Neufeld D S, Leader L P
Department of Physiology, University of Otago, Dunedin, New Zealand.
J Exp Biol. 1998 Jan;201(Pt 2):227-36. doi: 10.1242/jeb.201.2.227.
The ability of isolated Malpighian tubules from a freeze-tolerant insect, the New Zealand alpine weta (Hemideina maori), to withstand freezing was assessed by measuring post-freeze membrane potentials and rates of fluid secretion. The hemolymph of cold-acclimated Hemideina maori was found to contain relatively high concentrations of the cryoprotectants trehalose (>300 mmol l-1) and proline (41 mmol l-1). Survival of isolated Malpighian tubules was correspondingly high when a high concentration of trehalose was present in the bathing saline. Tubules allowed to recover for 20 min from a 1 h freeze to -5 degrees C in saline containing 400 mmol l-1 trehalose had a basolateral membrane potential of -53 mV compared with a potential of -63 mV in tubules not exposed to a freeze/thaw cycle. Fluid secretion in tubules that had experienced a freeze/thaw cycle in saline containing 400 mmol l-1 trehalose was 9.9+/-2.6 nl h-1 compared with 18.7+/-5.0 nl h-1 (means +/- s.e.m., N=18) in tubules that had not been frozen. Tubules frozen in saline containing a lower concentration of trehalose (200 mmol l-1) or in glucose (400 mmol l-1) showed a similar ability to survive freezing to -5 degrees C. In contrast, freezing for 1 h at -5 degrees C in saline containing 400 mmol l-1 sucrose produced a 57 % decrease in membrane potential and an 88 % decrease in secretion rate. Tubules held in saline lacking high concentrations of sugars showed no survival after freezing to -5 degrees C for 1 h. When frozen to -15 degrees C, tubules appeared to survive best in saline with the highest trehalose concentration (400 mmol l-1). Freezing damage was not simply the result of exposure to cold, since tubules chilled (unfrozen) to -5 degrees C for 1 h were not compromised even when the bathing saline lacked a high sugar concentration. Exposure of tubules to a combination of low temperature and high osmolality mimicked damage caused by actual freezing: the membrane potential showed a 60 % recovery when the test was performed in saline containing trehalose, but showed no recovery in saline containing sucrose.
通过测量冷冻后膜电位和液体分泌速率，评估了来自耐冻昆虫——新西兰高山沙螽（Hemideina maori）的离体马氏管的抗冻能力。发现经冷驯化的Hemideina maori的血淋巴中含有相对高浓度的低温保护剂海藻糖（>300 mmol l-1）和脯氨酸（41 mmol l-1）。当浴液盐水中存在高浓度海藻糖时，离体马氏管的存活率相应较高。在含有400 mmol l-1海藻糖的盐水中从-5℃冷冻1小时后恢复20分钟的小管，其基底外侧膜电位为-53 mV，而未经历冻融循环的小管电位为-63 mV。在含有400 mmol l-1海藻糖的盐水中经历冻融循环的小管的液体分泌速率为9.9±2.6 nl h-1，而未冷冻的小管为18.7±5.0 nl h-1（平均值±标准误，N = 18）。在含有较低浓度海藻糖（200 mmol l-1）的盐水中或在葡萄糖（400 mmol l-1）中冷冻的小管，显示出与在-5℃冷冻相似的存活能力。相比之下，在含有400 mmol l-1蔗糖的盐水中于-5℃冷冻1小时，膜电位降低57%，分泌速率降低88%。在缺乏高浓度糖类的盐水中保存的小管，在-5℃冷冻1小时后无存活。当冷冻至-15℃时，小管在海藻糖浓度最高（400 mmol l-1）的盐水中似乎存活得最好。冷冻损伤并非仅仅是暴露于寒冷的结果，因为即使浴液盐水缺乏高糖浓度，在-5℃冷却（未冷冻）1小时的小管也未受到损害。将小管暴露于低温和高渗的组合中会模拟实际冷冻造成的损伤：当在含有海藻糖的盐水中进行测试时，膜电位显示有60%的恢复，但在含有蔗糖的盐水中则无恢复。
