Adenosine 5'triphosphate transport and accumulation during the cold preservation of rat hepatocytes in University of Wisconsin solution.
OBJECTIVE
We used isolated hepatocytes to investigate how different concentrations of ATP in the University of Wisconsin (UW) solution affected both cellular ATP content and cell viability during the cold storage and the rewarming step. The mechanism involved in ATP transport and accumulation in hypothermia was also determined.


METHODS
The cells were preserved up to 72 h in different conditions: UW solution without ATP (a-group), UW+5 mmol/L ATP (b-group), and UW+10 mmol/L ATP (c-group). The ATP content and the cell viability (LDH release) were determined during the cold storage and the rewarming step. In the groups a and c, the respiratory function of the cells at rewarming was studied. In addition, the cell volume of hepatocytes and the mechanism involved in ATP transport and accumulation were assessed. The extracellular degradation of exogenous nucleotides during transport experiments was investigated by a HPLC technique.


RESULTS
After three days of cold storage a loss of cellular ATP content was observed in hepatocytes preserved either without nucleotides (a-group) or with 5 mmol/L ATP (b-group). In contrast, 10 mmol/L ATP (c-group) was able to maintain a normal ATP cellular content, with only a 6% diminution after 72 h of cold storage. The respiratory function was significantly different in hepatocytes preserved with 10 mmol/L ATP than without ATP. No significant change was detected for the three groups in cellular volume during the cold storage. We also report that the time course accumulation of (3H)-ATP by cold stored hepatocytes is a rapid process that is completed after 180 s with linear dependence on the extracellular ATP concentration (linear fitting results in a slope of 0.5624+/-0.1179 mmol/L ATP intracell/mmol/L ATP extracell).


CONCLUSIONS
Our results show that, during hypothermic storage in UW solution, hepatocytes are permeable to ATP by a diffusive mechanism. Also, we found that it is ATP the main extracellular nucleotide available for transport and it is not the breakdown products.
Mamprin
Maria E
ME
Farmacologia, Departamento Ciencias Fisiologicas, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina.
Vega
Felix
F
Rodriguez
Joaquin V
JV
D000241:Adenosine / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000493:Allopurinol / Q000494:pharmacology*; D000818:Animals; D019069:Cell Respiration; D002470:Cell Survival; D003080:Cold Temperature; D015925:Cryopreservation; D005978:Glutathione / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D006358:Hot Temperature; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D019188:Organ Preservation Solutions / Q000494:pharmacology*; D011887:Raffinose / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D014316:Tritium; D014882:Water-Electrolyte Balance
D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D014316:Tritium; D000493:Allopurinol; D000255:Adenosine Triphosphate; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose