Ringer Bicarbonate Solution Research Articles

Storage of washed platelets in BRS-A platelet additive solutions based on two types of clinically available bicarbonated Ringer's solutions with different electrolyte concentrations

BackgroundIn Japan, no platelet (PLT) additive solutions (PASs) are officially approved for clinical use although blood centers often receive requests for washed PLTs to reduce adverse reactions. Recently, we developed a novel PAS called BRS-A based on clinically available bicarbonated Ringer's solution (BRS), Bicanate and acid–citrate–dextrose formula A (ACD-A), which has been shown to maintain the in vitro properties of PLTs in the condition of <5% residual plasma during 7-day storage. The aim of this study was to evaluate whether another clinically available BRS, Bicarbon with different electrolyte concentrations can be used as a PAS. Study design and methodsTwo types of BRS-As were prepared by adding 25 mL of ACD-A to 500 mL of Bicanate or Bicarbon BRSs. Bicanate-based BRS-A and Bicarbon-based BRS-A contain 0.9 or 0.5 mmol/L of magnesium chloride, 95.2 or 100.1 mmol/L of sodium chloride, 4.2 or 5.1 mmol/L of trisodium citrate, and 26.6 or 23.8 mmol/L of sodium bicarbonate, respectively; the other components were identical. Apheresis PLTs stored in these solutions with less than 5% plasma for 7-day storage were compared with regard to their in vitro properties. ResultsThe pH levels of all units were above 7 throughout storage. The mean PLT volume, hypotonic shock response, glucose consumption, lactate production, swirling, and CD62P and CD42b expression were similar during 7-day storage. The bicarbonate levels in Bicarbon-based BRS-A were lower than those in Bicanate-based BRS-A. ConclusionDifferences in concentrations of electrolytes such as magnesium, sodium, citrate, and bicarbonate salts in BRS-A do not affect the in vitro properties of PLTs during 7-day storage. These results indicate that the use of another type of BRS-A based on Bicarbon as a PAS is feasible. Thus, BRS-A can be used in hospitals that do not stock Bicanate but have Bicarbon.

Low molecular weight pentastarch is more effective than crystalloid solution in goal-directed fluid management in patients undergoing major gastrointestinal surgery

This prospective observational study compared the volume effect between hydroxyethyl starch (HES) and crystalloid solution and its context dependency in intraoperative goal-directed fluid management. With institutional review board (IRB) approval, 35 patients undergoing major gastrointestinal surgery were enrolled. Fluid challenge consisting of 250 ml of either bicarbonate Ringer solution (BRS) or low molecular weight pentastarch (HES 70/0.5) was given to maintain stroke volume index >35 ml/m2. The context of fluid challenge was classified as related to either epidural block (EB) or blood loss (BL) or as nonspecific. The primary end point was the interval between index fluid challenge and the next fluid challenge, and the secondary end point was the hemodynamic parameter at the end of fluid challenge. Differences in these parameters in each clinical context were compared between BRS and HES 70/0.5. A p value <0.05 was considered statistically significant. Eighty-eight, 77, and 127 fluid challenges were classified as related to EB and BL and as nonspecific, respectively. In the nonspecific condition, the median (range) interval after fluid challenge with HES 70/0.5 and BRS was 45 (11-162) min and 18 (8-44) min, respectively, and the difference was statistically significant. Also, mean arterial pressure and stroke volume index significantly increased, whereas stroke volume variation significantly decreased after fluid challenge with HES 70/0.5 compared with BRS. Such differences were not observed in the other situations. HES 70/0.5 exerted larger volume effects than did crystalloid under nonspecific conditions. However, similar volume effects were observed during volume loss and extensive sympathetic blockade.

重炭酸リンゲル液を用いた血小板製剤の洗浄と保存

血小板製剤（PC）輸血時の非溶血性副作用発生頻度を低下させるためには，血漿の大部分を洗浄保存液に置換した洗浄血小板（WPC）が有効である．現在，日本では承認された洗浄保存液が無いため，市販の酢酸リンゲル液，重炭酸ナトリウム，硫酸マグネシウムなどの混液が使用されている．近年，アルカリ化剤として重炭酸塩が用いられた重炭酸リンゲル液（BRS）が開発された．本研究では，BRSが洗浄保存液として使用できるかどうか検討した． BRS 500mlにACD-A液25mlを添加し，その混液を除菌フィルターで処理して洗浄保存液（BRS-A）を調製した．BRS-Aで調製したWPC（血漿残存率5％未満）を7日間保存し，その機能を非洗浄PC（血漿浮遊）と比較した． 7日間保存中，洗浄血小板のpH，低浸透圧性ショック回復率，グルコース消費割合，乳酸産生割合，スワーリング，CD62P・CD42b発現率は非洗浄PCと同等かそれ以上に優れていた． 国内で認可されている2つの輸液を混合することにより，血小板機能を良好に維持する洗浄保存液を調製できた．今回の結果から，BRS-AはPCの洗浄保存液として有用であることが示された．

Isolation and biochemical analysis of vesicles from taurohyodeoxycholic acid-infused isolated perfused rat livers

To isolate biliary lipid-carrying vesicles from isolated perfused rat livers after taurohyodeoxycholic acid (THDC) infusion. Biliary lipid vesicles have been implicated in hepatic disease and THDC was used since it increases biliary phospholipid secretion. Rat livers were isolated and perfused via the hepatic portal vein with THDC dissolved in Krebs Ringer Bicarbonate solution, pH 7.4, containing 1 mmol/L CaCl2, 5 mmol/L glucose, a physiological amino acid mixture, 1% bovine serum albumin and 20% (v/v) washed human erythrocytes at a rate of 2000 nmol/min for 2 h. The livers were then removed, homogenized and subjected to centrifugation, and the microsomal fraction was obtained and further centrifuged at 350000 g for 90 min to obtain subcellular fractions. These were analyzed for total phospholipid, cholesterol, protein and alkaline phosphodiesterase I (PDE). No significant changes were observed in the total phospholipid, cholesterol and protein contents of the gradient fractions obtained from the microsomal preparation. However, the majority of the gradient fractions (ρ= 1.05-1.07 g/mL and ρ = 1.95-1.23 g/mL) obtained from THDC-infused livers had significantly higher PDE activity compared to the control livers. The low density gradient fraction (ρ = 1.05-1.07 g/mL) which was envisaged to contain the putative vesicle population isolated from THDC-perfused livers had relatively small amounts of phospholipids and protein when compared to the relevant control fractions; however, they displayed an increase in cholesterol and PDE activity. The phospholipids were also isolated by thin layer chromatography and subjected to fractionation by high performance liquid chromatography; however, no differences were observed in the pattern of the fatty acid composition of the phospholipids isolated from THDC and control perfused livers. The density gradient fractions (ρ = 1.10-1.23 g/mL) displayed an increase in all the parameters measured from both control and THDC-infused livers. No significant changes in biliary lipids were observed in the fractions from THDC-infused livers; however, PDE activity was significantly increased compared to the control livers.

Comparative in vitro evaluation of apheresis platelets stored with 100% plasma versus bicarbonated Ringer's solution with less than 5% plasma

The major strategy for reducing the frequency of adverse reactions to platelet (PLT) transfusions is PLT washing with PLT additive solutions (PASs). In Japan, a mixture of medical infusion solutions such as acetate Ringer's solution, sodium bicarbonate, magnesium sulfate, and ACD-A is currently used as a PAS because none of the common types of PASs are officially permitted for clinical use. Recently, a bicarbonated Ringer's solution (BRS) was developed using bicarbonate as an alkaline agent. The aim of this study was to evaluate whether a BRS can effectively be utilized as a PAS for clinical use. The washing and storage solution was prepared by adding 25 mL ACD-A to 500 mL of BRS (BRS-A), consisting of 95.2 mmol/L NaCl, 3.8 mmol/L KCl, 0.9 mmol/L MgCl2 ,1.4 mmol/L CaCl2 , 26.6 mmol/L NaHCO3 , 5.8 mmol/L glucose, 4.2 mmol/L trisodium citrate, and 1.8 mmol/L citric acid. The in vitro properties of apheresis PLTs suspended in BRS-A with low concentration of plasma (<5%) were compared with those suspended in 100% plasma during 7-day storage. The in vitro properties of pH, hypotonic shock response, glucose consumption rate, lactate production rate, swirling, CD62P, and CD42b expression in PLTs suspended in BRS-A were comparable or superior to those suspended in 100% plasma during 7-day storage. BRS-A, prepared by mixing the only two solutions permitted for clinical use in Japan, has a positive capability to maintain PLT function. These results indicate that PLT washing and storage with BRS-A is feasible.

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.

Two cases of urgent use of bicarbonate Ringer solution as replacement filtrate solution of continuous hemodiafiltration at the Great East Japan Earthquake

Two cases of urgent use of bicarbonate Ringer solution as replacement filtrate solution of continuous hemodiafiltration at the Great East Japan Earthquake

Restricted feeding improves postischemic recovery of Langendorff-perfused rat hearts

The goal of the present study was to assess the effects of a restricted feeding schedule (RFS) on postischemic contractile recovery in relation to triacylglycerol (TAG), glycogen, and ATP content. Glucose-6-phosphate dehydrogenase (G6PDH) activity, reduced/oxidized glutathione ratio (GSH/GSSG), and thiobarbituric acid reactive substances (TBARS) levels were also determined. Isolated rat hearts entrained to daily RFS (2 h food access starting at 1200) or fed ad libitum (FED) for 3 weeks were Langendorff-perfused (25 min ischemia, 30 min reperfusion) with Krebs-Ringer bicarbonate solution (10mmol/L glucose). RFS improved the recovery of contractility and reduced creatine kinase (CK) release upon reperfusion. Further, at the end of reperfusion, RFS hearts exhibited increased G6PDH activity and repletion of tissue glycogen, TAG, and ATP that was not observed in the FED hearts. GSH/GSSG at the end of reperfusion fell to the same value in both nutritional states, and TBARS levels were higher in the RFS hearts. In conclusion, RFS improved postischemic functional recovery, which was accompanied by a reduction in CK release and a striking energy recovery. Although enhanced G6PDH activity was displayed, RFS was unable to reduce lipid peroxidation, supporting a clear dissociation between protection against mechanical dysfunction and CK release on the one hand and oxidative damage on the other.

Selective Translocation of the Bordetella pertussis Adenylate Cyclase Toxin across the Basolateral Membranes of Polarized Epithelial Cells

The catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) translocates directly across the plasma membrane of mammalian cells to induce toxicity by the production of cAMP. Here, we use electrophysiology to examine the translocation of toxin into polarized epithelial cells that model the mucosal surfaces of the host. We find that both polarized T84 cell monolayers and human airway epithelial cultures respond to nanomolar concentrations of ACT when applied to basolateral membranes, with little or no response to toxin applied apically. The induction of toxicity is rapid and fully explained by increases in intracellular cAMP, consistent with toxin translocation directly across the basolateral membrane. Intoxication of T84 cells occurs in the absence of CD11b/CD18 or evidence of another specific membrane receptor, and it is not dependent on post-translational acylation of the toxin or on host cell membrane potential, both previously reported to be required for toxin action. Thus, elements of the basolateral membrane render epithelial cells highly sensitive to the entry of ACT in the absence of a specific receptor for toxin binding.

Contribution of Thromboxane A2 in Rat Common Carotid Artery Response to Serotonin

Serotonin is a vasoactive substance that in different blood vessels mostly induces vasoconstriction. Considering the important role of common carotid artery in brain blood supply, the aims of this study were to investigate the effect of serotonin on isolated rat common carotid artery and also to examine participation of intact endothelium, cyclooxygenase products, Ca++ channels and 5-HT2 receptors in serotonin-evoked action. Endothelium was mechanically removed from some vascular rings. Circular artery segments were placed in organ baths containing Krebs–Ringer bicarbonate solution. Cumulative concentration-contraction curves for serotonin were obtained in rings previously equilibrated at basal tone. Serotonin produced concentration-dependent contraction, which was unaltered by endothelial denudation. Serotonin-induced effect was notably and comparably reduced by indomethacin (cyclooxygenase inhibitor) or OKY–046 (thromboxane A2-synthase inhibitor) on intact or denuded rings. Nifedipine (Ca++ channel blocker) or ketanserin (5-HT2 receptor antagonist) strongly reduced serotonin-evoked effect. Our results suggest that serotonin produced concentration-dependent and endothelium-independent contraction of carotid artery, which was initiated by activation of 5-HT2 receptors located on smooth muscle cells and mediated via L-type Ca++ channels. Thromboxane A2 from smooth muscle cells notably contributed to the overall contraction of carotid artery induced by serotonin.

Optimal Fluid Resuscitation: Timing and Composition of Intravenous Fluids

Recent data suggest that the timing of fluid resuscitation and the type of fluid used to treat hemorrhagic shock contribute to the inflammatory response as well as cell death. Rats were bled of 40% of their total blood volume and then resuscitated in either early or delayed fashion. Treatment was assigned randomly and consisted of lactated Ringer's solution, normal saline, bicarbonate Ringer's solution, hypertonic saline, or no resuscitation. The first four groups were subdivided into early and late resuscitation. After a 5-h observation period, lung and liver samples were evaluated for apoptosis, and blood was collected for measurements of the cytokines interleukin (IL)-6, IL-10, and IL-1beta. The rats that were not resuscitated had significantly more apoptosis in liver tissue. In the lung, bicarbonate Ringer's solution, when given early, was associated with significantly less apoptosis. Non-resuscitated rats had significantly higher IL-6 concentrations than all other groups. Animals receiving hypertonic saline early had significantly higher IL-6 concentrations than those given any other fluid. The concentration of IL-1beta was significantly higher in the non-resuscitated rats than in those receiving bicarbonate Ringer's, lactated Ringer's, or normal saline for early resuscitation. Interleukin-10 was elevated significantly in non-resuscitated rats. Cellular destruction and a pro-inflammatory response follow hemorrhagic shock. Early resuscitation with isotonic crystalloid fluids decreases these responses.

INVOLVEMENT OF MITOCHONDRIAL PERMEABILITY TRANSITION, GLUTATHIONE STATUS, PENTOSE PHOSPHATE PATHWAY AND OXIDATIVE DAMAGE IN THE PROTECTIVE EFFECT OF FASTING ON THE ISCHAEMIC‐REPERFUSED RAT HEART

1. Fasting, which increases the catabolism of fatty acids, gives functional protection to the ischaemic-reperfused heart. To obtain further knowledge of this cardioprotective effect, changes in mitochondrial permeability transition (MPT) were measured by the entrapment of 2-deoxy-[(3)H]-glucose (2-DG). We also assessed whether MPT is associated with changes in glutathione status, the activity of glucose-6-phosphate-dehydrogenase (G6PDH) and tissue oxidative damage, estimated by the measurement of Thiobarbituric acid-reactive substances (TBARS). 2. Spontaneously beating hearts of fed and 24 h fasted rats were Langendorff perfused with Krebs'-Ringer bicarbonate solution (10 mmol/L glucose) and exposed to 25 min global ischaemia, followed by 30 min reperfusion. 3. Ischaemia-reperfusion resulted in a fourfold increase in mitochondrial entrapment of 2-DG in the fed group. This response was 29% lower in the fasted group, but there were no concomitant changes in total retention of 2-DG in the heart. Fasting increased the activity of G6PDH by a factor of 1.4 and caused a 2.8-fold increase in the ratio of reduced glutathione to oxidized glutathione (GSH:GSSG) at the end of the pre-ischaemic period. Ischaemia-reperfusion did not affect G6PDH activity, but reduced the GSH:GSSG ratio in both the fed and fasted groups by 50%. Therefore, the GSH:GSSG ratio remained higher in the fasted group. Fasting also decreased cellular levels of TBARS by approximately 25%. Lipolysis of endogenous triacylglycerol was increased during the pre-ischaemic period in the fasted group. 4. These data suggest that the enhancement of fatty acid catabolism that occurs in fasting activates mechanisms that tend to reduce oxidative damage and limit MPT.

Comparison of corneal endothelial cell loss during phacoemulsification using continuous anterior chamber infusion versus those using ophthalmic viscosurgical device: Randomized controlled trial

AimWe conducted this study to evaluate and compare corneal endothelial cell loss between phacoemulsification with continuous anterior chamber infusion using anterior chamber maintainer (ACM) and phacoemulsification using ophthalmic viscosurgical device (OVD).Materials and MethodsThis was a prospective, randomized controlled trial. Fifty eyes of 47 patients of senile cataract undergoing phacoemulsification were included. Patients were randomly allocated into two groups of 25 eyes each. Cataract surgery was performed by phacoemulsification with anterior chamber (AC) continuous infusion with balanced salt solution (BSS) plus and ACM without OVD in Group A, and in Group B, phacoemulsification was performed using OVD with BSS plus. Corneal endothelial cell count and pachymetry were performed preoperatively and postoperatively on day 1, day 7, and day 30.ResultsThe mean increase in pachymetry was 4.86%, 2.94%, and 1.94%, (Group A) and 5.95%, 3.94%, and 0.51%, (Group B) on first, seventh, and 30th postoperative day respectively. The difference between the percentage increase in pachymetry between the two groups was not significant at day 1 (P = 0.441), day 7 (P = 0.298), and day 30 (P = 0.174) postoperatively. The density of endothelial cells decreased postoperatively (day 30) by 7.38% (Group A) and 7.47% (Group B) without any significant statistical difference (P = 0.983) between two groups.ConclusionUse of ACM for continuous AC infusion and omission of OVD during phacoemulsification did not cause significant difference in corneal swelling or endothelial cell loss in the immediate postoperative period up to one month.

The effect of glycosaminoglycans on rat gametes in vitro and the associated signal pathway

The effects of adding the extracellular glycosaminoglycans (GAGs), hyaluronic acid (HA) and chondroitin sulphate (CS) to rat in vitro fertilisation (IVF) media were assessed. Metaphase II (MII) oocytes were also incubated in GAG-supplemented modified rat 1-cell embryo culture medium (mR1ECM+BSA) for 3 days. Cytoplasmic fragmentation was significantly reduced in mR1ECM+BSA with HA (39.0-48.0%) compared with the control (82.0%). In IVF experiments, neither HA (8.0-30.8%) nor CS (9.7-42.5%) improved fertilisation rates compared with controls fertilised in M16 (47.2%) or enriched Krebs-Ringer bicarbonate solution (61.5%). RT-PCR and Western blot were used to probe for CD44 mRNA and protein in Sprague-Dawley gametes and cumulus cells. CD44 was identified in cumulus cells, suggesting a role for oocyte maturation and cumulus expansion. The CD44 protein was also present on caudal epididymal spermatozoa that were highly stimulated by CS in vitro implicating a role in fertilisation for CS and CD44.

Transcorneal permeation of ciprofloxacin and diclofenac from marketed eye drops

The purpose of this research was to evaluate the in vitro permeation characteristics of various marketed eye drops of ciprofloxacin (0.3% w/v aqueous solution) and diclofenac (0.1% w/v aqueous solution) through isolated goat cornea. Effect of these drugs on isolated goat eye lenses was also evaluated. Permeation studies were conducted by putting 1 ml of formulation on the cornea fixed between the donor and receptor compartments of an all glass modified Franz diffusion cell and monitoring ciprofloxacin and diclofenac concentration in the receptor (containing normal saline or bicarbonate ringer solution under continuous stirring at 37±2°) spectrophotometrically at their respective absorption maxima, after 120 min. Paired isolated goat lenses (i.e. of same animal) were used to evaluate the effect of these drugs at selected concentrations against oxidative stress (1 mM hydrogen peroxide solution). After 24 h of incubation at 37°, the lens treated with test solution (hydrogen peroxide+drug in bicarbonate ringer solution) was estimated for soluble protein content and compared with control (only hydrogen peroxide). Among marketed eye drops of ciprofloxacin, Joxin (Jawa Pharmaceuticals) showed maximum in vitro transcorneal permeation (0.558%) while I-Gesic (Centaur Pharmaceuticals) showed maximum % in vitro permeation or in vitro ocular availability among diclofenac eye drops after 120 min of permeation. The soluble protein content estimation studies revealed that these drugs at selected concentrations (permeated after 120 min.) had no deleterious effect on eye lenses rather possessed protective effect, since all formulation showed more soluble protein content when compared with control.

Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes

We studied the effects of mouse embryonic fibroblasts (MEF) and canine embryonic fibroblasts (CEF) on IVM, IVF and IVC of canine oocytes. Cumulus-oocyte complexes were harvested from ovaries by slicing, and in vitro maturation was evaluated in three different conditions: culture media only (control), co-culture with MEF, or co-culture with CEF. The oocytes were cultured for 48 or 72 h. Only oocytes larger than 100 μm in diameter with a homogeneous dark cytoplasm and two or more layers of cumulus cells were used. The culture medium was TCM 199 + 10% fetal bovine serum (FBS) with 100 IU/mL penicillin and 100 μg/mL streptomycin. After 48 h of IVM, the oocytes were fertilized in vitro with fresh canine spermatozoa that had been selected by a swim-up method, and the oocytes and spermatozoa were co-cultured in modified Krebs–Ringer bicarbonate solution (TYH) for up to 20 h in 5% CO 2 in air at 38.5 °C. After insemination, oocytes were transferred to three different conditions (the same as for IVM) and were cultured. After 48 or 72 h of maturation in vitro, the maturation rate of MII oocytes cultured in co-culture of MEF and CEF was higher than for oocytes cultured in control ( P < 0.05). Although the rate that reached the MII stage was not different in the 48 and 72 h cultures, the percentage of degenerated oocytes was greater at 72 h in all three treatment groups. The proportion of monospermic and polyspermic oocytes was not different among the three treatment groups. Cleavage rates were higher in the MEF and CEF treatment groups than in the control group ( P < 0.05). Co-culture with CEF developed the embryo up to the 16-cell stage, and with MEF up to morula stage. In conclusion, co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes.

Pharmacological study of BRS, a new bicarbonated Ringer's solution, in haemorrhagic shock dogs

Sodium bicarbonate is the most physiological alkalinizing agent. The effect of a new bicarbonated Ringer's solution (BRS) containing Mg2+, on metabolic acidosis and serum magnesium abnormality were evaluated and compared with those of acetated Ringer's (ARS), lactated Ringer's (LRS) and Ringer's (RS) solutions in an experimental haemorrhagic shock model with dogs. Animals were randomly divided into six groups (n = 6 in each group), a sham-operated group, an operated group without infusion, and 4 operated groups with infusion (BRS, ARS, LRS and RS groups). Each RS was intravenously administered at 60 mL kg(-1) h(-1) for 1.5 h. Arterial blood gases, plasma electrolytes and cardiovascular parameters were analysed. BRS significantly improved blood base excess values, which were decreased by blood-letting, faster and more markedly than did LRS and RS (BRS--6.3 +/- 0.5 mEq L(-1); LRS--9.2 +/- 1.1 mEq L(-1); RS--12.4 +/- 1.0 mEq L(-1) at the end of infusion). The alkalinizing effect of BRS tended to be better than that of ARS but not significantly so. The serum Mg2+ concentration was well-maintained by BRS as compared to other RS (BRS 1.5 +/- 0.0 mgdL(-1); ARS 1.2 +/- 0.0mgdL(-1); LRS 1.1 +/- 0.0mgdL(-1); RS 1.3 +/- 0.1 mgdL(-1), at the end of infusion). These results suggest that BRS is a suitable perioperative solution for metabolic acidosis and serum electrolyte balance among RS tested.

Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.

Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry

Hypoxic pulmonary vasoconstriction (HPV) requires influx of extracellular Ca2+ in pulmonary arterial smooth muscle cells (PASMCs). To determine whether capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCCs) contributes to this influx, we used fluorescent microscopy and the Ca2+-sensitive dye fura-2 to measure effects of 4% O2 on intracellular [Ca2+] ([Ca2+]i) and CCE in primary cultures of PASMCs from rat distal pulmonary arteries. In PASMCs perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels (VOCCs), hypoxia markedly enhanced both the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] and the rate at which extracellular Mn2+ quenched fura-2 fluorescence. These effects, as well as the increased [Ca2+]i caused by hypoxia in PASMCs perfused with normal salt solutions, were blocked by the SOCC antagonists SKF-96365, NiCl2, and LaCl3 at concentrations that inhibited CCE >80% but did not alter [Ca2+]i responses to 60 mM KCl. In contrast, the VOCC antagonist nifedipine inhibited [Ca2+]i responses to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca2+]i caused by hypoxia was completely reversed by perfusion with Ca2+-free KRBS. LaCl3 increased basal [Ca2+]i during normoxia, indicating effects other than inhibition of SOCCs. Our results suggest that acute hypoxia enhances CCE through SOCCs in distal PASMCs, leading to depolarization, secondary activation of VOCCs, and increased [Ca2+]i. SOCCs and CCE may play important roles in HPV.

Inhibition of bicarbonate reabsorption in the rat proximal tubule by activation of luminal P2Y1 receptors.

The present study used a stationary microperfusion technique to investigate in vivo the effect of P2Y1 receptor activation on bicarbonate reabsorption in the rat proximal tubule. Proximal tubules were perfused with a bicarbonate Ringer solution before flow was stopped by means of an oil block. The recovery of lumen pH from the initial value (pH 8.0) to stationary values (pH approximately 6.7) was recorded by a H+-sensitive microelectrode inserted downstream of the perfusion pipette and oil block. The stationary pH value and the t(1/2) of pH recovery were used to calculate bicarbonate reabsorption (JHCO3). Both EIPA and bafilomycin A1 caused significant reductions in proximal tubule JHCO3, consistent with the established contributions of Na/H exchange and H+-ATPase to proximal tubule HCO3 reabsorption. The nucleotides ADP and, to a lesser extent, ATP reduced JHCO3 but AMP and UTP were without effect. 2MeSADP, a highly selective agonist of the P2Y1 receptor, reduced JHCO3 in a dose-dependent manner. MRS-2179, a P2Y1 receptor-specific antagonist, abolished the effect of 2MeSADP, whereas theophylline, an antagonist of adenosine (P1) receptors, did not. The inhibitory action of 2MeSADP was blocked by inhibition of protein kinase C and reduced by inhibition of protein kinase A. The effects of EIPA and 2MeSADP were not additive. The data provide functional evidence for P2Y1 receptors in the apical membrane of the rat proximal tubule: receptor activation impairs acidification in this nephron segment.