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Background: Cystic Fibrosis

Inhibition of type 5 Phosphodiesterase with sildenafil increases chloride secretion and augments the response to submaximal CNP in the rectal gland of the spiny dogfish, Squalus acanthias.

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Background: Cystic Fibrosis

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  1. Inhibition of type 5 Phosphodiesterase with sildenafil increases chloride secretion and augments the response to submaximal CNP in the rectal gland of the spiny dogfish, Squalus acanthias Megan H. Kelley1,2,5, August M. Melita1,2,5, Montana Morris1,5, Bob Tom Flynn,3,5 Hugo R. de Jonge4,5 and John N. Forrest Jr1,5. 1Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510 2University of Vermont, Burlington, VT 0540 3John Bapst Memorial High School, Bangor ME, 05501 4Department of Biochemistry, Erasmus University Medical Center, 3000CA Rotterdam, The Netherlands 5Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672

  2. Background: Cystic Fibrosis • Genetic Disease that causes a defect (thousands of mutations) in one’s Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) • CFTR is found in secretory cells (mucus, saliva, sweat…etc) • CFTR regulates chloride (Cl-) balance w/in the cell • Defect either: • Prevents CFTR from securing itself in the membrane • inhibits conductance by lowering its frequency of opening, thus the cell retains too much Cl- • a.k.a Cystic Fibrosis

  3. CFTR

  4. Background: Main CF Symptoms • The transport of chloride ions helps control the movement of water in tissues, which is necessary for the production of thin, freely flowing mucus. • In people with cystic fibrosis, the body produces mucus that is abnormally thick and sticky. • obstructs the airways, leading to severe problems with breathing and bacterial infections in the lungs. These infections cause chronic coughing, wheezing, and inflammation. Over time, mucus buildup and infections result in permanent lung damage, including the formation of scar tissue (fibrosis) and cysts in the lungs. • In people with cystic fibrosis, mucus blocks the ducts of the pancreas, preventing these enzymes from reaching the intestines to aid digestion. Problems with digestion can lead to diarrhea, malnutrition, poor growth, and weight loss. • Cystic fibrosis used to be considered a fatal disease of childhood. With improved treatments and better ways to manage the disease, many people with cystic fibrosis now live well into adulthood.

  5. Background: Main CF Symptoms

  6. Background: The Rectal Gland of the Spiny Dogfish Shark (Squalus acanthias) • Regulates salt (Cl-) in the intestine of the shark, balance essential to mucus formation • Cheap and Easy Analogue to Human Secretory Glands b/c • It is a very simple gland • Its size is desirable • The microbiology, the level on which cystic fibrosis is treated, is essentially the same as in humans

  7. Background: The Rectal Gland of the Spiny Dogfish Shark (Squalus acanthias) Artery Gland Duct Intestine Vein

  8. Background: Key Molecules in CF • Phosphodiesterase (PDE) - an enzyme that breaks a phosphodiester bond • There is a family of 11 isoforms • Key PDEs: • PDE-3 inhibited by cGMP; breaks down cAMP • PDE-5 breaks down cGMP; Inhibited by Sildenafil • In this case they breakdown cyclic nucleotides--> nucleotides whose concentrations fluctuate within the cell, and are critical components of the signal transduction that activates CFTR • Key cyclic nucleotides: • cyclic adenosine monophosphate (cAMP) • cyclic guanosine monophosphate (cGMP) • Protein Kinase A - (PKA) • C-type Natriuretic peptide (CNP) - the hormone that stimulates the pathway • NPR-B - the receptor for CNP • Forskolin - independently elevates cAMP

  9. Background: CNP CFTR Signaling Pathway (draw) • CNP binds to NPR-B on membrane • Stimulates an increase in cGMP levels • This increase down-regulates PDE-3’s breakdown of cAMP through cGMP’s competitive inhibition of PDE-3 (inhibiting the inhibitor) • cAMP levels increase as a result • This increase up-regulates PKA activity • Thus PKA’s phosphorylation of CFTR is increased

  10. Methods: Uh…How? • Main Method: Live Perfusion Experiments • Cannulate Live Gland’s vein, artery and duct • Perfuse it with Ringer’s solution (fake shark blood) (30 mins) • Record volume of secretion and chloride concentration from duct on 10 min intervals • Add experimental drug to solution, and record each minute --> usually looking for an increase in volume and/or concentration, indicating that CFTR is being activated, and chlorides are being secreted • Standardize gland for mass • Protein Assays: Snap Freeze Gland with Liquid Nitrogen at different points in experiment, and then analyze the blended tissue’s concentration of different molecules: ex. cGMP, cAMP…etc • Combine this data with knowledge of how these molecules behave in general (ex. cGMP inhibits PDE-3) • Deduce a plausible pathway • Also short circuit current experiments with oocytes and cultured SRG cells

  11. The Experiment • Sildenafil (Viagra) is a PDE-5 inhibitor • PDE-5 breaks down cGMP • Questions: • Is PDE-5 present in the SRG? • How does Sildenafil interact with the CNP pathway, based on the given information?

  12. Results: Live Perfusions • All n=7 except basal, n=3 • Error bars are SEM

  13. Results: Maximums Bar Graph • Error Bars are SEM • n=7 for all • Pvalue Red/Green =0.5681 • Pvalue Purple/Orange = 0.0876

  14. Results Takeaways • Sildenafil modestly stimulates Chloride Secretion on its own • A synergism is suggested between CNP and Sildenafil • Supported by: • The combination of the two yielded the highest hill on the live perfusion. Higher than each individually, and significantly higher than Sildenafil + Forskolin, a known strong stimulant, which operates through a different mechanism • The Bar Graph: The max of Sildenafil + CNP combined was almost statistically significantly higher than their additive effect (individual maxes combined), as well as the additive and combined effects of forskolin and Sildenafil.

  15. Discussion: Implications • Firstly, It is clear that PDE-5 is present in the SRG • A possible implication of the synergy, if it does indeed exist, is that Sildenafil is working through the same pathway as CNP • Sildenafil inhibits PDE-5, thus inhibiting the breakdown of cGMP, and raising cGMP levels, just like CNP

  16. Discussion: Major Errors • There are an enormous amount of non-standardized variables given variance amongst sharks and thus rectal glands • Ex. Natural vasodialation of the gland that is independent of drugs perfused • Such a difficult procedure, like canulating the gland, is prone to human error • The small sample size, as a result of the monetary and moral price of each experiment

  17. Discussion: Relation to the Disease • Choosing beneficial drug therapies is not as simple as pumping someone full of a CFTR stimulator, due to side-effects and over stimulation of CFTR • Effective treatment comes from understanding the disease from the ground up, and then pinpointing the best treatment drug • That being said, a synergistic effect between CNP and Sildenafil is potential grounds for treatment, due to its particularly effective ability to open CFTR

  18. Additional Sources: • http://www.cff.org/AboutCF/ • http://en.wikipedia.org/wiki/PDE5_inhibitor • http://ghr.nlm.nih.gov/condition/cystic-fibrosis • http://en.wikipedia.org/wiki/Cyclic_guanosine_monophosphate • http://en.wikipedia.org/wiki/Phosphodiester_bond

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