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Biomedical subjects

G N Pierce

Publications and source records attributed to G N Pierce.

At least 55 records · Page 3Linked to original sources
PubMed1996

Nuclear membrane cholesterol can modulate nuclear nucleoside triphosphatase activity.

Previous work has suggested that changes in nuclear membrane cholesterol may induce a stimulation in nuclear nucleoside triphosphatase (NTPase) activity. The purpose of the present study was to directly investigate if nuclear membrane cholesterol can stimulate nuclear NTPase activity. The cholesterol content of nuclei was altered with a liposomal methodology. The cholesterol content of nuclei isolated from hepatic tissue was relatively low in comparison to that typically exhibited by other membrane fractions. Because of this, it was difficult to further deplete the nuclear membrane of cholesterol, but we could successfully increase the cholesterol content after exposure to cholesterol-enriched liposomes. Nuclear NTPase activity was potently stimulated (approximately 150-200% of control) by an increase in the nuclear membrane cholesterol content. The Vmax of the NTPase activity in the presence of ATP or GTP was significantly increased after cholesterol enrichment without altering the affinity of the enzyme for these moieties. Mg2+ dependency of NTPase activity was also altered by cholesterol incorporation into the nuclear membrane. Cholesterol enrichment of the nuclear membrane also left the nuclei more susceptible to damage by salt-induced lysis than control nuclei. Our results clearly demonstrate that the cholesterol content of the nuclear membrane will have significant, direct effects on nuclear integrity and NTPase activity.

Acid Anhydride Hydrolases↗
PubMed1996

Nuclear cholesterol content and nucleoside triphosphatase activity are altered in the JCR:LA-cp corpulent rat.

A nuclear pore complex-associated nucleoside triphosphatase (NTPase) activity is believed to provide energy for nuclear export of poly(A)+ mRNA. This study was initiated to determine if nuclear membrane lipid composition is altered during chronic hyperlipidemia, and what effect this has on NTPase activity. The JCR:LA-cp corpulent rat model is characterized by severe hypertriglyceridemia and moderate hypercholesterolemia, and thus represents an ideal animal model in which to study nuclear cholesterol and NTPase activity. NTPase activity was markedly increased in purified hepatic nuclei from corpulent female JCR:LA-cp rats in comparison to lean control rats as a function of assay time, [GTP], [ATP], and [Mg2+]. Nuclear membrane cholesterol and phospholipid content were significantly elevated in the corpulent animals. Nuclei of corpulent animals were less resistant to salt-induced lysis than nuclei of lean animals, suggesting a change in relative membrane integrity. Together, these results indicate that altered lipid metabolism in a genetic corpulent animal model can lead to changes in nuclear membrane lipid composition, which in turn may alter nuclear membrane NTPase activity and integrity.

Acid Anhydride Hydrolases↗
PubMed1996

The presence and partitioning of calcium binding proteins in hepatic and cardiac nuclei.

Calcium is an important signal in key nuclear events, including cell cycle timing regulation of gene expression and activation of nuclear kinases and phosphatases. It is therefore important to identify calcium binding proteins in the nucleus which may play roles in these functions, and to determine whether these proteins are located in the nuclear envelope or in the nucleoplasm. Rat hepatic and pig cardiac nuclei were isolated and treated with a high salt solution to separate nucleoplasmic proteins from those associated with the nuclear envelope. The presence of calcium binding proteins was then revealed by Stains-All, staining of electrophoretic gels and 45Ca2+ overlays of Western blots. Four major calcium binding proteins were putatively identified in the pig cardiac nuclei, and another three in the rat hepatic nuclei. Proteins of 110, 93 and 35 kDa were observed in the pig cardiac nuclear envelope fraction, and another of 55 kDa in the pig cardiac high salt fraction. A 93-kDa protein was observed in the rat hepatic nuclear envelope fraction, and others of 120 and 110 kDa in the rat hepatic high salt fraction. A tentative identification has been made of the 93-kDa protein in each tissue type as calnexin, and of the cardiac 55 kDa protein as calsequestrin. This study, therefore, has putatively identified for the first time the presence of several calcium binding proteins which have distinct partitioning within hepatic and cardiac nuclei. This localization may play an important functional role within the nuclei.

Animals↗
PubMed1995

Na(+)-Ca2+ exchange and Ca2+ channel characteristics in bovine aorta and coronary artery smooth muscle sarcolemmal membranes.

Tension generation and Ca2+ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na(+)-Ca2+ exchanger and the Ca2+ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The Km and Vmax of Na(+)-dependent Ca2+ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [3H] PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher Kd and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [3H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca2+ channel characteristics in isolated sarcolemmal membranes.

Animals↗
PubMed1995

The contribution of ionic imbalance to ischemia/reperfusion-induced injury.

Recent data from a number of independent laboratories have implicated ionic disturbances within the myocardium as being a critical contributory factor in injury to the heart during ischemia/reperfusion. The present treatise discusses the data supporting a role for Ca2+, Na+ and K+ in ischemic/reperfusion injury. The mechanism whereby intracellular ionic homeostasis becomes altered is the focus of the review. In particular, the evidence in support of an involvement of abnormal ion movements through Na+/H+ exchange, Na+ channels and the ATP-sensitive K+ channel in ischemic/reperfusion injury is advanced.

Animals↗
PubMed1995

Adult cardiomyocytes express functional high-affinity receptors for basic fibroblast growth factor.

As a first step in addressing the question of function for basic fibroblast growth factor (bFGF) in the adult myocardium, expression of bFGF receptors by adult rat myocytes was investigated. Cross-linking of 125I-labeled bFGF to purified sarcolemmal vesicles from adult hearts indicated specific binding to 90- to 104-kDa proteins, whereas equilibrium binding studies revealed the presence of "low"-affinity (1 nM) and "high"-affinity (115 pM) sites. Adult myocytes were found to express short and long variants of bFGF receptor 1 (FGFR-1, tyrosine kinase) mRNA. Adult heart overall levels of FGFR-1 mRNA were decreased by about one-third of corresponding fetal values. Several lines of evidence indicated that bFGF receptors in adult cardiomyocytes in situ and/or in isolation are functional. Isolated adult myocytes were found to be capable of heparin-resistant internalization of 125I-labeled bFGF, to lose their viability after interaction with bFGF-saporin (a mitotoxin known to kill cells after entry via the bFGF receptor), and to respond to bFGF by activation of mitogen-activated protein kinase. In addition, introduction of exogenous bFGF into the myocardium by Langendorff perfusion resulted in stimulation of tyrosine phosphorylation in association with cardiomyocyte intercalated disks, as assessed by immunofluorescence. It is concluded that adult cardiomyocytes express functionally coupled high-affinity bFGF receptors and that they are capable of a biologic response to bFGF in vivo.

Animals↗
PubMed1995

Involvement of lipoproteins, free radicals, and calcium in cardiovascular disease processes.

Low density lipoproteins have the capacity to alter cell calcium concentrations of a wide variety of cell types. The precise mechanism of physical interaction of the LDL or its products with the cell is not defined yet. There is more agreement about the calcium transport pathway affected. Most studies show an ability of LDL to alter transsarcolemmal calcium flux, probably through the slow calcium channel. However, internal stores of calcium like the sarcoplasmic reticulum also appear to be altered. These effects of LDL on cellular calcium homeostasis have potential importance in both physiological and disease settings. Oxidised LDL has even more potential significance than the native LDL in disease conditions. Free radical mediated oxidation of LDL during atherosclerotic and ischaemic conditions creates a molecule with even greater potency for altering cell calcium and ultimately cell function and viability. The interaction of this trio of compounds--LDL, free radicals, and cellular calcium--may be critical for the mechanism of pathogenesis in specific cardiovascular diseases. It will be important in the future to identify new therapeutic strategies to prevent their interaction.

Animals↗
PubMed1995

Induction of expression of the sodium-hydrogen exchanger in rat myocardium.

OBJECTIVE: The aim was to examine the regulation of the cardiac Na+/H+ exchanger NHE-1 isoform mRNA in response to ischaemia and acidosis in the mammalian myocardium. METHODS: Male Sprague Dawley rat hearts were perfused in a non-circulated retrograde fashion according to the Langendorff method. Hearts were perfused for 3 h at flow rates of either 10 ml.min-1 (control), or 3, 1, or 0 ml.min-1 (ischaemia) followed by 5 min of reperfusion. Hearts were immediately frozen in liquid N2, and stored at -80 degrees C until ready for RNA isolation. Northern blot analysis was used to examine expression of the NHE-1 isoform of the Na+/H+ exchanger message in these isolated perfused hearts. Activity of the Na+/H+ exchanger was assessed in primary cultures of neonatal rat myocytes under either control conditions or after treatment with chronic, low external pH. RESULTS: A decrease in developed tension and an increase in resting tension was observed which was dependent upon the severity of the ischaemic episode. Low flow ischaemia of 3 ml.min-1 caused increased Na+/H+ exchanger message levels, while perfusion at more reduced flow rates eliminated the increase. Treatment of primary cultures of isolated myocytes with low external pH resulted in increased ability to recover from an acute acid load. CONCLUSIONS: Low flow ischaemia can increase the Na+/H+ exchanger message in the intact mammalian myocardium. More severe ischaemia prevents the increase, suggesting that severely damaged tissue may not be capable of the ischaemic response. Primary cultures of isolated myocytes can respond to chronic low external pH by increasing Na+/H+ exchanger activity.

Acidosis↗
PubMed1994

Calcium and calcium-binding proteins in the nucleus.

Calcium has long been known to play a role as a key cytoplasmic second messenger, but until relatively recently its possible involvement in nuclear signal transduction and the regulation of nuclear events has not been extensively studied. Evidence revealing the presence of transmembrane nuclear Ca2+ gradients and a variety of intranuclear Ca2+ binding proteins has fueled renewed interest in this key ion and its involvement in cell-cycle timing and division, gene expression, and protein activation. This review will offer an overview of the current state of knowledge and theory regarding calcium orchestration of nuclear functions and events and discuss possible future directions in this field of study.

Calcium↗
PubMed1994

Effect of high-dose verapamil administration on the Ca2+ channel density in rat cardiac tissue.

It is well known that beta-adrenergic receptors will down-regulate in the presence of high circulating levels of beta-adrenergic agonists over extended periods of time. However, less is known with respect to the effect of Ca2+ channel antagonist on their receptors. The purpose of this study was to determine if chronic administration of high dosages of verapamil (in the toxic range) could alter the density of Ca2+ channels in the heart as determined by [3H]PN 200-110 binding. A range of high verapamil concentrations was administered to rats via s.c. implantable slow-release pellets or s.c. injection. An increasing rate of mortality was observed as the dose of verapamil administered increased. Quantitation of serum verapamil concentrations demonstrated that the s.c. slow release implantable pellets were not releasing the drug evenly and instead released toxic quantities of drug during the first 24 h after implantation. Serum verapamil levels determined from verapamil-injected animals demonstrated a dose-dependent increase in circulating levels. No significant alterations in Ca2+ channel characteristics (Bmax and Kd) were noted in cardiac tissue obtained from either treatment regime. Our results demonstrate that implantable pellets are not a reliable administration method for verapamil and cardiac Ca2+ channels are unusually resistant to biochemical alterations even after administration of verapamil dosages in the toxic range.

Animals↗
PubMed1993

The action of oxidized low density lipoprotein on calcium transients in isolated rabbit cardiomyocytes.

The effect of oxidized low density lipoprotein (oxLDL) on the Ca2+ transients of isolated rabbit cardiomyocytes was assessed. LDL was oxidized by a free radical generating system in vitro. The systolic Ca2+ concentration in transients was significantly increased after treatment with 100 micrograms of oxLDL cholesterol/ml for 16 min without having any effect on the diastolic [Ca2+]. However, a toxic effect was observed when the concentration of oxLDL was increased to 1,000 micrograms/ml. Cells treated with 100 micrograms of oxLDL/ml were more sensitive to the blocking action of nicardipine on the Ca2+ transient than were control cardiomyocytes. Furthermore, oxLDL failed to produce a stimulatory effect on the transient when the extracellular [Ca2+] was lowered. The malondialdehyde content in the oxLDL correlated well with the change in systolic [Ca2+] of treated cells. Lazaroid, a novel antioxidant, completely inhibited malondialdehyde formation in the oxLDL and prevented the increment of systolic [Ca2+] in the treated cells. The data indicate that oxLDL can induce relatively rapid alterations in cellular Ca2+ transients via a modification of Ca2+ entry through the L-type Ca2+ channel. LDL oxidation induced by free radicals may play an important role in influencing cardiac contractile function during pathological conditions such as ischemia/reperfusion challenge.

Animals↗
PubMed1993

Identification and purification of a calcium-binding protein in hepatic nuclear membranes.

Recent evidence suggests that nuclei possess Ca2+ transport mechanisms to regulate nucleoplasmic/cytosolic Ca2+ gradients. We, therefore, investigated the possibility that Ca(2+)-binding proteins may also exist within the nucleus. Electrophoretic analysis revealed the presence of an acidic 93-kDa protein (p93) in the membranes of isolated nuclei. p93 stained blue with "Stains-All" in SDS-polyacrylamide gels and was the major 45Ca(2+)- and ruthenium red-binding nuclear envelope protein in electroblot overlays. p93 was resistant to extraction by 6 M urea but was solubilized in 2% Triton X-100. Citric acid was highly effective in removing the outer nuclear membrane (ER) with concomitant reduction (< 10-fold) of mannose-6-phosphatase activity, but not p93. 45Ca(2+)-binding assays of purified p93 revealed the presence of high capacity Ca(2+)-binding sites comparable to calreticulin. This evidence strongly suggests that p93 is a major Ca(2+)-binding protein of the inner nuclear envelope membrane. Partial amino acid sequence analysis revealed that p93 was close to 100% homologous with a recently identified ER Ca(2+)-binding protein known as calnexin. It is likely, therefore, that p93 is calnexin. However, mild CHAPS detergent treatment of nuclear envelopes and ER revealed distinctly different solubility properties of each membrane for the extraction of p93. This, together with the citrate data, strongly suggests that p93/calnexin, in isolated nuclear envelopes, is mostly bound to the inner membrane. It is possible that p93 may be involved with the regulation of Ca2+ transients between the nucleoplasm and perinuclear space.

Amino Acid Sequence↗
PubMed1993

The effects of low density lipoprotein on calcium transients in isolated rabbit cardiomyocytes.

The purpose of this study was to examine the effects of low density lipoprotein (LDL) on Ca2+ transients of isolated rabbit cardiomyocytes. Incubation of cardiomyocytes with > or = 1 mg of LDL cholesterol/ml of perfusion medium induced a slow (> or = 30 min) but significant increase (2-fold) in the cellular Ca2+ transient. The time course for the effect was similar to that observed for the accumulation of cholesterol in the cells. Using Dil- labeled LDL as a fluorescent marker for LDL interaction with the cardiomyocytes, it was concluded that LDL interacted via a receptor-mediated event, but probably this was not the primary mechanism whereby the lipid entered the cell. LDL-treated cells were resistant to the depressant actions for ryanodine, nicardipine, and dichlorobenzamil on the cellular Ca2+ transient. Lowering the extracellular Ca2+ concentration removed the stimulatory effect of LDL on the Ca2+ transient. It is concluded that LDL can induce an increase in the magnitude of the Ca2+ transient in isolated cardiomyocytes. This is a relatively slow process. The mechanism appears to involve a stimulation of a transsarcolemmal Ca2+ transport pathway. These findings have important implications for cardiac contractile function in hypercholesterolemic and drug-treated hypercholesterolemic subjects.

Amiloride↗
PubMed1993

Effect of amiloride and selected analogues on postischemic recovery of cardiac contractile function.

The purpose of the present study was to compare the protective effects of amiloride and three of its derivatives (which are selective inhibitors of Na(+)-H+ exchange) during postischemic reperfusion. Previously, amiloride has been shown to have a protective effect on ischemia-reperfusion injury. However, because of its nonselective actions, the mechanism of its effect is unclear. 5-(N,N-dimethyl)-amiloride (DMA) is also protective and appears to act via inhibition of the Na(+)-H+ exchanger. However, corroborative effects using other selective Na(+)-H+ exchange blockers are needed. Amiloride, DMA, ethylisopropyl amiloride (EIPA), and 5-(N,N-hexamethylene)-amiloride (HMA) were included for 10 min in the reperfusion period after 60 min of global ischemia in the rat right ventricular wall. Peak developed tension and the rates of tension generation and relaxation were significantly improved during reperfusion in the presence of 100 microM amiloride, 10 microM DMA, 2.5 microM HMA, or 1 microM EIPA compared with those of drug-untreated muscles. Contracture formation was significantly depressed in the presence of these drug concentrations as was release of creatine kinase from the ventricular wall into the coronary effluent. The efficacy of these drugs for protecting the right ventricular wall from postischemic contractile dysfunction correlates well with their potency as blockers of Na(+)-H+ exchange. The results provide further evidence in support of a role for Na(+)-H+ exchange. The results provide further evidence in support of a role for Na(+)-H+ exchange in determining ischemia-reperfusion injury in the heart.

Amiloride↗
PubMed1993

Ischemic cardioprotection by ATP-sensitive K+ channels involves high-energy phosphate preservation.

We previously demonstrated that ATP-sensitive K+ channels (KATP) protect the guinea pig myocardium against ischemia-reperfusion injury (Cole et al., Circ. Res. 69: 571-581, 1991), but the cellular alterations leading to ischemic injury affected by KATP remain to be defined. This study investigates the relationship between activation of KATP and preservation of high-energy phosphates during global no-flow ischemia in arterially perfused guinea pig right ventricular walls. Electrical and mechanical activity were recorded via intracellular microelectrodes and a force transducer. Glibenclamide (10 and 50 microM) and pinacidil (10 microM) were used to modulate KATP. ATP and creatine phosphate (CP) levels were determined at the end of no-flow ischemia by enzymatic analysis. Preparations were subjected to 1) 20 min no-flow +/- glibenclamide (10 or 50 microM), 2) 30 min no-flow +/- pinacidil (10 microM) or pinacidil (10 microM) and glibenclamide (50 microM), or 3) 40 or 50 min of control perfusion before rapid freezing in liquid nitrogen. Pinacidil (10 microM) enhanced ischemic shortening of action potential duration (APD) and early contractile failure, prevented ischemic contracture, and inhibited high-energy phosphate depletion during ischemia. Glibenclamide (50 microM) inhibited the effects of pinacidil (10 microM) on electromechanical function and preservation of ATP and CP. Glibenclamide (10 microM) alone inhibited the early decline in APD and produced earlier ischemic contracture but did not enhance ATP or CP depletion compared with untreated tissues during 20 min of no-flow. Glibenclamide (50 microM) produced a greater inhibition of APD shortening in early ischemia, further decreased the latency to ischemic contracture, and caused enhanced ischemic depletion of ATP. The data indicate the changes in electrical activity induced by KATP indirectly preserve high-energy phosphates and reduce injury associated with ischemia. However, the data also suggest the possible presence of additional mechanisms for cardioprotection by KATP.

Action Potentials↗
PubMed1993

Interference of N-(1-[4-(4-fluorophenoxy)butyl]-piperidinyl)-N-methyl- 2-benzothiazolamine with Na+/H+ exchange and Na+/Ca2+ exchange in purified cardiac sarcolemmal membranes.

The effect of R 56865 (N-[1-[4-(4-fluorophenoxy)butyl]-piperidinyl]-N-methyl-2- benzothiazolamine, CAS 104606-13-5) on Na+/H+ exchange and Na+/Ca2+ exchange was studied in isolated cardiac sarcolemmal vesicles. R 56865 inhibited Na+/H+ exchange with an ED50 of 180 mumol/l at a concentration of 0.05 mmol/l Na+. The potency of R 56865 decreased with increasing concentrations of Na+. Na+/Ca2+ exchange was also inhibited. The ED50 amounted to 45 mumol/l. This ED50 was close to that of the reference compound dichlorobenzamil (ED50 = 27 mumol/l). The inhibitory potency of R 56865 on Na+/H+ and Na+/Ca2+ exchange is far too low to explain the already reported beneficial effects of this inhibitor of Na+ and Ca+ overload during ouabain intoxication and ischemia and reperfusion.

Amiloride↗
PubMed1993

Modulation of cardiac performance by amiloride and several selected derivatives of amiloride.

Amiloride and its derivatives (benzamil, dichlorobenzamil, 5-(N,N-dimethyl)-amiloride, 5-(N-ethyl-N-isopropyl)-amiloride, (N,N-hexamethylene)- amiloride and 5-(N-methyl-N-isobutyl)-amiloride) are commonly used as selective blockers of Na+/Ca++ exchange or Na+/H+ exchange. Very little information is currently available regarding their effects on cardiac performance. It was observed that addition of amiloride or any of the selected derivatives to the coronary perfusate of the right ventricular wall produced a potent depressive effect on peak developed tension and the rates of tension generation and dissipation. The concentrations at which this occurred are those that are commonly used in ischemia or hypoxia studies. Significantly, the depressive action of the drugs increased with the perfusion duration and never achieved a stable level. An initial, transient positive inotropic effect was observed with some of the drugs. If the drug concentration and perfusion time was limited, the effects were reversible. All of the drugs except amiloride produced extra systoles. The drugs were capable of blocking Ca++ transients in isolated cardiomyocytes but had little effect on intracellular pH. The drugs lengthened the action potential duration and decreased the action potential amplitude and upstroke velocity. Their effects on cardiac performance may involve a complex inhibition of Ca++ influx and K+ efflux in addition to a stimulation of a nonselective cation current. It is concluded that amiloride and its analogs have striking effects on cardiac performance which may be unrelated to their capacity to inhibit Na+/Ca++ or Na+/H+ exchange. In summary, the use of these drugs is not normally recommended in cell or tissue perfusion experiments because of their nonselectivity. However, if the drug concentration and perfusion time is controlled carefully, interpretable data may be obtained in some cases.

Action Potentials↗