Adolescent high-fructose corn syrup consumption leads to dysfunction in adult affective behaviors and mesolimbic proteins in male Sprague-Dawley rats.

Adolescence is a critical period of development, during which the brain undergoes rapid maturation. Problematically, adolescents are the top consumers of high fructose corn syrup (HFCS) sweetened beverages and snacks, which may have neurodevelopmental consequences. While HFCS consumption has been linked to an increased likelihood of obesity and other physical health impairments, the link between HFCS and persistent behavioral changes is not yet fully established. The present study aimed to assess whether adolescent HFCS consumption could lead to alterations in adult behaviors and protein expression, following cessation. Adolescent HFCS-exposure contributed to deficits in learning and motivation on an effort-related T-Maze procedure, as well as increased immobility time in the forced swim paradigm during adulthood. Molecularly, protracted decreases in accumbal dopamine D1 and D2 receptors and protein kinase G (PKG), as well as increases in tyrosine hydroxylase and GluA2 receptor subunits, were observed following HFCS-exposure. Taken together, these data suggest that adolescent HFCS-consumption leads to protracted dysfunction in affective behaviors and alterations in accumbal proteins which persist following cessation of HFCS-consumption.

Vasorelaxing cell permeant phosphopeptide mimetics for subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm leads to vasospasm resulting in delayed cerebral ischemia. Therapeutic options are currently limited to hemodynamic optimization and nimodipine, which have marginal clinical efficacy. Nitric oxide (NO) modulates cerebral blood flow through activation of the cGMP-Protein Kinase G (PKG) pathway. Our hypothesis is that SAH results in downregulation of signaling components in the NO-PKG pathway which could explain why treatments for vasospasm targeting this pathway lack efficacy and that treatment with a cell permeant phosphopeptide mimetic of downstream effector prevents delayed vasospasm after SAH. Using a rat endovascular perforation model, reduced levels of NO-PKG pathway molecules were confirmed. Additionally, it was determined that expression and phosphorylation of a PKG substrate: Vasodilator-stimulated phosphoprotein (VASP) was downregulated. A family of cell permeant phosphomimetic of VASP (VP) was wasdesigned and shown to have vasorelaxing property that is synergistic with nimodipine in intact vascular tissuesex vivo. Hence, treatment targeting the downstream effector of the NO signaling pathway, VASP, may bypass receptors and signaling elements leading to vasorelaxation and that treatment with VP can be explored as a therapeutic strategy for SAH induced vasospasm and ameliorate neurological deficits.

Cannabinoid receptor type 1 in the bed nucleus of the stria terminalis modulates cardiovascular responses to stress via local N-methyl-D-aspartate receptor/neuronal nitric oxide synthase/soluble guanylate cyclase/protein kinase G signaling.

BACKGROUND: Endocannabinoid neurotransmission in the bed nucleus of the stria terminalis is involved in the control of cardiovascular responses to stress. However, the local mechanisms involved is this regulation are not known. AIMS: The purpose of this study was to assess an interaction of bed nucleus of the stria terminalis endocannabinoid neurotransmission with local nitrergic signaling, as well as to investigate the involvement of local N-methyl-D-aspartate glutamate receptor and nitric oxide signaling in the control of cardiovascular responses to acute restraint stress by bed nucleus of the stria terminalis endocannabinoid neurotransmission in rats. METHODS: The first protocol evaluated the effect of intra-bed nucleus of the stria terminalis microinjection of the selective cannabinoid receptor type 1 receptor antagonist AM251 in nitrite/nitrate content in the bed nucleus of the stria terminalis following restraint stress. The other protocols evaluated the impact of local pretreatment with the selective N-methyl-D-aspartate glutamate receptor antagonist LY235959, the selective neuronal nitric oxide synthase inhibitor Nω-propyl-L-arginine, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or the protein kinase G inhibitor KT5823 in restraint-evoked cardiovascular changes following bed nucleus of the stria terminalis treatment with AM251. RESULTS: Bilateral microinjection of AM251 into the bed nucleus of the stria terminalis increased local nitric oxide release during restraint stress. Bed nucleus of the stria terminalis treatment with the cannabinoid receptor type 1 receptor antagonist also enhanced the tachycardia caused by restraint stress, but without affecting arterial pressure increase and sympathetic-mediated cutaneous vasoconstriction. The facilitation of restraint-evoked tachycardia following bed nucleus of the stria terminalis treatment with the cannabinoid receptor type 1 receptor antagonist was completely inhibited by local pretreatment with LY235959, Nω-propyl-L-arginine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or KT5823. CONCLUSIONS: Our results provide evidence that bed nucleus of the stria terminalis endocannabinoid neurotransmission inhibits local N-methyl-D-aspartate/neuronal nitric oxide synthase/soluble guanylate cyclase/protein kinase G signaling, and this mechanism is involved in the control of the cardiovascular responses to stress.

PDE9 inhibition promotes proliferation of neural stem cells via cGMP-PKG pathway following oxygen-glucose deprivation/reoxygenation injury in vitro.

Cerebral ischemia is one of leading causes of death and long-term disability worldwide. Stem cell-based therapy is promising some valuable strategies for the structural and functional recovery after ischemic insult. The inhibition of phosphodiesterases (PDEs) has wide spectrum neuroprotective properties by stimulating proliferation of neural stem cells (NSCs). However, the potential role of PDE9 on NSCs proliferation after cerebral ischemia is not well investigated. The present study aimed to assess the contribution of PDE9 inhibition on the proliferation of NSCs and to determine the details of its underlying mechanisms against cerebral ischemia. The survival and proliferation of NSCs were assessed by CCK-8 assay and BrdU immunofluorescence staining, respectively. PDE9 activity and cGMP level were measured by ELISA kits. The protein expression of PKG and BDNF was detected by Western blot. Exposing NSCs of cultured primary hippocampus to oxygen-glucose deprivation/reoxygenation (OGD/R) significantly decreased the survival rate, but increased the proliferation of NSCs. Meanwhile, PDE9 activity was decreased, cGMP level was increased, PKG and BDNF protein expression was increased. PF-04447953, a PDE9 inhibitor, increased the survival rate of NSCs, moreover, PDE9 activity reduced more, and NSCs proliferation, cGMP level, PKG and BDNF protein expression were increased further, compared with OGD/R model group. These effects of PF-04447953, except for PDE9 activity and cGMP level, were reversed by treatment with KT5823, a PKG inhibitor. Taken together, the inhibition of PDE9 can promote the proliferation of NSCs following OGD/R injury, which may be, at least partly, mediated by cGMP-PKG pathway.

Sacubitril/Valsartan Decreases Cardiac Fibrosis in Left Ventricle Pressure Overload by Restoring PKG Signaling in Cardiac Fibroblasts.

Background Heart failure (HF) is invariably accompanied by development of cardiac fibrosis, a form of scarring that increases muscular tissue rigidity and decreases cardiac contractility. Cardiac fibrosis arises from a pathological attempt to repair tissue damaged during maladaptive remodeling. Treatment options to block or reverse fibrosis have proven elusive. Neprilysin is an endopeptidase that degrades vasoactive peptides, including atrial natriuretic peptide. Thus, neprilysin inhibition reduces hypertension, ultimately limiting maladaptive cardiac remodeling. LCZ696, which consists of an angiotensin receptor blocker (valsartan [VAL]) and a neprilysin inhibitor (sacubitril [SAC]), was shown to be well tolerated and significantly reduced the risk of death and hospitalization in HF patients with reduced ejection fraction. We hypothesized that SAC/VAL directly inhibits fibroblast activation and development of pathological fibrosis. Methods and Results We used a mouse model of left ventricle pressure overload coupled to in vitro studies in primary mouse and human cardiac fibroblasts (CFs) to study the impact of SAC/VAL on CF activation and cardiac fibrosis. SAC/VAL significantly ameliorated pressure overload-induced cardiac fibrosis by blocking CF activation and proliferation, leading to functional improvement. Mechanistically, the beneficial impact of SAC/VAL at least partially stemmed from restoration of PKG (protein kinase G) signaling in HF patient-derived CF, which inhibited Rho activation associated with myofibroblast transition. Conclusions This study reveals that SAC/VAL acts directly on CF to prevent maladaptive cardiac fibrosis and dysfunction during pressure overload-induced hypertrophy and suggests that SAC/VAL should be evaluated as a direct antifibrotic therapeutic for conditions such as HF with preserved ejection fraction.

Single Quantum Dot Tracking Reveals Serotonin Transporter Diffusion Dynamics are Correlated with Cholesterol-Sensitive Threonine 276 Phosphorylation Status in Primary Midbrain Neurons.

Serotonin transporter (SERT) terminates serotonin signaling in the brain by enabling rapid clearance of the neurotransmitter. SERT dysfunction has been associated with a variety of psychiatric disorders, including depression, anxiety, and autism. Visualizing SERT behavior at the single molecule level in endogenous systems remains a challenge. In this study, we utilize quantum dot (QD) single particle tracking (SPT) to capture SERT dynamics in primary rat midbrain neurons. Membrane microenvironment, specifically membrane cholesterol, plays a key role in SERT regulation and has been found to affect SERT conformational state. We sought to determine how reduced cholesterol content affects both lateral mobility and phosphorylation of conformationally sensitive threonine 276 (Thr276) in endogenous SERT using two different methods of cholesterol manipulation, statins and methyl-ß-cyclodextrin. Both chronic and acute cholesterol depletion increased SERT lateral diffusion, radial displacement along the membrane, mobile fraction, and Thr276 phosphorylation levels. Overall, this work has provided new insights about endogenous neuronal SERT mobility and its associations with membrane cholesterol and SERT phosphorylation status.

PKG-Dependent Cell Death in 661W Cone Photoreceptor-like Cell Cultures (Experimental Study).

In humans cone photoreceptors are responsible for high-resolution colour vision. A variety of retinal diseases can compromise cone viability, and, at present, no satisfactory treatment options are available. Here, we present data towards establishing a reliable, high-throughput assay system that will facilitate the search for cone neuroprotective compounds using the murine-photoreceptor cell line 661 W. To further characterize 661 W cells, a retinal marker study was performed, followed by the induction of cell death using paradigms over-activating cGMP-dependent protein kinase G (PKG). We found that 661 W cells may be used to mimic specific aspects of cone degeneration and may thus be valuable for future compound screening studies.

Protein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes.

Bone loss and fractures are underrecognized complications of type 1 diabetes and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal transduction pathway and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to the addition of O-linked N-acetylglucosamine to NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat-an NO-independent activator of oxidized guanylate cyclase-increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of antioxidant genes and downregulation of excess NADPH oxidase-4-dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

The bradykinin-cGMP-PKG pathway augments insulin sensitivity via upregulation of MAPK phosphatase-5 and inhibition of JNK.

Bradykinin (BK) promotes insulin sensitivity and glucose uptake in adipocytes and other cell types. We demonstrated that in rat adipocytes BK enhances insulin-stimulated glucose transport via endothelial nitric oxide synthase, nitric oxide (NO) generation, and decreased activity of the mitogen-activated protein kinase (MAPK) JNK (c-Jun NH2-terminal kinase). In endothelial cells, NO increases soluble guanylate cyclase (sGC) activity, which, in turn, activates protein kinase G (PKG) by increasing cGMP levels. In this study, we investigated whether BK acts via the sGC-cGMP-PKG pathway to inhibit the negative effects of JNK on insulin signaling and glucose uptake in rat adipocytes. BK augmented cGMP concentrations. The BK-induced enhancement of insulin-stimulated glucose uptake was mimicked by the sGC activator YC-1 and a cell-permeable cGMP analog, CPT-cGMP, and inhibited by the sGC inhibitor ODQ and the PKG inhibitor KT 5823. Transfection of dominant-negative PKG reduced the BK augmentation of insulin-induced Akt phosphorylation. The activation of JNK and ERK1/2 by insulin was attenuated by BK, which was mediated by the sGC-cGMP-PKG pathway. Whereas insulin-stimulated phosphorylation of upstream activators of JNK and ERK, i.e., MKK4 and MEK1/2, was unaffected, BK augmented insulin-mediated induction of MKP-5 mRNA and protein levels. Furthermore, zaprinast, a phosphodiesterase inhibitor, enhanced cGMP and MKP-5 and prolonged the action of BK. These data indicate that BK enhances insulin action by inhibition of negative feedback by JNK and ERK via upregulation of MKP-5, mediated by the sGC-cGMP-PKG signaling pathway.

sGC-cGMP-PKG pathway stimulation protects the healthy but not the failing right ventricle of rats against ischemia and reperfusion injury.

BACKGROUND: To investigate whether modulation of the sGC-cGMP-PKG pathway protects against ischemia and reperfusion injury in the healthy and the failing right ventricle (RV). METHODS: Hearts from male Wistar rats with a healthy RV (n=39) or a hypertrophic and failing RV induced by pulmonary trunk banding (n=57) were isolated and perfused in a pressure-controlled modified Langendorff setup. The isolated hearts were randomized to control, ischemic preconditioning (IPC, 2×5min of global ischemia), a phosphodiesterase-5 (PDE5) inhibitor vardenafil (66nM) alone and in combination with a cGMP-dependent protein kinase (PKG) blocker KT 5823 (1µM). Failing hearts were exposed to the same protocols and to soluble guanylate cyclase stimulation/activation, and phosphodiesterase 9 inhibition. All interventions were followed by 40min of global ischemia and 120min of reperfusion. The effects on the RV were evaluated by measurement of the infarct size/area-at-risk ratio (IS/AAR). RESULTS: In healthy hearts, IPC and pharmacological preconditioning with vardenafil reduced RV infarct size. PKG blockade by KT-5823 did not alter infarct size per se but abolished the cardioprotective effect of vardenafil. In the hypertrophic and failing hearts, none of the conditioning strategies altered RV infarct size. CONCLUSION: PDE-5 inhibition by vardenafil protects the healthy right ventricle against ischemia and reperfusion injury by a PKG dependent mechanism. Neither ischemic preconditioning nor pharmacologic stimulation of the sGC-cGMP-PKG pathway induces cardioprotection in the hypertrophic and failing RV.

Priming the proteasome by protein kinase G: a novel cardioprotective mechanism of sildenafil.

The proteasome mediates the degradation of most cellular proteins including misfolded proteins, pivotal to intracellular protein hemostasis. Proteasome functional insufficiency is implicated in a large subset of human failing hearts. Experimental studies have established proteasome functional insufficiency as a major pathogenic factor, rationalizing proteasome enhancement as a potentially new therapeutic strategy for congestive heart failure. Protein kinase G activation known to be cardioprotective was recently found to facilitate proteasomal degradation of misfolded proteins in cardiomyocytes; sildenafil was shown to activate myocardial protein kinase G, improve cardiac protein quality control and slow down the progression of cardiac proteinopathy in mice. This identifies the first clinically used drug that is capable of benign proteasome enhancement and unveils a potentially novel cardioprotective mechanism for sildenafil.

Protein kinase G increases antioxidant function in lung microvascular endothelial cells by inhibiting the c-Abl tyrosine kinase.

Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI (PKGI) posttranscriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKGI is mediated via inhibition of the c-Abl tyrosine kinase. We found that activation of PKGI with the cGMP analog 8pCPT-cGMP inhibited c-Abl activity and decreased c-Abl expression in wild-type but not PKGI(-/-) MLMVEC. Treatment of wild-type MLMVEC with atrial natriuretic peptide also inhibited c-Abl activation. Moreover, treatment of MLMVEC with the c-Abl inhibitor imatinib increased catalase and GPx-1 protein in a posttranscriptional fashion. In imatinib-treated MLMVEC, there was no additional effect of 8pCPT-cGMP on catalase or GPx-1. The imatinib-induced increase in antioxidant proteins was associated with an increase in extracellular H2O2 scavenging by MLMVEC, attenuation of oxidant-induced endothelial barrier dysfunction, and prevention of oxidant-induced endothelial cell death. Finally, in the isolated perfused lung, imatinib prevented oxidant-induced endothelial toxicity. We conclude that cGMP, through activation of PKGI, inhibits c-Abl, leading to increased key antioxidant enzymes and resistance to lung endothelial oxidant injury. Inhibition of c-Abl by active PKGI may be the downstream mechanism underlying PKGI-mediated antioxidant signaling. Tyrosine kinase inhibitors may represent a novel therapeutic approach in oxidant-induced ALI.

Cyclic guanosine monophosphate-enhancing reduces androgenic extracellular regulated protein kinases-phosphorylation/Rho kinase II-activation in benign prostate hyperplasia.

OBJECTIVES: To investigate whether 7-[2-[4-(2-chlorophenyl) piperazinyl] ethyl]-1,3-di-methylxanthine (KMUP-1) inhibits the effects of testosterone on the development of benign prostatic hyperplasia and sensitizes prostate contraction. METHODS: A benign prostatic hyperplasia animal model was established by subcutaneous injections of testosterone (3 mg/kg/day, s.c.) for 4 weeks in adult male Sprague-Dawley rats. Animals were divided into six groups: control, testosterone, testosterone with KMUP-1 (2.5, 5 mg/kg/day), sildenafil (5 mg/kg/day) or doxazosin (5 mg/kg/day). After 4 weeks, the animals were killed, and prostate tissues were prepared for isometric tension measurement and western blotting analysis. KMUP-1, Y27632, zaprinast, doxazosin or tamsulosin were used at various concentrations to determine the contractility sensitized by phenylephrine (10 µmol/L). RESULTS: KMUP-1 inhibited testosterone-induced phosphorylation of extracellular signal-regulated phosphorylated protein kinase and mitogen-activated protein kinase kinase and Rho kinase-II activation. Sildenafil and doxazosin significantly decreased benign prostatic hyperplasia-induced mitogen-activated protein kinase kinase and Rho kinase-II activation. The decreased expressions of soluble guanylate cyclase α1 was reversed by KMUP-1, doxazosin and sildenafil. Soluble guanylate cyclase ß1 and protein kinase G were increased by KMUP-1, doxazosin, and sildenafil in the testosterone-treated benign prostatic hyperplasia group. Phosphodiesterase-5A was increased by testosterone and inhibited by KMUP-1 (5 mg/kg/day) or sildenafil (5 mg/kg/day). KMUP-1 inhibited phenylephrine-sensitized prostate contraction of rats treated with testosterone. CONCLUSIONS: Mitogen-activated protein kinase 1/extracellular regulated protein kinases kinase, soluble guanylate cyclase/cyclic guanosine monophosphate, protein kinase/protein kinase G and Rho kinase-II are related to prostate smooth muscle tone and proliferation induced by testosterone. KMUP-1 inhibits testosterone-induced prostate hyper-contractility and mitogen-activated protein kinase 1/extracellular regulated protein kinases kinase-phosphorylation, and it inactivates Rho kinase-II by cyclic guanosine monophosphate, protein kinase and α1A-adenergic blockade. Thus, KMUP-1 might be a beneficial pharmacotherapy for benign prostatic hyperplasia.

Target specific-trisubstituted pyrrole inhibits Babesia bovis erythrocytic growth.

Babesiosis, a significant veterinary disease and an emerging zoonotic human infection, is caused by certain species of the protozoan parasite, Babesia. Here we report that a trisubstituted pyrrole is a potent inhibitor of Babesia bovis, a bovine parasite. Furthermore, B. bovis expresses the known target of the compound, the cGMP dependent protein kinase. Target conservation and the in vitro efficacy support further investigation of this compound and validation of Babesia cGMP dependent protein kinase as its in vivo target.

Cardiac role of cyclic-GMP hydrolyzing phosphodiesterase type 5: from experimental models to clinical trials.

Cyclic guanosine monophosphate (cGMP) and its primary signaling kinase, protein kinase G, play an important role in counterbalancing stress remodeling in the heart. Growing evidence supports a positive impact on a variety of cardiac disease conditions from the suppression of cGMP hydrolysis. The latter is regulated by members of the phosphodiesterase (PDE) superfamily, of which cGMP-selective PDE5 has been best studied. Inhibitors such as sildenafil and tadalafil ameliorate cardiac pressure and volume overload, ischemic injury, and cardiotoxicity. Clinical trials have begun exploring their potential to benefit dilated cardiomyopathy and heart failure with a preserved ejection fraction. This review discusses recent developments in the field, highlighting basic science and clinical studies.

Kaurenoic acid from Sphagneticola trilobata Inhibits Inflammatory Pain: effect on cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-α and IL-1ß. Furthermore, the analgesic effect of 1 was inhibited by l-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

Protein kinase G inhibits flow-induced Ca2+ entry into collecting duct cells.

The renal cortical collecting duct (CCD) contributes to the maintenance of K(+) homeostasis by modulating renal K(+) secretion. Cytosolic Ca(2+) ([Ca(2+)](i)) mediates flow-induced K(+) secretion in the CCD, but the mechanisms regulating flow-induced Ca(2+) entry into renal epithelial cells are not well understood. Here, we found that atrial natriuretic peptide, nitric oxide, and cyclic guanosine monophosphate (cGMP) act through protein kinase G (PKG) to inhibit flow-induced increases in [Ca(2+)](i) in M1-CCD cells. Coimmunoprecipitation, double immunostaining, and functional studies identified heteromeric TRPV4-P2 channels as the mediators of flow-induced Ca(2+) entry into M1-CCD cells and HEK293 cells that were coexpressed with both TRPV4 and TRPP2. In these HEK293 cells, introducing point mutations at two putative PKG phosphorylation sites on TRPP2 abolished the ability of cGMP to inhibit flow-induced Ca(2+) entry. In addition, treating M1-CCD cells with fusion peptides that compete with the endogenous PKG phosphorylation sites on TRPP2 also abolished the cGMP-mediated inhibition of the flow-induced Ca(2+) entry. Taken together, these data suggest that heteromeric TRPV4-P2 channels mediate the flow-induced entry of Ca(2+) into collecting duct cells. Furthermore, substances such as atrial natriuretic peptide and nitric oxide, which increase cGMP, abrogate flow-induced Ca(2+) entry through PKG-mediated inhibition of these channels.

Nitric oxide-sensitive guanylyl cyclase is dispensable for nitrergic signaling and gut motility in mouse intestinal smooth muscle.

BACKGROUND & AIMS: The nitric oxide-guanosine 3',5'-cyclic monophosphate (cGMP) signaling pathway has an important role in the control of smooth muscle tone. NO is produced by NO synthases and acts as a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. The main target, NO-sensitive guanylyl cyclase (NO-GC), is stimulated by NO to produce the intracellular messenger cGMP. We investigated the role of NO-GC in nitrergic relaxation and GI motility. METHODS: We tested relaxation of GI smooth muscle in mice that do not express NO-GC or mice with disruption of NO-GC specifically in smooth muscle cells. Different segments of the GI tract (fundus, lower esophageal sphincter, pyloric sphincter, and duodenum) were used in isometric force studies. NO donors and electrical field stimulation were used to assess nitrergic signaling. Whole-gut transit time was measured as an indicator of GI motility. RESULTS: Mice that lack NO-GC do not have NO-induced relaxation of GI smooth muscle. Gut transit time was increased, resulting in GI dysfunction. Surprisingly, in mice that lack NO-GC specifically in smooth muscle, NO-induced relaxation was reduced only slightly, and whole-gut transit time was unchanged compared with wild-type mice. CONCLUSIONS: Lack of NO-GC in smooth muscle cells does not impair NO-induced relaxation of GI tissues or GI motility. The NO receptor guanylyl cyclase in GI smooth muscle is therefore dispensable for nitrergic signaling in mice.

8-pCPT-cGMP stimulates alphabetagamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties.

Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(2)-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH(2)- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.

New nitric oxide donors based on ruthenium complexes.

Nitric oxide (NO) donors produce NO-related activity when applied to biological systems. Among its diverse functions, NO has been implicated in vascular smooth muscle relaxation. Despite the great importance of NO in biological systems, its pharmacological and physiological studies have been limited due to its high reactivity and short half-life. In this review we will focus on our recent investigations of nitrosyl ruthenium complexes as NO-delivery agents and their effects on vascular smooth muscle cell relaxation. The high affinity of ruthenium for NO is a marked feature of its chemistry. The main signaling pathway responsible for the vascular relaxation induced by NO involves the activation of soluble guanylyl-cyclase, with subsequent accumulation of cGMP and activation of cGMP-dependent protein kinase. This in turn can activate several proteins such as K+ channels as well as induce vasodilatation by a decrease in cytosolic Ca2+. Oxidative stress and associated oxidative damage are mediators of vascular damage in several cardiovascular diseases, including hypertension. The increased production of the superoxide anion (O2-) by the vascular wall has been observed in different animal models of hypertension. Vascular relaxation to the endogenous NO-related response or to NO released from NO deliverers is impaired in vessels from renal hypertensive (2K-1C) rats. A growing amount of evidence supports the possibility that increased NO inactivation by excess O2- may account for the decreased NO bioavailability and vascular dysfunction in hypertension.