1. Module B – Shark Rectal Gland Robyn Levine, Roberto Rupcich, Keais Pope, Sam Harrington

2. K+K+ Na + BL SideApical Side Na + K+K+ 2 Cl - CFTR Cl- K+K+ -70mV TAL vs the shark rectal gland Absorption v. Secretion K+K+ Na + BL SideApical Side K+K+ Na + K+K+ 2 Cl - Cl- Barttin ClC-Kb

3. Quick Refresher on the shark rectal gland cell K+K+ Na + Basolateral Side (Artery) Apical Side (Duct Lumen) Na + K+K+ 2 Cl - CFTR Cl- K+K+ BaCl 2 + cAMP 5’AMP C ATP PDE K+ Bumetanide Ouabain Furosemide (Lasix) Perfusion Solution

4. Set-up ARTERY VEIN DUCT

7. Meanwhile…

8. Forskolin and IBMX Basolateral Side Apical Side CFTR Cl- cAMP 5’AMP C ATP PDE IBMX Forskolin Necessary to stimulate Chloride secretion. Included in the perfusion solution throughout the experiment. Forskolin - activates enzyme adenylyl cyclase and increases intracellular levels of cAMP + IBMX: phosphodiesterase inhibitor ( ∴ ↑cAMP) Phosphodiesterase: degrades cAMP

9. Sharky 1 Experiment Perfusion SolutionTime (min) Basal (Ringer + Glucose) 30 F + I 30-45 High K+ (50mmol) & (F+I) 45-65 F + I 65-80 Barium (BaCl 2 ) & (F+I) 80-95 F+I 95-110

10. Experiment 1 - Hypothesis 1. High K + (50mmol) Perfusion Increased Extracellular K +  Less K + released thru K channel If Less K + released  Depolarization Depolarization  Decreased Cl - secretion due to diminished electrochemical gradient 2.Barium (BaCl 2 ) perfusion Barium blocks TASK  Less K + released thru K channel If Less K + released  Depolarization Depolarization  Decreased Cl - secretion due to diminished electrochemical gradient

11. Experiment 1 - Hypothesis K+K+ Na + Basolateral Side Apical Side Na + K+K+ 2 Cl - CFTR Cl- K+K+ K+ Cl- -70mv -15mv Ringer + (F & I) Solution (5mmol K+)

12. Experiment 1 - Hypothesis 1. ↑ K + K+K+ Na + Basolateral Side (Artery) Apical Side (Duct) Na + K+K+ 2 Cl - CFTR Cl- K+K+ K+ Cl- K+ 2. BaCl 2 + Cell depolarizes

13. Experiment 1

15. Experiment 2 Hypothesis 1. Low K + (1 mmol) perfusion solution (compared to ringer K + 5mmol) low basolateral K + available for the Na/K/2Cl contransporter  cotransporter decreases Cl- transport into cell  low levels Cl- secreted Or could hyperpolarize the cell resulting in increased Cl- secretion Low basolateral K+  increased polarization  increased Cl- secretion

16. Experiment 2 - Hypothesis 1. ↓ K + K+K+ Na + Basolateral Side (Artery) Apical Side (Duct) Na + K+K+ 2 Cl - CFTR Cl- K+K+ K+ Cl- K+

17. Experiment 2

18. Conclusions Experiment 1 Both High K+ and BaCl2 act to inhibit Cl- secretion High K+ inhibits secretion by diminishing the electrochemical gradient to depolarize the cell BaCl2 likely inhibits secretion by blocking the K+ channel to depolarize the cell Experiment 2 Low K+ also acts to inhibit Cl- secretion Inhibition likely occurs via the Na+/K+/2Cl- co-transporter Low K+ decreases the available K+ needed to drive the co- transporter, limiting the concentration of Cl- inside the cell

19. Future Experiments Measure potential difference to determine to what extent the low K (1mmol) perfusion results in cell depolarization Gradually change potassium levels in perfusion solution to determine a more precise concentration at which Cl- secretion is inhibited. Search and identify other basolateral potassium channels

21. Questions?