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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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

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Background Cystic Fibrosis

• Genetic Disease that causes a defect (thousands
  of mutations) in ones Cystic Fibrosis
  Transmembrane Conductance Regulator (CFTR)
• CFTR is found in secretory cells (mucus, saliva,
  sweatetc)
• 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

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CFTR
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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.

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Background Main CF Symptoms
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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

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Background The Rectal Gland of the Spiny Dogfish
Shark (Squalus acanthias)
Artery
Gland
Duct
Intestine
Vein
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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--gt
  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

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Background CNP CFTR Signaling Pathway (draw)

1. CNP binds to NPR-B on membrane
2. Stimulates an increase in cGMP levels
3. This increase down-regulates PDE-3s breakdown of
   cAMP through cGMPs competitive inhibition of
   PDE-3 (inhibiting the inhibitor)
4. cAMP levels increase as a result
5. This increase up-regulates PKA activity
6. Thus PKAs phosphorylation of CFTR is increased

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Methods UhHow?

• Main Method Live Perfusion Experiments
• Cannulate Live Glands vein, artery and duct
• Perfuse it with Ringers 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 --gt 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 tissues concentration
  of different molecules ex. cGMP, cAMPetc
• 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

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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?

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Results Live Perfusions

• All n7 except basal, n3
• Error bars are SEM

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Results Maximums Bar Graph

• Error Bars are SEM
• n7 for all
• Pvalue Red/Green 0.5681
• Pvalue Purple/Orange 0.0876

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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.

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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

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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

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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

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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_mono
  phosphate
• http//en.wikipedia.org/wiki/Phosphodiester_bond