Immediate improvement of dysfunctional myocardial segments after coronary revascularization: detection by intraoperative transesophageal echocardiography.

To ascertain the immediate effects of coronary artery bypass grafting on regional myocardial function, intraoperative transesophageal two-dimensional echocardiograms were obtained in 20 patients using a 3.5 MHz phased array transducer at the tip of a flexible gastroscope. Cross-sectional images of the left ventricle were obtained at multiple levels before skin incision and were repeated serially before and immediately after cardiopulmonary bypass. Using a computer-aided contouring system, percent systolic wall thickening was determined for eight anatomic segments in each patient at similar loading conditions (four each at mitral and papillary muscle levels). Of the 152 segments analyzed, systolic wall thickening improved from a prerevascularization mean value (+/- SEM) of 42.7 +/- 2.9% to a postrevascularization mean value of 51.6 +/- 2.6% (p less than 0.001). Thickening improved most in those segments with the worst preoperative function (p less than 0.001). Chest wall echocardiograms obtained 8.4 +/- 2.3 days after operation showed no deterioration or further improvement in segmental motion compared with transesophageal echocardiograms obtained after revascularization. Thus: regional myocardial function frequently improves immediately after bypass grafting, with increases in regional thickening being most marked in those segments demonstrating the most severe preoperative dysfunction, and this improvement appears to be sustained; and in some patients, chronic subclinical ischemic dysfunction is present which can be improved by revascularization.

Alteration of voltage-dependent calcium channels in canine brain during global ischemia and reperfusion.

Elevated intracellular calcium (iCa2+) plays an important role in the pathophysiology of ischemic brain damage. The mechanisms by which iCa2+ increases are uncertain. Recent evidence implicates the voltage-dependent calcium channel (VDCC) as a likely site for the alteration in Ca2+ homeostasis during ischemia. The purpose of this study was to determine whether VDCCs are altered by global ischemia and reperfusion in a canine cardiac arrest, resuscitation model. We employed the radioligand, [3H]PN200-110, to quantitate the equilibrium binding characteristics of the VDCCs in the cerebral cortex. Twenty-five adult beagles were separated into four experimental groups: (a) nonischemic controls, (b) those undergoing 10-min ventricular fibrillation and apnea, (c) those undergoing 10-min ventricular fibrillation and apnea followed by spontaneous circulation and controlled respiration for 2 and (d) 24 h. Brain cortex samples were taken prior to killing of the animal, frozen immediately in liquid nitrogen, and crude synaptosomal membranes isolated by differential centrifugation/filtration. After 10 min of ischemia the maximal binding (Bmax) of [3H]PN200-110 increased to greater than 250% of control values (control Bmax 11.16 +/- 0.98; ischemic 28.35 +/- 2.78 fmol/mg protein; p less than 0.05). Bmax returned to near control values after 2 h of reperfusion but remained significantly greater than the control at 24 h. Although the affinity constant (Kd) (control = 0.12 +/- 0.03 nM) appeared to increase with ischemia and normalize with reperfusion, the changes were not statistically significant. We conclude that the binding of [3H]PN200-110 to L-type VDCCs is increased after 10 min of global ischemia/anoxia produced by ventricular fibrillation and apnea in the dog.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-dependent translocation of sorcin to membranes: functional relevance in contractile tissue.

Sorcin, a 22 kDa calcium binding protein present in abundance in cardiac tissue and in multi-drug resistant cells and previously described as a soluble protein, is now shown to undergo a calcium-dependent translocation process from the cytosol to cellular membranes in both systems. The translocation process takes place also in E. coli BL21 cells that express recombinant sorcin, r-sorcin, and can be exploited in the purification of the protein. Calcium binding to purified r-sorcin occurs at micromolar concentrations of the metal and is accompanied by a conformational change that renders the protein soluble in the non-ionic detergent Triton X-114. This finding suggests that lipids are the target of sorcin on cellular membranes. The possible significance of the calcium-dependent translocation of sorcin in the specialized functions of sorcin-expressing cells is discussed.

Effect of cardiolipin oxidation on solid-phase immunoassay for antiphospholipid antibodies.

Diagnostic assays for antiphospholipid antibodies are routinely performed on microtitre plates coated with cardiolipin. Here we show that contact between cardiolipin and NUNC-Immuno plates leads to extensive oxidation, generating a series of peroxy-cardiolipins which were identified by electrospray ionization mass spectrometry. To investigate the impact of oxidation on the antibody assay. cardiolipin was resolved into 12 molecular species, including oxidized species and non-oxidized species with different degrees of unsaturation. All 12 species reacted under anaerobic conditions with serum from patients with primary antiphospholipid syndrome. Immune reactivity was similar for tetralinoleoyl-cardiolipin, trilinoleoyl-oleoyl-cardiolipin, and peroxycardiolipins, but somewhat lower for tristearoyl-oleoyl-cardiolipin. Oxidative treatment of cardiolipin with air, cytochrome c, or Cu2+/tert-butylhydroperoxide, either before or during the assay, did not enhance immune reactivity. Similar results were obtained with a monoclonal IgM from lupus-prone mice, that binds cardiolipin in the absence of protein cofactors. We conclude that the solid-phase assay for antiphospholipid antibodies can be supported by various oxidized and non-oxididized molecular species of cardiolipin.

Lonidamine-mediated respiratory changes in rat heart myocytes: a re-examination of the functional response of mitochondrial cytochrome c oxidase.

Respiratory activity of intact cardiac myocytes isolated from rats treated with lonidamine (LND) has been examined under conditions where cytochrome oxidase turns over at its maximal rate. Compared to myocytes isolated from control rat hearts, those treated with LND displayed a 60% increase in the cytochrome oxidase-dependent rate of respiration; electron microscopy revealed, in agreement with the literature, that the membrane structure of the mitochondrion had become disorganized. The increase in the rate of oxygen consumption was correlated with the (partial) impairment of the membrane ability to maintain the proton electrochemical potential gradient which normally inhibits oxidase activity. Results are discussed with reference to previous reports showing no effect of LND on cytochrome c oxidase activity. The evidence reported better clarifies the contribution of cytochrome oxidase to the demonstrated energetic failure displayed by cells treated with LND.

CNS voltage-dependent Na(+) channel expression and distribution in an undifferentiated and differentiated CNS cell line.

Upon serum removal, CAD-R1 cells undergo neurite outgrowth and an increase in voltage-dependent Na(+) current (VDNaC) density without changing their activation and inactivation properties. Insulin and endothelial cell growth supplement inhibited the increase in VDNaC density but not the neurite outgrowth. RI, RII, RIII Na(+) channel proteins were expressed in CAD-R1 cells. These proteins exhibited both similar and different distribution and clustering patterns which suggested the channel's structural differences play a role in channel distribution.

Advances and strategies for spinal cord regeneration.

Although a cure for spinal cord injuries does not currently exist, advances have been made in the field of spinal cord regeneration. This article discusses the pathophysiology of spinal cord injury, animal models, and strategies for restoration and regeneration of the spinal cord.

Satellite glia cells in dorsal root ganglia express functional NMDA receptors.

Satellite glia cells (SGCs), within the dorsal root ganglia (DRG), surround the somata of most sensory neurons. SGCs have been shown to interact with sensory neurons and appear to be involved in the processing of afferent information. We found that in rat DRG various N-methyl-D-aspartate receptor (NMDAr) subunits were expressed in SGCs in intact ganglia and in vitro. In culture, when SGCs were exposed to brief pulses of NMDA they evoked transient increases in cytoplasmic calcium that were inhibited by specific NMDA blockers (MK-801, AP5) while they were Mg²âº insensitive indicating that SGCs express functional NMDAr. The percentage of NMDA responsive SGCs was similar in mixed- (SGCs plus neurons) and SGC-enriched cultures. The pattern of the magnitude changes of the NMDA-evoked response was similar in SGCs and DRG neurons when they were in close proximity, suggesting that the NMDA response of SGCs and DRG neurons is modulated by their interactions. Treating the cultures with nerve growth factor, and/or prostaglandin E2 did not alter the percentage of SGCs that responded to NMDA. Since glutamate appears to be released within the DRG, the detection of functional NMDAr in SGCs suggests that their NMDAr activity could contribute to the interactions between neurons and SGCs. In summary we demonstrated for the first time that SGCs express functional NMDAr.

The N-methyl-D-aspartate-evoked cytoplasmic calcium increase in adult rat dorsal root ganglion neuronal somata was potentiated by substance P pretreatment in a protein kinase C-dependent manner.

The involvement of substance P (SP) in neuronal sensitization through the activation of the neurokinin-1-receptor (NK1r) in postsynaptic dorsal horn neurons has been well established. In contrast, the role of SP and NK1r in primary sensory dorsal root ganglion (DRG) neurons, in particular in the soma, is not well understood. In this study, we evaluated whether SP modulated the NMDA-evoked transient increase in cytoplasmic Ca2+ ([Ca2+]cyt) in the soma of dissociated adult DRG neurons. Cultures were treated with nerve growth factor (NGF), prostaglandin E2 (PGE2) or both NGF+PGE2. Treatment with NGF+PGE2 increased the percentage of N-methyl-D-aspartate (NMDA) responsive neurons. There was no correlation between the percentage of NMDA responsive neurons and the level of expression of the NR1 and NR2B subunits of the NMDA receptor or of the NK1r. Pretreatment with SP did not alter the percentage of NMDA responsive neurons; while it potentiated the NMDA-evoked [Ca2+]cyt transient by increasing its magnitude and by prolonging the period during which small- and some medium-sized neurons remained NMDA responsive. The SP-mediated potentiation was blocked by the SP-antagonist ([D-Pro4, D-Trp7,9]-SP (4-11)) and by the protein kinase C (PKC) blocker bisindolylmaleimide I (BIM); and correlated with the phosphorylation of PKCε. The Nk1r agonist [Sar9, Met(O2)11]-SP (SarMet-SP) also potentiated the NMDA-evoked [Ca2+]cyt transient. Exposure to SP or SarMet-SP produced a rapid increase in the labeling of phosphorylated-PKCε. In none of the conditions we detected phosphorylation of the NR2B subunit at Ser-1303. Phosphorylation of the NR2B subunit at Tyr1472 was enhanced to a similar extent in cells exposed to NMDA, SP or NMDA+SP, and that enhancement was blocked by BIM. Our findings suggest that NGF and PGE2 may contribute to the injury-evoked sensitization of DRG neurons in part by enhancing their NMDA-evoked [Ca2+]cyt transient in all sized DRG neurons; and that SP may further contribute to the DRG sensitization by enhancing and prolonging the NMDA-evoked increase in [Ca2+]cyt in small- and medium-sized DRG neurons.

Volatile anesthetic action on muscle Ca2+ homeostasis.

It is proposed that volatile anesthetics act through the modification of Ca2+ homeostasis in excitable cells. To test this hypothesis, cardiac and skeletal muscles were used as models to examine Ca2+ response, and Ca2+ regulatory and delivery mechanisms. I found that halothane did not alter Ca2+ binding to cardiac troponin C. However, halothane and isoflurane reversibly decreased the Ca2+ affinity of calmodulin at low anesthetic concentration, and irreversibly increased the Ca2+ affinity of calmodulin at high anesthetic concentration. The volatile anesthetics also increased the permeability of light fraction of sarcoplasmic reticulum (SR) to Ca2+. I conclude that volatile anesthetics alter calcium homeostasis in cardiac and skeletal muscles. This work was in part performed in collaboration with Giovanni Salviati and the author benefited from Salviati's work in similar areas.

Thiopental does not alter Ca2+ uptake by cardiac sarcoplasmic reticulum.

The effect of thiopental on Ca2+ uptake by cardiac sarcoplasmic reticulum (SR) isolated from the rabbit was examined to clarify the role of the sarcoplasmic reticulum in the negative inotropic action of thiopental. Thiopental, from 0 to 378 microM, did not alter the rate of Ca2+ uptake by the SR. We also compared the ATP dependence of Ca2+ uptake in the presence and absence of 284 microM thiopental. The Km for ATP and the Vmax of Ca+ uptake were unaffected by thiopental. It is concluded that thiopental does not alter Ca2+ uptake by the SR and that the negative inotropic effects of thiopental occur at other sites in the myocardial cell.

Calcium transport by cardiac sarcoplasmic reticulum: modulation of halothane action by substrate concentration and pH.

The response of calcium transport to halothane by the cardiac sarcoplasmic reticulum (SR) was investigated to determine whether the SR is a site for anesthetic depression of the myocardium. It was observed that halothane could both stimulate (by 800%) and inhibit (by 500%) calcium transport. The varied effects are dependent on adenosine triphosphate (ATP) and calcium and hydrogen ion concentrations. At 2.25% halothane, the Km for ATP is decreased from 2.35 to 0.712 mM and Vmax is decreased from 292 to 149 nmoles/mg/2min. It was found that the steady-state level of calcium in the SR was decreased by 33% by halothane at pH 6.9, whereas halothane had no effect at pH 7.3. It was concluded that the SR is an unlikely site of halothane-induced myocardial depression in the normal heart when substrate concentrations and pH are maintained. In the ischemic heart in which the pH and substrate concentration have decreased, the interaction of halothane with the SR might contribute to a decrease in Ca2+ for contraction.

Measurement of halothane by ultraviolet spectroscopy.

Halothane absorbs strongly in the ultraviolet region of the spectrum. This property has been employed to measure the concentration of halothane in samples in which the effect of halothane on enzyme kinetics was being studied. Halothane can be completely extracted into heptane, displays a concentration-dependent linear increase in absorbance over a broad concentration range, and has a molar extinction coefficient of 447 M cm-1 at 208 nm. The procedure described for the measurement of halothane will enable other investigators who do not have a gas chromatograph to measure the concentration of halothane.

Halothane and isoflurane alter the Ca2+ binding properties of calmodulin.

BACKGROUND: Ca2+ plays an important role in signal transduction and anesthetic mechanisms. To date, no one has observed a direct effect of volatile anesthetics on a Ca(2+)-binding protein. We therefore examined the effects of halothane and isoflurane on the Ca(2+)-binding properties of bovine brain calmodulin. METHODS: The fluorescence emission of calmodulin was obtained over a range of Ca2+ concentrations (10(-7)-10(-4)M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KC1, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid at pH 7.0 and 37 degrees C. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography. RESULTS: The presence of volatile anesthetics altered the Ca(2+)-binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Ca2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Ca2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin-Ca2+ binding curve for Ca2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane. CONCLUSIONS: These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Ca(2+)-binding protein, undergoes a conformational shift when binding Ca2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act.

Creatine kinase activity and temperature in children after cardiac surgery.

The preoperative and postoperative serum creatine kinase (CK) activity and postoperative temperatures were studied in children undergoing surgery for congenital heart disease. Using multiple linear and logistic regression and analysis of variance, associations were found between postoperative CK activity (>2000 IU/L) and the use of succinylcholine, aortic cross-clamp time (>30 minutes), cardiopulmonary bypass time (>60 minutes), the development of fever (>38.5 degrees C), and complications. Complications were defined as hemodynamic instability, poor peripheral perfusion, metabolic acidosis, and eventual multiple organ failure. Associations were also found between postoperative fever and the development of complications. The results suggest that children who develop serum CK elevations greater than 2000 IU/L and fever greater than 39.5 degrees C during the early postoperative period after cardiac surgery more often develop serious complications.