Module B – Shark Rectal Gland Robyn Levine, Roberto Rupcich, Keais Pope, Sam Harrington
Dec 17, 2015
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
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