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Biomedical subjects

G N Pierce

Publications and source records attributed to G N Pierce.

At least 37 records · Page 2Linked to original sources
PubMed1999

The importance of lipid solubility in antioxidants and free radical generating systems for determining lipoprotein proxidation.

The oxidative modification of low-density lipoprotein (LDL) plays an important role in atherosclerosis. Protecting LDL from oxidation has been shown to reduce the risk of coronary heart disease. In this study, we compared the protective effects of two lipophilic antioxidants (vitamin E and lazaroid) with two hydrophilic antioxidants (trolox and vitamin C) in the presence of several different free radical generating systems. Vitamin E (IC50 = 5.9 microM) and lazaroid (IC50 = 5.0 microM) were more effective in inhibiting lipid peroxidation caused by a Fe-ADP free radical generating system than vitamin C (IC50 = 5.2 x 10(3) microM) and trolox (IC5 = 1.2 x 10(3) microM). Preincubation of lipoproteins with a lipophilic antioxidant increased the protective effect against various free radicals. Preincubation with hydrophilic antioxidants did not have an effect. We also tested the efficacy of the antioxidants when the free radicals were generated within the lipid or the aqueous environment surrounding the LDL. For this purpose, we used the peroxyl generating azo-compounds AMVN (2,2'-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2'azobis(2-amidinopropane) dihydrochloride). All of the antioxidants tested were more effective against free radicals generated in a water soluble medium than they were against free radicals generated in a lipid environment. In conclusion, our data demonstrate that lipid solubility is an important factor for both the antioxidant and the free radical generating systems in determining the extent of lipid peroxidation in LDL. Our data also demonstrate that antioxidant efficacy in one set of experimental conditions may not necessarily translate into a similar degree of protection in another set of conditions where lipophilicity is a variable.

Animals↗
PubMed1999

Cardiac myofibrillar and sarcoplasmic reticulum function are not depressed in insulin-resistant JCR:LA-cp rats.

Depressed myofibrillar Ca2+-ATPase activity and sarcoplasmic reticulum (SR) Ca2+ uptake are important mechanisms that are responsible for the cardiac dysfunction exhibited by insulin-deficient (type I) diabetic animals. The JCR:LA-cp rat is a model for type II non-insulin-dependent diabetes mellitus (NIDDM). This rat is insulin resistant, obese, and has high levels of circulating glucose, cholesterol, insulin, and triglycerides. The purpose of this study was to determine whether changes in cardiac myofibrillar, SR, and cardiomyocyte function exist in this model of type II diabetes. Myofibrils and SR were isolated from hearts by differential centrifugation. Surprisingly, we found that myofibrillar Ca2+-ATPase activities were unaltered in these animals. Ca2+ uptake in isolated SR fractions was increased in diabetic cp/cp rats, whereas Ca2+-ATPase activity and ryanodine binding were unchanged. Cardiomyocytes isolated from hearts of control and experimental animals had similar active cell shortening and intracellular Ca2+ concentration under basal conditions and in response to caffeine. Our data argue against the presence of a cardiomyopathy in this diabetic model and suggest that insulin may be an important factor in the cardiomyopathy observed in type I diabetic models.

Animals↗
PubMed1999

A model of low-flow ischemia and reperfusion in single, beating adult cardiomyocytes.

The present study was undertaken to comprehensively characterize low-flow ischemia and reperfusion in single adult cardiomyocytes and to determine whether it is important to control contractile activity. The ischemia-mimetic solution was hypoxic, acidic (pH 6.0), and deficient in glucose but contained elevated KCl. Cardiomyocytes were stimulated to contract throughout ischemia and during reperfusion with control perfusate. After the ischemia-reperfusion insult, cells exhibited poor recovery of active cell shortening, a decrease in passive cell length, increased frequency of necrosis, lower ATP content, and evidence of the generation of oxygen-derived free radicals within the cells. Intracellular lactate concentration increased, pH decreased, and Ca(2+) transients were depressed during the ischemic insult, but the latter two parameters recovered partially on reperfusion. Basal intracellular Ca(2+) concentration was elevated during ischemia and early into reperfusion. Recovery was attenuated in cells that were electrically stimulated to contract throughout ischemia. The duration of ischemia, stimulation frequency, and composition of the ischemia-mimetic solution were important variables. The inclusion of 10 mM lactate in the ischemia-mimetic solution significantly aggravated all the parameters examined above. Our data demonstrate that 1) an ischemia-mimetic solution administered to single, isolated adult cardiomyocytes can reproduce many of the responses observed in whole hearts, 2) caution should be used in adding lactate to an ischemic solution, and 3) it is important to stimulate contractile activity throughout ischemia to reproduce the effects of ischemia in whole hearts.

Animals↗
PubMed1999

Oxidized low-density lipoprotein induces cytoskeletal disorganization in smooth muscle cells.

Vascular smooth muscle cells in atherosclerotic vessels proliferate and change from a contractile to a synthetic phenotype. To determine whether oxidized low-density lipoprotein (oxLDL) is involved in this transformation, we chronically incubated cultured smooth muscle cells with native and oxidized LDL. Western blot analysis detected a decrease in actin and myosin content in treated cells. This was dependent on the time and concentration of oxLDL employed. Confocal microscopic images of cells immunostained for smooth muscle-specific alpha-actin and myosin showed a normal, elongated alignment of myofilaments in cells after incubation with native LDL. Surprisingly, when the cells were treated with oxLDL, actin and myosin filaments underwent a striking process of disorganization and accumulation into ball-shaped aggregates. These changes were dependent on the duration and concentration of oxLDL employed. Our results demonstrate that oxLDL has the capacity to decrease the content of myofilaments in smooth muscle cells. The loss in myosin and actin protein may be associated with an unusual formation of large cellular aggregates that appear to be in the process of being expelled from the cell.

Actin Cytoskeleton↗
PubMed1999

The atherogenic effects of chlamydia are dependent on serum cholesterol and specific to Chlamydia pneumoniae.

Epidemiological investigations have linked Chlamydia pneumoniae infection to atherosclerosis. It is not clear, however, whether C. pneumoniae infection plays a causal role in the development of atherosclerosis. Mice with low-density lipoprotein receptor deficiency were induced to develop atherosclerotic lesions in aorta with a cholesterol-enriched diet that increased serum cholesterol by two- to threefold. Using this mouse model, we found that the chlamydial infection alone with either the C. pneumoniae AR39 or the C. trachomatis MoPn strain failed to induce any significant atherosclerotic lesions in aorta over a period of nine months. However, in the presence of a high-cholesterol diet, infection with the C. pneumoniae AR39 strain significantly exacerbated the hypercholesterolemia-induced atherosclerosis, demonstrating that a hypercholesterolemic condition is required for the C. pneumoniae to aggravate the development of atherosclerosis. Although both AR39 and MoPn antigens were detected in aorta of mice infected with the corresponding strains, only mice infected with the C. pneumoniae strain AR39 displayed enhanced atherosclerotic lesions, suggesting that the C. pneumoniae species may possess a unique atherogenic property. This study may provide a model for further understanding the mechanisms of C. pneumoniae atherogenesis and evaluating chlamydial intervention strategies for preventing the advancement of atherosclerotic lesions enhanced by C. pneumoniae infection.

Animals↗
PubMed1998

Susceptibility of LDL to oxidative stress in obstructive sleep apnea.

Cardiovascular diseases are more common in patients with obstructive sleep apnea (OSA) than in the general population. We hypothesized that severe hypoxemia during sleep in these patients may cause an imbalance between reactive oxygen species and the antioxidant reserve that is important for the detoxification of these molecules. We tested the hypothesis that low-density lipoproteins (LDL) in hypoxic OSA patients may be more susceptible to oxidative stress than LDL of nonhypoxic OSA patients and normal controls. Fifteen OSA patients were included in this study, six with severe hypoxia (hypoxic group) who spent more than 10 minutes during sleep with SaO2 < 85% (mean 96 minutes), and nine OSA patients (nonhypoxic group) who spent less than 10 minutes during sleep with SaO2 < 85% (mean 1.1 minutes). Six healthy nonsmoking males of the same age group were included as a control group. The susceptibility of each individual's LDL to oxidative stress was examined after free-radical challenge in vitro by assessing changes in levels of conjugated dienes. The LDL in OSA patients with severe hypoxia was not more susceptible to oxidative stress compared to the LDL of nonhypoxic OSA patients and normal controls. After 6 hours of exposure to an oxidative agent, the changes in the mean conjugated diene were not different among the three groups (p = 0.75). The time required to reach 50% of maximal absorbance was also not different, p = 0.199. Glutathione peroxidase and catalase activities in red blood cells in the hypoxic and nonhypoxic patient groups were not significantly different. One night of CPAP therapy in each patient group did not significantly change the level of the antioxidant enzymes. Our results did not show any difference in the susceptibility to oxidative stress between hypoxic and nonhypoxic OSA patients and normal controls.

Adult↗
PubMed1998

Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells.

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 microM lyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31-8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate.

Arachidonic Acid↗
PubMed1998

Protection against myocardial ischemic/reperfusion injury by inhibitors of two separate pathways of Na+ entry.

Previous work has demonstrated that drugs which inhibit Na+ entry through voltage-sensitive Na+ channels, or via Na(+)-H+ exchange protect the heart from ischemic reperfusion damage. The purpose of our study was to determine whether these drugs in combination will have an additive protective effect in Langendorff-perfused hearts. During reperfusion following 30 min of ischemia, developed tension and resting tension were 24 +/- 3 and 162 +/- 5%, respectively, of pre-ischemic values in non-treated ischemic hearts. The administration of HOE-642 to inhibit Na+/H+ exchange increased active developed tension (DT) to 58 +/- 2% of pre-ischemic levels and decreased resting tension (RT) to 111 +/- 3% of pre-ischemic levels. The administration of tetrodotoxin (TTX) to block the Na+ channel increased DT to 56 +/- 3% of the pre-ischemic level and reduced the RT to 126 +/- 12% of the pre-ischemic level. Together, HOE-642 and TTX increased recovery of DT to 63 +/- 2% of pre-ischemic levels and improved RT to 116 +/- 4% of pre-ischemic levels after 30 min of reperfusion. All drug treatment protocols significantly lowered the creatine phosphokinase activity measured in the coronary effluent in comparison to that observed in the non-treated hearts. These data demonstrate that inhibition of Na+ entry through either Na(+)-H+ exchange or the Na+ channel protects the heart from ischemic injury, but there is no additional benefit of blocking both routes of Na+ entry simultaneously. This suggests that a threshold level of Na+i may be a critical factor in ischemic cardioprotection.

Animals↗
PubMed1998

FGF-2-induced negative inotropism and cardioprotection are inhibited by chelerythrine: involvement of sarcolemmal calcium-independent protein kinase C.

Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

Alkaloids↗
PubMed1997

Regulation of intracellular Ca2+ in the heart during diabetes.

Cardiovascular disease is a significant medical problem. The diabetic population is even more susceptible to cardiovascular complications and heart failure than non-diabetic patients. Atherosclerotic complications, a neuropathy and microvascular lesions have all been implicated causally in the accelerated cardiovascular disease during diabetes. However, one mechanism which may participate in the abnormalities in heart performance demonstrated during diabetes and may also contribute to heart failure in the diabetic is a derangement in the capacity of the myocardial cell to regulate its [Ca2+]. The purpose of this treatise is to identify the current controversies and conclusions available regarding the specific defects in Ca2+ flux thought to contribute to these cardiac defects during diabetes mellitus.

Animals↗
PubMed1997

Oxidation of nuclear membrane cholesterol inhibits nucleoside triphosphatase activity.

Oxygen derived free radicals can oxidize membrane cholesterol. We have previously shown that cholesterol in the nuclear membrane can modulate nuclear nucleoside triphosphatase (NTPase) activity. Nucleocytoplasmic transport of peptides and mRNA via the nuclear pore complex may be regulated by the NTPase. The purpose of the present study was to determine if oxidation of nuclear cholesterol could alter NTPase activity. Nuclear membrane cholesterol was oxidized in situ with cholesterol oxidase (to selectively oxidize cholesterol) and NTPase activity measured. HPLC analysis confirmed the formation of cholesterol oxides. The activity of the NTPase was strikingly inhibited by cholesterol oxidase treatment. The Vmax of the NTPase was significantly decreased after cholesterol oxidase treatment but the Km value was unchanged. The sensitivity of NTPase activity to varying cholesterol oxidase concentrations also suggested that cholesterol located in the inner leaflet of the nuclear membrane appeared to be more important in the modulation of NTPase activity than that in the cytoplasmic leaflet. Our results indicate that oxidation of nuclear membrane cholesterol inhibits NTPase activity. These results have implications for peptide and mRNA flux across the nuclear membrane during conditions where lipid oxidation may be expected.

Acid Anhydride Hydrolases↗
PubMed1997

Postischemic cardiac performance in the insulin-resistant JCR:LA-cp rat.

Hearts from hyperinsulinemic, insulin-resistant JCR:LA-cp rats do not properly regulate intracellular Ca2+ concentration. We hypothesized, therefore, that these hearts may be unusually sensitive to ischemic insults in which Ca2+ overload would be expected. We investigated the response to global ischemia of hearts from JCR: LA-cp animals at three different ages. At 3 mo of age, isolated hearts from insulin-resistant cp rats were mildly resistant to both mild and severe ischemic insults in comparison to the lean control rat hearts. However, at 6 and 9 mo of age, the cp rats demonstrated a poorer recovery of developed tension after ischemia and/or a higher level of resting tension during reperfusion than the lean controls. Postischemic glycogen and ATP contents were significantly lower and lactate content was higher in hearts from 6-mo-old cp rats compared with controls. The results demonstrate that an insulin-resistant animal model exhibits an increased sensitivity to ischemic myocardial injury that develops with advancing age. The mechanism responsible for the enhanced sensitivity may involve augmented glycolytic metabolism. The data also emphasize the importance of the type of diabetes when cardiac dysfunction is examined.

Adenosine Triphosphate↗
PubMed1997

Myocardial dysfunction is associated with activation of Na+/H+ exchange immediately during reperfusion.

Amiloride analogs block Na+/H+ exchange and thereby protect the heart from myocardial ischemia-reperfusion injury. It is unclear whether drugs must be present before ischemia to be cardioprotective. After 60 min of global ischemia in the coronary-perfused right ventricular wall (RVW), as little as 1 min of exposure to dimethyl amiloride (DMA) immediately at the time of reperfusion protected the RVW. Delaying the drug attenuated the cardioprotection. If DMA was introduced in an ischemic solution near the end of ischemia, the cardioprotective effects were augmented. If the drug was washed out of the RVW vascular space before ischemia, cardioprotection was not observed. In contrast, in whole hearts, preischemic perfusion of the drug was necessary for cardioprotection and the cardioprotection remained even if the drug was washed out before ischemia. We conclude that Na+/H+ exchange is active and contributes to contractile dysfunction during the first seconds of reperfusion. This is difficult to detect in the perfused whole heart, and the washout data suggest that this may be due to a limitation in drug delivery across the vascular wall. The data also suggest that the exchanger is not as active during ischemia itself as it is during reperfusion.

Amiloride↗
PubMed1997

Cardiovascular dysfunction in insulin-dependent and non-insulin-dependent animal models of diabetes mellitus.

Morbidity in the diabetic population is primarily a result of cardiovascular dysfunction and failure. Ischemic heart disease is a serious complication in the diabetic population. Recent data indicate that hearts from insulin-dependent models of diabetes differ significantly in their response to ischemia from hearts of models of non-insulin-dependent diabetes. Intrinsic cardiac factors may be responsible for the altered sensitivities to ischemia in the different types of diabetes. However, vascular dysfunction is also clearly present in the diabetic animal models. Endothelial cell damage and dysfunction play a prominent role in the contractile abnormalities and lesion formation during diabetes. The present treatise focuses upon insulin as a causative factor in cardiovascular disease and the differences between insulin-dependent and non-insulin-dependent models of diabetes.

Animals↗
PubMed1997

The nuclear membrane integrity assay.

A novel technique is described for the evaluation of membrane integrity in isolated nuclei. Membrane integrity is assessed by measuring nuclear fragility in response to high salt conditions. Salt-induced disruption of the nuclear membrane is followed by spectrophotometric monitoring of released nucleotides. The assay is based on determining the amount of salt necessary to induce release of 50% of the total pool of releasable nucleotides. This allows semiquantitative comparison of relative nuclear membrane strength or integrity of different samples in response to high salt conditions. In this manner, the effects of altered nuclear membrane composition or metabolism on membrane integrity may be monitored.

Animals↗
PubMed1997

Na(+)-H+ exchange inhibition at reperfusion is cardioprotective during myocardial ischemia-reperfusion; 31P NMR studies.

To help resolve the controversy as to whether or not Na(+)-H+ exchange is functioning during reperfusion of the ischemic myocardium we assessed the effects of dimethylamiloride (DMA, an amiloride analogue possessing selectivity for inhibition of the Na(+)-H+ exchanger) on cardiac function and intracellular pH during ischemia-reperfusion. Studies were performed in the presence of bicarbonate (modified Krebs-Henseleit buffer) or in the nominal absence of bicarbonate (HEPES buffer) in order to determine if similar cardioprotection and effects on intracellular pH were observed in the presence and absence of bicarbonate dependent transport processes. Isovolumic rat hearts were perfused in the Langendorff mode at a constant pressure of 80 mm Hg and subjected to 28 min total global ischemia at 37 degrees C. Intracellular pH was determined from the pH dependent shift of the inorganic phosphate peak in 31P nuclear magnetic resonance spectra. DMA (20 microM) was infused for either 2.5 min before ischemia, for the initial 5 min of reperfusion, or at both time intervals. DMA had no effect on the intracellular pH during ischemia. Intracellular pH returned to pre-ischemic levels within 2.5 min of reperfusion in bicarbonate buffer. This normalization of pH was slower in HEPES perfusate. In both bicarbonate and HEPES perfused hearts all drug dosing regimens caused a significant increase in the recovery of mechanical function after reperfusion and slowed the recovery of intracellular pH during reperfusion. These results suggest that the Na(+)-H+ exchanger is activated during reperfusion of the ischemic myocardium, that this activation of the exchanger contributes to ischemia-reperfusion induced cardiac dysfunction and that administration of an inhibitor of Na(+)-H+ exchange at reperfusion significantly attenuates the deleterious effects of exchanger activation.

Amiloride↗
PubMed1997

Age- and sex-related differences in nuclear lipid content and nucleoside triphosphatase activity in the JCR:LA-cp corpulent rat.

The putative role of the nuclear nucleoside triphosphatase (NTPase) is to provide energy to the nuclear pore complex for poly A(+) mRNA export. Previous work has demonstrated that liver nuclear NTPase activity is greater in 6 month old corpulent (cp/cp) female JCR:LA rats, a hyperlipidemic rat model, compared to lean (+/?) animals. This increase appeared to be related to increases in nuclear membrane cholesterol content. The current study extended these initial data to compare NTPase activity as a function of age and sex in isolated JCR:LA-cp rat liver nuclei, to further test the hypothesis that nuclear membrane cholesterol may modulate NTPase activity. NTPase activity was increased in cp/cp female animals compared to +/? females at all ages studied, with Vmax values increased by 60-176%. Membrane integrity of cp/cp female nuclei was reduced compared to +/? female nuclei. Nuclear membrane cholesterol levels increased linearly with age by 50, 150 and 250% in 3, 6 and 9 month old cp/cp females over leans. In contrast, nuclei from cp/cp males exhibited only minor, isolated changes in NTPase activity. Furthermore, there were no significant changes in nuclear cholesterol content or membrane integrity in the less hyperlipidemic male animals at any age. These data suggest that altered lipid metabolism may lead to changes in nuclear membrane structure, which in turn may alter NTPase activity and functioning of the nuclear pore complex.

Acid Anhydride Hydrolases↗