloroadenosine-3',5'-cyclic monophosphate (8-Cl-cAMP) and PKA type I-selective cAMP analogs [equimolar combination of the 8-piperidinoadenosine-3',5'-cyclic monophosphate (8-PIP-cAMP) and 8-hexylaminoadenosine-3',5'-cyclic monophosphate (8-HA-cAMP) in MTC cell lines (TT and MZ-CRC-1)]. 8-Cl-cAMP and the PKA I-selective cAMP analogs showed a potent anti-proliferative effect in both cell lines. In detail, 8-Cl-cAMP blocked significantly the transition of TT cell population from G2/M to G0/G1 phase and from G0/G1 to S phase and of MZ-CRC-1 cells from G0/G1 to S phase. Moreover, 8-Cl-cAMP induced apoptosis in both cell lines, as demonstrated by FACS analysis for annexin V-FITC/propidium iodide, the activation of caspase-3 and PARP cleavage. On the other hand, the only effect induced by PKA I-selective cAMP analogs was a delay in G0/G1-S and S-G2/M progression in TT and MZ-CRC-1 cells, respectively. In conclusion, these data demonstrate that cAMP analogs, particularly 8-Cl-cAMP, significantly suppress in vitro MTC proliferation and provide rationale for a potential clinical use of cAMP analogs in the treatment of advanced MTC.

Kolaviron‐Induced Inhibition of H1299 Lung Cancer Cells Growth and Survival via PKA/P13K Pathways

Kolaviron (KV), a natural biflavonoid obtained from the seeds of Garcinia kola, has been noted for its diverse pharmacological actions such as its antioxidative, antiproliferative, and anti‐inflammatory properties. We have evaluated the antiproliferative and cytotoxic effects of KV on H1299 lung cancer cell line. H1299 Cell was cultured in modified medium fortified with 10% FBS using 96 well plates and MTT assay was performed to determine effects of KV (0.025‐100 mg/ml) on cell proliferation 24 hrs. or 48 hrs. (cytotoxic/apoptosis) after plating. MTT assay indicates that KV dose‐and time ‐dependently inhibited H1299 cell proliferation as well as causing cell death in the cytotoxic studies. These effects were significantly different from the control. The cytotoxic effects of KV were only partially reversed at 24 and 48 hrs. following treatment with cell division/ cell growth promoter, 8‐bromoadenosine 3':5'‐cyclic monophosphate (1mM) analog of cAMP. This observation indicates that KV is probably mediating its cytotoxic effects via inhibition of pro‐survival and pro‐apoptotic signals mediated by cAMP modulated downstream effectors through PKA/PI3K, pathways. These preliminary results suggest that KV has therapeutic potential against lung cancer and the mechanisms of actions need further investigation.

Sildenafil stimulates human trophoblast invasion through nitric oxide and guanosine 3′,5′-cyclic monophosphate signaling

To determine the effect of sildenafil, a phosphodiesterase type 5 inhibitor, on trophoblast invasiveness. Laboratory investigation. Academic medical center. Placental tissues discarded after first-trimester terminations were obtained from patients with informed consent. A cell line, HTR-8/SVneo, established from first-trimester cytotrophoblast, and villous explants, was treated with or without sildenafil, guanosine 3',5'-cyclic monophosphate (cGMP) analog, cGMP inhibitor, or L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) and cultured on fibronectin or Matrigel. Integrins α6β4 and α1β1 were detected by immunocytochemistry. Trophoblast outgrowth from villous tips, cytotrophoblast cell invasion, and integrin immunostaining were assessed in cytotrophoblast and explant cultures. Integrin expression in trophoblast cells ex vivo switched from α6 to α1, and invasiveness increased, when exposed to sildenafil or cGMP agonist. Either cGMP antagonist or L-NAME blocked integrin switching and invasion induced by sildenafil. Elevation of nitric oxide pharmacologically induced invasion, but not when cGMP antagonist was present. Sildenafil altered trophoblast phenotype through a process dependent on nitric oxide availability and cGMP accumulation. In addition to its vasoactivity, sildenafil directly stimulates trophoblast extravillous differentiation, which would be favorable for implantation and reduce risk for adverse pregnancy outcomes.

Genetic ablation of solute carrier family 7a3a leads to hepatic steatosis in zebrafish during fasting.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder caused by abnormal lipid metabolisms, such as reduced hepatic fatty acid oxidation (FAO), but intracellular control of FAO under physio- and pathological conditions remains largely undefined. Here, we demonstrate that deprivation of Slc7a3a leads to hepatic steatosis in fasted zebrafish as a result of defects in arginine-dependent nitric oxide (NO) synthesis. Fast-induced hepatic steatosis in slc7a3a-null mutants can be rescued by treatments with NO donor, cyclic guanosine monophosphate analog, adenosine-monophosphate-activated protein kinase (AMPK) activator, or peroxisome proliferator-activated receptor alpha (PPAR-α) agonist. In contrast, inhibitors of NO synthases, AMPK, or soluble guanylate cyclase and liver-specifically expressed dominant negatives of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and PPAR-α are sufficient to induce hepatic steatosis in fasted wild-type larvae. Moreover, knockdown of Slc7a3 in mice or SLC7A3 in human liver cells impaired AMPK-PPAR-α signaling and resulted in lipid accumulation under fasting or glucose starvation, respectively. These findings have revealed a NO-AMPK-PPAR-α-signaling pathway that is crucial for the control of hepatic FAO in vertebrates.

Nitric-oxide inhibits nyctinastic closure through cGMP in Albizia lophantha leaflets

Nitric oxide (NO) is a highly reactive radical that acts as a direct or indirect cellular signalling molecule in plant growth, development and environmental responses. Here we studied the contribution of NO to the control of leaflet movements during nyctinastic closure. For this purpose, we tested the effect of NO donors and an NO scavenger, all supplied in light, on Albizia lophantha leaflet closure after transferral to darkness. Exogenous NO, applied as four donors [sodium nitroprusside (SNP), diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (DEA-NONOate), S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GS-NO)], inhibited nyctinastic leaflet closure while the application of an NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)] plus SNP cancelled the effect of the latter. The inclusion of Nω-nitro-l-arginine methyl ester (l-NAME) or sodium tungstate in the incubation media enhanced nyctinastic closure and also resulted in a decrease in the nitrate plus nitrite released by leaflets into the incubation solution. These results support the notion that NO is involved in regulating the nyctinastic closure of A. lophantha leaflets. Cellular perception of NO did not appear to be mediated by calcium. Pharmacological application of inhibitors of soluble guanylate cyclase (sGC) [1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ) and 6-anilino-5,8-quinolinequinone (Ly83583)], phosphodiesterase type 5 (PDE5) (Sildenafil) and the cyclic guanosine monophosphate (cGMP) analogue 8-bromoguanosine-3',5'-cyclomonophosphate sodium salt (8-Br-cGMP) indicated that cGMP was downstream of the NO signalling cascade during nyctinastic closure.

Stimulation of the soluble guanylate cyclase (sGC) inhibits fibrosis by blocking non-canonical TGFβ signalling

ObjectivesWe have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between...

Sildenafil Inhibits Hypoxia-Induced Transient Receptor Potential Canonical Protein Expression in Pulmonary Arterial Smooth Muscle via cGMP-PKG-PPARγ Axis

Transient receptor potential canonical (TRPC) proteins play important roles in chronically hypoxic pulmonary hypertension (CHPH). Previous results indicated that sildenafil inhibited TRPC1 and TRPC6 expression in rat distal pulmonary arteries (PAs). However, the underlying mechanisms remain unknown. We undertook this study to investigate the downstream signaling of sildenafil's regulation on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia-exposed rats (10% O2 for 21 d) and rat distal PASMCs (4% O2 for 60 h) were taken as models to mimic CHPH. Real-time PCR, Western blotting, and Fura-2-based fluorescent microscopy were performed for mRNA, protein, and Ca(2+) measurements, respectively. The cellular cyclic guanosine monophosphate (cGMP) analogue 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate sodium salt (CPT-cGMP) (100 μM) inhibited TRPC1 and TRPC6 expression, store-operated Ca(2+) entry (SOCE), and the proliferation and migration of PASMCs exposed to prolonged hypoxia. The inhibition of CPT-cGMP on TRPC1 and TRPC6 expression in PASMCs was relieved by either the inhibition or knockdown of cGMP-dependent protein kinase (PKG) and peroxisome proliferator-activated receptor γ (PPARγ) expression. Under hypoxic conditions, CPT-cGMP increased PPARγ expression. This increase was abolished by the PKG antagonists Rp8 or KT5823. PPARγ agonist GW1929 significantly decreased TRPC1 and TRPC6 expression in PASMCs. Moreover, hypoxia exposure decreased, whereas sildenafil treatment increased, PKG and PPARγ expression in PASMCs ex vivo, and in rat distal PAs in vivo. The suppressive effects of sildenafil on TRPC1 and TRPC6 in rat distal PAs and on the hemodynamic parameters of CHPH were inhibited by treatment with the PPARγ antagonist T0070907. We conclude that sildenafil inhibits TRPC1 and TRPC6 expression in PASMCs via cGMP-PKG-PPARγ-dependent signaling during CHPH.

Protein kinase G increases AMPA receptor GluR1 phosphorylation at serine 845 after repeated cocaine administration in the rat nucleus accumbens

The regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR1 subunit phosphorylation at serine 845 (GluR1-Ser845) by protein kinase G (PKG) activation was investigated in the nucleus accumbens (NAc) after repeated cocaine administration. Intra-NAc injection of the cyclic guanosine monophosphate (cGMP) analog, Rp-8-Br-PET-cGMPS (5nmol) and the PKG inhibitor, KT5823 (2nmol), prior to the final drug injection significantly decreased GluR1-Ser845 phosphorylation elevated by repeated systemic injections of cocaine (20mg/kg) once a day for seven consecutive days. The inhibition of PKG also attenuated Ca2+-calmodulin-dependent protein kinases II (CaMKII) phosphorylation, however inhibition of CaMKII with KN62 (20nmol) did not alter the phosphorylation state of GluR1-Ser845. Similarly, inhibition of cGMP or PKG attenuated the repeated cocaine-induced increase in locomotor activity. These findings suggest that the AMPA receptor provides a PKG-sensitive phosphorylation site on GluR1-Ser845 in the NAc after repeated cocaine, thus contributing to behavioral alterations.

Testosterone/Estradiol Ratio Regulates NO‐Induced Bladder Relaxation and Responsiveness to PDE5 Inhibitors

The efficacy of phosphodiesterase type 5 inhibitors (PDE5i) in treating lower urinary tract symptoms is supported by the extremely high expression and activity of PDE5 in male bladder. Although bladder function regulation is similar among genders, no data are available on PDE5 expression and activity in female bladder. To investigate sex differences in PDE5 expression and biological activity in female bladder, as opposed to the male counterpart. Gene and protein expression and enzymatic activity of PDE5. We studied gene and protein expression, and enzymatic activity of PDE5 in bladder of male and female rats. A subgroup of female rats was ovariectomized and alternatively replaced with estradiol (E2), progesterone, and testosterone (T) alone or in combination with letrozole to completely abrogate T-induced E formation. As a readout of PDE5 activity, we studied vardenafil efficacy in potentiating sodium nitroprusside (SNP)-induced relaxation in bladder of the different experimental groups. SNP was three-log unit less potent in relaxing the male bladder than the female one. On the contrary, the PDE5-resistant cyclic guanosine monophosphate (cGMP) analog (Bromo-β-phenyl-1, N(2) -ethenoguanosine-3', 5'-cyclic monophosphorothioate, Sp-isomer [SP-8-Br-PET-cGMPS]) was equipotent in relaxing male and female bladder. Vardenafil was more effective in potentiating SNP-induced bladder relaxation in male than in female. Accordingly, the cGMP-hydrolyzing activity of PDE5 was higher in male vs. female homogenates. In ovariectomized female rats, with or without sex-steroid replacement, vardenafil activity in potentiating SNP-induced bladder relaxation was associated with an increased T/E2 ratio. In particular, masculinization of ovariectomized rats--by the administration of T + letrozole--dramatically increased vardenafil capacity to potentiate SNP-induced relaxation. In this study, we demonstrated that PDE5 activity is more pronounced in male as compared with female bladder and that T/E ratio positively regulates responsiveness to PDE5i, thus suggesting that male bladder is a more suitable target for PDE5i than the female counterpart.