Biomedical subjects

G Fiskum

Publications and source records attributed to G Fiskum.

At least 55 records · Page 3Linked to original sources

PubMed1995

Bcl-2 protects neural cells from cyanide/aglycemia-induced lipid oxidation, mitochondrial injury, and loss of viability.

The protooncogene bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that the mechanism of action of Bcl-2 involves antioxidant activity. The involvement of free radicals in ischemia/reperfusion injury to neural cells has led us to investigate the effect of Bcl-2 in a model of delayed neural cell death. We have examined the survival of control and bcl-2 transfectants of a hypothalamic tumor cell line, GT1-7, exposed to potassium cyanide in the absence of glucose (chemical hypoxia/aglycemia). After 30 min of treatment, no loss of viability was evident in control or bcl-2 transfectants; however, Bcl-2-expressing cells were protected from delayed cell death measured following 24-72 h of reoxygenation. Under these conditions, the rate and extent of ATP depletion in response to treatment with cyanide in the absence of glucose and the rate of recovery of ATP during reenergization were similar in control and Bcl-2-expressing cells. Bcl-2-expressing cells were protected from oxidative damage resulting from this treatment, as indicated by significantly lower levels of oxidized lipids. Mitochondrial respiration in control but not Bcl-2-expressing cells was compromised immediately following hypoxic treatment. These results indicate that Bcl-2 can protect neural cells from delayed death resulting from chemical hypoxia and reenergization, and may do so by an antioxidant mechanism. The results thereby provide evidence that Bcl-2 or a Bcl-2 mimetic has potential therapeutic application in the treatment of neuropathologies involving oxidative stress, including focal and global cerebral ischemia.

Animals↗

PubMed1994

Autoradiographic analysis of L- and N-type voltage-dependent calcium channel binding in canine brain after global cerebral ischemia/reperfusion.

Binding of antagonists to L- and N-type voltage-dependent calcium channels (VDCC) was measured in canine brain following global ischemia and reperfusion. Ischemia was induced by 10 min cardiac arrest, followed by restoration of spontaneous circulation for periods of up to 24 h. Binding of [3H]PN200-110 and [125I]omega-conotoxin GVIA to frozen sections from hippocampus, striatum, parietal cortex and temporal cortex was analyzed using quantitative receptor autoradiography. The binding patterns of the two radioligands were similar in cortex and striatum, but differed in hippocampus. In the latter tissue, [125I]omega-conotoxin GVIA binding was dense over synaptic regions, especially the presynaptic polymorph layer of the dentate gyrus, but was virtually absent over cell body layers. In contrast, [3H]PN200-110 binding was more homogenously distributed, with highest binding in the molecular layer of the dentate gyrus. The binding of [125I]omega-conotoxin GVIA was not different from sham controls at any time point following cardiac arrest. [3H]PN200-110 binding was decreased in each region immediately following ischemia, recovering within 30 min of recirculation. These findings are in contrast to earlier findings of rapid increases in L-type VDCC binding to membrane fractions obtained from cortex and striatum in this model, and suggest that the previously detected increases may be due to a redistribution of channels from subcellular compartments to the plasma membrane during ischemia.

Animals↗

PubMed1994

Acetoacetate metabolism in AS-30D hepatoma cells.

Metabolic characteristics of experimental hepatoma cells include elevated rates of glycolysis and lipid synthesis. However, pyruvate derived from glucose is not redily oxidized, and the source of acetyl CoA for lipid synthesis in As-39D cells has not been characterized. In this study ketone bodies were examined as a possible source of acetyl CoA in AS-30D hepatoma cells. The major findings were: 1. Acetoacetate was utilized by AS-30D cells, with 14C-lipid and 14CO2 as major products of [3-14C] acetoacetate. 2. Lipid synthesis from acetoacetate was dependent on the presence of glucose in the medium. 3. Acetoacetate supported rapid respiration by AS-30D mitochondria in the presence of 0.1 mM malate. 4. Succinyl CoA acetoacetyl CoA transferase activity in AS-30D mitochondria was approximately 40 fold greater than that found in rat liver mitochondria. 5. Addition of acetoacetate, but not beta-hydroxybutyrate decreased conversion of [1-14C] acetate to 14CO2, presumably by diluting the specific radioactivity of the acetyl CoA derived from the acetate tracer. 6. In the presence of glucose, approximately one fourth of acetoacetate utilized was converted to lipid. This result is consistent with elevated lipogenesis postulated by the truncated TCA cycle hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetoacetates↗

PubMed1994

Postischemic inhibition of cerebral cortex pyruvate dehydrogenase.

Postischemic, mitochondrial respiratory impairment can contribute to prolonged intracellular lactic acidosis, secondary tissue deenergization, and neuronal cell death. Specifically, reperfusion-dependent inhibition of pyruvate dehydrogenase (PDH) may determine the degree to which glucose is metabolized aerobically vs. anaerobically. In this study, the maximal activities of pyruvate and lactate dehydrogenase (LDH) from homogenates of canine frontal cortex were measured following 10 min of cardiac arrest and systemic reperfusion from 30 min to 24 h. Although no change in PDH activity occurred following ischemia alone, a 72% reduction in activity was observed following only 30 min of reperfusion and a 65% inhibition persisted following 24 h of reperfusion. In contrast, no significant alteration in LDH activity was observed in any experimental group relative to nonarrested control animals. A trend toward reversal of PDH inhibition was observed in tissue from animals treated following ischemia with acetyl-L-carnitine, a drug previously reported to inhibit brain protein oxidation, and lower postischemic cortical lactate levels and improve neurological outcome. In vitro experiments indicate that PDH is more sensitive than LDH to enzyme inactivation by oxygen dependent free radical-mediated protein oxidation. This form of inhibition is potentiated by either elevated Ca2+ concentrations or substrate/cofactor depletion. These results suggest that site-specific protein oxidation may be involved in reperfusion-dependent inhibition of brain PDH activity.

Analysis of Variance↗

PubMed1994

Increased activation of L-type voltage-dependent calcium channels is associated with glycine enhancement of N-methyl-D-aspartate-stimulated dopamine release in global cerebral ischemia/reperfusion.

We investigated the relationships among N-methyl-D-aspartate, glycine, L-type voltage-dependent calcium channels, and [3H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [3H]PN200-110 ([3H]isradipine) to L-type voltage-dependent calcium channels, that open as a consequence of N-methyl-D-aspartate-induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage-sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N-methyl-D-aspartate-stimulated [3H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N-methyl-D-aspartate-stimulated [3H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N-methyl-D-aspartate-stimulated release in all treatment groups. The enhancement of N-methyl-D-aspartate-stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30-min reperfused animals. These data suggest that glycine may facilitate opening of the voltage-dependent calcium channels activated by N-methyl-D-aspartate and that this facilitation is blocked by the antagonist nitrendipine.

Animals↗

PubMed1993

Non-ionophoretic elevation of intracellular Ca2+ by Lonidamine.

Lonidamine is an antispermatogenic and anticancer drug that is believed to act by inhibition of energy metabolism. In this study, the effects of Lonidamine on the concentration of intracellular free Ca2+ of several tumor cell lines were assessed because of the important role that cytosolic Ca2+ plays in cell viability and proliferation. The presence of 300 microM Lonidamine resulted in large elevations of cytosolic Ca2+ (> 100 nM) in AS-30D rat ascites hepatoma cells and in cultured EMT6 murine mammary adenocarcinoma cells but had little effect on cultured NCI-H345 human small cell lung cancer cells. The apparent EC50 for Lonidamine was approximately 175 microM. The source of elevated cytosolic Ca2+ was primarily intracellular stores, and the effects of Lonidamine on Ca2+ efflux from these stores did not appear to be due to an ionophoretic action of this compound or to a decline in the level of cellular ATP. These results indicate that the Ca2+ homeostasis of certain lines of tumor cells is specifically altered by Lonidamine at concentrations known to affect cell proliferation.

Animals↗

PubMed1993

Dopamine release from canine striatum following global cerebral ischemia/reperfusion.

The elevation of extracellular dopamine (DA) levels in the striatum of experimental animals subjected to ischemic insult has been well documented. The contribution of excessive DA to neuronal damage can be inferred from the ability of DA antagonists, as well as selective destruction of dopaminergic tracts, to confer neuroprotection in models of ischemia. In the current study, we report an enhanced releasability of preloaded [3H]DA in response to either elevated potassium or N-methyl-D-aspartate (NMDA) from striatal slices of beagles that had experienced 10 min of ischemia induced by cardiac arrest. The elevation in sensitivity to potassium stimulation was transient, approaching control levels after 30 min of reperfusion. In contrast, release stimulated by NMDA was elevated immediately after cardiac arrest and remained elevated for as long as 24 h of reperfusion. Release stimulated by NMDA was enhanced by glycine (Gly) and inhibited by MK801, consistent with mediation through the NMDA receptor/channel complex. The increased sensitivity of DA release, coupled with the high levels of excitatory amino acids (EAAs), including glutamate (Glu), aspartate (Asp) and Gly in ischemic brain, probably contribute to the extensive neuronal cell damage.

Animals↗

PubMed1993

Inhibition of postcardiac arrest brain protein oxidation by acetyl-L-carnitine.

Free radical mediated, site-specific protein oxidation has been implicated in the pathophysiology of ischemia/reperfusion brain injury. The purpose of this study was to determine whether this form of molecular damage could be detected in a clinically relevant model employing 10-min cardiac arrest in dogs followed by restoration of spontaneous circulation for up to 24 h. The effects of postischemic acetyl-L-carnitine administration on protein oxidation were also tested due to its previously reported improvement of brain energy metabolism and neurological outcome in this model. Following the experimental period, soluble proteins were extracted from a sample of frontal cortex and reacted with dinitrophenylhydrazine for spectrophotometric measurement of protein carbonyl groups. The most important results of this study were that brain protein carbonyl groups were significantly elevated following 2 and 24 h of reperfusion compared to nonischemic controls, and that postischemic IV administration of acetyl-L-carnitine eliminated the increase in carbonyl groups observed at the 24-h period. These results indicate that brain protein oxidation does occur in a clinically relevant model of complete global cerebral ischemia and reperfusion, and that oxidation is inhibited under treatment conditions that improve neurological outcome.

Acetylcarnitine↗

PubMed1992

Prevention of post-ischemic brain lipid conjugated diene production and neurological injury by hydroxyethyl starch-conjugated deferoxamine.

Hydroxyethyl starch conjugated deferoxamine (DFO) was administered to rats following resuscitation from 6.5 min cardiac arrest (CA) in an attempt to prevent the iron-catalyzed production of oxygen free radicals which may lead to neurologic injury and ultimately death following restoration of spontaneous circulation (ROSC). Brain conjugated dienes were analyzed spectrophotometrically 4 and 24 hr following ROSC, and were found to be significantly elevated when compared to non-ischemic controls. Hydroxyethyl starch-DFO treated rats demonstrated no increased conjugated diene production at either period. Neurologic injury was significantly less in drug treated rats surviving 24 or 72 hours when compared to controls. While mortality was similar in drug treated or control rats for the first 24 hours following ROSC, delayed mortality (days 1-10) was significantly less in drug treated animals, presumably as a result of neurologic protection afforded by post-ischemic drug administration. Administration of DFO conjugated to hydroxyethyl starch appears to modulate the neurologic injury which occurs during brain ischemia and reperfusion.

Alkenes↗

PubMed1992

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)

Animals↗

PubMed1992

Calcium independence of phosphoinositide hydrolysis-induced increase in cyclic AMP accumulation in SK-N-SH human neuroblastoma cells.

Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin-sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)-mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK-N-SH human neuroblastoma cells while blocking the IP3-mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the carbachol-mediated intracellular calcium release without abolishing the carbachol-mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist-induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor-mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.

Calcium↗

PubMed1992

Differential sensitivity of AS-30D rat hepatoma cells and normal hepatocytes to anoxic cell damage.

A substantial fraction of cells present within hard tumors experience extremely hypoxic and hypoglycemic conditions that can lead to phenotypic alterations such as increased metastatic potential and chemotherapeutic drug resistance. Little is known regarding the influence of anoxic aglycemia on tumor cell energy metabolism and viability, and no direct comparisons have been made between the effects of this form of metabolic stress on tumor cells and their tissue of origin. In this study, the effects of in vitro aglycemic incubation under N2 (with or without iodoacetate) on trypan blue exclusion, lactate dehydrogenase release, cell surface blebbing, ATP levels, and mitochondrial respiratory capacity of rat AS-30D ascites hepatoma cells and normal hepatocytes were measured. Under anoxic-aglycemic conditions, the period of incubation during which 50% viability was lost was 2 h for hepatocytes and 6-8 h for AS-30D cells. In contrast, the rate of anoxia-induced loss of ATP was comparable for the two cell types, and mitochondrial damage was actually accelerated in the tumor cells. These findings suggest that tumor cells are more resistant to anoxic cell death because of their greater ability to withstand deenergization and subcellular injury.

Adenosine Triphosphate↗

PubMed1992

Prevention of postischemic canine neurological injury through potentiation of brain energy metabolism by acetyl-L-carnitine.

BACKGROUND AND PURPOSE: Mechanisms of ischemia/reperfusion brain injury include altered patterns of energy metabolism that may be amenable to pharmacological manipulation. The purpose of this study was to test the effectiveness of postischemic acetyl-L-carnitine administration on potentiation of metabolic recovery and prevention of neurological morbidity in a clinically relevant model of complete, global cerebral ischemia and reperfusion. METHODS: Neurological deficit scoring as well as spectrophotometric and fluorescent assays of frontal cortex lactate and pyruvate levels were used in a canine model employing 10 minutes of cardiac arrest followed by restoration of spontaneous circulation for 2 or 24 hours. RESULTS: Dogs treated with acetyl-L-carnitine exhibited significantly lower neurological deficit scores (p = 0.0037) and more normal cerebral cortex lactate/pyruvate ratios than did vehicle-treated control animals. CONCLUSIONS: Postischemic administration of acetyl-L-carnitine potentiates normalization of brain energy metabolites and substantially improves neurological outcome in a clinically relevant model of global cerebral ischemia and reperfusion.

Acetylcarnitine↗

PubMed1992

Ca(2+)-regulated serine protease associated with the nuclear scaffold.

The nuclear scaffold (NS) is a proteinaceous network of orthogonally arrayed intermediate filament proteins, termed lamins, which is responsible for nuclear structure. Recent work has demonstrated that a subset of lamins A/C is proteolytically cleaved to produce an ATP-binding protein. This proteolytic cleavage is accomplished by a NS protease activity, which shows a considerable selectivity for lamins A/C and is stringently regulated by Ca2+ in vitro, suggesting that it might also participate in control of NS breakdown in various scenarios. Here, we identify the major NS protease as a novel serine protease with a predominantly chymotryptic-like substrate preference, and we show that even transient perturbations in cytosolic Ca2+ have significant effects on the NS protease activity. This NS protease activity shows extensive similarities to the multicatalytic proteinase complex. In addition to a potential role in control of NS breakdown at mitosis and/or under pathological conditions, this NS protease is also strategically located for other functions, such as inactivation of various oncogenic proteins or maturation-promoting factor.

Amino Acid Sequence↗

PubMed1991

Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver.

Peroxisomes were isolated from AS-30D hepatoma and compared to normal rat liver cells for the purpose of investigating the cholesterol accumulation in the hepatoma cells. Cholesterol was found to be approximately 10-fold higher relative to protein in AS-30D peroxisomes as compared to peroxisomes from normal liver. The peroxisomes from the hepatoma cells were found to be more stable; catalase was not released from these peroxisomes during isolation or osmotic shock of the peroxisomal fraction. The elevated cholesterol level may stabilize the peroxisomal membrane. Sterol carrier protein-2 (SCP-2) levels were measured using a radioimmunoassay (RIA), which indicated the highest concentration of SCP-2 to be in peroxisomes. Hepatoma peroxisomes had a lower concentration of SCP-2 (2.5 micrograms/mg) than normal liver peroxisomes (8 micrograms/mg). Approximately half of all SCP-2 detected was found to be soluble in both hepatoma and normal rat liver cells. Immunoblots from both rat liver and AS-30D fractions demonstrated the presence of the 14-kDa form of SCP-2. The liver fractions also had a 57-kDa immunoreactive protein, which was barely detectable in the AS-30D fractions. The low abundance of the high molecular weight form of SCP-2 from hepatoma peroxisomes and the lower amounts of SCP-2 detected in the AS-30D peroxisomes may be related to the accumulation of cholesterol in the cells.

Animals↗

PubMed1990

Submicromolar Ca2+ regulates phosphorylating respiration by normal rat liver and AS-30D hepatoma mitochondria by different mechanisms.

The stimulation of 2-oxoglutarate and NAD(+)-isocitrate dehydrogenase by Ca2+ in mitochondria from normal tissues has been proposed to mediate partially the activation of oxidative energy metabolism elicited by physiological elevations in cytosolic Ca2+. This mode of regulation may also occur in tumor cells in which several aspects of mitochondrial metabolism are known to be altered. This study provides a comparison of the stimulation by submicromolar concentrations of Ca2+ on the rates of ATP-generating (state 3) respiration under physiologically realistic conditions by mitochondria isolated from normal rat liver and from highly malignant rat AS-30D ascites hepatoma cells. The K0.5 for activation of glutamate-dependent state 3 respiration by Ca2+ in the presence of ATP at 37 degrees C was determined to be 0.70 +/- 0.05 (S.E.) microM for hepatoma mitochondria and 0.90 +/- 0.03 microM for rat liver mitochondria. This activation was also reflected by a Ca2(+)-induced shift in the oxidation-reduction state of hepatoma mitochondrial pyridine nucleotides to a more reduced level and Ca2+ stimulation of 14CO2 production from [1-14C]glutamate. Whereas the Ca2+ sensitivity of state 3 respiration by hepatoma mitochondria can be explained by the activation of 2-oxoglutarate and possibly NAD(+)-isocitrate dehydrogenases, the Ca2+ sensitivity of liver mitochondrial respiration appears to be predominantly mediated by activation of electron flow through ubiquinone and Complex III of the electron transport chain, as indicated by the specificity of the effects of Ca2+ on respiration with different oxidizable substrates. Although rat liver and hepatoma mitochondria employ different modes of Ca2(+)-activated ATP generation, these results support the hypothesis that changes in cytosolic Ca2+ play a significant role in the potentiation of energy production in tumor, as well as normal tissue.

Animals↗

PubMed1990

Regulation of nuclear scaffold protease activity by calcium.

Recent investigations have disclosed protease activity which is intimately associated with the nuclear scaffold. This protease activity shows a marked selectivity for lamins A/C, and from them may produce the 46-kDa NTPase thought to participate in nucleocytoplasmic RNA transport. Preliminary characterizations suggested that the protease activity might require Ca2+. In this report, we present evidence showing that nuclear scaffold protease activity is stringently regulated by Ca2+ within the physiologic range. Further, calmodulin antagonists such as calmidazolium and chlorpromazine produce marked inhibition of the protease activity, suggesting modulation by calmodulin or calmodulin-like factors.

Animals↗

PubMed1989

Cholecystokinin elevates cytosolic calcium in small cell lung cancer cells.

The ability of cholecystokinin (CCK) to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. CCK-8 elevated the cytosolic Ca2+ levels in cell line NCI-H345 in a dose dependent manner. Nanomolar concentration of CCK-8 elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent CCK agonists such as gastrin-1 and nonsulfated CCK-8 but not inactive compounds such as CCK-27-32-NH2 elevated cytosolic Ca2+ levels. These data suggest that CCK receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.

Bombesin↗