Kunihiko Hiraoka

Both stimulatory and inhibitory GDPGTP exchange proteins, smg GDS and rho GDI, are active on multiple small GTP-binding proteins☆

Six peaks of small GTP-binding proteins (G proteins) were separated by column chromatographies from the cytosol fraction of the differentiated HL-60 cells: two peaks of rho p21, one peak of smg/rap1 p21, two peaks of rac1 p21, and one peak of an unidentified small G protein with a Mr of about 20,000 (20 KG). smg GDS, previously thought to be a stimulatory GDP/GTP exchange protein for smg p21, Ki-ras p21, and rho p21, but not for Ha-ras p21 or smg p25A, was also active on rac1 p21. rho GDI, previously thought to be an inhibitory GDP/GTP exchange protein specific for rho p21, was also active on rac1 p21. These results indicate that both smg GDS and rho GDI are active on multiple small G proteins.

Pancreas preservation by the 2-layer cold storage method before islet isolation protects isolated islets against apoptosis through the mitochondrial pathway

BACKGROUND Apoptosis in isolated islets has been implicated in primary nonfunction or early graft failure after islet transplantation. Recently, pancreas preservation by the 2-layer method (TLM) before islet isolation has been proved to improve the islet yield, quality, and transplant results not only in experimental models, but also in clinical settings. We examined the influence of TLM on apoptosis of isolated islets. METHOD Rat islets freshly isolated and after pancreas preservation by TLM or conventional cold storage in University of Wisconsin solution (UW) were examined and compared. Islet apoptosis was assessed by TUNEL and annexin V assays. The apoptosis pathways involved were investigated by measurement of caspase 3, 8, and 9 activities and by immunoblotting for total and phosphorylated c-Jun NH2-terminal kinase (JNK) and p38. RESULTS Islet apoptosis in the UW group was significantly increased compared with the fresh and TLM groups. Both caspase 3 and 9 activities in the UW group were higher than in the fresh and TLM groups with an approximate increase of 2- to 3-fold. On the other hand, there was no significant difference in caspase 8 activity among these 3 groups. JNKs were strongly activated both in the TLM and UW groups; although they were not activated in the fresh group, p38 was activated to almost the same levels in these 3 groups. CONCLUSIONS Pancreas preservation by TLM before islet isolation protects isolated islets against apoptosis mainly through the mitochondrial pathway. Pancreas storage before islet isolation even with TLM triggers activation of JNKs in isolated islets.

Role of adenosine in preservation by the two-layer method of ischemically damaged canine pancreas

The purpose of this study was to clarify the role of adenosine in preservation of ischemically damaged pancreas by the two-layer (Euro-Collins solution [EC]/perfluorochemical [PFC]) method using a canine model. Twenty-four-hour preservation of the pancreas graft subjected to 60-min warm ischemia was successful by the two-layer (EC with adenosine/PFC) method (4/5, 80%), but neither simple cold storage in EC (0/5, 0%), nor EC with adenosine (1/5, 20%), nor the two-layer (EC/PFC) method (0/3, 0%) was successful. Tissue ATP concentrations at the end of preservation by the two-layer (EC with adenosine/PFC) method were significantly higher compared with the two-layer (EC/PFC) method (7.23 +/- 2.17 vs. 1.56 +/- 0.40 mumol/g dry weight, P < 0.01). Studies with [2-3H]adenosine demonstrated that only part of adenosine was converted to inosine, hypoxanthine, and adenine, whereas the remainder was incorporated into adenine nucleotides in the pancreas graft. In addition, hypoxanthine, inosine, and adenine did not substitute for adenosine. We conclude that provision of adenosine to ischemically damaged pancreas during preservation by the two-layer (EC/PFC) method allows ATP synthesis within the graft via direct phosphorylation of adenosine. Metabolic processes vital to repair damaged cells and maintain cellular integrity can be maintained, which makes it possible to preserve ischemically damaged pancreas.

Resuscitation of ischemically damaged pancreas during short-term preservation at 20°C by the two-layer (University of Wisconsin solution/perfluorochemical) method

We have shown that 24-hr preservation by a two-layer (University of Wisconsin solution [UW]/perfluorochemical [PFC]) cold storage method allows tissue ATP synthesis and makes it possible to resuscitate a canine pancreas subjected to 90 min of warm ischemia. The purpose of this study was to examine whether increasing preservation temperature to 20 degrees C makes it possible to shorten a preservation period for recovery of ischemically damaged pancreas grafts. After 90 min of warm ischemia, canine pancreas grafts were preserved using the two-layer (UW/PFC) method for 1 to 8 hr at 20 degrees C, and then autotransplanted. A K-value of intravenous glucose tolerance test more than 1.0 at 2 weeks after transplantation was considered graft survival. ATP tissue levels were measured by high performance liquid chromatography at the end of preservation. Pancreatic tissue perfusions were measured using an H2 clearance technique after 30 min to 4 hr of reperfusion. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5, control group). However, 3- and 5-hr preservations made it possible to recover the ischemically damaged pancreas (3/5 and 5/5, respectively), although 1- and 8-hr preservations were not successful (0/3 and 0/3, respectively). ATP tissue levels in 1-hr-preserved grafts were 2.55 +/- 0.38 mumol/g dry weight and were significantly lower compared with the levels in 5- and 8-hr-preserved grafts, 9.40 +/- 2.09 (P < 0.01) and 7.37 +/- 1.06 (P < 0.01), respectively. On the other hand, pancreatic tissue perfusions in 8-hr-preserved grafts after 2 hr of reperfusion were 28.50 +/- 7.52 ml/100 g/min and were significantly lower than the values in 1- and 5-hr-preserved grafts, 66.0 +/- 11.22 (P < 0.01) and 57.10 +/- 4.40 (P < 0.01), respectively. It was suggested that 1-hr-preservation was not enough to synthesize ATP, which was essential to repair damaged cells, although vascular microcirculation at reperfusion was maintained and 8-hr preservation incurred microcirculatory disturbances, although ATP for repairing damaged cells was synthesized. We conclude that 3- to 5-hr preservation at 20 degrees C by the two-layer (UW/PFC) method accelerates ATP synthesis, which is essential for repairing damaged cells and protects vascular microcirculation. This makes it possible to resuscitate ischemically damaged pancreases faster. This method holds promise for pancreas-kidney transplantation from cardiac arrest donors.

Protective effect of preservation of canine pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) method against rewarming ischemic injury during implantation.

Rewarming ischemia during implantation severely compromises posttransplant pancreas graft survival because the graft has already been subjected to warm and cold ischemia before implantation. The purpose of this study was to examine whether preservation of the pancreas graft by the two-layer method ameliorates rewarming ischemic injury of the graft during implantation using a canine model. After flushing with cold University of Wisconsin solution (UW), the pancreas grafts were preserved by the two-layer (UW/perfluorochemical [PFC]) method (group 1) or simple cold storage in UW (group 2) for 24 hr and then autotransplanted. In control, the pancreas grafts were flushed out with cold UW and immediately autotransplanted without preservation (group 3). After completion of vascular anastomosis, vascular clamp was not released until 90, 120, or 150 min of rewarming ischemia, including anastomosis time, had elapsed. After 90 min of rewarming ischemia, graft survival rates were 5/5, 100%, 5/5, 100%, and 5/5, 100%, in groups 1, 2, and 3, respectively. After 120 min, all the grafts in groups 2 and 3 failed (0/5, 0%, and 0/5, 0%, respectively); however, all the grafts in group 1 survived (5/5, 100%). Even after 150 min, 1 of 3 grafts in group 1 survived (1/3, 33%). After 24 hr preservation, tissue ATP levels of the grafts in group 1 were about 2-fold the reference values before harvesting (8.23 +/- 0.72 vs. 4.44 +/- 0.49 mumol/g dry weight, P < 0.05) and significantly higher compared with group 2 (8.23 +/- 0.72 vs. 1.76 +/- 0.52 mumol/g dry weight, P < 0.01). After 120 min of rewarming ischemia, tissue ATP levels in group 1 were 84% of the reference values and significantly higher compared with group 2 (3.75 +/- 0.25 vs. 1.57 +/- 0.48 mumol/g dry weight, P < 0.05). Two hours after reperfusion, ATP levels in group 1 were 42% of reference values but significantly higher compared with group 2 (1.86 +/- 0.36 vs. 1.03 +/- 0.18 mumol/g dry weight, P < 0.05). We conclude that the two-layer (UW/PFC) method ameliorates rewarming ischemic injury of the pancreas graft during implantation by increasing tissue ATP contents during preservation and consequently maintaining tissue ATP levels during implantation.

Difference in energy metabolism between fresh and warm ischemic canine pancreases during preservation by the two-layer method.

Abstract We have demonstrated that adenosine triphosphate (ATP) is synthesized within a canine pancreas during preservation by the two‐layer method and there is a direct correlation between a high ATP tissue level and good post‐transplant outcome. The purpose of this study was to examine the difference in energy metabolism between fresh and warm ischemic pancreases during preservation by this method. First, fresh pancreases were preserved with simple cold storage in Euro‐Collins solution (EC; group 1 A), or by the two‐layer method using EC (group 1B), EC with 2,4 dinitrophenol (DNP; group 1C), an uncoupler of oxidative phosphorylation, or modified EC (ECM; group 1 d), which contained mannitol in place of glucose for 48 h. ATP tissue concentrations in group 1B were significantly higher than in group 1A (7.91 ± 1.21 vs. 1.21 ±0.31 μmol/g dry weight, P < 0.01) but almost equal to group 1d (7.91 ±1.21 vs. 7.59 ±0.97 μmol/g dry weight, NS). DNP (group 1C) caused a significant decrease in tissue ATP levels in group 1A (0.61 ± 0.07 vs. 7.91 ± 1.21 μmol/g dry weight, P<0.01). Second, pancreases subjected to 60 min of warm ischemia were preserved by simple cold storage with EC (group 2 A) or the two‐layer method using EC (group 2B) or EC with adenosine (group 2C) for 24 h. ATP tissue levels in groups 2 A and 2B after preservation were 1.40 ± 0.46 and 1.56 ±0.40 μmol/g dry weight and graft survival rates were 0/5 (0%) and 0/3 (0%), respectively. However, tissue ATP levels in group 2C after preservation were significantly higher compared with the value before preservation (7.23 ± 2.17 vs. 1.90±0.53/g dry weight, P <0.01) and graft survival rate was 4/5, 80%. Other nucleosides, hypoxanthine, inosine, and adenine did not substitute for adenosine. In addition, studies with [2–3 H] adenosine demonstrated that almost all of the adenosine was converted to adenine nucleotides. This study clearly demonstrated that fresh grafts synthesize, ATP mainly via mitochondrial 6xidative phosphorylation using endogenous substrates. However, after significant warm ischemia, pancreases produce ATP mainly via direct phosphorylation of exogenous adenosine during preservation by the two‐layer method.

Metabolic intervention to affect canine pancreas recovery following ischemia during preservation by the two-layer method

Abstract We have demonstrated that a high adenosine triphosphate (ATP) level in a canine pancreas during preservation by the two‐layer method is an important determinant for the ultimate success of pancreatic transplantation. In this study, we investigated (a) the effect of factors that seemed to have an influence on energy metabolism in the canine pancreas at the tissue ATP level and (b) graft viability during preservation by the two‐layer method. ATP tissue concentration was determined by high‐performance liquid chromatography and graft viability was assessed on the basis of survival rate following autotransplantation. First, the pancreas was harvested from either 72‐h‐fasted (n= 5) or fed dogs (n= 5) and preserved by the two‐layer Euro‐Collins solution (EC)/perfluorochemical (PFC) method for 24 h. All the pancreatic grafts were viable in both fed and fasted groups. There was also no significant difference in ATP tissue concentration between the two groups (7.48 ± 0.55 vs. 7.03 ± 0.74 μmol/g dry weight, NS). Second, the pancreatic grafts subjected to 60 min of warm ischemia were preserved by either the two‐layer (EC/PFC) or (EC + adenosine/PFC) method for 24 h. Without adenosine, ATP tissue concentration did not recover (1.62 ± 0.26 after warm ischemia vs. 1.56 ± 0.40 μmol/g dry weight after preservation, NS) and all the pancreatic grafts failed. However, provision of adenosine led to restoration of ATP tissue levels (1.90 ± 0.53 vs. 7.23 ± 2.17 μmol/g dry weight, P < 0.01) and four of five grafts functioned immediately and maintained normoglycemia after transplantation. These results clearly demonstrated that the nutritional state of the pancreatic graft before procurement had no influence on ATP tissue level as well as graft viability during 24‐h preservation by the two‐layer method. On the other hand, provision of adenosine during 24‐h preservation enhanced ATP synthesis of the pancreatic tissue, thereby improving viability of the ischemically damaged pancreas.