This study investigated the effects of epinephrine on aquaglyceroporin HC-3 expression and localization in erythrocytes of the freeze tolerant treefrog Hyla chrysoscelis. The researchers found that treating erythrocytes with epinephrine for 30-60 minutes increased both native and glycosylated HC-3 expression levels and resulted in HC-3 membrane localization. Pretreating cells with the PKC inhibitor calphostin C prevented epinephrine-induced HC-3 membrane localization and reduced native HC-3 expression. This suggests epinephrine regulates HC-3 through a PKC-dependent pathway. Epinephrine treatment also increased phosphorylated PKC levels, supporting the involvement of

1. Connor Ratycz1, Philip Nickell1, James Frisbie2, David L. Goldstein2, Carissa M. Krane1
1Department of Biology, University of Dayton, Dayton, OH
2Department of Biological Sciences, Wright State University, Dayton OH
Abstract
B
E
B
Peptide Block
Cope’s gray treefrog, Hyla chrysoscelis, accumulates and distributes glycerol as a cryoprotectant
in anticipation of freezing. Transmembrane glycerol and water flux in H. chrysoscelis
erythrocytes likely occurs through HC-3, an ortholog of mammalian aquaporin 3. HC-3 protein
is in higher abundance and is preferentially localized to the plasma membrane in RBCs from
cold-acclimated treefrogs as compared to warm-acclimated animals. It is hypothesized that
neuroendocrine agonists via receptor mediated second messenger pathways integrate signals
derived from fasting, dehydration, diurnal, and/or temperature changes during cold-acclimation
to regulate HC-3 expression as part of the mechanism of freeze tolerance. In this study, cultured
H. chrysoscelis erythrocytes were exposed to 1 uM epinephrine for 30 and 60 minutes. Native
HC-3 expression increased 3 fold at 30 minutes and 5.5-fold at 60 minutes relative to controls,
whereas glycosylated HC-3 expression increased by 1.1-fold at 30 minutes and by 2 -fold at 60
minutes relative when exposed to epinephrine. Moreover, epinephrine treatment resulted in
membrane localization as compared to cytosolic distribution in control cells. Erythrocytes pre-
treated with Calphostin C, a PKC inhibitor, showed no additional HC-3 membrane localization,
and native HC-3 expression was reduced by 5% relative to controls and 3 -fold relative to
epinephrine-treated cells. Thus, epinephrine begins a PKC-dependent mechanism that results in
an increase in HC-3 abundance, HC-3 membrane localization, and enhanced glycosylation in
erythrocytes. These regulatory mechanisms are consistent with the in vivo regulation of HC-3
expression observed in erythrocytes from cold-acclimated treefrogs. This research was
supported by NSF Research Grant IOS-1121457, UD University Honors Program and American
Physiological Society 2013 UGSRF.
This research was supported by NSF Research Grant IOS-1121457, the University of Dayton
University Honors Program, and the American Physiological Society’s 2013 UGSRF.
Thanks to my advisor, Dr. Carissa Krane , as well as Phil Nickell and other Krane lab members.
We hypothesize that neuroendocrine hormones, which are responsible for
behavioral/physiological changes in H. chrysoscelis during cold-acclimation, act via second
messenger pathways to regulate HC-3 expression as part of the mechanism of freeze
tolerance.
Hypothesis
Epinephrine regulates aquaglyceroporin HC-3 expression and subcellular localization in cultured
erythrocytes from the freeze tolerant anuran, Cope’s gray treefrog, Hyla chrysoscelis
Glycerol, cAMP, and Epinephrine Induce HC-3 Translocation to the Cell Membrane
Acknowledgements
Conclusions and Significance
Aquaporins and Aquaglyceroporins
Aquaporins (AQPs), members of Major Intrinsic Protein (MIP) family, are integral membrane
proteins that span across the cell membrane and increase the water permeability of the plasma
membrane. Aquaglyceroporins (GLPs) are another class belonging to the MIP family that
facilitate the movement of both water and glycerol across the cell membrane. Because GLPs
function in the plasma membrane, the trafficking of GLPs is important to understand the process
of glycerol movement and freeze tolerance. These experiments attempt to understand the
agonist-induced mechanisms of localization and expression of aquaglyceroporin HC-3 in H.
chrysoscelis erythrocytes.
Figure 3. HC-3 subcellular localization in Hyla chrysoscelis erythrocytes exposed to cAMP
and epinephrine.
Cells cultured in CCCM (A), CCCM with 150mM glycerol for 48 hours (B), and cells in
CCCM for 48 hours then exposed to cAMP (D,E,K), or epinephrine (L,M) were fixed on 2%
gelatinized slides. Fluorescent immunocytochemistry using a primary antibody directed against
HC-3 and a fluorescein conjugated secondary antibody was used to examine HC-3 (green)
subcellular localization in erythrocytes (A-E, K-M). Nuclei were stained with ToPro (red).
Semi-quantitative analysis of the fluorescent intensity of HC-3 immunofluorescence was
measured via ImageJ software (F-J, N-Q).
Future Directions
B
C
C
Figure 1. Aquaporin 1 structure
The integral membrane protein composed of six
transmembrane domains which form a
homotetramer, cytosolic amino and carboxy
termini, intracellular Loop B and extracellular
Loop E which interact with each other to form the
water-selective pore. Image taken from Krane &
Goldstein (2007) Mammalian Genome; 18: 452-
462.
HC-3 translocation
HC-3
Neuroendocrine Hormones
Second Messenger
Pathway?
Figure 2. Hypothesized Mechanism
The translocation of aquaglyceroporin HC-
3 from intracellular membranous vesicles to
the plasma membrane of Hyla chrysoscelis
erythrocytes via second messenger pathway
induced by neuroendocrine hormones.
PKC Inhibition of Proposed Epinephrine-Induced PKC Pathway
Epinephrine Induces Increased Native HC-3 Expression and Enhanced Glycosylation
βγ Gα
Epinephrine
PLC
PKC
PIP2
DAG
Calphostin C
Agonist treatment of H. chrysoscelis erythrocytes revealed
epinephrine induces HC-3 membrane translocation at 30 and
60 minutes. To investigate the epinephrine-induced signaling
mechanism, calphostin C, a PKC inhibitor, was used to inhibit
PKC activity and observe effects on HC-3 translocation.
Figure 4. Proposed PKC-Dependent Mechanism of HC-3
translocation.
Binding of epinephrine to GpCR leads to activation of
phospholipase C (PLC) which leads to the activation of PKC.
PKC activation leads to downstream events such as HC-3
membrane localization. Calphostin C, PKC inhibitor, should
prevent HC-3 trafficking by inhibiting PKC activity.
Calphostin C Inhibits HC-3 Membrane Translocation In Presence of Epinephrine
Figure 5. Immunocytochemistry of H. chrysoscelis exposed to epinephrine and calphostin C.
Erythrocytes were cultured in CCCM (A) or CCCM with 150mM glycerol (B) for 48 hours, and
cells cultured in CCCM for 48 hours then exposed to 1μM calphostin C (C), 1μM epinephrine
(D), or 60 min. preincubation with calphostin C followed by 60 min. exposure to epinephrine (E).
Fluorescent intensity was measured using ImageJ (F-J).
Figure 6. Abundance of Glycosylated and Native HC-3 in Hyla chrysoscelis erythrocytes
exposed to epinephrine and calphostin C.
Western blotting was used to quantify the expression of HC-3 protein in erythrocytes cultured
in CCCM and CCCM with glycerol for 48 hours, and cells cultured in CCCM for 48 hours then
treated with calphostin C, epinephrine, or preincubation with calphostin C followed by
epinephrine treatment (A). Glycosylated HC-3 appeared as bands near 75-150 kDa. and native
HC-3 appeared at 31kDa. Densitometry was used to compare expression levels of native and
glycosylated HC-3. Values were normalized to β-actin, and expressed as a percentage of the
control (B).
Increased PKC Phosphorylation at Ser. 660 with Epinephrine Exposure
Figure 7. Abundance of Glycosylated and Native HC-3 in Hyla chrysoscelis erythrocytes
exposed to epinephrine and calphostin C.
Western blotting was used to quantify the expression of HC-3 protein in erythrocytes cultured
in CCCM and CCCM with glycerol for 48 hours, and cells cultured in CCCM for 48 hours then
treated with calphostin C, epinephrine, or preincubation with calphostin C followed by
epinephrine treatment (A). Phosphorylated PKC at Thr. 514 (A) and Ser. 660 (B) residues
appeared as a band at 85 kDa., while PKC appeared as a band at 80 kDa. Densitometry was
used to compare levels P-PKC at Thr. 514 (C) and Ser. 660 (D) residues against total PKC
levels. Values were normalized to β-actin, and expressed as a percentage of the control.
• Epinephrine initiates a PKC-dependent mechanism that results in increased HC-3
abundance, localization, and enhanced glycosylation in Hyla chrysoscelis erythrocytes.
• Epinephrine may also lead to upregulation of native HC-3 through PKC and subsequent
PKC-dependent transcriptional/translational mechanism.
• Epinephrine leads to HC-3 membrane localization through a mechanism different than that
of glycerol.
• Investigate epinephrine-induced temporal change of phosphorylated PKC and how this
parallels HC-3 membrane localization.
• Activate PKC and observe affect on translocation and native HC-3 upregulation over time.
K
N
A B C D E
F G H I J
L M
O P Q
A B
A B C D E
F G H I JμM
A B
DC