Ethanol inhibition of phagocytosis and superoxide anion production by microglia

A case of a symptomatic colonic lipoma causing recurrent abdominal pain and intestinal obstruction, not treated is reported. Lipomas are the most common mesenchymal benign tumors that can be found in the colon and are second as frequence only to the adenomatous polyps. In 65% of cases the lipomas are located in the large bowel and represent the most common cause of intestinal intussusception in the adult. Lipomas are most frequent in the right colon (40%-68%) an opposite distribution in comparison with adenocarcinomas and adenomatous polyps. When their diameter is more than 3 cm, lipomas become symtomatic. In lipomas less then 2 cm in diameter it is possible an endoscopic removal while for bigger sizes the surgical laparoscopic approach is recommended.     

Superoxide anion production in influenza protein-activated NADPH oxidase of human polymorphonuclear leukocytes

There are conflicting reports regarding superoxide anion (O2-) production by human polymorphonuclear leukocytes (PMNL) that have been activated by influenza virus. In the present study, the output of O2- was determined by measuring superoxide dismutase-inhibitable cytochrome c reduction. Incubation of PMNL with purified influenza matrix (M) protein, neuraminidase (NA), or hemagglutinin (HA) enhanced the production of O2-: 4.93 nmol of O2-/4 x 10(5) cells/15 min was produced with M protein, 5.20 with NA, and 6.89 with HA. These values were significantly higher (P < .05) than that for untreated PMNL (1.51). Both nonglycosylated and glycosylated proteins had the potential to generate O2- in human PMNL. Neither the hemagglutinating activity of HA nor the enzymatic activity of NA were necessary for viral protein activation of PMNL.     

Distinct phospholipases A2 regulate the release of arachidonic acid for eicosanoid production and superoxide anion generation in neutrophils

Arachidonic acid (AA) released from membrane phospholipids by phospholipase A2 (PLA2) is important as a substrate for eicosanoid formation and as a second messenger for superoxide anion (O2-) generation in neutrophils. Different isoforms of PLA2 in neutrophils might mobilize AA for different functions. To test this possibility, we sought to characterize the PLA2s that are activated by the neutrophil stimuli, Aroclor 1242, a mixture of polychlorinated biphenyls, and A23187, a calcium ionophore. Both Aroclor 1242 and A23187 caused release of [3H]AA; however, O2- production was seen only in response to Aroclor 1242. Eicosanoids accounted for >85% of the radioactivity recovered in the supernatant of A23187-stimulated cells but <20% of the radioactivity recovered from cells exposed to Aroclor 1242. Omission or chelation of calcium abolished A23187-induced AA release, but did not alter AA release in Aroclor 1242-stimulated neutrophils. AA release and O2- production in response to Aroclor 1242 were inhibited by bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2. BEL, however, did not alter Calcium-independent activity was inhibited >80% by BEL, whereas calcium-dependent activity was inhibited <5%. Furthermore, calcium-independent, but not calcium-dependent, PLA2 activity was significantly enhanced by Aroclor 1242. These data suggest that Aroclor 1242 and A23187 activate distinct isoforms of PLA2 that are linked to different functions: Aroclor 1242 activates a calcium-independent PLA2 that releases AA for the generation of O2-, and A23187 activates a calcium-dependent PLA2 that mobilizes AA for eicosanoid production.     

Impaired posthypoxic relaxation in single cardiac myocytes: role of intracellular pH and inorganic phosphate

OBJECTIVE: The aim was to examine the effects of alterations in intracellular pH and inorganic phosphate concentration (known to influence myofilament kinetics and to change rapidly during hypoxia) on cell contraction, relaxation, and the Ca2+ transient in normoxic and hypoxic myocytes. METHODS: Single adult rat ventricular myocytes were electrically stimulated (0.2 Hz) and cell length (photodiode array), intracellular Ca2+ (indo-1 fluorescence), or intracellular pH (SNARF-1 fluorescence) measured. Hypoxia was induced in a special open chamber in which a laminar layer of argon prevented the back diffusion of atmospheric oxygen. RESULTS: Electrically stimulated contraction was preserved during exposure to hypoxia. At reoxygenation 10 minutes later the time from the stimulus to the peak of contraction (TPK) increased by 30(SEM 9)% and the time from the peak of contraction to 50% recovery of cell length (RT50) increased by 59(13)% relative to prehypoxic values (n = 8). These changes were not accompanied by a change in the kinetics of the Ca2+ transient. pHi fell from a baseline of 7.33(0.04) to 7.25(0.03) during hypoxia and then overshot to 7.44(0.03) at reoxygenation (n = 5). Since an intracellular alkalosis can slow myofilament relaxation, proton extrusion routes were blocked to examine posthypoxic relaxation in the absence of an alkalosis. Despite inhibition of the pHi overshoot, posthypoxic relaxation remained impaired. Intracellular inorganic phosphate levels were manipulated in two protocols (2-deoxyglucose to "trap" phosphate and Tris(hydroxymethyl)-aminomethane to buffer phosphate) and both TPK and RT50 increased in normoxic cells. Having established that these two interventions, which would be expected to decrease intracellular inorganic phosphate, result in a slowing of relaxation, myocytes were first phosphate loaded (exposed to 5.0 mM phosphate) and then made hypoxic and reoxygenated after 10 min to blunt the expected fall in phosphate accompanying reoxygenation. This led to a reduction in the slowing of contraction and relaxation following reoxygenation [TPK increased by 7(5)% and RT50 by 17(9)%, n = 8; p < 0.05 v cells studied in control buffer]. CONCLUSIONS: Impaired posthypoxic relaxation is not the result of changes in pHi but is attenuated by phosphate loading of cells and may be due to a rapid decrease in intracellular phosphate accompanying the resynthesis of high energy phosphates at reoxygenation.     

Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production

Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.     

NMDA-induced superoxide production and neurotoxicity in cultured rat hippocampal neurons: role of mitochondria

Excitotoxic mechanisms are believed to be involved in the death of neurons after trauma, epileptic seizures and cerebral ischaemia. We investigated the role of mitochondrial superoxide production in excitotoxic cell death of cultured rat hippocampal neurons. Brief exposure to the selective glutamate agonist N-methyl-D-aspartate (NMDA; 100-300 microM, 10 min) induced significant neuronal death, which was sensitive to cycloheximide (1 microM) and the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone (10 microM). Intracellular superoxide production was monitored semiquantitatively on sister cultures from the same platings using the oxidation-sensitive probe, hydroethidine. Brief exposures to toxic NMDA concentrations induced significant increases in superoxide production which correlated with the degree of neuronal injury. However, subtoxic NMDA exposures also produced moderate, yet statistically significant increases in superoxide production. Both NMDA-induced superoxide production and neurotoxicity were reduced by inhibition of mitochondrial electron transport using either sodium cyanide (1 mM), or a combination of rotenone (2 microM) and oligomycin (2 microM). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 1 microM) mimicked the effect of NMDA on mitochondrial superoxide production. Both NMDA-induced superoxide production and neurotoxicity were potentiated by FCCP (1 microM). Exposure to FCCP alone (1-10 microM, 10 min), however, failed to produce any toxicity. Our data suggest that mitochondrial superoxide production per se is not sufficient to trigger the degeneration of cultured hippocampal neurons, but that manipulation of mitochondrial activity alters NMDA-induced superoxide production and neurotoxicity.     

Glucocorticoids inhibit superoxide anion production and p22 phox mRNA expression in human aortic smooth muscle cells

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the concentration of extracellular Ca2+. In this study, two novel phenylalkylamine compounds, NPS 467 and NPS 568, were examined for effects on Xenopus laevis oocytes expressing the bovine or human parathyroid Ca2+ receptors. Increases in chloride current (ICl) were elicited in oocytes expressing the bovine Ca2+ receptor when the extracellular Ca2+ concentration was raised above 1.5 mM, whereas Ca2+ concentrations > 3 mM were generally necessary to elicit responses in oocytes expressing the human Ca2+ receptor. NPS 467 and NPS 568 potentiated the activation of ICl by extracellular Ca2+ in oocytes expressing either Ca2+ receptor homolog, and this resulted in a leftward shift of the Ca2+ concentration-response curve. Neither compound was active in the absence of extracellular Ca2+. Certain inorganic and organic cations known to activate the Ca2+ receptor were substituted for elevated levels of extracellular Ca2+ to increase ICl and the effects of these agonists were also potentiated by NPS 568 or NPS 467. The effects of NPS 568 were stereoselective and the R-enantiomer was about 10-fold more potent than the corresponding S-enantiomer. Neither NPS 467 nor 568 affected ICl in water-injected oocytes or in oocytes expressing the substance K receptor or the metabotropic glutamate receptor 1a. These results provide compelling evidence that NPS 467 and NPS 568 act directly upon the parathyroid Ca2+ receptor to increase its sensitivity to activation by extracellular Ca2+. This activity suggests that these compounds are positive allosteric modulators of the Ca2+ receptor. As such, these compounds define a new class of pharmacological agents with potent and selective actions on the Ca2+ receptor.     

TNF-induced superoxide anion production in adherent human neutrophils involves both the p55 and p75 TNF receptor

TNF, a potent activator of neutrophil granulocytes, acts via two cell-surface receptors: the p55-TNF receptor (TNF-R55) and the p75-TNF receptor (TNF-R75), which can be cleaved from the cell surface and thus form soluble TNF-binding proteins (TNF-BP). The role of the two receptors in activation of the neutrophil respiratory burst was investigated. Two mAbs reacting with TNF-R55 (H398 and TBP2) induced O2 release in a similar manner but to a lesser extent than TNF. TBP2, however, required preincubation at 4 degrees C to exert its effect. Preincubation of neutrophils (both at 4 and 37 degrees C) with mAb to TNF-R75 decreased TNF-induced superoxide anion production by 67 and 64%, respectively, indicating the essential role also for TNF-R75 in neutrophil activation. This inhibitory effect could not be explained by cross-down-regulation of TNF-R55 because the TNF-R75 mAb had no effect on TNF binding to TNF-R55 as determined by binding of 125I-labeled TNF or release of TNF-R55-BP as measured by ELISA. Furthermore, the TNF-R75 mAb did not decrease superoxide anion generation induced by the TNF-R55 mAb H398, thus ruling out that the inhibitory effect of the TNF-R75 mAb is due to inhibition of the signaling pathway downstream of TNF-R55. In contrast to the TNF-R75 mAb, TNF-R55 mAbs induced down-regulation of TNF-R75 and shedding of both TNF-R55-BP and TNF-R75-BP. We conclude that both TNF-R55 and TNF-R75 are involved in TNF-induced activation of the neutrophil respiratory burst.     

Beta-2 adrenergic activation of L-type Ca++ current in cardiac myocytes

The whole-cell patch-clamp and intracellular perfusion techniques were used for studying the effects of a beta-2 adrenergic receptor activation on the L-type Ca current (ICa) in frog ventricular myocytes. The beta-2 adrenergic agonist zinterol increased ICa in a concentration-dependent manner with an EC50 (i.e., the concentration of zinterol at which the response was 50% of the maximum) of 2.2 nM. The effect of zinterol was essentially independent of the membrane potential. The stimulatory effect of zinterol was competitively antagonized by ICI 118,551, a beta-2 adrenergic antagonist. The maximal stimulatory effect of zinterol was comparable in amplitude to the effect of a saturating concentration (1 or 10 microM) of isoprenaline, a nonselective beta adrenergic agonist. Moreover, 3-isobutyl-1-methylxanthine (100 microM), a nonselective phosphodiesterase inhibitor, or forskolin (10 microM), a direct activator of adenylyl cyclase, had no additive effects in the presence of 0.1 microM zinterol. Zinterol had a long lasting action on frog ICa because after washout of the drug, ICa returned to basal level with a time constant of 17 min. An application of acetylcholine (1 microM) during this recovery phase promptly reduced ICa back to its basal level suggesting a persistent activation of adenylyl cyclase due to a slow dissociation rate constant of zinterol from its receptor. Zinterol also increased ICa in rat ventricular and human atrial myocytes, and the maximal effect was obtained at 10 and 1 microM, respectively. In all three preparations, intracellular perfusion with 20 microM PKI(15-22), a highly selective peptide inhibitor of cAMP-dependent protein kinase, completely antagonized the stimulatory effect of zinterol on ICa. We conclude that beta-2 adrenergic receptor activation produces a strong increase in ICa in frog, rat and human cardiac myocytes which is due to stimulation of adenylyl cyclase and activation of cAMP-dependent phosphorylation.     

Generation of superoxide anion and induction of superoxide dismutase and peroxidase in bean leaves infected with pathogenic fungi

PURPOSE: The purpose of this study was to review management strategies with respect to systemic therapy, radiation therapy treatment techniques, and patient outcome (local regional control, distant metastases, and overall survival) in patients undergoing conservative surgery and radiation therapy (CS + RT) who had four or more lymph nodes involved at the time of original diagnosis. METHODS AND MATERIALS: Of 1040 patients undergoing CS + RT at our institution prior to December 1989, 579 patients underwent axillary lymph node dissection. Of those patients undergoing axillary lymph node dissection, 167 had positive nodes and 51 of these patients had four or more positive lymph nodes involved and serve as the patient population base for this study. All patients received radiation therapy to the intact breast using tangential fields with subsequent electron beam boost to the tumor bed to a total median dose of 64 Gy. The majority of patients received regional nodal irradiation as follows: 40 patients received RT to the supraclavicular region without axilla to a median dose of 46 Gy, 10 patients received radiation to the supraclavicular region and axilla to a median dose of 46 Gy. Thirty of the 51 patients received a separate internal mammary port with a mixed beam of photons and electrons. One patient received radiation to the tangents alone without regional nodal irradiation. Adjuvant systemic therapy was used in 49 of the 51 patients (96%) with 27 patients receiving chemotherapy alone, 14 patients receiving cytotoxic chemotherapy and tamoxifen, and 8 patients receiving tamoxifen alone. RESULTS: As of December 1994, with a minimum evaluable follow-up of 5 years and a median follow-up of 9.29 years, there have been 18 distant relapses, 2 nodal relapses, and 5 breast relapses. Actuarial statistics reveal a 10-year distant metastases-free rate of 65%, 10-year nodal recurrence-free rate of 96%, and a 10-year breast recurrence-free rate of 82%. All five patients who sustained a breast relapse were successfully salvaged with mastectomy. Both patients with nodal relapses (one supraclavicular and one axillary/supraclavicular) failed within the irradiated volume. Of the 40 patients treated to the supraclavicular fossa (omitting complete axillary radiation), none failed in the dissected axilla. With a median follow-up of nearly 10 years, 29 of the 51 patients (57%) remain alive without evidence of disease, 15 (29%) have died with disease, 2 (4%) remain alive with disease, and 5 (10%) have died without evidence of disease. Overall actuarial 10-year survival for these 51 patients is 58%. CONCLUSIONS: We conclude that in patients found to have four or more positive lymph nodes at the time of axillary lymph node dissection, conservative surgery followed by radiation therapy to the intact breast with appropriate adjuvant systemic therapy results in a reasonable long-term survival with a high rate of local regional control. Omission of axillary radiation in this subset of patients appears appropriate because there were no axillary failures among the 41 dissected but unirradiated axillae.     

Interleukin-8 and GRO alpha prime human neutrophils for superoxide anion production and induce up-regulation of N-formyl peptide receptors

Interleukin-8 (IL-8) and GRO alpha are leukocyte-attracting peptides of the chemokine family. To study the priming potential of these chemokines, we measured superoxide anion production and up-regulation of N-formyl peptide receptors in human neutrophils. IL-8 and GRO alpha themselves did not stimulate production of significant amounts of superoxide anions but potentiated N-formyl peptide-induced superoxide anion production in a concentration-dependent manner. Binding measurements by flow cytometry at 37 degrees C with fluorescein-labeled N-formyl peptide revealed enhanced total N-formyl peptide binding after pretreatment of neutrophils with IL-8 and GRO alpha. Binding measurements performed at 4 degrees C indicated that the chemokines stimulated the up-regulation of N-formyl peptide receptors at the cell surface but did not alter their affinity for the ligand. This study indicates that IL-8 and GRO alpha, in addition to their known chemotactic activity, prime neutrophils for superoxide anion production, presumably by up-regulating the number of receptors for strong superoxide-anion-triggering stimuli.     

Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes

Neuregulins (i.e. neuregulin-1 (NRG1), also called neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor-inducing activity) are known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG1 or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG1 (recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/ml) provoked an approximate 2-fold increase in embryonic cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic growth in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic factor and skeletal alpha-actin. Moreover, NRG1 mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG1 expression in these cells was increased by endothelin-1, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.     

Force regulation by Ca2+ in skinned single cardiac myocytes of frog

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.     

Spark-to-wave transition: saltatory transmission of calcium waves in cardiac myocytes

In order to investigate a possible regional specificity of activation asymmetries of different parts of the cortex and the stability of these asymmetries across time and conditions, EEG was recorded during rest and stimulation conditions in a large sample of right-handed university students (n = 60). Recordings were made in two sessions (interval between sessions 2-4 weeks). In the first session two rest periods were conducted (interval 30 min) and one under stimulation. In the second session EEG was again recorded at rest. Reliability analyses show that the long-term stability of orbitofrontal asymmetries is markedly lower than that of dorsolateral, temporal, and parietal scores. Intercorrelations of EEG asymmetries at different electrode positions suggest that anterior and posterior EEG asymmetries are largely independent measures of cortical laterality. Both this partial independence of activation asymmetries and the differences in temporal stability underscore the significance of the anterior-posterior dimension in laterality research and may be one reason for several contradictory observations in studies on brain laterality.     

Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1

The cellular source(s) and mechanisms of generation of reactive oxygen species (ROS) in nonphagocytic cells stimulated by cytokines are unclear. In this study, we demonstrate that transforming growth factor beta 1 (TGF-beta 1, 1 ng/ml) induces the release of H2O2 from human lung fibroblasts within 8 h following exposure to this cytokine. Elevation in H2O2 release peaked at 16 h (approximately 22 pmol/min/10(6) cells) and gradually declined to undetectable levels at 48 h after TGF-beta 1 treatment. NADH consumption by these cells was stimulated by TGF-beta 1 while that of NADPH remained unchanged. NADPH oxidase activity as measured by diphenyliodonium (DPI)-inhibitable NADH consumption in TGF-beta 1-treated cells followed a time course similar to that of H2O2 release. DPI, an inhibitor of the NADPH oxidase complex of neutrophils and other flavoproteins, also inhibited the TGF-beta 1-induced H2O2 production. Inhibitors of other enzymatic systems involving flavoproteins that may be responsible for the production of H2O2 in these cells, including xanthine oxidase, nitric oxide synthase, and both mitochondrial and microsomal electron transport systems, failed to inhibit TGF-beta 1-induced NADH oxidation and H2O2 production. The delay (> 4 h) following TGF-beta 1 exposure along with the inhibition of this process by cycloheximide and actinomycin D suggest the requirement of new protein synthesis for induction of NADH oxidase activity in TGF-beta 1-stimulated fibroblasts.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Structural remodeling and mechanical dysfunction of cardiac myocytes in heart failure

End stage heart failure due to ischemic (ICM) or dilated (DCM) cardiomyopathy is characterized by a dilated, relatively thin-walled ventricle. The hypothesis has been proposed that the structural basis of ventricular expansion is due to side-to-side slippage of myocytes within the wall. Although this represents one potential mechanism for the observed phenomena of chamber dilatation and subsequent wall thinning, the degree of slippage claimed is not necessarily in harmony with the magnitude of chamber enlargement and mural thinning. Moreover, sarcomere extension was not examined in the base to the apical regions of the heart, leaving open the question as to the role of changes in resting sarcomere length in acute chamber dilatation. In this regard, an alternative etiology for the detrimental cardiac architectural rearrangement seen in dilated failure can be supplied by postulating the occurrence of maladaptive remodeling of cardiac myocyte morphology. In this model, myocytes increase in length by an increase in the number of sarcomeres in series, thus increasing chamber diameter in an attempt to maintain cardiac output. However, these cells do not enlarge to any significant degree in the transverse diameter preventing the heart from developing adequate force. This hypothesis is supported by recent evidence from patients with ICM and DCM indicating that myocyte lengthening alone could account for all the dilatation observed. Furthermore, it appears that the thinning of the ventricular wall in failure is due to inadequate transverse growth of cardiac myocytes coupled with scattered myocyte cell loss throughout the ventricular wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of the contractile assembly and Ca2+-activated tension in adenovirus infected adult cardiac myocytes

Rats were given suction lesions of the presumptive frontal cortex on embryonic day 18 (E18) and subsequently tested, as adults, on tests of spatial navigation (Morris water task, radial arm maze), motor tasks (Whishaw reaching task, beam walking), and locomotor activity. Frontal cortical lesions at E18 affected cerebral morphogenesis, producing unusual morphological structures including abnormal patches of neurons in the cortex and white matter as well as neuronal bridges between the hemispheres. A small sample of E18 operates also had hydrocephaly. The animals with E18 lesions without hydrocephalus were behaviorally indistinguishable from littermate controls. The results demonstrate that animals with focal lesions of the presumptive frontal cortex have gross abnormalities in cerebral morphology but the lesions leave the functions normally subserved by the frontal cortex in adult rats unaffected. The results are discussed in the context of a hypothesis regarding the optimal times for functional recovery from cortical injury.     

Cardiotoxicity of chlorodibromomethane and trichloromethane in rats and isolated rat cardiac myocytes

The cardiovascular effects were investigated after acute and subacute treatment with chlorodibromomethane (CDBM; 0.4 to 3.2 mmol/kg p.o.), trichloromethane (TCM; 0.31 and 1.25 mmol/kg p.o.) and mixtures of CDBM and TCM (acute, 0.8 mmol CDBM/kg + 1.25 mmol TCM/kg p.o.; subacute, 0.4 mmol CDBM/kg + 0.31 mmol TCM/kg p.o.) in conscious and urethane anaesthetized male Wistar rats (n = 6-10 per treatment). Furthermore it was observed whether cardiovascular responses were modified in CDBM or TCM treated rats after administration of exogenous catecholamines (epinephrine, 1 microg/kg; norepinephrine, 2 microg/kg) and underpinned with in vitro alterations of Ca2+ dynamics in cardiac myocytes. The present findings demonstrated that single and subacute oral administration of CDBM or TCM and mixtures of CDBM and TCM resulted in arrhythmogenic and negative chronotropic and dromotropic effects in conscious and urethane anaesthetized rats. The atrioventricular conduction time and the intraventricular extension time were extended. A slight shortening of the repolarization velocity was observed. The myocardial contractility was depressed and the heart was sensitized to the arrhythmogenic effects of epinephrine. After catecholamine injection the adrenergic cardiovascular responses in urethane anesthetized rats were modified: increased hypertensive epinephrine and norepinephrine action as well as augmentation of negative chronotropic and negative dromotropic cardiac effects of catecholamines were observed. The positive inotropic adrenergic response was diminished. The present in vivo findings, myocardial depression after acute CDBM treatment, as determined by different indices of contractility, correlate well with the observed inhibitory actions of CDBM on Ca2+ dynamics in isolated cardiac myocytes. All cardiovascular alterations found after CDBM or TCM treatment were not intensified after treatment with mixtures of CDBM and TCM. The effects observed were distinctly stronger after TCM (1.25 and 0.31 mmol/kg) treatment compared to CDBM (0.8 and 0.4 mmol/kg) treatment.