POCU1b, the n-Butanol Soluble Fraction of Polygoni Cuspidati Rhizoma et Radix, Attenuates Obesity, Non-Alcoholic Fatty Liver, and Insulin Resistance via Inhibitions of Pancreatic Lipase, cAMP-Dependent PDE Activity, AMPK Activation, and SOCS-3 Suppression.

This study investigated the effects of the n-BuOH soluble fraction of Polygoni Cuspidati 80% ethanol extract (POCU1b) on high-fat diet (HFD)-induced obesity, non-alcoholic fatty liver (NAFL), and insulin resistance (IR) to find a safe and more effective agent. HPLC profiling of POCU1b identified seven marker compounds. POCU1b increased glycerol release, cyclic adenosine monophosphate (cAMP) level, and inhibited phosphodiesterase (PDE) activity. Seven weeks of POCU1b treatment decreased body weight gain, weight and adipocyte size in fat tissues, serum lipids, and triglyceride and lipid droplets in the livers of HFD-fed rats. POCU1b improved blood glucose, insulin sensitivity, and impaired insulin secretion in the pancreas. Further, POCU1b ameliorated adiponectin, leptin, IL-6 and TNF-α levels, increased AMPK and p-ACC expression, activated CPT-1 activity, and suppressed FAS mRNA, SOCS-3 protein expression, and NF-κB DNA-binding activity. When compared with the Xenical®-treated group, a positive group, the action of POCU1b on body weight was more effective than that of Xenical. POCU1b did not show side effects, such as oily spotting and loss of appetite. These results suggest that POCU1b possesses therapeutic or preventive potential for obesity, NAFL and IR via inhibitions of pancreatic lipase and cAMP-dependent PDE activity, AMPK activation, and SOCS-3 suppression, without oily spotting and loss of appetite.

Development of a Novel Intraocular-Pressure-Lowering Therapy Targeting ATX.

Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.

Inhibitory Effect of New Semisynthetic Usnic Acid Derivatives on Human Tyrosyl-DNA Phosphodiesterase 1.

Usnic acid, a lichen secondary metabolite produced by a whole number of lichens, has attracted the interest of researchers owing to its broad range of biological activity, including antiviral, antibiotic, anticancer properties, and it possessing a certain toxicity. The synthesis of new usnic acid derivatives and the investigation of their biological activity may lead to the discovery of compounds with better pharmacological and toxicity profiles. In this context, a series of new usnic acid derivatives comprising a terpenoid moiety were synthesized, and their ability to inhibit the catalytic activity of the human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 was investigated. The most potent compounds (15A, 15B, 15G: , and 16A, 16B, 16G: ) had IC50 values in the range of 0.33 - 2.7 µM. The inhibitory properties were mainly dependent on the flexibility and length of the terpenoid moiety, but not strongly dependent on the configuration of the asymmetric centers. The synthesized derivatives showed low cytotoxicity against human cell lines in an MTT assay. They could be used as a basis for the development of more effective anticancer therapies when combined with topoisomerase 1 inhibitors.

Identification and pharmacological characterization of a novel inhibitor of autotaxin in rodent models of joint pain.

OBJECTIVE: Autotaxin is a secreted lysophospholipase that mediates the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator. Autotaxin levels in plasma and synovial fluid correlate with disease severity in patients with knee osteoarthritis (OA). The goal of this study was to develop and characterize a novel small molecule inhibitor of autotaxin to inhibit LPA production in vivo and determine its efficacy in animal models of musculoskeletal pain. DESIGN: Compound libraries were screened using an LPC coupled enzyme assay that measures the amount of choline released from LPC by the action of autotaxin. Hits from this assay were tested in a plasma assay to assess inhibition of endogenous plasma autotaxin and subsequently tested for their ability to lower plasma LPA levels upon oral dosing of rats. The best compounds were then tested in animal models of musculoskeletal pain. RESULTS: Compound screening led to the identification of compounds with nanomolar potency for inhibition of autotaxin activity. Studies in rats demonstrated a good correlation between compound exposure levels and a decrease in LPA levels in plasma. The leading molecule (compound-1) resulted in a dose dependent decrease in joint pain in the mono-sodium iodoacetate (MIA) and meniscal tear models and a decrease in bone fracture pain in the osteotomy model in rats. CONCLUSION: We have identified and characterized a novel small molecule inhibitor of autotaxin and demonstrated its efficacy in animal models of musculoskeletal pain. The inhibitor has the potential to serve as an analgesic for human OA and bone fracture.

(±)-Torreyunlignans A-D, rare 8-9' linked neolignan enantiomers as phosphodiesterase-9A inhibitors from Torreya yunnanensis.

(±)-Torreyunlignans A-D (1a/1b-4a/4b), four pairs of new 8-9' linked neolignan enantiomers featuring a rare (E)-2-styryl-1,3-dioxane moiety, were isolated from the trunk of Torreya yunnanensis. The structures were determined by combined spectroscopic and chemical methods, and the absolute configurations were elucidated by ECD calculations. The compounds were screened by using tritium-labeled adenosine 3',5'-cyclic monophosphate ([(3)H]-cGMP) as a substrate for inhibitory affinities against phosphodiesterase-9A (PDE9A), which is a potential target for the treatment of diabetes and Alzheimer's disease. All of the enantiomers exhibited inhibition against PDE9A with IC50 values ranging from 5.6 to 15.0 µM. This is the first report of PDE9A inhibitors from nature.

Development of a plate-based optical biosensor fragment screening methodology to identify phosphodiesterase 10A inhibitors.

We describe the development of a novel fragment screening methodology employing a plate-based optical biosensor system that can operate in a 384-well format. The method is based on the "inhibition in solution assay" (ISA) approach using an immobilized target definition compound (TDC) that has been specifically designed for this purpose by making use of available structural information. We demonstrate that this method is robust and is sufficiently sensitive to detect fragment hits as weak as KD 500 µM when confirmed in a conventional surface plasmon resonance approach. The application of the plate-based screen, the identification of fragment inhibitors of PDE10A, and their structural characterization are all discussed in a forthcoming paper.

Synthesis and in vitro evaluation of new analogues as inhibitors for phosphodiesterase 10A.

A series of analogues were synthesized by optimizing the structure of papaverine. The in vitro PDE10A binding affinity (IC(50)) values for these new analogues were measured; for compounds that have IC(50) value less than 60 nM for PDE10A, the binding affinities (IC(50) value) for PDE3A and PDE3B were tested. Of these analogues, compounds 6a, 6b, 6n, 8b, 8c and 11 displayed relatively higher PDE10A potency with IC(50) value in the range of 28-60 nM. The most potent compound 1-(4-(2-(2-fluoroethoxy)ethoxy)-3-methoxybenzyl)-6,7-dimethoxyisoquinoline (8c) has the IC(50) value of 28 ± 1.2 nM for PDE10A, 2200 ± 437 nM for PDE3A and 2520 ± 210 nM for PDE3B. Compared to papaverine, compound 8c displayed similar PDE10A potency but improved selectivity to PDE10A versus PDE3A and PDE3B. To identify high potent PDE10A inhibitor, further optimization of the structures of these analogues is necessary.

Anti-obesity effect of Morus bombycis root extract: anti-lipase activity and lipolytic effect.

AIM OF THE STUDY: This study evaluated anti-obesity effect of the ethanolic extract of Morus bombycis root on lipase activity and lipolysis in adipocytes and adipose tissues. MATERIALS AND METHODS: Lipase (triacylgycerol acylhydrolase, EC 3.1.1.3) activity was determined by measuring the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol at 405 nm. Lipolytic effects were assayed in fully differentiated 3T3-L1 adipocytes and adipose tissues. In vitro, phosphodiesterase (PDE) activity was also measured. RESULTS: Morus bombycis root extract exhibited strong anti-lipase activity, with an IC50 value of 2.07 microg/mL.In differentiated adipocytes and adipose tissues, the extract increased lipolytic effects such as decreased intracellular triglyceride and the release of glycerol. Further, the extract inhibited PDE activity in a dose-dependent manner. CONCLUSION: The present study suggests that Morus bombycis root extract might be of therapeutic interest with respect to the treatment of obesity.

Epimedium brevicornum Maxim extract relaxes rabbit corpus cavernosum through multitargets on nitric oxide/cyclic guanosine monophosphate signaling pathway.

Epimedium brevicornum Maxim (EbM) has been reputed to have sexual stimulation effects on males. The study is aimed to test the hypothesis that EbM extracts relaxed the corpus cavernosum (CC) smooth muscle through activation of multitargets on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway. Water extract of EbM and its subfraction (EP-20) were prepared and standardized by high-performance liquid chromatography. Isolated rabbit CC strips were mounted in organ baths and isometric tension was recorded in the presence or absence of specific inhibitors related to NO/cGMP signaling such as L-N(G)-nitro-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ, a guanylyl cyclase inhibitor) or phosphodiesterase 5 (PDE 5) inhibitors. cGMP level was determined in EP-20-treated CC strips. The results showed that EP-20 enriched the content of L-arginine in the process of purification and relaxed the CC smooth muscle precontracted with phenylephrine (PE, 1 microM) in a concentration-dependent manner. Besides, EP-20 increased the amount of cGMP production in rabbit CC tissues. Coincubation with EP-20 and L-NAME or ODQ significantly decreased EP-20-induced relaxation whereas EP-20 increased sodium nitroprusside-induced relaxation in PE-precontracted CC strips. Besides, EP-20 increased the potency and the duration of the relaxation effects caused by electrical field stimulation. Finally, EP-20 could potentiate PDE 5 inhibitors in relaxation of PE-precontracted CC strips. We concluded that extract of EbM relax the CC smooth muscle through multitargets in NO/cGMP/PDE 5 pathway and might bring into perspective the treatment strategy for those patients with erectile dysfunction.

Simultaneous determination of synthetic phosphodiesterase-5 inhibitors found in a dietary supplement and pre-mixed bulk powders for dietary supplements using high-performance liquid chromatography with diode array detection and liquid chromatography-electrospray ionization tandem mass spectrometry.

A high-performance liquid chromatography-diode array detection (HPLC-DAD) method and a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method were developed to screen for the presence of synthetic phosphodiesterase-5 (PDE-5) inhibitors and their analogues, namely sildenafil, vardenafil, tadalafil, homosildenafil, acetildenafil and hydroxyhomosildenafil. The methods were applied to pre-market samples submitted to the Health Sciences Authority of Singapore (HSA) for testing. One sample was in the form of capsules while six other samples were pre-mixed bulk powder samples for dietary supplements to be repackaged or formulated into the final dosage forms (usually capsules). Identification of PDE-5 inhibitors and their analogues was achieved by comparing individual peak retention times, UV spectra and mass spectra with those of reference standards. The seven samples were found to contain at least one of the following compounds: sildenafil, vardenafil, hydroxyhomosildenafil, homosildenafil and acetildenafil. The five compounds were simultaneously determined by LC-ESI-MS/MS in multiple reactions monitoring (MRM) scan mode. The method has been validated for accuracy, precision, linearity and sensitivity.

A new chemical tool for exploring the physiological function of the PDE2 isozyme.

Oxindole (2) is a potent and selective PDE2 inhibitor with a favorable ADME, physiochemical and pharmacokinetic profile to allow for use as a chemical tool in elucidating the physiological role of PDE2.

Implication of cyclic nucleotide phosphodiesterase inhibition in the vasorelaxant activity of the citrus-fruits flavonoid (+/-)-naringenin.

The potential vasorelaxant, antioxidant and cyclic nucleotide phosphodiesterase (PDE) inhibitory effects of the citrus-fruit flavonoids naringin and (+/-)-naringenin were comparatively studied for the first time in this work. (+/-)-Naringenin (1 microM - 0.3 mM) did not affect the contractile response induced by okadaic acid (OA, 1 microM). However, (+/-)-naringenin relaxed, in a concentration-dependent manner, the contractions elicited by phenylephrine (PHE, 1 microM) or by a high extracellular KCl concentration (60 mM) in intact rat aortic rings. Mechanical removal of endothelium and/or pretreatment of aorta rings with glibenclamide (GB, 10 microM) or tetraethylammonium (TEA, 2 mM) did not significantly modify the vasorelaxant effects of this flavanone. (+/-)-Naringenin (10 microM - 0.1 mM) did not alter the basal uptake of 4) Ca2+ but decreased the influx of 45Ca2+ induced by PHE and KCl in endothelium-containing and endothelium-denuded rat aorta. (+/-)-Naringenin (10 microM - 0.1 mM) was ineffective to scavenge superoxide radicals (O*2-) generated by the hypoxanthine (HX)-xanthine oxidase (XO) system and/or to inhibit XO activity. (+/-)-Naringenin (0.1 mM) significantly increased the production of cGMP and cAMP decreased by PHE (1 microM) and high KCl (60 mM) in cultured rat aortic myocytes. (+/-)-Naringenin preferentially inhibited calmodulin (CaM)-activated PDE1, PDE4 and PDE5 isolated from bovine aorta with IC50 values of about 45 microM, 60 microM and 68 microM, respectively. In contrast, the 7-rhamnoglucoside of (+/-)-naringenin, naringin (1 microM - 0.3 mM), was totally inactive in all experiments. These results indicate that the vasorelaxant effects of (+/-)-naringenin seem to be basically related to the inhibition of PDE1, PDE4 and PDE5 activities.

DA-8159 reverses selective serotonin reuptake inhibitor-induced erectile dysfunction in rats.

OBJECTIVES: To assess the efficacy of the phosphodiesterase 5 inhibitor, DA-8159, in selective serotonin reuptake inhibitor (SSRI)-induced rat erectile dysfunction model by measuring intracavernous pressure (ICP). METHODS: Erectile dysfunction was induced by oral administration of either paroxetine or fluoxetine in rats. The changes in ICP and mean arterial pressure (MAP) were simultaneously recorded throughout electrostimulation of the cavernous nerve with 2 or 10 Hz after intravenous injection of DA-8159 (1 mg/kg). Statistical analysis was performed on the ICP/MAP ratio and the area under the curve of the ICP/MAP ratio. RESULTS: Although the reduction in the ICP responses after acute paroxetine or fluoxetine administration was statistically significant, the electrical stimulation of the cavernous nerve induced a statistically significant, frequency-dependent increase in the ICP/MAP ratio after DA-8159 administration. The differences in the ICP/MAP ratio and corresponding area under the curve values from the SSRI-treated group were statistically significant. CONCLUSIONS: The results of the present study have demonstrated that DA-8159 reverses the decrease in ICP induced by SSRI treatment, suggesting that DA-8159 may be a potential therapeutic agent for the treatment of erectile dysfunction associated with the use of SSRIs.

Effects of a new phosphodiesterase enzyme type V inhibitor (UK 343-664) versus milrinone in a porcine model of acute pulmonary hypertension.

BACKGROUND: Perioperative pulmonary hypertension remains a clinical challenge. The phosphodiesterase enzyme type III inhibitor milrinone produces pulmonary vasodilation but lacks selectivity. Sildenafil, a phosphodiesterase enzyme type V inhibitor, can also induce relaxation of the pulmonary vasculature; however, only the oral formulation is presently available. This study evaluated the effects of a new intravenous sildenafil analogue--UK 343-664--compared with milrinone during acute pulmonary hypertension in a porcine model of thromboxane-induced pulmonary hypertension. METHODS: After acute pulmonary hypertension, 24 adult swine were randomized to 3 groups. Group 1 (n = 9) received an intravenous dose of 500 microg of UK 343-664, group 2 (n = 8) received milrinone 50 mg/kg, and group 3 (n = 7) received 10 mL of normal saline solution. All agents were administered for more than 5 minutes. Data were recorded continuously for 30 minutes. RESULTS: Both milrinone and UK 343-664 partially reversed thromboxane-induced pulmonary hypertension, with a notable decrease in mean pulmonary artery pressure and pulmonary vascular resistance and a concomitant increase in cardiac output. In addition, milrinone improved right ventricular contractility but produced marked systemic vasodilatation. In contrast, the administration of UK 343-664 was associated with pulmonary vasodilatation, without appreciable changes in systemic arterial pressure or vascular resistance. CONCLUSIONS: Milrinone and UK 343-664 were equally effective as pulmonary vasodilators; however, only UK 343-664 exhibited a high degree of pulmonary selectivity. Potential uses for this new phosphodiesterase enzyme type V inhibitor warrant further study.

Signaling molecules in overcirculation-induced pulmonary hypertension in piglets: effects of sildenafil therapy.

BACKGROUND: The phosphodiesterase type-5 (PDE-5) inhibitor sildenafil has been reported to improve pulmonary arterial hypertension (PAH), but the mechanisms that account for this effect are incompletely understood. Severe pulmonary hypertension has been characterized by defects in a signaling pathway involving angiopoietin-1 and the bone morphogenetic receptor-2 (BMPR-2). We investigated the effects of sildenafil on hemodynamics and signaling molecules in a piglet overcirculation-induced model of early PAH. METHODS AND RESULTS: Thirty 3-week-old piglets were randomized to placebo or sildenafil therapy 0.75 mg/kg TID after anastomosis of the left subclavian artery to the pulmonary arterial trunk or after a sham operation. Three months later, the animals underwent a hemodynamic evaluation followed by pulmonary tissue sampling for morphometry, immunohistochemistry or radioimmunoassay, and real-time quantitative-polymerase chain reaction. Chronic systemic-to-pulmonary shunting increased pulmonary mRNA for angiopoietin-1, endothelin-1 (ET-1), angiotensin II, inducible nitric oxide synthase, vascular endothelial growth factor, and PDE-5. Pulmonary messenger RNA for BMPR-1A and BMPR-2 decreased. Pulmonary angiotensin II, ET-1, and vascular endothelial growth factor proteins increased. Pulmonary artery pressure increased from 20+/-2 to 33+/-1 mm Hg, and arteriolar medial thickness increased by 91%. The expressions of angiopoietin-1, ET-1, and angiotensin II were tightly correlated to pulmonary hypertension. Sildenafil prevented the increase in pulmonary artery pressure, limited the increase in medial thickness to 41%, and corrected associated biological perturbations except for the angiopoietin-1/BMPR-2 pathway, PDE-5, and angiotensin II. CONCLUSIONS: Sildenafil partially prevents overcirculation-induced PAH and associated changes in signaling molecules. Angiotensin II, PDE-5, and angiopoietin-1/BMPR-2 signaling may play a dominant role in the early stages of the disease.

Effects of phosphodiesterase V inhibition on nitric oxide-mediated relaxation responses in guinea pig trachea.

In the present study, we aimed to evaluate the effects of PDE V inhibition on NO-mediated relaxation responses in isolated guinea pig trachea. Under the NANC conditions, tracheal preparations were contracted with histamine (100 microm/l). When contraction had reached a plateau, relaxation responses to electrical field stimulation (EFS, 60 V, 0.5 ms, 5-10 Hz) were determined before and after incubation of the tracheal ring with L-NAME (1 mmol/l), a NO synthase inhibitor. L-NAME significantly inhibited the relaxation responses and this inhibitory effect was reversed by L-arginine (1 mmol/l), a precursor of NO, but was not affected by D-arginine. In addition, cumulative application of the NO donors, 3-morpholino-sydnonimine (SIN-1) and sodium nitroprusside (SNP), caused concentration-dependent relaxation of tissues precontracted with histamine. The selective PDE type V inhibitor zaprinast at EC50 concentration (30 micromol/l) significantly potentiated EFS-induced NANC relaxations and relaxant responses to SIN-1 and SNP. In conclusion, these data support the hypothesis that NO is a mediator of NANC relaxations of guinea pig tracheal rings and PDE V inhibition potentiates NO-mediated relaxation.

Spasmolytic effects, mode of action, and structure-activity relationships of stilbenoids from Nidema boothii.

A CH(2)Cl(2)-MeOH (1:1) extract prepared from the whole plant of Nidema boothii inhibited spontaneous contractions (IC(50) = 6.26 +/- 2.5 microg/mL) of the guinea-pig ileum. Bioassay-guided fractionation of the active extract led to the isolation of the novel spiro compound 1, which was given the trivial name nidemone, and the new dihydrophenanthrene 3, characterized as 1,5,7-trimethoxy-9,10-dihydrophenanthrene-2,6-diol. In addition, the known stilbenoids aloifol II (2), 1,5,7-trimethoxyphenanthrene-2,6-diol (4), ephemeranthoquinone (5), gigantol (6), ephemeranthol B (7), 2,4-dimethoxyphenanthrene-3,7-diol (8), lusianthridin (9), and batatasin III (10) were obtained. The isolates were characterized structurally by spectroscopic data interpretation. Compounds 2-6, 9, and 10 induced notable concentration-dependent inhibition of the spontaneous contractions of the guinea-pig ileum with IC(50) values that ranged between 0.14 and 2.36 microM. Bibenzyl analogues 23-35 were synthesized and tested pharmacologically. The results indicated that for maximum spasmolytic activity the bibenzyls should have oxygenated substituents on both aromatic rings; on the other hand, methylation of free hydroxyl groups as well as the increment of oxygenated groups in relation to compounds 6 and 10 decreased the smooth muscle relaxant activity. It was also demonstrated that bibenzyls 6 and 10 might exert their spasmolytic action not only by a nitrergic mechanism but also by inhibiting CaM-mediated processes.

Cardiovascular assessment of ER-118585, a selective phosphodiesterase 5 inhibitor.

The aim of this study was to assess the cardiovascular effects of a selective phosphodiesterase 5 inhibitor ER-118585, 4-[(3-chloro-4-methoxybenzyl)amino]-1-(2-hydroxy-7-azaspiro[3.5]non-7-yl)-6-phthalazinecarbonitrile monohydrochloride. The present results indicated that 1) ER-118585 significantly inhibited the human ether-a-go-go related gene (HERG) tail current at 10 nM and above with an IC(50) value of 40.7 nM in human embryonic kidney 293 cells transfected with HERG cDNA; 2) ER-118585 at 100 and 1000 nM significantly increased the action potential duration (APD) at 50% and 90% repolarization in isolated papillary muscles of guinea pig; and 3) intravenous infusion of ER-118585 at 10 microg/kg/min significantly prolonged the QT interval by 10.5+/-1.6% from 281+/-2 ms to 311+/-6 ms in six anesthetized dogs subjected to atrial pacing. In consideration of both the plasma concentration of ER-118585 (984+/-78 nM, n=3) and its protein binding fraction (99.0+/-0.1%, n=5), the free plasma concentration was estimated at 9.8+/-0.8 nM, which is consistent with the minimum concentration of HERG current inhibition. In conclusion, these evaluation methods demonstrated that ER-118585 could prolong the QT interval via APD prolongation, attributable to the inhibition of the HERG potassium current.

Protein kinase A-mediated phosphorylation of HERG potassium channels in a human cell line.

OBJECTIVE: To investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line. METHODS: HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody. RESULTS: Elevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated. CONCLUSIONS: PKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.