Anemia increases the risk of renal cortical and medullary hypoxia during cardiopulmonary bypass.

INTRODUCTION: Anemia is an independent predictor of acute kidney injury (AKI) following cardiopulmonary bypass (CPB), possibly due to inadequate renal oxygen delivery. The objective of this study was to investigate the effects of CPB and anemia on tissue oxygen tension (pO2) and blood flow in the renal cortex and medulla. METHODS: Rats (n=6/group) underwent 1 hr of normothermic cardiopulmonary bypass (CPB), with target hemoglobin concentrations (Hb) of 10 g/dL (CPB) or 6.5 g/dL (anemia-CPB). Renal blood flow (RBF) and tissue PO2 were measured before, during and after 1 hr of CPB. To confirm the observed differences in renal cortical and medullary PO2, HIF-1α (ODD) luciferase mice were exposed to 8% O2 (hypoxia) and HIF-1α dependent luminescence was measured in the renal cortex and medulla (n=5). RESULTS: Renal tissue PO2 values decreased initially and returned towards baseline, however, values at the end of CPB. Anemia-CPB resulted in a significant increase in both renal cortical and medullary blood flow, PO2 remained significantly reduced throughout anemia-CPB. Renal medullary HIF-1α-dependent luminescence confirmed a greater degree of hypoxia in the renal medulla. DISCUSSION: During CPB, renal O2 delivery was transiently jeopardized, but recovered after 1 hr. Anemia-CPB resulted in a dramatic and sustained reduction in renal cortical and medullary PO2, which suggests an increased risk of renal hypoxic injury with anemia. CONCLUSION: The clear difference in the degree of hypoxia in the renal cortex and medulla may be useful in understanding the progress of medullary hypoxia during CPB with anemia and the potential development of AKI. Further studies should aim at identifying early markers of medullary hypoxia and potential agents that may decrease the work and O2 consumption in the renal medulla to reduce the risk of hypoxic damage during CPB and anemia.

Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca(2+) handling.

To explain that bronchial smooth muscle undergoes sustained agonist-induced contractions in a Ca(2+)-free medium, we hypothesized that caveolae in the plasma membrane (PM) contain protected Ca(2+). We isolated caveolae from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Detergent-resistant membranes were enriched in caveolin-1, a specific marker for caveolae as well as for L-type Ca(2+) channels and Ca(2+) binding proteins (calsequestrin and calreticulin) as determined by Western blotting. Also, the PM Ca(2+) pump was present but not connexin 43 (a noncaveolae PM protein), the sarcoplasmic reticulum (SR) Ca(2+) pump, or the type 1 inositol 1,4, 5-trisphosphate receptor, supporting the idea that SR-derived membranes were not present. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin or calreticulin. Thus some of the cellular calsequestrin and calreticulin associated with caveolin on the cytoplasmic face of each caveola. Immunohistochemistry of tracheal smooth muscle crysosections confirmed the localization of caveolin and the PM Ca(2+) pump to the cell periphery, whereas the SR Ca(2+) pump was located deeper in the cell. The presence of L-type Ca(2+) channels, the PM Ca(2+) pump, and the Ca(2+) bindng proteins calsequestrin and calreticulin in caveolin-enriched membranes supports caveola involvement in airway smooth muscle Ca(2+) handling.

Intrafamilial homicide committed by juveniles: examination of a sample with recommendations for prevention.

The current study examines a sample of 112 adolescents convicted of murder. From this sample, 11 adolescents who murdered a member of their family were chosen for in-depth examination. All participants were convicted as an adult for murder and received a prison sentence. Characteristics of the crimes were explored, including demographic information, relationship to victim, motive, and weapon used in the commission of the crime. Additionally, DSM diagnosis information was available for some participants. A majority of the homicides (N = 8) were committed with a gun that was available in the home. More than half of the subjects murdered a parent or parental figure and the most salient characteristic was the presence of a chaotic family life prior to the homicide. No single motive was dominant, indicating that intrafamilial homicide is the culmination of a number of factors, including abuse and psychopathology. Based on the findings, recommendations are provided to address the needs of youth who are at risk for committing violent crimes such as intrafamilial homicide.

Selective inhibition of oxalate-stimulated Ca2+ transport by cyclopiazonic acid and thapsigargin in smooth muscle microsomes.

45Ca2+ uptake and efflux studies were performed on membranes prepared from dog mesenteric artery and rat vas deferens. Oxalate-stimulated, ATP-dependent Ca2+ uptake in microsomal vesicles, a property characteristic of sarcoplasmic reticulum, was completely inhibited in a concentration-dependent manner by cyclopiazonic acid (0.1-30 microM) and thapsigargin (10 nM-10 microM). Using discontinuous sucrose gradient centrifugation, rat vas deferens microsomes were separated into two fractions, one enriched in plasma membrane (F2), the other enriched in sarcoplasmic reticulum (F3). The F3 fraction had a major increase in Ca2+ uptake in the presence of oxalate, which was completely inhibited by either cyclopiazonic acid or thapsigargin. In the F2 fraction Ca2+ uptake in the presence of oxalate was lower than in F3 and was not completely inhibited by thapsigargin and cyclopiazonic acid. Instead, the F2 fraction had a thapsigargin-insensitive and cyclopiazonic acid insensitive, saponin-sensitive component of uptake, which probably represents Ca2+ uptake by plasma membrane. In the absence of oxalate, the inhibition of Ca2+ uptake by saponin and cyclopiazonic acid or thapsigargin was additive in the F2 and F3 fractions, suggesting that cyclopiazonic acid and thapsigargin selectively inhibited sarcoplasmic reticulum derived Ca2+ uptake and did not affect plasma membrane derived Ca2+ uptake. Measurement of the initial rate of Ca2+ uptake in the presence and absence of oxalate by rat vas deferens microsomes demonstrated selective inhibition of oxalate-stimulated Ca2+ uptake by cyclopiazonic acid and thapsigargin. Ca2+ efflux from rat vas deferens microsomes actively loaded with 45Ca2+ either in presence or the absence of oxalate was not increased by cyclopiazonic acid or thapsigargin, showing that the inhibition of Ca2+ accumulation was not due to an increase in Ca2+ efflux. In both rat vas deferens and dog mesenteric artery, the maximal inhibitory effects of cyclopiazonic acid developed rapidly, whereas for maximal inhibition thapsigargin required pretreatment of microsomes prior to measurement of Ca2+ uptake. In rat vas deferens microsomes the inhibitory effects of cyclopiazonic acid could be quickly and completely reversed, whereas the effects of thapsigargin were not easily reversed. Collectively, these results suggest selectivity of cyclopiazonic acid and thapsigargin for the sarcoplasmic reticulum Ca2+ pump. Their selective inhibitory properties and differences in onset and offset of inhibition make cyclopiazonic acid thapsigargin useful pharmacological tools in the study of the physiological and pathophysiological roles of the sarcoplasmic reticulum Ca2+ pump in regulating smooth muscle Ca2+.

Use of calcium pump inhibitors in the study of calcium regulation in smooth muscle.

The study of the relative roles of the plasma membrane (PM) and the sarcoplasmic reticulum (SR) Ca2+ pumps in the regulation of internal Ca2+ has been limited in the past due in part to the lack of selective inhibitors for either Ca2+ pump. Recently, three compounds have been discovered which appear to be selective SR Ca2+ pump inhibitors: thapsigargin (TSG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl)-1,4-benzohydroquinone. Contractility studies in various smooth muscle tissues have demonstrated that all three compounds inhibit repletion of the intracellular Ca2+ store, presumably due to inhibition of the SR Ca2+ pump. These functional studies however provided only indirect evidence for Ca2+ pump inhibition. Using the microsomal membrane fraction isolated from the smooth muscle of rat vas deferens, we investigated the effects of CPA and TSG of ATP-dependent Ca2+ uptake in order to obtain direct evidence about the mechanism of action of CPA and TSG on smooth muscle Ca2+ pumps. CPA and TSG potently inhibited oxalate-stimulated Ca2+ uptake in a concentration-dependent manner. Since oxalate-stimulated Ca2+ uptake is generally considered to be a property of the SR and not the PM Ca2+ pump, this directly demonstrates that CPA and TSG act by inhibiting the SR Ca2+ pump. We also demonstrated that inhibition of Ca2+ uptake in the presence of oxalate by CPA could be completely reversed while the effects of TSG could only be partially reversed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of thapsigargin and ryanodine on vascular contractility: cross-talk between sarcoplasmic reticulum and plasmalemma.

Thapsigargin and ryanodine are proposed to interfere with Ca2+ storage in sarcoplasmic reticulum by different mechanisms. Thapsigargin inhibits Ca2+ transport into and ryanodine enhances Ca2+ out of the sarcoplasmic reticulum. Contractility studies were performed in the rat aorta and dog mesenteric artery. Ryanodine was found to reduce phenylephrine-induced (10 microM) contraction in Ca(2+)-free medium of rat aorta and dog mesenteric artery in a concentration-dependent manner. Each agent alone caused a slow contraction in the rat aorta. In this tissue, the tension caused by ryanodine (30 microM) but not that by thapsigargin (1 microM) was found to be dependent on the status of the sarcoplasmic reticulum: prior stimulation with K+ (60 mM) enhanced the rate of development of ryanodine-induced tension compared with when the sarcoplasmic reticulum was previously depleted with phenylephrine stimulation in Ca(2+)-free medium. Sodium nitroprusside (1 microM) or isoproterenol (1 microM) fully antagonized the contraction induced by ryanodine or phenylephrine. However, thapsigargin-induced contraction was antagonized fully by sodium nitroprusside and only partially by isoproterenol. This result suggests that cAMP elevation by isoproterenol required a functioning sarcoplasmic reticulum Ca2+ pump for its relaxant effect while cGMP elevation by sodium nitroprusside did not. These findings are consistent with the view that ryanodine promotes Ca2+ release from the sarcoplasmic reticulum and that thapsigargin inhibits the ability of cAMP to stimulate Ca2+ uptake into the store by blocking its Ca2+ pump. In the dog mesenteric artery, when the phenylephrine-sensitive Ca2+ pool was emptied and thapsigargin was added to block Ca2+ uptake into the store, restoration of Ca2+ in the Ca(2+)-free medium caused a transient contraction (absent in controls). This contraction was replaced by a significantly larger amplitude and more sustained contraction in low Na+ medium indicating the involvement of the Na+/Ca2+ exchanger in the homeostasis of cytosolic [Ca2+]. In the presence of nifedipine (2 microM), repletion of the phenylephrine-sensitive store was inhibited. It is possible that refilling occurs in part through L-type Ca2+ channels.

Thapsigargin inhibits repletion of phenylephrine-sensitive intracellular Ca++ pool in vascular smooth muscles.

Thapsigargin (TSG), a putative selective Ca(++)-ATPase inhibitor, has been used to study Ca++ mobilization in many non-excitable cell types. This study aims to determine whether TSG is effective as a selective microsomal Ca++ uptake inhibitor by studying its ability to affect repletion of the phenylephrine (PE)-sensitive Ca++ pool in rat aorta and dog mesenteric artery evaluated by contractility studies. TSG caused a concentration-dependent contraction that was dependent on the concentration of extracellular Ca++. Ca++ influx promoted by TSG was found to occur mostly through L-type Ca++ channels in the dog mesenteric artery but not in the rat aorta. When arterial rings, depleted of their PE-sensitive internal store, were allowed to replete their stores in normal Krebs' solution or in the presence of elevated K+ levels, it was found that repletion was significantly enhanced in the presence of elevated K+. In TSG-treated rings, however, repletion was significantly inhibited under both conditions as indicated by the subsequent PE-induced contraction in Ca(++)-free medium. While the rate of contraction induced by elevated K+ levels was slow immediately after pool depletion in controls, it was rapid in TSG-treated arterial rings. The slow onset of K+ contraction may reflect Ca++ uptake into the pool which was absent in TSG-treated arteries. Differences in the behavior of the two arteries were noted and these may reflect differences in the size of their Ca++ store and their coupling to the extracellular space. Single cells isolated from the dog mesenteric artery were also found to shorten in response to TSG to an amount comparable with that obtained from whole tissue experiments.(ABSTRACT TRUNCATED AT 250 WORDS)