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

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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

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C

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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.

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