cGMP Regulation Research Articles

The Mayo Clinic Cellular Therapy Fellowship: Training the Next Generation of Cellular Therapy Leaders

Background & Aim The field of cellular therapy is rapidly evolving with promising clinical outcomes and multiple potential therapeutic targets. This rapid expansion, while exciting, has quickly led to a deficit of qualified directors for burgeoning human cellular therapy laboratories. Unlike conventional drug therapies, cellular based therapies require an added layer of expertise in policy, good manufacturing practices (cGMP) and regulatory elements. Academic medical centers, both nationally and internationally, are in desperate need of well-trained, experienced leaders capable of directing the labs which both oversee and produce these therapeutic agents. To meet this need, we have established the Mayo Clinic School of Graduate Medical Education (MSGME) Cellular Therapy Fellowship; an intensive one-year program for M.D. and Ph.D. candidates consisting of didactic, experiential, and project-driven training with an emphasis on laboratory management and graduated responsibility. Mayo Clinic is a multidisciplinary academic medical and research institution, which houses a lab that directly supports the bone marrow transplant service in addition to a multifaceted immune and progenitor cell program focusing on investigational cell therapies such as mesenchymal stem cells, dendritic cells, and CAR T cells. The program is robust performing more than 500 clinical stem cell transplants annually and encompassing more than 25 active investigator-initiated trials in addition to greater than 30 external manufacturing protocols. Our aim is to produce scientific and clinical leaders fluent in cGMP regulations, quality control and regulatory components, clinical laboratory management, current methodologies in cell therapy manufacturing, IND preparation and clinical trial oversight. Methods, Results & Conclusion Our curriculum mirrors this aim with rotations in the BMT and progenitor cell laboratories, our commercial processing unit, CAR-T manufacturing, quality, apheresis and infusions and leadership and management each with varying levels of graduated responsibility and on-call duties. The fellows actively participate in director level conversations and decisions, which, in combination with the aforementioned experiences and their research projects, provides a thorough and comprehensive training experience for the next generation of cell therapy leaders.

A Localized Scaffold for cGMP Increase Is Required for Apical Dendrite Development

SUMMARYStudies in cultured neurons have established that axon specification instructs neuronal polarization and is necessary for dendrite development. However, dendrite formation in vivo occurs when axon formation is prevented. The mechanisms promoting dendrite development remain elusive. We find that apical dendrite development is directed by a localized cyclic guanosine monophosphate (cGMP)-synthesizing complex. We show that the scaffolding protein Scribble associates with cGMP-synthesizing enzymes soluble-guanylate-cyclase (sGC) and neuronal nitric oxide synthase (nNOS). The Scribble scaffold is preferentially localized to and mediates cGMP increase in dendrites. These events are regulated by kinesin KifC2. Knockdown of Scribble, sGC-b1, or KifC2 or disrupting their associations prevents cGMP increase in dendrites and causes severe defects in apical dendrite development. Local cGMP elevation or sGC expression rescues the effects of Scribble knockdown on dendrite development, indicating that Scribble is an upstream regulator of cGMP. During neuronal polarization, dendrite development is directed by the Scribble scaffold that might link extracellular cues to localized cGMP increase.

P2863Regional specific remodeling of cGMP pathway in human atrial fibrillation

Abstract Background Atrial fibrillation (AF) is accompanied by a profound remodeling of membrane receptors and alterations in cyclic nucleotides-dependent regulation of Ca2+-handling. Thus, while basal ryanodine receptors activity is upregulated, L-type calcium current (ICa,L) density is diminish in AF, due to local microdomain-specific cAMP dynamics. The same seems true for cGMP regulation in AF. In AF cGMP-mediated increase in ICa,L is blunted but NO-mediated attenuation of β-adrenoceptors stimulation-mediated increase is preserved. However, although the role of cGMP in controling atrial function and pathophysiology is controversial, no study has been ever performed in human myocytes to measure cGMP directly. Methods We isolated myocytes from the right and/or left atrium of 27 patients in sinus rhythm (SR), and with AF. Cells were then transfected with adenovirus to express the cytosolic FRET-based cGMP sensor red-cGES-DE5 and cultured for 48 hours. Förster resonance energy transfer (FRET) was used to measure cGMP in 61 living human atrial myocytes. We stimulated cells with the C-type natriuretic peptide CNP (100 nM and 1 μM), and the non-selective phosphodiesterases (PDEs) inhibitor IBMX (100 μM). Additionally, PDE specific inhibitors for PDE2 (Bay 60–7550, 100 nM) and PDE3 (Cilostamide, 10 μM) as well as inhibitor of the soluble guanylyl cyclase (ODQ, 50 μM) were used. We also measured PDE2 and PDE3 mRNA levels in atrial tissue samples from both groups of patients using RT-qPCR. Results We could show that stimulation with CNP increased cGMP levels in human atrial myocytes. However, in myocytes from patients with AF global cGMP responses to CNP and to IBMX was reduced compared to SR. Additionally, there was a difference in response to CNP and IBMX in patients with AF between the right and the left atria. Whereas in the right atria IBMX could further increase cGMP levels in the cell, in the left atria leaded to a reduction in cGMP levels. RT-qPCR showed a tendency of PDE3 to be reduced in AF. On the other hand, PDE2A gene expression was upregulated in the left atria. Conclusions We have shown that PDEs contributes cGMP signaling in the human atria and that they are involved in atrial pathophysiology. Now our data clearly show differences in cGMP regulation in cardiomyocytes isolated from left and right atrium from patients in atrial fibrillation and sinus rhythm. We observe a major role of PDEs, regulating cGMP pathway promoted by the reduced responses in AF, especially PDE2 in the left atria.

Unified electronic traceability and data storage system

Background & Aim In a field that requires complete visibility and traceability, we are still relying on hand written forms and documents that introduce the possibility of fraud, human error, and other complications. I propose that soon, we as an industry will be able to electronically record patient information, sample data, logistics tracking, manufacturing batch records, and patient treatment data with little to no need for manual input. This software will use a series of patient, sample, and product barcodes to maintain complete traceability of the process from start to finish. All equipment used throughout the process will integrate into the software and automatically record values and calculations into the appropriate forms or batch records Methods, Results & Conclusion With this technology physicians and patients will have complete visibility into the treatment or therapy that is being developed and manufacturers will be able to track data trends and monitor incoming and outgoing materials in real time along with receiving updates and notifications related to critical events within the process. Privacy is also a serious concern within the cell therapy industry because all parties need the necessary information to produce a quality product all while respecting the regulations set in place by organizations like the FDA and HIPAA. With this technology only certain participants in the process will be given access to this sensitive information. A system like I am describing will streamline the FDA approval process that previously took years to complete by centralizing all necessary information. This will allow regulatory committees to locate specific pieces of data with a simple search on their computer instead of sifting through pages upon pages of documents allowing the company to get to market much faster. With date and time stamps attached to each piece of recorded information things such as deviation investigations and chain of custody will be expedited and made much easier to complete. This integrated software will also allow for real time communications between collection and manufacturing sites that will decrease the occurrence of miscommunication-based errors. Many collection and infusion sites do not have personnel that are aptly trained on things like cGMP regulations or proper technique to administer your product. In a field that requires complete visibility and traceability, we are still relying on hand written forms and documents that introduce the possibility of fraud, human error, and other complications. I propose that soon, we as an industry will be able to electronically record patient information, sample data, logistics tracking, manufacturing batch records, and patient treatment data with little to no need for manual input. This software will use a series of patient, sample, and product barcodes to maintain complete traceability of the process from start to finish. All equipment used throughout the process will integrate into the software and automatically record values and calculations into the appropriate forms or batch records With this technology physicians and patients will have complete visibility into the treatment or therapy that is being developed and manufacturers will be able to track data trends and monitor incoming and outgoing materials in real time along with receiving updates and notifications related to critical events within the process. Privacy is also a serious concern within the cell therapy industry because all parties need the necessary information to produce a quality product all while respecting the regulations set in place by organizations like the FDA and HIPAA. With this technology only certain participants in the process will be given access to this sensitive information. A system like I am describing will streamline the FDA approval process that previously took years to complete by centralizing all necessary information. This will allow regulatory committees to locate specific pieces of data with a simple search on their computer instead of sifting through pages upon pages of documents allowing the company to get to market much faster. With date and time stamps attached to each piece of recorded information things such as deviation investigations and chain of custody will be expedited and made much easier to complete. This integrated software will also allow for real time communications between collection and manufacturing sites that will decrease the occurrence of miscommunication-based errors. Many collection and infusion sites do not have personnel that are aptly trained on things like cGMP regulations or proper technique to administer your product.

Diallyl disulfide potentiates anti-obesity effect of green tea in high-fat/high-sucrose diet-induced obesity

Obesity is a major problem in developed countries and a burden on social health care systems. Several epidemiological studies showed the protective effects of green tea against obesity-related diseases. Cyclic guanosine monophosphate (cGMP) acts as a mediator for the physiological effects of (−)-epigallocatechin-3-O-gallate, the major constituent of green tea. Here, we showed that the level of phosphodiesterase 5, a negative regulator of cGMP, was up-regulated in adipose tissues of high-fat/high-sucrose (HF/HS) diet-fed mice and that this up-regulation was ameliorated by diallyl disulfide (DADS), the major organosulfur in garlic. A green tea extract (GT) and DADS in combination attenuated HF/HS diet-induced adipose increase and triglyceride accumulation in the liver. In these mechanisms, the combination regimen suppressed the HF/HS diet-induced up-regulation of fatty acid synthesis-related enzymes including sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase, and stearoyl-CoA desaturase-1. Moreover, this combination diet up-regulated thermogenesis-related genes including peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha and uncoupling proteins in both white and brown adipose tissues. In conclusion, we identified DADS as an enhancer of the anti-obesity effect of GT accompanied by the suppression of SREBP-1 and activation of PPAR axis. The combination diet is a novel and easily applicable approach against obesity-related diseases.

Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure

Heart failure (HF) is a shared manifestation of several cardiovascular pathologies, including hypertension and myocardial infarction, and a limited repertoire of treatment modalities entails that the associated morbidity and mortality remain high. Impaired nitric oxide (NO)/guanylyl cyclase (GC)/cyclic guanosine-3',5'-monophosphate (cGMP) signaling, underpinned, in part, by up-regulation of cyclic nucleotide-hydrolyzing phosphodiesterase (PDE) isozymes, contributes to the pathogenesis of HF, and interventions targeted to enhancing cGMP have proven effective in preclinical models and patients. Numerous PDE isozymes coordinate the regulation of cardiac cGMP in the context of HF; PDE2 expression and activity are up-regulated in experimental and human HF, but a well-defined role for this isoform in pathogenesis has yet to be established, certainly in terms of cGMP signaling. Herein, using a selective pharmacological inhibitor of PDE2, BAY 60-7550, and transgenic mice lacking either NO-sensitive GC-1α (GC-1α-/-) or natriuretic peptide-responsive GC-A (GC-A-/-), we demonstrate that the blockade of PDE2 promotes cGMP signaling to offset the pathogenesis of experimental HF (induced by pressure overload or sympathetic hyperactivation), reversing the development of left ventricular hypertrophy, compromised contractility, and cardiac fibrosis. Moreover, we show that this beneficial pharmacodynamic profile is maintained in GC-A-/- mice but is absent in animals null for GC-1α or treated with a NO synthase inhibitor, revealing that PDE2 inhibition preferentially enhances NO/GC/cGMP signaling in the setting of HF to exert wide-ranging protection to preserve cardiac structure and function. These data substantiate the targeting of PDE2 in HF as a tangible approach to maximize myocardial cGMP signaling and enhancing therapy.

171 - Human platelet lysate as a media supplement for ex vivo expansion of immune cells

Adoptive immunotherapy with ex vivo-derived T lymphocytes (T cells) and natural killer cells (NK cells) has emerged as a very effective therapeutic strategy to treat various cancer and autoimmune diseases. Adoptive cellular therapy involves the ex vivoexpansion of lymphocytes under cGMP regulations. The use of fetal bovine serum (FBS) as a supplement for cell culture carries a risk of pathogen transmission as well as xenoimmunization against bovine antigens. Human AB serum, yet another cell culture option for T cells and NK cells, has supply limitations and therefore may not be sufficient to meet the expected demand for immunotherapies. Platelet lysate (HPL) obtained from outdated human donor platelets is widely recognized as a valuable, alternative to both FBS and human AB serum for cell culture. This study explores the potential use of HPL to expand T cells and NK cells. Primary human CD3+ and CD56+ cells were isolated from peripheral blood mononuclear cells using negative and positive immunomagnetic separation, respectively. Both T and NK cells were expanded in vitro using basal media supplemented with either FBS, AB serum or HPL with the addition of CD3/CD28 activator plus interleukin-2 for T cells and interleukin-2 plus interleukin-15 for NK cells. The growth kinetics and viability of T cells expanded with the three different media supplements were comparable, whereas higher expansion rate and better viability was observed for NK cells expanded with HPL compared to FBS and AB serum. This study demonstrates the feasibility to use human HPL as a successful alternative to FBS and human AB serum to effectively propagate primary human lymphocytes.

Discovery of 2-[(E)-2-(7-Fluoro-3-methylquinoxalin-2-yl)vinyl]-6-pyrrolidin-1-yl-N-(tetrahydro-2H-pyran-4-yl)pyrimidin-4-amine Hydrochloride as a Highly Selective PDE10A Inhibitor.

Phosphodiesterase (PDE) 10A is a dual hydrolase of cAMP and cGMP and highly expressed in striatal medium spiny neurons. Inhibition of PDE10A modulates the activity of medium spiny neurons (MSN) via the regulation of cAMP and cGMP. Signal control of MSN is considered associated with psychotic symptoms. Therefore PDE10A inhibitor is expected as a therapeutic method for psychosis disease such as schizophrenia. Avanafil (1) is a PDE5 inhibitor (treatment for erectile dysfunction) discovered by our company. We paid attention to the homology of PDE10A and PDE5 and took advantage of PDE5 inhibitor library to discover PDE10A inhibitors, and found a series of compounds that exhibit higher potency for PDE10A than PDE5. We transformed the afforded derivatives, which had weak inhibitory activity against PDE10A, and discovered stilbene as a PDE10A inhibitor. Brain penetration of this compound was improved by further conversion of N-containing heterocycles and their substituents. The afforded dimethylaminopyrimidine was effective for rat conditioned avoidance response (CAR) test; however, it did not exhibit good brain penetration. We performed in-depth optimization focusing on substituents of the quinoxaline ring, and produced 3-methyl-7-fluoro quinoxaline. This compound was the most effective in rat CAR test due to its strong PDE10A inhibitory activity and good pharmacokinetics.

Functional Overview of Process Validation of Tablets - A Critical Review

Purpose : Validation is a main tool in achieving and maintaining the quality and safety of the final product as per cGMP(current Good Manufacturing Practice). The purpose of this present work is to give introduction, general overview and how to plan, develop, execute process validation of pharmaceutical manufacturing process especially tablet manufacturing process. Approach : When we consider any product, quality is always an imperative pre requisite hence a highest quality levels must be included in the manufacturing of the drug. Findings : To assure the identity, purity, safety, efficacy and also maintaining the quality of final product process validation is the key element. Three consecutive batches were considered to execute the process validation activity. Conclusion : Process validation is a major requirement of cGMP's regulation. Process validation is a key element to maintain product quality, safety, identity and efficacy.

Management of Out of Specification (OOS) for Finished Product

Background of the study: Difficult lies at the core of drug producer successful operation. Laboratory testing, which is compulsory by the CGMP regulations are required to check that components, containers and closures, in-process materials, and finished products conform to specifications, including stability specifications. Objective of the study: The objective of the investigation procedure should clearly state when the investigation is required, and define OOS, OOT, and aberrant results. OOS results are most often generated due to laboratory or manufacturing-related errors, the setting of inappropriate specifications, or poor method development. Materials and Methods: The current work is an effort to deliberate several aspects of finding the root cause for the OOS during the finished product analysis by using HPLC. Results and Discussion: Product’s individual unknown impurity was not in specification limit and, hence study carried out to find the root cause. Conclusion: After conducting detail investigation it was proved that an analyst conducted the analysis of the product after the due date to expiry.

Control of Neuropeptide Expression by Parallel Activity-dependent Pathways in Caenorhabditis elegans

Monitoring of neuronal activity within circuits facilitates integrated responses and rapid changes in behavior. We have identified a system in Caenorhabditis elegans where neuropeptide expression is dependent on the ability of the BAG neurons to sense carbon dioxide. In C. elegans, CO2 sensing is predominantly coordinated by the BAG-expressed receptor-type guanylate cyclase GCY-9. GCY-9 binding to CO2 causes accumulation of cyclic GMP and opening of the cGMP-gated TAX-2/TAX-4 cation channels; provoking an integrated downstream cascade that enables C. elegans to avoid high CO2. Here we show that cGMP regulation by GCY-9 and the PDE-1 phosphodiesterase controls BAG expression of a FMRFamide-related neuropeptide FLP-19 reporter (flp-19::GFP). This regulation is specific for CO2-sensing function of the BAG neurons, as loss of oxygen sensing function does not affect flp-19::GFP expression. We also found that expression of flp-19::GFP is controlled in parallel to GCY-9 by the activity-dependent transcription factor CREB (CRH-1) and the cAMP-dependent protein kinase (KIN-2) signaling pathway. We therefore show that two parallel pathways regulate neuropeptide gene expression in the BAG sensory neurons: the ability to sense changes in carbon dioxide and CREB transcription factor. Such regulation may be required in particular environmental conditions to enable sophisticated behavioral decisions to be performed.

Effects of a novel phosphodiesterase 10A inhibitor in non-human primates: A therapeutic approach for schizophrenia with improved side effect profile

Schizophrenia symptoms are associated with alterations in basal ganglia-cortical networks that include the cyclic nucleotides (cAMP/cGMP) signaling pathways. Phosphodiesterase 10A (PDE10A) inhibitors have been considered as therapeutic agents for schizophrenia because the regulation of cAMP and cGMP in the striatum by PDE10A plays an important role in the signaling mechanisms of the striatal-cortical network, and thereby in cognitive function. In the present study we assessed in non-human primates (NHPs) the effects of a novel PDE10A inhibitor (FRM-6308) that has demonstrated high potency and selectivity for human recombinant PDE10A in vitro. The behavioral effects of FRM-6308 in a dose range were determined in rhesus monkeys using a standardized motor disability scale for primates, motor tasks, and the “drug effects on the nervous system” (DENS) scale. The neuronal metabolic effects of FRM-6308 were determined with [(18)F]-fluorodeoxyglucose PET imaging. Results showed that FRM-6308 did not have any specific effects on the motor system at s.c. doses up to 0.32 mg/kg in NHPs, which induced a significant increase in the FDG-SUV in striatum (F 16.069, p < 0.05) and cortical (F 15.181, p < 0.05) regions. Higher doses induced sedation and occasional involuntary movements with clear development of tolerance after repeated exposures. These findings suggest that FRM-6308 has the adequate pharmacological profile to advance testing in clinical trials and demonstrate antipsychotic efficacy of PDE10A inhibition for the treatment of schizophrenia patients.

Cyclic nucleotide regulation of cardiac sympatho-vagal responsiveness.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are now recognized as important intracellular signalling molecules that modulate cardiac sympatho-vagal balance in the progression of heart disease. Recent studies have identified that a significant component of autonomic dysfunction associated with several cardiovascular pathologies resides at the end organ, and is coupled to impairment of cyclic nucleotide targeted pathways linked to abnormal intracellular calcium handling and cardiac neurotransmission. Emerging evidence also suggests that cyclic nucleotide coupled phosphodiesterases (PDEs) play a key role limiting the hydrolysis of cAMP and cGMP in disease, and as a consequence this influences the action of the nucleotide on its downstream biological target. In this review, we illustrate the action of nitric oxide-CAPON signalling and brain natriuretic peptide on cGMP and cAMP regulation of cardiac sympatho-vagal transmission in hypertension and ischaemic heart disease. Moreover, we address how PDE2A is now emerging as a major target that affects the efficacy of soluble/particulate guanylate cyclase coupling to cGMP in cardiac dysautonomia.

The Endothelium-Dependent Nitric Oxide-cGMP Pathway.

Nitric oxide (NO)-cyclic 3'-5' guanosine monophosphate (cGMP) signaling plays a critical role on smooth muscle tone, platelet activity, cardiac contractility, renal function and fluid balance, and cell growth. Studies of the 1990s established endothelium dysfunction as one of the major causes of cardiovascular diseases. Therapeutic strategies that benefit NO bioavailability have been applied in clinical medicine extensively. Basic and clinical studies of cGMP regulation through activation of soluble guanylyl cyclase (sGC) or inhibition of cyclic nucleotide phosphodiesterase type 5 (PDE5) have resulted in effective therapies for pulmonary hypertension, erectile dysfunction, and more recently benign prostatic hyperplasia. This section reviews (1) how endothelial dysfunction and NO deficiency lead to cardiovascular diseases, (2) how soluble cGMP regulation leads to beneficial effects on disorders of the circulation system, and (3) the epigenetic regulation of NO-sGC pathway components in the cardiovascular system. In conclusion, the discovery of the NO-cGMP pathway revolutionized the comprehension of pathophysiological mechanisms involved in cardiovascular and other diseases. However, considering the expression "from bench to bedside" the therapeutic alternatives targeting NO-cGMP did not immediately follow the marked biochemical and pathophysiological revolution. Some therapeutic options have been effective and released on the market for pulmonary hypertension and erectile dysfunction such as inhaled NO, PDE5 inhibitors, and recently sGC stimulators. The therapeutic armamentarium for many other disorders is expected in the near future. There are currently numerous active basic and clinical research programs in universities and industries attempting to develop novel therapies for many diseases and medical applications.

Interaction between phosphodiesterases in the regulation of the cardiac β-adrenergic pathway

In cardiac myocytes, the second messenger cAMP is synthesized within the β-adrenergic signaling pathway upon sympathetic activation. It activates Protein Kinase A (PKA) mediated phosphorylation of multiple target proteins that are functionally critical to cardiac contractility. The dynamics of cAMP are also controlled indirectly by cGMP-mediated regulation of phosphodiesterase isoenzymes (PDEs). The nature of the interactions between cGMP and the PDEs, as well as between PDE isoforms, and how these ultimately transduce the cGMP signal to regulate cAMP remains unclear. To better understand this, we have developed mechanistically detailed models of PDEs 1–4, the primary cAMP-hydrolyzing PDEs in cardiac myocytes, and integrated them into a model of the β-adrenergic signaling pathway. The PDE models are based on experimental studies performed on purified PDEs which have demonstrated that cAMP and cGMP bind competitively to the cyclic nucleotide (cN)-binding domains of PDEs 1, 2, and 3, while PDE4 regulation occurs via PKA-mediated phosphorylation. Individual PDE models reproduce experimentally measured cAMP hydrolysis rates with dose-dependent cGMP regulation. The fully integrated model replicates experimentally observed whole-cell cAMP activation-response relationships and temporal dynamics upon varying degrees of β-adrenergic stimulation in cardiac myocytes. Simulations reveal that as a result of network interactions, reduction in the level of one PDE is partially compensated for by increased activation of others. PDE2 and PDE4 exert the strongest compensatory roles among all PDEs. In addition, PDE2 competes with other PDEs to bind and hydrolyze cAMP and is a strong regulator of PDE interactions. Finally, an increasing level of cGMP gradually out-competes cAMP for the catalytic sites of PDEs 1, 2, and 3, suppresses their cAMP hydrolysis rates, and results in amplified cAMP signaling. These results provide insights into how PDEs transduce cGMP signals to regulate cAMP and how PDE interactions affect cardiac β-adrenergic response.

Cyclic GMP and Cilia Motility.

Motile cilia of the lungs respond to environmental challenges by increasing their ciliary beat frequency in order to enhance mucociliary clearance as a fundamental tenant of innate defense. One important second messenger in transducing the regulable nature of motile cilia is cyclic guanosine 3′,5′-monophosphate (cGMP). In this review, the history of cGMP action is presented and a survey of the existing data addressing cGMP action in ciliary motility is presented. Nitric oxide (NO)-mediated regulation of cGMP in ciliated cells is presented in the context of alcohol-induced cilia function and dysfunction.

Adrenomedullin in Vascular Endothelial Injury and Combination Therapy: Time for a New Paradigm.

Adrenomedullin is a highly conserved vasoactive peptide participating in a variety of physiological and pathophysiological processes including pregnancy, embryonic development, tumor progression and regulation of vascular tone. In particular, adrenomedullin expression is widely distributed throughout the cardiovascular system including heart, lungs, blood vessels and kidneys. The observation that adrenomedullin co-localizes with its receptors suggests that the peptide may act as an autocrine and/or paracrine factor in the regulation of cardiovascular function. In this review, we described the pathophysiological changes in plasma and local adrenomedullin associated with vascular endothelial function and the signaling mechanisms involved. Recent evidence has depicted a unique role of adrenomedullin in the regulation of vascular endothelial function in part through regulation of cAMP, cGMP and intracellular Ca(2+) mobilization. This review summarized vascular endothelial actions of adrenomedullin under both physiological and pathophysiological conditions in an effort to provide guidance for the clinical application of adrenomedullin in vascular diseases.

Mo1886 Regulation of Linaclotide-Induced cGMP and Electrolyte Secretion by Multidrug Resistance-Associated Protein 4 (MRP4)

Mo1886 Regulation of Linaclotide-Induced cGMP and Electrolyte Secretion by Multidrug Resistance-Associated Protein 4 (MRP4)

Identification of potent phosphodiesterase inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites.

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis, respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds of host cell invasion, replication, and egress (exit), destroying host cell tissue in the process. Previous work has identified important roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress and invasion, yet little is understood about the regulation of cyclic nucleotides, cGMP and cAMP, that activate these enzymes. To address this, we have focused upon the development of inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) to block the breakdown of cyclic nucleotides. This was done by repurposing human PDE inhibitors noting various similarities of the human and apicomplexan PDE binding sites. The most potent inhibitors blocked the in vitro proliferation of P. falciparum and T. gondii more potently than the benchmark compound zaprinast. 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) was found to be a potent inhibitor of recombinant P. falciparum PfPDEα and activated PKG-dependent egress of T. gondii and P. falciparum, likely by promoting the exocytosis of micronemes, an activity that was reversed by a specific Protein Kinase G inhibitor. BIPPO also promotes cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion, suggesting that the compound inhibits single or multiple PDE isoforms that regulate both cGMP and cAMP levels. BIPPO is therefore a useful tool for the dissection of signal transduction pathways in apicomplexan parasites.

Cellular Sensing System for Green Tea Polyphenol Epigallocatechin Gallate

The green tea catechin (‐)-epigallocatechin-3-O-gallate (EGCG) is known to exhibit various biological and pharmacological properties. We have identified 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor that confers EGCG responsiveness to many cancer cells at physiological concentrations. Here we provide an overview of several pathways that sense and manage the activities of EGCG. EGCG has been shown to rescue mice from lipopolysaccharide (LPS)-induced lethal endotoxemia and downregulate inflammatory responses in macrophages. LPS is one of the most powerful activators of toll-like receptor (TLR)4 signaling and is also well known to induce production of inflammatory mediators. A negative regulator of TLR signaling, Toll-interacting protein (Tollip), is indispensable for mediating the anti-inflammatory action of EGCG, and its protein expression level is upregulated by EGCG through 67LR. Additionally, EGCG can reduce the expression of TLR4 via 67LR. Using a direct genetic screen, eukaryotic translation elongation factor 1A (eEF1A) is identified as a component responsible for the anti-melanoma activity of EGCG. EGCG induces the dephosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) at Thr-696 and activates myosin phosphatase through both eEF1A and 67LR. Silencing of 67LR, eEF1A, or MYPT1 in tumor cells results in abrogation of EGCG-induced tumor growth inhibition in vivo. Additionally, we found that eEF1A is up-regulated by EGCG through 67LR. EGCG has been shown to be able to induce apoptotic cell death in multiple myeloma cells through the 67LR, while having no significant effect on peripheral blood mononuclear cells (PBMCs). The expression of 67LR was significantly elevated in myeloma cells compared to PBMCs. We found that the apoptosis-inducing activity of EGCG in multiple myeloma cells is attributable to the activation of acid sphingomyelinase (ASM) that hydrolyzes sphingomyelin into ceramide. EGCG induces ASM translocation to the plasma membrane and protein kinase C delta (PKCδ) phosphorylation at Ser 664 , which was necessary for ASM/ceramide signaling, via 67LR. Furthermore, EGCG activates PKCδ/ASM/ceramide pathway by activating Akt/eNOS/NO/cGMP signaling through 67LR. We also found that the upregulation of cGMP is a rate-determining process of this cell death pathway, and cancer-overexpressed negative regulator of cGMP, PDE5 attenuates the cell death induced by EGCG. Vardenafil, one of the PDE5 selective inhibitors used for treating erectile dysfunction potentiates the anti-cancer effect of EGCG. These results demonstrate that cGMP elevation caused by targeting the overexpressed 67LR and PDE5 in cancer cells may be a useful approach for cancer-specific chemotherapy.