Module B – Shark Rectal GlandRobyn Levine, Roberto Rupcich, Keais Pope, Sam Harrington

K+Na+

BL SideApical Side

Na+

K+

2 Cl-

CFTRCl-

K+

-70mV

TAL vs the shark rectal glandAbsorption v. Secretion

K+Na+

BL SideApical Side

K+

Na+

K+

2 Cl-

Cl-

Barttin

ClC-Kb

Quick Refresher on the shark rectal gland cell

K+Na+

Basolateral Side(Artery)

Apical Side(Duct Lumen)

Na+

K+

2 Cl-

CFTRCl-

K+ BaCl2 +

cAMP

5’AMPC

ATP

PDE

K+

Bumetanide

Ouabain

Furosemide (Lasix)

Perfusion Solution

Set-upARTERY

VEIN

DUCT

Meanwhile…

Forskolin and IBMXBasolateral SideApical Side

CFTRCl-

cAMP

5’AMPC

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

Sharky 1 Experiment

Perfusion Solution Time (min)

Basal (Ringer + Glucose)30

F + I30-45

High K+ (50mmol) & (F+I)45-65

F + I65-80

Barium (BaCl2) & (F+I)80-95

F+I95-110

Experiment 1 - Hypothesis

• 1. High K+ (50mmol) Perfusion

• Increased Extracellular K+Less K+ released thru K channel• If Less K+ releasedDepolarization • DepolarizationDecreased Cl- secretion due to diminished

electrochemical gradient

• 2.Barium (BaCl2) perfusion• Barium blocks TASKLess K+ released thru K channel• If Less K+ releasedDepolarization • DepolarizationDecreased Cl- secretion due to diminished

electrochemical gradient

Experiment 1 - Hypothesis

K+Na+

Basolateral SideApical Side

Na+

K+

2 Cl-

CFTRCl-

K+

K+

K+

K+

K+

K+

Cl-

Cl-

Cl-

-70mv

-15mv

Ringer + (F & I) Solution(5mmol K+)

Experiment 1 - Hypothesis

1. ↑K+

K+Na+

Basolateral Side(Artery)

Apical Side(Duct)

Na+

K+

2 Cl-

CFTRCl-

K+

K+

K+

K+

K+

K+

Cl-

Cl-

Cl-

K+

K+

K+

K+K+

K+

K+

K+

K+

K+

K+

2. BaCl2 +

Cell depolarizes

0 10 20 30 40 50 60 70 80 90 100 1100

500

1000

1500

2000

R6

Time (min)

Ch

lori

de S

ecre

tion

(µ

Eq

/h/g

)

F+I F+IF+IHigh K+

(50mmol) Barium

Experiment 1

Sharky 2 Experiment

Perfusion Solution Time (min)

Basal 30

F + I only 30-45

Low K+ (1.0mMol) + (F+I) 45-65

F + I only 65-80

High K+ (50mmol) + (F+I) 80-100

F + I only 100-115

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

Experiment 2 - Hypothesis

1. ↓K+

K+Na+

Basolateral Side(Artery)

Apical Side(Duct)

Na+

K+

2 Cl-

CFTRCl-

K+

K+

K+

Cl-

Cl-

K+

K+

0 10 20 30 40 50 60 70 80 90 100 1100

500

1000

1500

2000

2500

R8

Time (min)

Ch

lori

de S

ecre

tion

(µ

Eq

/h/g

)

BASAL

l

F+I

F+I F+I

Low K(1mmol)

High K+

(50mmol)

Experiment 2

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

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

Questions?