Temporal profile of changes in brain tissue extracellular space and extracellular ion (Na(+), K(+)) concentrations after cerebral ischemia and the effects of mild cerebral hypothermia

Cerebral ischemic cellular swelling occurs primarily in astrocytes. This water influx into the intracellular space is believed to result from osmotic water movement after disruption of membrane ionic homeostasis. However, cellular swelling occurs earlier than expected after ischemia and new ionic and water channels have been discovered. This study examined the temporal profile of the water and ionic movement across the cell membrane after global ischemia by measuring the changes in extracellular space (ECS), extracellular K(+) and Na(+) ion concentrations ([K(+)](e) and [Na(+)](e)) using a high resolution tissue impedance probe and ion selective micropipettes in the rat cortex. The effect of mild cerebral hypothermia (31.5 +/- 2.6 degrees C brain temperature) on these parameters was also examined. The ECS started to decrease at 34 +/- 8 sec after global ischemia and reached half the maximum change at 61 +/- 17 sec. [K(+)](e) started to increase initially at 33 +/- 11 sec (phase 1) and then increased rapidly at 62 +/- 25 sec (phase 2), and [Na(+)](e) started to decrease at 88 +/- 27 sec after ischemia. With mild hypothermia, the ECS started to decrease at 75 +/- 35 sec after ischemia and reached half the maximum change at 123 +/- 44 sec, [K(+)](e) started to increase initially at 80 +/- 24 sec (phase 1) and then increased rapidly at 120 +/- 32 sec (phase 2), and [Na(+)](e) started to decrease at 172 +/- 70 sec. The present study shows that ischemic cellular swelling (decreased ECS) occurs concomitantly with the phase 1 increase of [K(+)](e) but precedes the disruption of ionic membrane homeostasis (phase 2). Mild hypothermia prolongs the onset of these phenomena but does not affect the magnitude of the changes in ECS and ion concentrations.     

Chronic neutral phosphate supplementation induces sustained, renal metabolic alkalosis.

The aim of the present study was to test whether intravenous neutral phosphate supplementation, recently shown in our laboratory to acutely stimulate proton secretion in the distal nephron, was able to induce a sustained metabolic alkalosis. Neutral Na and K phosphate supplementation for seven days, with equivalent reduction in chloride supply and unchanged intake of sodium and potassium, in ADX rats receiving fixed physiological doses of aldosterone and dexamethasone (group 1, N = 7), was responsible for a severe metabolic alkalosis (MA; delta [HCO3] 11 +/- 1.3 mM, and delta pH 0.11 +/- 0.06 unit). Metabolic alkalosis was at least in part of renal origin, since net acid excretion (NAE) transiently increased, principally due to an increment in titratable acid excretion rate. Balances were equilibrated for sodium and negative for chloride and potassium, which may have contributed to the severity of the MA. Chronic i.v. neutral Na phosphate, without change in potassium and chloride supply, in ADX rats receiving the same doses of steroids (group 2, N = 5), was responsible for a less severe MA (delta [HCO3] 7.5 +/- 0.9 mM, and delta pH 0.07 +/- 0.01 unit), also of renal origin. In this group, balances were positive for chloride and sodium and equilibrated for potassium. Finally, neutral Na and K phosphate supplementation with reduction in chloride supply in intact rats (group 3, N = 4) was also able to induce a MA (delta [HCO3] 5.5 +/- 1.8 mM, and delta pH 0.06 +/- 0.01 unit) of renal origin, with balances negative for chloride and equilibrated for potassium and sodium. In all groups, the generation and maintenance of MA probably resulted from stimulated proton secretion in the distal nephron, as suggested by the observed increase of PCO2 over HCO3 concentration ratio in the urine and a fall in urine pH despite augmented urinary buffer content throughout the phosphate infusion period. Glomerular filtration rate did not significantly vary in any group. In conclusion, chronic supplementation of neutral phosphate appears to stimulate per se proton secretion in the distal nephron, independently of sodium, chloride, and potassium balances, and adrenal steroid secretion. Thus neutral phosphate supplementation should be added to the previously known factors able to induce MA.     

Propofol Increases Contractility during α1a-Adrenoreceptor Activation in Adult Rat Cardiomyocytes

Background: The objective of this study was to identify the extent to which propofol alters intracellular free Ca2+ concentration ([Ca2+]i), myofilament Ca2+ sensitivity, and contraction of individual cardiomyocytes during activation of [alpha]1a adrenoreceptors and to determine the cellular mechanism of action.

Methods: Freshly isolated ventricular myocytes were obtained from adult rat hearts. Myocyte shortening and [Ca2+]i were simultaneously monitored in individual cardiomyocytes exposed to phenylephrine after treatment with chloroethylclonidine ([alpha]1b-adrenoreceptor antagonist) and BMY 7378 ([alpha]1d-adrenoreceptor antagonist). Data are reported as mean +/- SD.

Results: Phenylephrine increased myocyte shortening by 124 +/- 9% (P = 0.002), whereas peak [Ca2+]i only increased by 8 +/- 3% (P = 0.110). Inhibition of phospholipase A2 and phospholipase C attenuated the phenylephrine-induced increase in shortening by 84 +/- 11% (P = 0.004) and 15 +/- 6% (P = 0.010), respectively. Inhibition of protein kinase C (PKC) and Rho kinase attenuated the phenylephrine-induced increase in shortening by 17 +/- 8% (P = 0.010) and 74 +/- 13% (P = 0.006), respectively. In the presence of phenylephrine, propofol increased shortening by 40 +/- 6% (P = 0.002), with no concomitant increase in [Ca2+]i. PKC inhibition prevented the propofol-induced increase in shortening. Selective inhibition of PKC[alpha], PKC[delta], PKC[varepsilon], and PKC[zeta] reduced the propofol-induced increase in shortening by 12 +/- 5% (P = 0.011), 36 +/- 8% (P = 0.001), 32 +/- 9% (P = 0.007), and 19 +/- 5% (P = 0.008), respectively. Na+-H+ exchange inhibition reduced the propofol-induced increase in shortening by 56 +/- 7% (P = 0.001).     

Airway Occlusion Pressure to Titrate Positive End-expiratory Pressure in Patients with Dynamic Hyperinflation

Background: Although the use of external positive end-expiratory pressure (PEEP) is recommended for patients with intrinsic PEEP, no simple method exists for bedside titration. We hypothesized that the occlusion pressure, measured from airway pressure during the phase of ventilator triggering (P0.1t), could help to indicate the effects of PEEP on the work of breathing (WOB).

Methods: Twenty patients under assisted ventilation with chronic obstructive pulmonary disease were studied with 0, 5, and 10 cm H2O of PEEP while ventilated with a fixed level of pressure support.

Results: PEEP 5 significantly reduced intrinsic PEEP (mean +/- SD, 5.2 +/- 2.4 cm H2O at PEEP 0 to 3.6 +/- 1.9 at PEEP 5;P < 0.001), WOB per min (12.6 +/- 6.7 J/min to 9.1 +/- 5.9 J/min;P = 0.003), WOB per liter (1.2 +/- 0.4 J/l to 0.8 +/- 0.4 J/l;P < 0.001), pressure time product of the diaphragm (216 +/- 86 cm H2O [middle dot] s-1 [middle dot] min-1 to 155 +/- 179 cm H2O [middle dot] s-1 [middle dot] min-1;P = 0.001) and P0.1t (3.3 +/- 1.5 cm H2O to 2.3 +/- 1.4 cm H2O;P = 0.002). At PEEP 10, no further significant reduction in muscle effort nor in P0.1t (2.5 +/- 2.1 cm H2O) occurred, and transpulmonary pressure indicated an increase in end-expiratory lung volume. Significant correlations were found between WOB per min and P0.1t at the three levels of PEEP (P < 0.001), and between the changes in P0.1tversus the changes in WOB per min (P < 0.005), indicating that P0.1t and WOB changed in the same direction. A decrease in P0.1 with PEEP indicated a decrease in intrinsic PEEP with a specificity of 71% and a sensitivity of 88% and a decrease in WOB with a specificity of 86% and a sensitivity of 91%.     

A clinico-physiological comparison of ileal pouch-anal and straight ileoanal anastomoses

The ileal pouch-anal anastomosis improves clinical results after colectomy and mucosal proctectomy compared to the straight ileoanal anastomosis. The question was what physiologic changes brought about by the pouch led to the improvement. Among 124 patients who had had ileoanal anastomosis, 25 volunteered for a detailed clinicophysiologic evaluation. Fourteen had had the ileal pouch-anal operation a mean of 8 months previously, and 11 had the straight ileoanal operation a mean of 25 months previously. Both groups of patients had satisfactory anal sphincter resting pressures (mean +/- SEM, pouch = 68 +/- 8 cm H2O, straight = 65 +/- 9 cm H2O, p greater than 0.05) and neorectal capacities (pouch = 278 +/- 26 ml, straight = 233 +/- 36 ml, p less than 0.05), and all could evacuate spontaneously. However, the pouch patients had a more distensible neorectum (delta V/delta P pouch = 9.5 +/- 1.3 ml/cm H2O, straight = 4.9 +/- 0.9 ml/cm H2O, p less than 0.05) and smaller amplitude neorectal contractions (pouch = 36 +/- 5 cm H2O, straight = 90 +/- 13 cm H2O; p less than 0.05). We concluded that the pouch-anal anastomosis increased the distensibility of the neorectum and decreased its propulsive drive, and so improved clinical results.     

Interrelationships of bladder compliance with age, detrusor instability, and obstruction in elderly men with lower urinary tract symptoms

Data on the interrelationships of bladder compliance (BC), detrusor instability (DI), and bladder outflow obstruction (BOO) in elderly men with lower urinary tract symptoms (LUTS) are scarce and were therefore assessed in this study. Principle inclusion criteria for this study were men aged > or = 50 years suffering from LUTS as defined by an International Prostate Symptoms Score (IPSS) of > or = 7 and a peak flow rate (Qmax) of < or = 15 ml/sec. Patients with previous surgery of the bladder, prostate, or urethra as well as a pathological neurourological status were excluded from this study. The following parameters were studied in all patients: IPSS, prostate volume calculated by transrectal ultrasonography, free uroflow study, post-void residual volume determined by transurethral catheterization, and a multichannel pressure flow study (pQS). A group of 170 men were included in the analysis. The mean BC in the overall group was 32 +/- 2 ml/cm H2O (mean +/- standard error of the mean [SEM]; range, 4-100 ml/cm H2O). In 36.5% of patients, BC was significantly reduced (< or = 20 ml/cm H2O), and in a further 37.1%, it ranged from 20 to 40 ml/cm H2O. BC decreased statistically significantly (p < 0.05) in patients with advanced age, lower Qmax, higher voiding pressures, and larger prostates. In men with DI (n = 61), mean BC was significantly lower (22 +/- 3 ml/cm H2O) compared to those without (37 +/- 3 ml/cm H2O; p = 0.001; n = 109). Patients with severe BOO as defined by a linear passive urethral resistance relationship of > or = 3 (n = 109), had a significantly lower BC (23 +/- 2 ml/cm H2O) compared to those without or minimal obstruction only (39 +/- 3 ml/cm H2O; p = 0.0002; n = 61). Stepwise logistic regression analysis revealed that DI, a low bladder capacity, and a high maximum detrusor pressure were independent predictors of markedly reduced BC (< 20 ml/cm H2O). BC is decreased in elderly men with high voiding pressures, BOO, and DI. The mechanism leading to the reduction of BC under these circumstances is largely unknown and could result from cytostructural alterations of the detrusor and changes in detrusor innervation.     

Signal Transduction of Opioid-induced Cardioprotection in Ischemia-Reperfusion

Background: Morphine reduces myocardial ischemia-reperfusion injury in vivo and in vitro. The authors tried to determine the role of opioid [delta]1 receptors, oxygen radicals, and adenosine triphosphate-sensitive potassium (KATP) channels in mediating this effect.

Methods: Chick cardiomyocytes were studied in a flow-through chamber while pH, flow rate, oxygen, and carbon dioxide tension were controlled. Cell viability was quantified by nuclear stain propidium iodide, and oxygen radicals were quantified using molecular probe 2',7'-dichlorofluorescin diacetate.

Results: Morphine (1 [mu]m) or the selective [delta]-opioid receptor agonist BW373U86 (10 pm) given for 10 min before 1 h of ischemia and 3 h of reoxygenation reduced cell death (31 +/- 5%, n = 6, and 28 +/- 5%, n = 6 [P < 0.05], respectively, 53 +/- 6%, n = 6, in controls) and generated oxygen radicals before ischemia (724 +/- 53, n = 8, and 742 +/- 75, n = 8 [P < 0.05], respectively, vs. 384 +/- 42, n = 6, in controls, arbitrary units). The protection of morphine was abolished by naloxone, or the selective [delta]1-opioid receptor antagonist 7-benzylidenenaltrexone. Reduction in cell death and increase in oxygen radicals with BW373U86 were blocked by the selective mitochondrial KATP channel antagonist 5-hydroxydecanoate or diethyldithiocarbamic acid (1000 [mu]m), which inhibited conversion of O2- to H2O2. The increase in oxygen radicals was abolished by the mitochondrial electron transport inhibitor myxothiazol. Reduction in cell death was associated with attenuated oxidant stress at reperfusion.     

Long-term myocardial preservation: beneficial and additive effects of polarized arrest (Na+-channel blockade), Na+/H+-exchange inhibition, and Na+/K+/2Cl- -cotransport inhibition combined with calcium desensitization

BACKGROUND: Polarized arrest, induced by tetrodotoxin (TTX) at an optimal concentration of 22 micromol/L, has been shown to reduce ionic imbalance and improve myocardial preservation compared with hyperkalemic (depolarized) arrest. Additional pharmacologic manipulation of ionic changes (involving inhibition of Na+ influx by the Na+/H+ exchanger [HOE694] and Na+/K+/2Cl- cotransporter [furosemide], and calcium desensitization [BDM]) may further improve long-term preservation. In this study, we (i) established optimal concentrations of each drug, (ii) determined additive effects of optimal concentrations of each drug and (iii) compared our optimal preservation solution to an established depolarizing cardioplegia (St Thomas' Hospital solution No 2: STH2) used during long-term hypothermic storage for clinical transplantation. METHODS: The isolated working rat heart, perfused with Krebs Henseleit (KH) buffer was used; cardiac function was measured after 20 min aerobic working mode perfusion. The hearts (n=6/group) were arrested with a 2 ml infusion (for 30 sec) of the polarizing (control) solution (22 micromol/L TTX in KH) or control+drug and subjected to 5 hr or 8 hr of storage at 7.5 degrees C in the arresting solution. Postischemic function during reperfusion was measured (expressed as percentage of preischemic function). RESULTS: Dose-response studies established optimal concentrations of HOE694 (10 micromol/L), furosemide (1.0 micromol/L) and BDM (30 mmol/L) in the polarizing (control) solution. Sequential addition to the control solution (Group I) of optimal concentrations of HOE694 (Group II), furosemide (Group III), and BDM (Group IV) were compared with STH2 (Group V); postischemic recovery of aortic flow was 29+/-7%, 49+/-6%*, 56+/-2%*, 76+/-3%*, and 25+/-6%, respectively (*P<0.05 vs. I and V). Creatine kinase leakage was lowest, and myocardial ATP content was highest in Group IV. CONCLUSIONS: A polarizing preservation solution (KH+TTX) containing HOE694, furosemide, and BDM significantly enhanced long-term preservation compared with an optimized depolarizing solution (STH2) used clinically for long-term donor heart preservation.     

Functional integrity of proximal tubule cells. Effects of hypoxia and ischemia.

Effects of warm hypoxia and ischemia on electrophysiologic properties of isolated perfused mouse proximal straight tubules were studied. Oxyrase (5 to 10 microliters/mL) was added to the hypoxic and ischemic solutions to lower the oxygen tension to 5 mm Hg. The ischemic solution also simulated acidosis, K+ and lactate accumulation, and substrate deprivation. Twenty-minute tubular perfusion with the hypoxic and ischemic solutions (lumen and bath) at 37 degrees C did not significantly alter basolateral membrane potential, basolateral K+ transference number, or intracellular Na+ activity from control values of -69 +/- 1 mV (N = 91), 0.71 +/- 0.01 (N = 15), and 15.2 +/- 0.8 mM (N = 12), respectively. However, the hypoxic and ischemic perfusions decreased transepithelial potential by 40% (hypoxia: -1.7 +/- 0.1 to -1.1 +/- 0.1 mV [N = 30; P < 0.001]; ischemia: -1.4 +/- 0.1 to -0.82 +/- 0.05 mV [N = 17; P < 0.001]). A similar extent of reduction in transepithelial resistance was observed (hypoxia: 14.3 +/- 1.0 to 9.2 +/- 1.1 omega.cm2 [N = 7; P < 0.005]; ischemia: 12.6 +/- 1.2 to 8.1 +/- 1.0 omega.cm2 [N = 6; P < 0.03]). In addition, neither apical (R(ap)) nor basolateral (Rbl) cell membrane resistances were significantly altered after the ischemic perfusion (control: R(ap) = 369 +/- 48 omega.cm2; Rbl = 92 +/- 11 omega.cm2 [N = 63]; reperfusion: R(ap) = 454 +/- 88 omega.cm2; Rbl = 101 +/- 16 omega.cm2 [N = 21]). It was concluded that tubular cells are able to maintain their electrogenic ionic transport after short-term exposure to hypoxic or ischemic conditions. However, cell-to-cell junctions are damaged by these insults, which could possibly increase leakage and decrease the efficiency of the active transport.     

Hydrocortisone Preserves the Vascular Barrier by Protecting the Endothelial Glycocalyx

Background: Hydrocortisone protects against ischemia-reperfusion injury, reduces paracellular permeability for macromolecules, and is routinely applied in the prevention of interstitial edema. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, suggesting that the glycocalyx is a target for hydrocortisone action.

Methods: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer. Hydrocortisone was applied in a stress dose (10 [mu]g/ml) before inducing 20 min of ischemia (37[degrees]C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure, 70 cm H2O) with Krebs-Henseleit buffer or Krebs-Henseleit buffer plus 2 g% hydroxyethyl starch (130 kd). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Hearts were perfusion fixed to visualize the glycocalyx.

Results: Ischemia-induced degradation of the glycocalyx enhanced coronary perfusion pressure (118.8 +/- 17.3 cm H2O) and increased vascular permeability (8 +/- 0.2 [mu]l [middle dot] min-1 [middle dot] cm H2O-1 at baseline vs. 34 +/- 3.3 [mu]l [middle dot] min-1 [middle dot] cm H2O-1 after reperfusion). Enzymatic digestion of the glycocalyx (heparinase) elicited similar effects. Hydrocortisone reduced postischemic oxidative stress, perfusion pressure (86.3 +/- 6.4 cm H2O), and transudate formation (11 +/- 0.6 [mu]l [middle dot] min-1 [middle dot] cm H2O-1). Applying colloid augmented this (70.6 +/- 5.6 cm H2O and 9 +/- 0.5 [mu]l [middle dot] min-1 [middle dot] cm H2O-1). Postischemic shedding of syndecan-1, heparan sulfate, and hyaluronan was inhibited by hydrocortisone, as was release of histamine from resident mast cells. Electron microscopy revealed a mostly intact glycocalyx after hydrocortisone treatment, but not after heparinase treatment.     

烫伤及内毒素/脂多糖对豚鼠胃肠动力的影响

目的探讨烫伤及内毒素/脂多糖(LPS)对豚鼠胃肠动力障碍发生的机制。方法将30只健康豚鼠随机分为烫伤组(造成30%TBSA深Ⅱ度烫伤)、LPS组(腹腔注射LPS)及对照组(腹腔注射等渗盐水),每组10只。3组豚鼠处理后30min予碳素墨水灌胃,然后测定其胃肠道碳素墨水推进距离;并取肠道组织匀浆检测其降钙素基因相关肽(CGRP)、Na -K -腺苷三磷酸(ATP)酶、Mg2 -ATP酶、Ca2 -ATP酶、Ca2 -Mg2 -ATP酶活性及结肠袋平滑肌细胞线粒体膜电位(△ψm).结果烫伤组及LPS组豚鼠肠道碳素墨水推进距离[(53±9)、(91±10)cm]较对照组[(142±11)cm]明显缩短(P<0·01);烫伤组与LPS组比较,肠道推进距离更短(P<0·01).烫伤组及LPS组豚鼠肠道组织中CGRP的含量为(52.0±39.0)、(20.0±23.0)μg/L,均高于对照组(0.8±2.0)μg/L(P<0.05或0·01)。而烫伤组与LPS组比较,其增高幅度更大(P<0.05).烫伤组、LPS组大鼠Na -K -ATP酶、Mg2 -ATP酶、Ca2 -ATP酶、Ca2 -Mg2 -ATP酶活性和线粒体膜电位与对照组比较呈不同程度降低(P<0·05),但烫伤组与LPS组各种ATP酶和线粒体膜电位改变程度比较,差异无统计学意义(P>0·05).结论烫伤及LPS对豚鼠肠道动力功能有明显抑制作用,尤以烫伤明显;重度烫伤时,引起肠道平滑肌细胞线粒体膜电位改变的主要因素可能是肠道LPS.     

Respiratory Mechanics during Xenon Anesthesia in Pigs: Comparison with Nitrous Oxide

Background: Because of its high density and viscosity, xenon (Xe) may influence respiratory mechanics when used as an inhaled anesthetic. Therefore the authors studied respiratory mechanics during xenon and nitrous oxide (N2O) anesthesia before and during methacholine-induced bronchoconstriction.

Methods: Sixteen pentobarbital-anesthetized pigs initially were ventilated with 70% nitrogen-oxygen. Then they were randomly assigned to a test period of ventilation with either 70% xenon-oxygen or 70% N2O-oxygen (n = 8 for each group). Nitrogen-oxygen ventilation was then resumed. Tidal volume and inspiratory flow rate were set equally throughout the study. During each condition the authors measured peak and mean airway pressure (Pmax and Pmean) and airway resistance (Raw) by the end-inspiratory occlusion technique. This sequence was then repeated during a methacholine infusion.

Results: Both before and during methacholine airway resistance was significantly higher with xenon-oxygen (4.0 +/- 1.7 and 10.9 +/- 3.8 cm H2O [middle dot] s-1 [middle dot] l-1, mean +/- SD) when compared to nitrogen-oxygen (2.6 +/- 1.1 and 5.8 +/- 1.4 cm H2O [middle dot] s-1 [middle dot] l-1, P < 0.01) and N2O-oxygen (2.9 +/- 0.8 and 7.0 +/- 1.9, P < 0.01). Pmax and Pmean did not differ before bronchoconstriction, regardless of the inspired gas mixture. During bronchoconstriction Pmax and Pmean both were significantly higher with xenon-oxygen (Pmax, 33.1 +/- 5.5 and Pmean, 11.9 +/- 1.6 cm H2O) when compared to N2O-oxygen (28.4 +/- 5.7 and 9.5 +/- 1.6 cm H2O, P < 0.01) and nitrogen-oxygen (28.0 +/- 4.4 and 10.6 +/- 1.3 cm H2O, P < 0.01).     

Hypotonic stress-induced release of KHCO3 in fused renal epitheloid (MDCK) cells

Mechanisms of cell volume regulation induced by the reduction of the osmolality of the Ringer solution by one-third were studied in fused Madin-Darby canine kidney (MDCK) cells. Intracellular HCO3-, K+ and Cl- concentrations [ion]i in parallel with cell membrane potential (PD), cell membrane conductance (Gm) and conductances of individual ions (Gmion) were evaluated with microelectrode techniques. Fused cells regulate their cell volume by about 50%. Gm increased from 0.43 +/- 0.03 mS/cm2 in isotonic Ringer solution to 4.3 +/-0.3 mS/cm2 in the steady state phase of cell swelling. GmCl was 0.31 +/- 0.03 mS/cm2 in isotonic Ringer solution and thus was the dominant individual ion conductance. In the initial phase of cell swelling GmK increased transiently 64-fold to 0.32 +/- 0.03 mS/cm2, and consequently PD hyperpolarized. At peak hyperpolarization GmCl transiently decreased by 15%. Cell swelling increased GmCl 11-fold and GmHCO3 28-fold to 0.95 +/- 0.1 mS/cm2 in the steady state phase of cell swelling. In this phase GmCl and GmHCO3 were dominating, whereas GmK was only slightly increased compared to isotonic conditions. The hyperpolarization of PD was paralleled by cytoplasmic acidification. At peak acidification [HCO3-]i decreased by 6.4 mmol/kg H2O. Cl- extrusion was not detectable in the initial phase of cell swelling. In isotonic Ringer solution [K+]i was 125 +/- 5 mmol/kg H2O. During the initial phase of cell swelling 23 +/- 5 mmol/kg H2O K+ was extruded, indicating that yet unknown anions participated in cell volume regulation in this phase of cell swelling. In the steady state phase of cell swelling [pH]i was normalized by replenishing [HCO3-]i, whereas Cl- was extruded. We conclude that fused renal epitheloid cells acutely release KHCO3 in response to hypotonicity, but then regain pH homeostasis in the steady state phase of cell swelling.     

Functional role of the Na+/H+ exchanger in the regulation of cerebral arteriolar tone in rats

OBJECT: In vascular smooth-muscle cells, the Na+/H+ exchanger (NHE) is involved in the regulation of [Na+]i, pHi through [H+], and cell volume. Recently, investigations have determined that this exchanger contributes to ischemia and reperfusion injury in coronary circulation. Nonetheless, there is limited information on this glycoprotein in cerebral circulation, especially microcirculation. Thus, the authors in the present study examined the role of NHE in the regulation of cerebral arteriolar tone and its related mechanisms in vitro. METHODS: The internal diameter of isolated pressurized intracerebral arterioles in rats was monitored with the aid of a microscope. To examine the basal activity of NHE two kinds of Na+/H+ exchange inhibitors (FR183998 and 5-[N,N-hexamethylene]amiloride) were administered in the arterioles. Furthermore the authors studied the effects of nitric oxide (NO) synthase inhibitor (NG methyl-L-arginine), Na+/K+ -adenosine triphosphatase (NKA) inhibitor (ouabain), and the Na+/Ca++ exchange inhibitor (SEA0400) on the vascular response induced by either of the Na+/H+ exchange inhibitors. Both of the Na+/H+ exchange inhibitors constricted the arteriole. Subsequent application of NO synthase inhibitor further decreased the diameter of the arterioles. The Na+/H+ exchange inhibitor-induced constriction was completely abolished in the presence of ouabain and SEA0400. CONCLUSIONS: The NHE is active in the basal condition and regulates cerebral arteriolar tone through NKA and the Na+/Ca++ exchanger. Endogenous NO is not related to the activity of NHE in basal conditions.     

Luminal P2Y2 receptor-mediated inhibition of Na+ absorption in isolated perfused mouse CCD.

Extracellular nucleotides regulate renal transport. A luminal P2Y2 receptor in mouse cortical collecting duct (CCD) principal cells has been demonstrated elsewhere. Herein the effects of adenosine triphosphate (ATP) and uridine triphosphate (UTP) on electrogenic Na+ absorption in perfused CCD of mice kept on a low-NaCl diet were investigated. Simultaneously, transepithelial voltage (V(te)), transepithelial resistance (R(te)), and fura-2 [Ca2+]i fluorescence were measured. Baseline parameters were V(te), -16.5 +/- 1.2 mV; R(te), 80.8 +/- 7.1 Omega cm2; and equivalent short-circuit current (I(sc)), -261.0 +/- 25.1 microA/cm2 (n = 45). Amiloride (10 microM) almost completely inhibited I(sc) to -3.9 +/- 3.8 microA/cm2 (n = 10). Luminal ATP (100 microM) reduced V(te) from -16.5 +/- 2.1 to -12.5 +/- 1.93 and increased R(te) from 113.1 +/- 16.2 to 123.8 +/- 16.7 Omega cm2, which resulted in a 31.7% inhibition of amiloride-sensitive I(sc) (n = 12). Similarly, luminal UTP reversibly reduced V(te) from -22.0 +/- 2.1 to -13.6 +/- 2.1 mV and increased R(te) from 48.4 +/- 5.3 to 59.2 +/- 7.1 Omega cm2, which resulted in 49.1% inhibition of Na+ absorption (n = 6). In parallel, luminal ATP and UTP elevated [Ca2+]i in CCD, increasing the fura-2 ratio by 2.7 +/- 0.7 and 4.0 +/- 1.2, respectively. Basolateral ATP and UTP (100 microM) also inhibited amiloride-sensitive I(sc) by 21.8 (n = 14) and 20.1% (n = 8), respectively. Inhibition of luminal nucleotide-induced [Ca2+]i increase by Ca2+ store depletion with cyclopiazonic acid (3 microM) did not affect nucleotide-mediated inhibition of Na+ transport (n = 7). No evidence indicated the activation of a luminal Ca2+-activated Cl- conductance, a phenomenon previously shown in M-1 CCD cells (J Physiol 524: 77-99, 2000). In essence, these data indicate that luminal ATP and UTP, most likely via P2Y2 receptors, mediate inhibition of amiloride-sensitive I(sc) in perfused mouse CCD. This inhibition appears to occurs independently of an increase of cytosolic Ca2+.     

Propofol increases contractility during alpha1a-adrenoreceptor activation in adult rat cardiomyocytes

BACKGROUND: The objective of this study was to identify the extent to which propofol alters intracellular free Ca2+ concentration ([Ca2+]i), myofilament Ca sensitivity, and contraction of individual cardiomyocytes during activation of alpha1a adrenoreceptors and to determine the cellular mechanism of action. METHODS: Freshly isolated ventricular myocytes were obtained from adult rat hearts. Myocyte shortening and [Ca2+]i were simultaneously monitored in individual cardiomyocytes exposed to phenylephrine after treatment with chloroethylclonidine (alpha1b-adrenoreceptor antagonist) and BMY 7378 (alpha1d-adrenoreceptor antagonist). Data are reported as mean +/- SD. RESULTS: Phenylephrine increased myocyte shortening by 124 +/- 9% (P = 0.002), whereas peak [Ca2+]i only increased by 8 +/- 3% (P = 0.110). Inhibition of phospholipase A2 and phospholipase C attenuated the phenylephrine-induced increase in shortening by 84 +/- 11% (P = 0.004) and 15 +/- 6% (P = 0.010), respectively. Inhibition of protein kinase C (PKC) and Rho kinase attenuated the phenylephrine-induced increase in shortening by 17 +/- 8% (P = 0.010) and 74 +/- 13% (P = 0.006), respectively. In the presence of phenylephrine, propofol increased shortening by 40 +/- 6% (P = 0.002), with no concomitant increase in [Ca2+]i. PKC inhibition prevented the propofol-induced increase in shortening. Selective inhibition of PKCalpha, PKCdelta, PKCepsilon, and PKCzeta reduced the propofol-induced increase in shortening by 12 +/- 5% (P = 0.011), 36 +/- 8% (P = 0.001), 32 +/- 9% (P = 0.007), and 19 +/- 5% (P = 0.008), respectively. Na+ - H+ exchange inhibition reduced the propofol-induced increase in shortening by 56 +/- 7% (P = 0.001). CONCLUSION: Activation of alpha1a adrenoreceptors increases cardiomyocyte shortening primarily via a phospholipase A2-dependent, Rho kinase-dependent increase in myofilament Ca2+ sensitivity. Propofol further increases myofilament Ca2+ sensitivity and shortening via a PKC-dependent pathway and an increase in Na+ - H+ exchange activity.     

Aerodynamic evaluation of crystalloid and colloid flush perfusion for lung preservation

To assess the effectiveness of pulmonary perfusion we evaluated the lung mechanics of 36 canine lungs in an isolated perfused working lung (IPWL) model. Four groups of lungs (n = 9 each) were preserved by pulmonary artery flushing with either high-potassium colloid (UW), high-potassium crystalloid (EuroCollins', EC), low-potassium crystalloid control (lactate), or low-potassium substrate-enhanced crystalloid (RPMI) followed by 130 +/- 10 min of cold storage. Ventilation remained constant (TV 10 ml/kg at 14 breaths/min with 5 cm H2O PEEP). Assessed data included lung resistance (R), timed expiratory volume (EV0.3 sec as %TV), lung compliance (C), elastic work (Wel), and flow-resistive work (Wres). Immediately following storage, R and Wel were similar for all groups (16 +/- 3 cm H2O/liter/sec and 149 +/- 18 gm/min). UW preserved lungs were less compliant (1.5 +/- 0.1 X 10(-2) liter/cm H2O) and required more inspiratory work (Wres 5.8 +/- 0.8 gm/min) compared to the low-potassium crystalloid (Lactate) group (2.0 +/- 0.1 X 10(-2) liter/cm H2O and 3.4 +/- 0.6 gm/min, respectively, P less than 0.05). For 3 hr of reperfusion, crystalloid lungs showed no significant change in R, C, Wel, or Wres. In contrast, R of the UW group increased significantly to 32 +/- 5 and 40 +/- 8 cmH2O/liter/sec at 1 and 3 hr, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pressure support ventilation versus continuous positive airway pressure ventilation with the ProSeal laryngeal mask airway: a randomized crossover study of anesthetized pediatric patients

Continuous positive airway pressure (CPAP) and pressure support ventilation (PSV) improve gas exchange in adults, but there are little published data regarding children. We compared the efficacy of PSV with CPAP in anesthetized children managed with the ProSeal laryngeal mask airway. Patients were randomized into two equal-sized crossover groups and data were collected before surgery. In Group 1, patients underwent CPAP, PSV, and CPAP in sequence. In Group 2, patients underwent PSV, CPAP, and PSV in sequence. PSV comprised positive end-expiratory pressure set at 3 cm H(2)O and inspiratory pressure support set at 10 cm H(2)O above positive end-expiratory pressure. CPAP was set at 3 cm H(2)O. Each ventilatory mode was maintained for 5 min. The following data were recorded at each ventilatory mode: ETco(2), Spo(2), expired tidal volume, peak airway pressure, work of breathing patient (WOB), delta esophageal pressure, pressure time product, respiratory drive, inspiratory time fraction, respiratory rate, noninvasive mean arterial blood pressure, and heart rate. In Group 1, measurements for CPAP were similar before and after PSV. In Group 2, measurements for PSV were similar before and after CPAP. When compared with CPAP, PSV had lower ETco(2) (46 +/- 6 versus 52 +/- 7 mm Hg; P < 0.001), slower respiratory rate (24 +/- 6 versus 30 +/- 6 min(-1); P < 0.001), lower WOB (0.54 +/- 0.54 versus 0.95 +/- 0.72 JL(-1); P < 0.05), lower pressure time product (94 +/- 88 versus 150 +/- 90 cm H(2)O s(-1)min(-1); P < 0.001), lower delta esophageal pressure (10.6 +/- 7.4 versus 14.1 +/- 8.9 cm H(2)O; P < 0.05), lower inspiratory time fraction (29% +/- 3% versus 34% +/- 5%; P < 0.001), and higher expired tidal volume (179 +/- 50 versus 129 +/- 44 mL; P < 0.001). There were no differences in Spo(2), respiratory drive, mean arterial blood pressure, and heart rate. We conclude that PSV improves gas exchange and reduces WOB during ProSeal laryngeal mask airway anesthesia compared with CPAP in ASA physical status I children aged 1-7 yr.     

Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney

INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.