The functional effects of suppression of hypothermia-induced cell swelling in liver preservation by cold storage

It is known that cellular edema and functional impairment develop during anaerobic cold storage of organs. The extent of both is related to the storage time and the composition of the preservation solution used. We studied hypothermia-induced cell swelling and its effect on liver function after cold storage preservation with either Eurocollins (EC), a number of modified EC solutions in which glucose was replaced by various concentrations of raffinose, or UW solution. After 24 h storage, tissue swelling as determined by total tissue water (TTW) in rat liver tissue slices was most pronounced in slices incubated in Eurocollins, whereas the TTW was only moderately increased in slices stored in modified Eurocollins containing 90 to 120 mM raffinose. In contrast, slices incubated in UW solution had a TTW equal to normal rat liver tissue. Furthermore, intact rabbit livers preserved with Eurocollins had an increase in the whole organ weight, while there was no weight change after preservation with the modified solution containing 120 mM raffinose (M120). In contrast, a pronounced weight loss was observed after preservation with UW solution. After cold storage, the livers were reperfused for 2 h at 38 degrees C in an isolated perfusion circuit (IPL) with an acellular perfusate. Bile flow was significantly greater in livers preserved in M120 than in those preserved with the conventional Eurocollins. However, the bile flow in the livers stored in M120 was inferior to that in the livers preserved with UW solution, which in turn was equal to that in control livers. The release of alanine-aspartate-aminotransferase into the perfusate was higher in livers preserved with Eurocollins, with or without modification, than in the livers preserved with UW solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of cold storage and perfusion of dog livers on function of tissue slices

We compared how two methods of hypothermic preservation affect physiological functions of tissue slices of dog liver. Livers were preserved by either (i) cold storage (CS) in Collins' solution or (ii) continuous perfusion (P) with a perfusate, containing hydroxyethyl starch, sodium gluconate, adenosine, and potassium phosphate, recently developed in our laboratory. Livers were cold stored for 6 to 8, 24, or 48 hr, and perfused for 24 or 72 hr. Tissue slices of preserved livers were incubated at 30 degrees C and analyzed for volume control, electrolyte-pump activity (K and Na), and adenine nucleotide concentration. Also, mitochondria were isolated after preservation to quantify respiratory activity. Slice functions of livers preserved for short periods (6 to 8 hr by CS and 24 hr by P) were similar to those for control livers. After normothermic incubation, the mean (+/- SD) water content of tissue (expressed per unit dry mass of tissue) was 2.3 +/- 0.3 kg/kg for control, 2.6 +/- 0.4 kg/kg for 6- to 8-hr CS, and 2.5 +/- 0.5 kg/kg for 24-hr P. Longer periods of preservation resulted in cell swelling, and water content was 3.3 +/- 0.4 kg/kg for 24- to 48-hr CS and 2.8 +/- 0.3 kg/kg for 72-hr P. The mean (+/- SD) K/Na ratio was nearly normal for livers preserved for short periods: 3.7 +/- 0.5 for control, 4.1 +/- 0.2 for 6- to 8-hr CS, and 3.3 +/- 0.4 for 24-hr P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hyperosmotic medium on hepatocyte volume, transmembrane potential and intracellular K+ activity

Hepatocyte transmembrane potential (Vm) behaves as an osmometer and varies with changes in extracellular osmotic pressure created by altering the NaCl concentration in the external medium (Howard, L.D. and Wondergem, R. (1987) J. Membr. Biol. 100, 53). We now have demonstrated similar effects on Vm by increasing external osmolality with added sucrose and not altering ionic strength. We also have demonstrated that hyperosmotic stress-induced depolarization of Vm results from changes in membrane K+ conductance, gK, rather than from changes in the K+ equilibrium potential. Vm and aKi of hepatocytes in liver slices were measured by conventional and ion-sensitive microelectrodes, respectively. Cell water vols. were estimated by differences in wet and dry weights of liver slices after 10-min incubations. Effect of hyperosmotic medium on membrane transference number for K+, tK, was measured by effects on Vm of step-changes in external [K+]. Hepatocyte Vm decreased 34, 52 and 54% when tissue was superfused with medium made hyperosmotic with added sucrose (50, 100 and 150 mM). Correspondingly, aKi increased 10, 18 and 29% with this hyperosmotic stress of added sucrose. Tissue water of 2.92 +/- 0.10 kg H2O/kg dry weight in control solution decreased to 2.60 +/- 0.05, 2.25 +/- 0.06 and 2.22 +/- 0.05 kg H2O/kg dry weight with additions to medium of 50, 100 and 150 mM sucrose, respectively. Adding 50 mM sucrose to medium decreased tK from 0.20 +/- 0.01 to 0.05 +/- 0.01. Depolarization by 50% with hyperosmotic stress (100 mM sucrose) also occurred in Cl-free medium where Cl- was substituted with gluconate. We conclude that hepatocytes shrink during hyperosmotic stress, and the aKi increases. The accompanying decrease in Vm is opposite to that expected by an increase in aKi, and at least in part results from a concomitant decrease in gK. Changes in membrane Cl- conductance most likely do not contribute to osmotic stress-induced depolarization, since equivalent decreases in Vm occurred with added sucrose in cells depleted of Cl- by superfusing tissue with Cl-free medium.     

Effect of sugars in the preservation solution on liver storage in rats.

We have performed 128 rat liver transplants in order to examine the effect of sugars in preservation solutions on cold storage of rat livers. Glucose (Mw. 180), sucrose (Mw. 348), and raffinose (Mw. 594) were tested. Rat livers were preserved at 4 degrees C for 12, 16, 18, and 24 h in standard Eurocollins solution (EC solution) (solution A) or in one of three modified EC solutions in which 194 mM/liter glucose in standard EC solution was replaced by 140 mM/liter of glucose (solution B), sucrose (solution C), or raffinose (solution D). The osmolarity of the modified solutions (solution B-D) was 320 mOsm/liter. Using standard EC solution (solution A), the 1-week survival rate of rats receiving livers preserved for 12, 16, 18, or 24 h was 6/8, 4/8, 1/8, and 0/4, respectively. With solution B, in which 194 mM/liter glucose was replaced by 140 mM/liter glucose, 1 week survivors following transplantation of livers preserved for 12, 16, 18 or 24 h were 4/8, 3/8, 2/8 and 0/4, respectively. Solution C, which was identical to solution A except for the replacement of 194 mM/liter glucose by 140 mM/liter sucrose, gave the following 1-week survival rates: 5/8 for 12 h, 5/8 for 16 h, 2/8 for 18 h, and 0/4 for 24 hours preservation, respectively. Using solution D, which differed from A in the replacement of glucose by 140 mM/liter raffinose, the 1-week survival rates of rats grafted with livers preserved for 12, 16, 18, and 24 h were 6/8, 5/8, 3/8 and 0/4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bronchial circulation in pulmonary artery occlusion and reperfusion

Obstruction of pulmonary arterial blood flow results in minimal biochemical and/or morphological changes in the involved lung. If the lung is reperfused, a syndrome of leukopenia and lung edema occurs. We used the radiolabeled microsphere technique to measure the response of the bronchial circulation in rabbits to acute pulmonary artery occlusion (PAO) and to pulmonary artery reperfusion. We found that the bronchial blood flow (Qbr) decreased from a base line of 0.37 +/- 0.10 to 0.09 +/- 0.04 (SE) ml.min-1.g dry lung-1 (P less than or equal to 0.05) after 4 h of PAO. In a separate group of animals, Qbr 24 h after PAO remained low (0.20 +/- 0.07 ml.min-1.g dry lung-1, P = 0.06). Qbr during PAO was inversely correlated with the wet-to-dry ratio after reperfusion (r = -0.68, P = 0.06). Qbr did not change during 4 h of reperfusion. We speculate that a critical level of Qbr may be necessary during PAO to prevent ischemia/reperfusion injury from occurring.     

Effect of drug treatment on liver-slice function following 72-hour hypothermic perfusion

The viability of hypothermically perfused dog liver was evaluated with a tissue-slice technique. After being preserved for 72 hr, slices of liver were incubated at 30 degrees C for as long as 2 hr; then water content, K+/Na+ ratio, and ATP concentration were measured. Dog livers were assigned to the following experimental groups: Group 1 (no preservation; control); Group 2 (livers preserved for 72 hr); Group 3 (donor animals pretreated with 3.5 mg/kg of chlorpromazine (CPZ) and 20 mg/kg of methylprednisolone (MP), and livers preserved for 72 hr); Group 4 (livers pretreated with 2-deoxycoformycin (2-DOC), 50 mg/liter, and preserved for 72 hr); and Group 5 (combination of Group 3 and Group 4 treatments). Livers in Groups 2, 3, and 4 lost K+ during preservation, and the mean K+/Na+ ratio significantly decreased from a control value of 4.2 +/- 0.4 to 1.5-1.9 (P less than 0.05). Group 5 livers did not lose K+; mean K+/Na+ ratio was 3.9 +/- 0.5. Fresh livers (no preservation) rapidly reaccumulated K+ when the tissue slices were incubated for 2 hr at 30 degrees C; mean K+/Na+ ratio was 3.7 +/- 0.5. Tissue slices from Group 2 livers (72 hr preservation), and livers pretreated with CPZ-MP (Group 3) or pretreated with 2-DOC (Group 4) did not significantly reaccumulate K+ at 30 degrees C; mean K+/Na+ ratio was 1.7-2.1. Only slices prepared from liver pretreated with both CPZ-MP and 2-DOC reaccumulated K+; mean K+/Na+ ratio was 4.6 +/- 1.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galactosyl-sucrose metabolism and UDP-galactose pyrophosphorylase from Cucumis melo L. fruit

In muskmelon ( Cucumis melo L.), sink tissues receive stachyose, raffinose and sucrose through phloem translocation of carbohydrates that are formed as products of leaf photosynthesis. Melon fruits accumulate sucrose massively during the final stages of maturation. This sucrose is derived partially from the catabolism of raffinose saccharides. Rapid galactose metabolism is required, because liberation of free galactose is the first step in the metabolic utilization of the raffinose sugars. The current study demonstrates that the enzyme UDP-glucose-hexose-1-P uridylyltransferase (EC 2.7.7.12), the central enzyme in the classical Lelior pathway, is not the central enzyme in galactose metabolism in muskmelon fruit. Rather, a broad substrate specificity UDP-galactose pyrophosphorylase (PPase) serves the same functional role. This enzyme accepts either UDP-galactose or UDP-glucose as a substrate and is different from a UDP-glucose PPase with more strict substrate specificity for UDP-glucose that is also present in melon tissue. UDP-galactose PPase was purified 113-fold from melon tissue and was shown to be a 54 kDa (size exclusion chromatography) to 68 kDa (SDS-PAGE) protein that is enzymatically active as a monomer. We also present evidence that the enzyme likely accepts UDP-galactose and UDP-glucose at the same catalytic site. Polyclonal antibodies prepared against this protein reacted with numerous other antigens in melon extracts, apparently as a result of the presence of common antigenic epitopes.     

The response of liver to lactobionate/raffinose (University of Wisconsin--UW) solution during hypothermic preservation: a study using 31phosphorus nuclear magnetic resonance

31P nuclear magnetic resonance spectroscopy has been used to study rat livers following flushing with the University of Wisconsin (UW) lactobionate/raffinose solution (N. Jamieson, R. Sundberg, S. Lindell, J. Southard, and F.O. Belzer, Cryobiology 24, 573-574, 1987; M. Kalayoglu, H. Sollinger, R. Stratta, A. D'Alessandro, R. Hoffman, J. Pirsch, and F. O. Belzer, Lancet 1, 617-619, 1988). These studies have revealed that despite the improved storage properties that have been reported for this solution, hepatic ATP and ADP declined at a rate similar to that seen in the more commonly used Marshall's or Collins' solutions. However, there was a significant inhibition of the developing acidosis, such that by 5 hr postflush, the intracellular pH was 7.17 +/- 0.06 (mean +/- SD, n = 5) compared to 6.90 +/- 0.06 for Marshall's solution (4 hr postflush) and 6.94 +/- 0.04 for Collins' solution (4 hr postflush). This did not appear to be due to a buffering effect of the solution, as this was found to be relatively low, but was probably due to a modification of hepatic metabolism caused by the solution itself.     

Comparison of the effect of 3- and 5-day hypothermic perfusion of dog kidneys on metabolism of tissue slices

Hypothermic perfusion effectively preserves the viability of kidneys for 3 days. Long-term preservation (5 days or greater) has not been consistently obtained. In this study, the differences between kidneys perfused for 3 and 5 days were compared by determining the "integrated-metabolic" capabilities of tissue slices incubated in vitro at 30 degrees C. The "integrated-metabolic" parameters determined include (1) respiration rates, (2) cell volume regulation [total tissue water (TTW) and saccharide permeable space], (3) rate of reaccumulation of K+ and pumping of Na+, (4) maintenance of ATP concentrations, and (5) mitochondrial functions. Conditions that result in high and low concentrations of ATP following perfusion of kidneys for 5 days were also compared for effects on tissue slice metabolism. The results indicate that energy metabolism in tissue slices is well preserved under all conditions and times of perfusion of kidneys. This includes average respiration rates (315 +/- 50, 275 +/- 35, and 255 +/- 45 mumol O2/hr/g dry wt at 0, 3, and 5 days, respectively, mitochondrial function [respiratory control ratio (RCR) = 4.6, 4.0, and 4.1 for 0, 3, and 5 days, respectively], and steady-state concentration of ATP in slices after incubation (4.0 +/- 1.45, 3.9 +/- 1.28, and 3.3 +/- 0.81 mumol/g/dry wt, for 0, 3, and 5 days, respectively). The primary differences between 3- and 5-day perfused kidneys were the capability of the slices to regulate cell volume and reaccumulate K+. Slices from kidneys perfused for 3 days maintained the TTW at 3.8 kg/kg dry wt, a value similar to that of control tissue slices. However, slices from 5-day perfused kidneys remained swollen (TTW = 4.6 kg/kg dry wt). Also, slices from the 5-day perfused kidney pumped K+ at less than one-half the rate found in slices from control or 3-day preserved kidneys. No significant differences were apparent in the permeability properties of the tissue slices from kidneys perfused for 3 and 5 days to radiolabeled saccharides. The defects in membrane-linked transport functions, resulting from long-term kidney perfusion, were reduced in kidneys containing a high concentration of ATP. The results suggest that one factor which may limit successful preservation of kidneys is the increased membrane permeability (to electrolytes) which is partially prevented by maintaining elevated concentrations of tissue ATP during perfusion.     

Pancreas preservation with TP-IV: a hyperosmolar colloid solution

This study compares the efficacy of a new hyperosmolar colloid solution (TP-IV) with Euro-Collins solution for long-term (72 hr) hypothermic storage of canine pancreas autografts. Four experimental recipient groups and their survival (30-day study period) results were as follows: Gr. I (n = 6) pancreatectomized controls, without autotransplant (X +/- SD = 5.83 +/- 3.06 days); Gr. II (n = 6) fresh nonpreserved autografts (X +/- SD = 23.83 +/- 10.12 days, 5 of 6 greater than 30 days); and Gr. III (n = 7) and Gr. IV (n = 5) receiving pancreas autografts stored at 4 degrees C for 72 hr in either Euro-Collins or TP-IV, respectively (Gr. III, 13.85 +/- 9.04 days; Gr. IV, 21.2 +/- 12.37 days). The results appear to indicate that TP-IV is superior to Euro-Collins solution for 72-hr hypothermic storage of pancreas grafts. In fact, survival in the TP-IV-presented group was comparable to that of fresh, non-preserved autografts.     

Protective effect of a low K+ cardioplegic solution on myocardial Na,K-ATPase activity.

Long duration ischemia in hypothermic conditions followed by reperfusion alters membrane transport function and in particular Na,K-ATPase. We compared the protective effect of two well-described cardioplegic solutions on cardiac Na,K-ATPase activity during reperfusion after hypothermic ischemia. Isolated perfused rat hearts (n = 10) were arrested with CRMBM or UW cardioplegic solutions and submitted to 12 hr of ischemia at 4 degrees C in the same solution followed by 60 min of reperfusion. Functional recovery and Na,K-ATPase activity were measured at the end of reperfusion and compared with control hearts and hearts submitted to severe ischemia (30 min at 37 degrees C) followed by reflow. Na,K-ATPase activity was not altered after 12 hr of ischemia and 1 hr reflow when the CRMBM solution was used for preservation (55 +/- 2 micromolPi/mg prot/hr) compared to control (53 +/- 2 micromol Pi/mg prot/hr) while it was significantly altered with UW solution (44 +/- 2 micromol Pi/mg prot/hr, p < 0.05 vs control and CRMBM). Better preservation of Na,K-ATPase activity with the CRMBM solution was associated with higher functional recovery compared to UW as represented by the recovery of RPP, 52 +/- 12% vs 8 +/- 5%, p < 0.05 and coronary flow (70 +/- 2% vs 50 +/- 8%, p < 0.05). The enhanced protection provided by CRMBM compared to UW may be related to its lower K+ content.     

A comparison of cold storage solutions for hepatic preservation using the isolated perfused rabbit liver

Rabbit livers were stored cold for periods of 6 or 24 hr and tested using the isolated perfused liver model. Five solutions were tested: Eurocollins (EC), Ross and Marshall's hypertonic citrate (HC), modified plasma protein fraction (Cambridge PPF), Ringer lactate, and the recently developed "University of Wisconsin" (UW) solution. After storage livers were perfused with an erythrocyte-free oxygenated Krebs-Henseleit solution containing 4% bovine serum albumin at 38 degrees C for 2 hr. Bile production proved to be the most sensitive index of liver function for discriminating between the various storage solutions and the different preservation times. After 6 hr of cold storage, bile production was similar to control liver bile production (9.8 +/- 2.4 ml/2 hr/100 g) in livers stored in HC (8.8 +/- 2 ml), PPF (9.9 +/- 2.2 ml), and UW (10.3 +/- 1.9 ml); it was slightly depressed in EC (6.7 +/- 2.5 ml, P = 0.06), and markedly depressed in Ringer lactate (4.3 +/- 0.8 ml, P less than 0.05). After 24 hr of cold storage bile production in UW-stored livers was near normal (9.3 +/- 0.7 ml) but significantly depressed (3.5-6.2 ml) in all other solutions tested. Release of enzymes into the normothermic perfusate was also measured (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). In this small series the differences between cold storage solutions did not always reach statistical significance although the trend was for less enzyme release in livers stored in UW solution. This technique permits rapid assessment and refinement of new storage methods and new solutions for liver preservation prior to testing in a large animal transplant model. The results suggest that UW solution is superior to other preservation solutions and would permit successful 24-hr storage of livers.     

Microvascular changes of the liver preserved in UW solution. Pathological and immunohistochemical examination.

Rat livers preserved in University of Wisconsin (UW) solution for 24 h were compared with those preserved in Euro-Collins (EC) solution before and after liver transplantation using an immunohistochemical method. Tissue ATP and total tissue adenine nucleotide (TAN) were measured using HPLC. The levels of TAN in the UW group or the EC group were significantly low compared with the control group (no preservation) after 24-h storage. In the EC group, the levels of tissue adenine nucleotides (TAN) decreased 1 h after reperfusion and never reached control levels. In the UW group, the levels of TAN increased a little 1 h after reperfusion and increased more 3 h after reperfusion. After 24-h preservation, the expression of factor VIII-related antigen (FRA) in endothelial cells of central veins was weak in the EC group; in the UW group, FRA was clearly detected in these cells. After reperfusion, although severe endothelial cell damage to the central veins and numerous FRA-positive substances were observed in EC group, endothelial cells of central veins retained their normal structure and FRA-positive substances were rarely noted in the UW group. In both groups, no endothelial changes were detected in portal veins. From these results, it is concluded that UW solution prevents endothelial cell damage and microcirculatory injury in zone III during the preservation period resulting in prevention of initial graft nonfunction. Also, measurement of the TAN level after reperfusion is useful to predict the function of the graft.     

Local hypothermia during early reperfusion protects skeletal muscle from ischemia-reperfusion injury

Amputated tissue maintained in a hypothermic environment can endure prolonged ischemia and improve replantation success. The authors hypothesized that local tissue hypothermia during the early reperfusion period may provide a protective effect against ischemia-reperfusion injury similar to that seen when hypothermia is provided during the ischemic period. A rat gracilis muscle flap model was used to assess the protective effects of exposing skeletal muscle to local hypothermia during ischemia only (p = 18), reperfusion only (p = 18), and both ischemia and reperfusion (p = 18). Gracilis muscles were isolated and exposed to hypothermia of 10 degrees C during 4 hours of ischemia, the initial 3 hours of reperfusion, or both periods. Ischemia-reperfusion outcome measures used to evaluate muscle flap injury included muscle viability (percent nitroblue tetrazolium staining), local edema (wet-to-dry weight ratio), neutrophil infiltration (intramuscular neutrophil density per high-power field), neutrophil integrin expression (CD11b mean fluorescence intensity), and neutrophil oxidative potential (dihydro-rhodamine oxidation mean fluorescence intensity) after 24 hours of reperfusion. Nitroblue tetrazolium staining demonstrated improved muscle viability in the experimental groups (ischemia-only: 78.8 +/- 3.5 percent, p < 0.001; reperfusion-only: 80.2 +/- 5.2 percent, p < 0.001; and ischemia-reperfusion: 79.6 +/- 7.6 percent, p < 0.001) when compared with the nonhypothermic control group (50.7 +/- 9.3 percent). The experimental groups demonstrated decreased local muscle edema (4.09 +/- 0.30, 4.10 +/- 0.19, and 4.04 +/- 0.31 wet-to-dry weight ratios, respectively) when compared with the nonhypothermic control group (5.24 +/- 0.31 wet-to-dry weight ratio; p < 0.001, p < 0.001, and p < 0.001, respectively). CD11b expression was significantly decreased in the reperfusion-only (32.65 +/- 8.75 mean fluorescence intensity, p < 0.001) and ischemia-reperfusion groups (25.26 +/- 5.32, p < 0.001) compared with the nonhypothermic control group (62.69 +/- 16.93). There was not a significant decrease in neutrophil CD11b expression in the ischemia-only group (50.72 +/- 11.7 mean fluorescence intensity, p = 0.281). Neutrophil infiltration was significantly decreased in the reperfusion-only (20 +/- 11 counts per high-power field, p = 0.025) and ischemia-reperfusion groups (23 +/- 3 counts, p = 0.041) compared with the nonhypothermic control group (51 +/- 28 counts). No decrease in neutrophil density was observed in the ischemia-only group (40 +/- 15 counts per high-power field, p = 0.672) when compared with the nonhypothermic control group (51 +/- 28 counts). Finally, dihydrorhodamine oxidation was significantly decreased in the reperfusion-only group (45.83 +/- 11.89 mean fluorescence intensity, p = 0.021) and ischemia-reperfusion group (44.30 +/- 11.80, p = 0.018) when compared with the nonhypothermic control group (71.74 +/- 20.83), whereas no decrease in dihydrorhodamine oxidation was observed in the ischemia-only group (65.93 +/- 10.3, p = 0.982). The findings suggest a protective effect of local hypothermia during early reperfusion to skeletal muscle after an ischemic insult. Inhibition of CD11b expression and subsequent neutrophil infiltration and depression of neutrophil oxidative potential may represent independent protective mechanisms isolated to local tissue hypothermia during the early reperfusion period (reperfusion-only and ischemia-reperfusion groups). This study provides evidence for the potential clinical utility of administering local hypothermia to ischemic muscle tissue during the early reperfusion period.     

Carbohydrate Changes during Maturation of Cucumber Fruit : Implications for Sugar Metabolism and Transport

Changes in the carbohydrate profiles in the mesocarp, endocarp, and seeds of maturing cucumber (Cucumis sativus, L.) fruit were analyzed. Fruit maturity was measured by a decrease in endocarp pH, which was found to correlate with a loss in peel chlorophyll and an increase in citric acid content. Concentrations of glucose and fructose (8.6-10.3 milligrams per gram fresh weight, respectively) were found to be higher than the concentration of sucrose (0.3 milligrams per gram fresh weight) in both mesocarp and endocarp tissue. Neither raffinose nor stachyose were found in these tissues. The levels of glucose and fructose in seeds decreased during development, but sucrose, raffinose, and stachyose accumulated during the late stages of maturation. Both raffinose and stachyose were found in the seeds of six lines of Cucumis sativus L. This accumulation of raffinose saccharides coincided with an increase in galactinol synthase activity in the seeds. Funiculi from maturing fruit were found to be high in sucrose concentration (4.8 milligrams per gram fresh weight) but devoid of both raffinose and stachyose. The results indicated that sucrose is the transport sugar from the peduncle to seed, and that raffinose saccharide accumulation in the seed is the result of in situ biosynthesis and not from direct vascular transport of these oligosaccharides into the seeds.     

Sugar metabolism in developing lupin seeds is affected by a short-term water deficit

A short-term water deficit (WD) imposed during the pre-storage phase of lupin seed development [15-22 d after anthesis (DAA)] accelerated seed maturation and led to smaller and lighter seeds. During seed development, neutral invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) have a central role in carbohydrate metabolism. Neutral invertase is predominant during early seed development (up to 40 DAA) and sucrose synthase during the growing and storage phase (40-70 DAA). The contribution of acid invertase is marginal. WD decreased sucrose synthase activity by 2-fold and neutral invertase activity by 5-6-fold. These changes were linked to a large decrease in sucrose ( approximately 60%) and an increase of the hexose:sucrose ratio. Rewatering restored sucrose synthase activity to control levels while neutral invertase activity remained depressed (30-60%). A transient accumulation of starch observed in control seeds was abolished by WD. Despite the several metabolic changes the final seed composition was largely unaltered by WD except for approximately 60% increase in stachyose and raffinose (raffinose family oligosaccharides). This increase in raffinose family oligosaccharides appears as the WD imprinting on mature seeds.     

Effects of short-term hypothermic perfusion and cold storage on function of the isolated-perfused dog kidney

The isolated-perfused dog kidney was used as a model to measure the effects of short-term hypothermic preservation on renal function and metabolism. Kidneys were cold-stored in Collins' solution, hypotonic citrate, or phosphate-buffered sucrose for 4 and 24 hr, or were continuously perfused for 4 and 24 hr with a synthetic perfusate. Following preservation kidneys were perfused with an albumin-containing perfusate at 37 degrees C for 60 min for determination of renal function. The results indicate that many of the effects of short-term preservation on renal function in dog kidneys are similar to results reported for rat and rabbit kidneys. Cold storage for 4 hr resulted in a large decrease in GFR (57%), but only a small decrease in Na reabsorption (from 97 to 87%). Cold storage for 24 hr caused a further decline in renal function (GFR = 95% decrease, Na reabsorption = 49-64%). Results were similar for all cold storage solutions tested. Perfusion for 4 hr was less damaging to renal function than cold storage. The GFR decreased only 14% and urine formation and Na reabsorption were practically normal. After 24 hr of hypothermic perfusion, the GFR was reduced by 79%, urine flow was normal, and Na reabsorption was 78%. There were no obvious biochemical correlates (adenine nucleotides, tissue edema, or electrolyte concentration) with the loss of renal function during short-term preservation. The results suggest that the isolated-perfused dog kidney can be used to test the effects of preservation on renal function, and yields results similar to those obtained using small animal models.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy

Metabolism of [3H] arachidonic acid ([3H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 microCi of [3H] AA and with 200 micrograms radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [3H] AA to PGE2, PGF2 alpha, PGFM, 6-keto-PGF1 alpha, and to unidentified compounds in three chromatographic regions. One of these regions (region I) contained triglycerides. Endometrial slices metabolized only small amounts of the [3H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF2 alpha (32.1 +/- 17.9), PGFM (8.4 +/- 6.2), PGE2 (12.3 +/- 7.5) and 6-keto-PGF1 alpha (41.4 +/- 4.8), whereas day 16 conceptuses released more (P less than .05) PGF2 alpha (9.0 +/- 4.1) and 6-keto-PGF1 alpha (15.9 +/- 2.7) than PGE2 (0.9 +/- 0.2) or PGFM (0.5 +/- 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P less than .05) PGFM (3.0 +/- 0.2) and 6-keto-PGF1 alpha (4.0 +/- 0.4) than PGF2 alpha (0.5 +/- 0.08) or PGE2 (0.05 +/- 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [3H] AA to determine the effects of indomethacin on [3H] AA metabolism. In general, indomethacin (Id; 4 X 10(-4) M) reduced (P less than .05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [3H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [3H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embryonal survival and development, were synthesized in vitro by ovine conceptuses.     

SEA0400, a novel Na+/Ca2+ exchanger inhibitor, reduces calcium overload induced by ischemia and reperfusion in mouse ventricular myocytes

Given the potential clinical benefit of inhibiting Na+/Ca2+ exchanger (NCX) activity during myocardial ischemia reperfusion (I/R), pharmacological approaches have been pursued to both inhibit and clarify the importance of this exchanger. SEA0400 was reported to have a potent NCX selectivity. Thus, we examined the effect of SEA0400 on NCX currents and I/R induced intracellular Ca2+ overload in mouse ventricular myocytes using patch clamp techniques and fluorescence measurements. Ischemia significantly inhibited inward and outward NCX current (from -0.04+/-0.01 nA to 0 nA at -100 mV; from 0.23+/-0.08 nA to 0.11+/-0.03 nA at +50 mV, n=7), Subsequent reperfusion not only restored the current rapidly but enhanced the current amplitude obviously, especially the outward currents (from 0.23+/-0.08 nA to 0.49+/-0.12 nA at +50 mV, n=7). [Ca2+]i, expressed as the ratio of Fura-2 fluorescence intensity, increased to 138+/-7% (P<0.01) during ischemia and to 210+/-11% (P<0.01) after reperfusion. The change of NCX current and the increase of [Ca2+]i during I/R can be blocked by SEA0400 in a dose-dependent manner with an EC50 value of 31 nM and 28 nM for the inward and outward NCX current, respectively. The results suggested that SEA0400 is a potent NCX inhibitor, which can protect mouse cardiac myocytes from Ca2+ overload during I/R injuries.     

Xanthine oxidase and activated neutrophils cause oxidative damage to skeletal muscle after contractile claudication

We previously showed oxidative damage and edema within skeletal muscle after contractile claudication. To investigate the sources of this oxidative damage in the gastrocnemius muscle, we administered allopurinol (Allo, to inhibit xanthine oxidase) and cyclophosphamide (Cyclo, to deplete neutrophils) before inducing contractile claudication in male Sprague Dawley rats. Contractile claudication (ligated stimulated, LS) caused a significant increase in xanthine oxidase activity [sham ligated stimulated (SS) = 2.57 +/- 0.07; LS = 3.22 +/- 0.07] and neutrophil infiltration (SS = 0.47 +/- 0.03; LS = 0.91 +/- 0.10) compared with controls (SS), and this was associated with increased lipid peroxidation, protein oxidation, muscle damage, and edema. Pretreatment with Allo attenuated the increase in xanthine oxidase activity and attenuated lipid hydroperoxides (control LS = 12.85 +/- 0.50; Allo LS = 9.96 +/- 0.71), muscle damage, and neutrophil infiltration (control LS = 0.91 +/- 0.10; Allo LS = 0.61 +/- 0.07). This latter finding suggests that xanthine oxidase-derived oxidants are chemotactic to neutrophils. Pretreatment with Cyclo reduced neutrophil infiltration (control LS = 0.91 +/- 0.10; Cyclo LS = 0.55 +/- 0.02) and attenuated lipid peroxidation (control LS = 12.85 +/- 0.50; Cyclo LS = 6.462 +/- 0.62), protein oxidation (control LS = 2.59 +/- 0.47; Cyclo LS = 1.77 +/- 0.60), muscle damage, and edema. Together, these data indicate that contractile claudication causes an increase in xanthine oxidase activity and neutrophils in muscle and that inhibition of these oxidant sources protects against oxidative stress, muscle damage, and edema.