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POSSIBLE ROLE OF PHOSPHODIESTERS IN COLD TOLERANCE
Marina Marjanovic1, Barbara A. Lawrence2,
Nicholas C. Wright2,
Jon P. Costanzo3 and Richard E. Lee, Jr.3
1 Department of Biological Sciences, Eastern Illinois
University
2 Chemistry Department, Eastern Illinois University
3 Miami University, Oxford, OH
Introduction
Ectothermic animals living in the northern regions are well
adapted to survive exposure to extreme cold. Several species of insects,
amphibians and reptiles are freeze-tolerant. During the freezing process in
these animals, ice crystals are forming only in the extracellular environment,
while intracellular water remains in the liquid state. Although several
mechanisms contributing to this phenomenon have been described, physiological
basis for natural freeze tolerance is not completely understood.
This study involved representatives of each of the major taxa for which
freeze-tolerance has been described: wood frog (Rana sylvatica), tree
frog (Pseudacris crucifer), painted turtle (Chrycemys picta), and
goldenrod gall fly (Eurosta solidignis). Leopard frog (Rana pipiens)
was used as a control, freeze-intolerant species.
Previous studies showed that some of these species have significant
concentrations of cytoplasmic phosphodiesters (PDEs), that are not component of
the cellular membranes. Using non-invasive
31P-Nuclear Magnetic Resonance (NMR), we investigated
phosphorus metabolites in both isolated tissues and whole bodies of these
animals (Table 1). The goal was to establish biochemical differences that could
further explain the cellular mechanisms of freeze-tolerance.
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Table 1. Levels of total PDEs
expressed as percent of the total phosphorus signal. PDE values are
averages of number of samples given in parenthesis. |
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Methods
31P-NMR spectroscopy. Samples were placed
in NMR tubes and spectra were obtained with the 300 MHz G.E. QE300 FT-NMR
spectrometer. The chemical shift and relative areas under the peaks in the
spectra were used to identify and determine relative concentration of the
metabolites. The identity of the compounds was confirmed by “spiking” the
extracts of the tissues with a suspected compound and comparing its chemical
shift with the chemical shift of peaks in the spectrum.
Results
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Freeze-tolerant wood frog shows higher level of PDEs
in the skeletal muscle compared to freeze-intolerant leopard frog
(Figure 1). The levels of PDEs (glycerophosphorylethanolamine,
glycerophosphorylcholine and serine ethanolamine phosphate) and
phosphomonoesters (glucose-6-phosphate and phosphoenolpyruvate) in
wood frog skeletal muscle increase with cold acclimation and with
freezing (Figure 1). |
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Figure 1.
Phosphorus spectra of isolated skeletal muscles (m. gastocnemii)
from leopard frog and cold-acclimated wood frog at 21oC.
Lower spectrum: wood frogs were frozen for 36-42 h at –3oC
and muscles were dissected without thawing. Numbers indicate
chemical shifts of compounds given in the legend. |
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Induced freezing in cold-acclimated wood frog also
increases the level of PDEs at the whole body level (Figure 2). |
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Figure 2.
Phosphorus spectra of the wood frog in vivo. Upper spectrum:
cold-acclimated wood frog. Lower spectrum: wood frog was kept frozen
for 36 hours at
–3oC
and then thawed at room temperature. Freezing increased the level of
cytoplasmic PDEs at the whole body level. |
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All investigated freeze-tolerant species have similar
PDEs (Figures 3 and
4) |
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Figure 3. Whole
body spectra of cold-acclimated wood frog, tree frog and hatchlings
of painted turtle show the presence of cytoplasmic PDEs. |
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Figure 4.
Phosphorus spectra of gall fly larvae after transfer from -4oC
to the environmental chamber at +20oC for 0, 2, 4 and 9
days. PDEs show significant decrease with warm-acclimation. |
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PDE levels decrease significantly in gall fly larvae
with warm-acclimation within days (Figure 4). |
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Conclusions
Based on our findings that cytoplasmic PDEs accumulate in the
cytoplasm during cold acclimation, and during freezing, they could have an
important role in adaptation of organisms to low temperature.
Possible role of PDEs.
- Endogenous inhibitors of lysophospholipase (by
decreasing the turnover of membrane phospholipids during dormancy; Burt and
Ribolow, 1984, 1994);
- Osmoregulation (non-perturbing osmolytes; Nakanishi and Burg, 1989);
- Precursors of membrane phospholipids;
- Changes in cytoplasmic PDEs may reflect alterations in membrane
phospholipids during cold stress (Thebault et al., 1989);
References
Burt C.T. and Ribolow H. Biochem. Med. 31:20, 1984
Burt C.T. and Ribolow H. Comp. Biochem. Physiol. 108B: 11, 1994
Nakanishi T. and Burg M.B. Am. J. Physiol. 257: C795, 1989
Van den Thillart G. et al. Am. J. Physiol. 258:R922, 1989
Acknowledgements
Supported by NSF grant IOB 0416750
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