Depletion Of Cellular ATP Research Articles

Oxygen deprivation and reoxygenation augment prostacyclin synthesis in cultured ventricular myocytes

Prostacyclin production in cultured cardiomyocytes is not induced by cellular ATP depletion per se, suggesting that the mechanism of ischemic injury is more complex. In the present study we subjected cultured ventricular myocytes to ‘simulated ischemia’ followed by reoxygenation. A slight increase in 6-keto-PGF 1α (the stable metabolite of PGI 2) was found during ‘ischemia’, which continued to increase markedly during reoxygenation. PGE 2 levels were pronouncedly enhanced during ischemia but decreased during reoxygenation, and TXB 2 levels remained undetectable throughout. These findings reflect a cardiomyocyte response to anoxic injury, suggesting that they act to protect against cardiac injury by producing the potent vasodilators PGI 2 and PGE 2 during ischemia and reoxygenation.

ATP depletion causes translational immobilization of cell surface transferrin receptors in K562 cells.

We have used quantitative fluorescence microscopy and fluorescence photobleaching recovery to examine the role of metabolic energy in the translational movement of transferrin receptors in the plasma membrane of K562 erythroleukemia cells. Cellular ATP depletion caused a significant decrease in the translational mobility of cell surface transferrin receptors and a significant increase in the number of receptors on the cell surface. ATP repletion restored receptor translational mobility and cell surface expression to control values. Inhibition of ATP hydrolases by orthovanadate also immobilized cell surface transferrin receptors and altered cell surface receptor expression, in a concentration-dependent manner. Vanadate-induced changes in receptor mobility and cell surface expression were reversible upon washing out the drug. Cellular ATP depletion did not affect the translational mobility of plasma membrane glycophorins or a fluorescent phospholipid analogue. We conclude that the translational movement of cell surface transferrin receptors specifically requires metabolic energy and ATP hydrolysis.

Alterations in reactive oxygen, pH, and calcium in astrocytoma cells during lethal injury.

Exposure of cultured human astrocytoma cells to iodoacetic acid results in rapid depletion of cellular ATP and cell death. Pathophysiological changes in the injured cells, including formation of reactive oxygen species (ROS), cell viability, glutathione, pH, and cytosolic calcium, were characterized at the cellular level via fluorescence microscopy. After iodoacetic acid treatment, cellular ATP and intracellular glutathione fell sharply to undetectable levels within 2 h. ROS, as detected by the oxidation of dichlorofluorescein, appeared in 20 min and reached a maximum before the loss of membrane integrity. Cells became acidotic within 10 min. Cytosolic free calcium concentration exhibited a slow increase and then a sharp influx shortly before the rupture of the cell membrane. The addition of lipophilic antioxidants, nordihydroguaiaretic acid or the troloxamine U-78517F, eliminated the accumulation of ROS and delayed the onset of cell death without affecting other parameters observed in the early phase of the injury. We conclude that ROS is formed and may play important roles during lethal cell injury caused by energy depletion.

Efflux of lithocholate and its sulfate and glucuronide from isolated rat hepatocytes is normal in EHBR and is not affected by cellular ATP depletion

The efflux of lithocholate and its sulfate and glucuronide and taurocholate was studied in isolated rat hepatocytes. The velocity of the initial efflux rate from hepatocytes was in the order of taurocholate > lithocholate > lithocholate-3- O-glucuronide > lithocholate-3-sulfate. The efflux rate of these bile acids did not change in hepatocytes isolated from EHBR (Eisai hyperbilirubinemic mutant rat). Cellular ATP-depletion by fructose or rotenone did not affect the efflux rate of these bile acids. These findings seem to indicate that the efflux of these bile acids from isolated rat hepatocytes mainly reflects the sinusoidal efflux rather than the canalicular excretion, and that the efflux rate of bile acids is reduced by glucuronidation and sulfation.

Hypothesis on cellular ATP depletion and adenosine release as causes of heart failure and vasodilatation in cardiovascular beriberi

Cardiovascular beriberi is a syndrome caused by thiamine deficiency and characterized by systemic vasodilatation, heart failure and lactic acidosis. The occurrence of heart failure and vasodilatation is yet unexplained: neither theoretical nor experimental data are known. In this article, it is suggested that a fall of cellular ATP levels causes heart failure and that the release of adenosine is the cause of vasodilatation.

Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein.

The eukaryotic organism Dictyostelium discoideum has become one of the organisms of choice for the overexpression of recombinant myosins and myosin fragments. Here, we describe a protocol that facilitates the screening of cells that have been transformed with myosin expression constructs and allows the rapid purification of recombinant myosins. Depletion of cellular ATP is used to recruit most of the endogenous and recombinant myosin into a rigor-like complex with actin. Following cell lysis the insoluble actomyosin complex is precipitated by centrifugation, washed, and Mg(2+)-ATP is added to extract the recombinant protein from the pellet. More than 90% of the protein in the resulting supernatant corresponds to actin, myosin, and the recombinant myosin fragments. Therefore, it is easy to detect any differences in expression level between individual myosin constructs on SDS-polyacrylamide gels. Additionally, the dependence of expression on external factors, such as cell density, can be readily determined. Furthermore, the presence of a band corresponding to the recombinant protein indicates that the overexpressed protein has at least some of the functional properties that are characteristic for a myosin motor. This rapid and selective extraction protocol can also be utilized to facilitate the purification of recombinant myosin motors on a preparative scale and has proved particularly useful in the purification of myosin head fragments, that are tagged with histidine residues, by Ni(2+)-chelate affinity chromatography.

Fusion of cationic liposomes with mammalian cells occurs after endocytosis

The interaction of cationic liposomes prepared using either dioleoyltrimethylammonium propane (DOTAP) or 3β-( N-( N′ , N′-dimethylaminoethane)carbamoyl)cholesterol (DC-CHOL) with model membranes and with cultured mammalian cells was examined using an assay developed for monitoring virus-cell fusion (Stegmann et al. (1993) Biochemistry 32, 11330–11337). Lipid mixing between cationic liposomes and liposomes composed of DOPE/dioleoylphosphatidylglycerol (DOPG) or dioleoylphosphatidylcholine (DOPC)/DOPG was insensitive to pH in the range of pH 4.5–7.0 and was not affected by sodium chloride concentration in the range of 0–150 mM. Lipid mixing was dependent on dioleoylphosphatidylethanolamine (DOPE), since cationic liposomes prepared using dioleoylphosphatidylcholine (DOPC) were incapable of lipid mixing with DOPC/DOPG liposomes. The interaction of cationic liposomes with Hep G-2 and CHO D − cells was also studied. For both cell types, liposome-cell lipid mixing was rapid at 37° C, beginning within minutes and continuing for up to 1 hour after uptake. The extent of lipid mixing was decreased at 15° C, especially at later (≥ 20 min) time points. This suggests that at least part of the observed lipid mixing occurred after reaching cellular lysosomes. No lipid mixing was seen at 4° C. Monensin inhibited lipid mixing between cationic liposomes and the cells, despite having no effect on liposome uptake. Inhibition of endocytic uptake of liposomes, either by incubation in hypertonic media or by depletion of cellular ATP with sodium azide and 2-deoxyglucose abolished liposome-cell fusion in both cell types. These data demonstrate that binding to the cell surface is insufficient for cationic liposome-cell fusion and that uptake into the endocytic pathway is required for fusion to occur.

Effects of organic anions and bile acids on hepatocellular uptake of lithocholate-3-sulfate

We previously reported that hepatic uptake of lithocholate-3-sulfate is mediated by a Na +-independent system, as has been reported with various organic anions and unconjugated bile acids. Herein, we examined the effects of organic anions and bile acids on hepatic lithocholate-3-sulfate uptake using isolated rat hepatocytes. Lithocholate-3-sulfate uptake was inhibited by sulfobromophthalein, indocyanine green and rose bengal. By contrast, lithocholate-3-sulfate uptake was not inhibited by dibromosulfophthalein, the uptake of which has been reported to be ATP-dependent, or by cholate. Cellular ATP depletion had no effect on lithocholate-3-sulfate uptake. These findings suggest that hepatic lithocholate-3-sulfate uptake is mediated by a Na +-independent system common for organic anions such as sulfobromophthalein, which is not facilitated by ATP.

ATP-dependent Regulation of Sodium-Calcium Exchange in Chinese Hamster Ovary Cells Transfected with the Bovine Cardiac Sodium-Calcium Exchanger

Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na+]i and stimulate Ca2+ influx by Na/Ca exchange. Depletion of cellular ATP inhibited 45Ca uptake by 40% or more and reduced the half-maximal Na+ concentration for inhibition of 45Ca uptake from 90 to 55 mM. ATP depletion also reduced the rate of rise in [Ca2+]i when [Na+]o was reduced and inhibited the decline in [Ca2+]i when high [Na+]o was restored. The effects of ATP depletion were either absent or reduced in cells expressing a mutant exchanger missing most of the cytosolic hydrophilic domain. We were unable to detect a phosphorylated form of the exchanger in immunoprecipitates from 32P-labeled cells. ATP depletion caused a breakdown in the actin cytoskeleton of the cells. Treatment of the cells with cytochalasin D mimicked the effects of ATP depletion on the [Na+] inhibition profile for 45Ca uptake. Thus, ATP depletion inhibits both the Ca2+ influx and Ca2+ efflux modes of Na/Ca exchange, and may alter the competitive interactions of extracellular Na+ and Ca2+ with the transporter. The latter effect appears to be related to changes in the actin cytoskeleton.

Flow cytometric analysis of transmembrane phospholipid movement in bull sperm.

Fluorescent phospholipids are useful to investigate phospholipid dynamics in biological membranes. We used flow cytometry to investigate transbilayer phospholipid movement in live sperm cells. Acyl-labeled N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) -phosphatidylcholine (-PC), -phosphatidylethanolamine (-PE), or -phosphatidylserine (-PS) were incorporated into sperm cells, and the transbilayer location was determined by extraction of probe from cell with excess bovine serum albumin (BSA) or by chemical destruction of probe by sodium dithionite. Using these methods, we have measured the head group specific outer leaflet to inner leaflet movement (flip) of the aminophospholipids NBD-PS and NBD-PE. The fluorescent phospholipids moved inward across the plasma membrane with half-times of 1.8, 2.5, and 11.2 min, for NBD-PS, NBD-PE, and NBD-PC and reached apparent equilibrium levels of 88%, 94%, and 32% inside, respectively. The inward movement of NBD-PE was inhibited by sulfhydryl reagents, elevated intracellular Ca2+, and depletion of cellular ATP. Analysis of the kinetics of NBD-PE and -PS extraction by BSA allows determination of the rates for outward movement (flop) across the plasma membrane. Half-times for flop were 4.7 and 4.5 min for NBD-PS and -PE, respectively. Based on these measurements, a simple model of NBD-phospholipid equilibria was developed and fit to the kinetic data. Computer-generated fits reflected major features of the experimental data and provide a potential tool for predicting the dynamics of endogenous lipids.

The roles of multiple pathways in regulating bombesin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts.

The regulation of bombesin-stimulated phospholipase D (PLD) activity in Swiss 3T3 fibroblasts was examined. Increasing protein-tyrosine phosphorylation by using pervanadate to inhibit tyrosine phosphatases was found to stimulate protein kinase C (PKC)-independent [3H]phosphatidylbutanol ([3H]PtdBut) accumulation within 5 min, which continued to increase up to 30 min. The stimulation of PLD activity in response to submaximal [bombesin] could be decreased by approx. 50% by the tyrosine kinase inhibitor genistein, whereas pretreatment with genistein and the PKC inhibitor Ro-31-8220 completely abolished the generation of [3H]PtdBut in response to a maximal concentration of bombesin. The addition of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) into permeabilized cells resulted in an increase in [3H]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 microM GTP[S] did not increase the stimulation of PLD activity by any [bombesin] tested, whereas it was synergistic with that stimulated in response to phorbol 12-myristate 13-acetate. These findings suggest that bombesin-stimulated PLD activity is indirectly regulated by G-proteins, possibly through a kinase intermediate. Furthermore, activation of protein tyrosine kinases is proposed to account for the PKC-independent arm of bombesin-stimulated PLD activity. No evidence was obtained for a form of PLD directly regulated by tyrosine phosphorylation.

Intracellular pH in vascular smooth muscle: regulation by sodium-hydrogen exchange and multiple sodium dependent HCO3− mechanisms

The aim was to determine the mechanisms, particularly bicarbonate dependent mechanisms, of intracellular pH (pHi) recovery from various acidoses in vascular smooth muscle and to explore the ATP dependency of the respective mechanisms. Experiments were conducted in rat aortic smooth muscle cells grown in primary culture and synchronised in a non-growing state by serum deprivation. pHi was measured in cells loaded with the pH sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF). Chloride efflux was studied by determination of the rate of efflux of 36Cl over 5 min. Cells were ATP depleted by substitution of glucose in the medium by 2-deoxyglucose. Acidoses were induced by CO2 influx and NH3 efflux techniques. In the absence of HCO3-, the 5-(N-ethyl-N-isopropyl) amiloride (EIPA) sensitive Na+/H+ exchange accounted for the recovery from intracellular acidosis. In the presence of HCO3- ions the response to respiratory acidosis (CO2 influx) was predominantly via activation of Na+/H+ exchange and an EIPA sensitive Na+ and HCO3- dependent mechanism. A 4-acetamido-4'-isothiocyanostilbene-2',2'-sulphonic acids (SITS) sensitive Na+ dependent Cl-/HCO3- mechanism which is also sensitive to EIPA makes a small contribution during severe intracellular acidosis. Under such conditions HCO3- dependent mechanisms contributed about 40% to the overall pHi regulating capacity of vascular smooth muscle cells. However, under conditions which deplete cellular ATP these pHi regulating mechanisms account for virtually all of theses cells' ability to regulate pHi. The inability of Na+/H+ exchange to participate in pHi recovery under these circumstances, reduces the ability of vascular smooth muscle cells to recover pHi by approximately 50-60%. Chloride efflux was approximately linear over 5 min and was increased by 36% in the presence of extracellular HCO3-. Efflux in the presence of HCO3- was inhibited similarly by both SITS and EIPA. At least three transporters contribute to recovery from acidosis in vascular smooth muscle: Na+/H+ exchange, an Na(+)-HCO3- cotransporter which is sensitive to EIPA, and an Na+ dependent HCO3-/Cl- exchange sensitive to both SITS and EIPA. The Na(+)-HCO3- cotransporter appears to be similar to that described in human vascular smooth muscle. When the Na+/H+ exchanger is attenuated by cellular ATP depletion, the alternative pathways, particularly the Na(+)-HCO3- cotransporter, ensure that substantial pHi regulatory capacity is maintained.

Signal transduction for nuclear factor-kappa B activation. Proposed location of antioxidant-inhibitable step.

Reactive oxygen species are thought to be messengers for nuclear factor (NF)-kappa B activation because its activation can be abrogated by antioxidants. However, this study identifies, for the first time, NF-kappa B activators that are insensitive to antioxidants. NF-kappa B activation that is induced by either calyculin A or okadaic acid (inhibitors of serine/threonine protein phosphatases 1 and 2A) is not blocked by N-acetylcysteine or dihydrolipoate in Jurkat and U937 cells. Nonetheless, these antioxidants block induction by TNF-alpha, lymphotoxin, and PMA. Unlike okadaic acid and calyculin A, neither TNF-alpha, lymphotoxin, nor PMA inhibited activities of phosphatases 1 and 2A. NF-kappa B activation induced by okadaic acid or calyculin A was not blocked by a myosin light chain kinase inhibitor, but was prevented by a protease inhibitor. The mitochondrial inhibitor, rotenone, also inhibited NF-kappa B activation by calyculin A; however, this inhibition was accompanied by a depletion of cellular ATP. These results suggest that 1) phosphatase inhibitors either target a component of signal transduction, which occurs downstream to an antioxidant-sensitive step or use distinct signaling pathways; 2) inhibition of phosphatases 1 and 2A is not a step in the pathway of TNF-alpha-, lymphotoxin-, or PMA-induced NF-kappa B activation; 3) myosin light chain kinase does not participate in NF-kappa B activation; and 4) activation of NF-kappa B by phosphatase inhibitors is controlled by proteases.

Decreased protein phosphorylation induced by anoxia in proximal renal tubules.

Anoxia-induced depletion of cellular ATP may affect the degree of protein phosphorylation due to kinase inhibition. In this study, protein phosphorylation was measured in rabbit kidney proximal tubules under normoxic or anoxic conditions in a medium containing 32P. During the first 20 min of normoxia, phosphate incorporation was linear, averaging 17 +/- 5 pmol.mg protein-1.min-1 and was 70% inhibited by the protein kinase C inhibitor chelerythrine chloride. Phosphorylation measurements initiated simultaneously with anoxic conditions (95% N2-5% CO2) significantly reduced the initial rate to 58% of control, saturating after 15 min, and reaching 28 +/- 5% of the normoxic value after 60 min of incubation. The phosphatase inhibitor calyculin A did not affect the initial rate of phosphate incorporation by anoxic tubules but increased phosphate incorporation at 60 min to 43 +/- 4% of normoxia. Addition of 32P after 15 min of anoxia abolished phosphate incorporation, demonstrating that kinase activity was completely inhibited. Cellular phosphate uptake was measured and found not to be rate limiting for phosphorylation. Chelerythrine chloride increased lactate dehydrogenase (LDH) release during normoxia, and calyculin A decreased anoxia-induced LDH release, suggesting that protein phosphorylation events may control plasma membrane permeability.

Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation.

Four distinct isoforms of the mammalian Na+/H+ exchanger (NHE) have been identified by molecular cloning. Three of these (NHE-1, NHE-2, and NHE-3) have been shown to be functionally active by heterologous expression. Their kinetic and pharmacological properties are well documented, yet comparatively little is known about their regulation. In this report, rat NHE-1, NHE-2, and NHE-3 were stably transfected into antiporter-deficient Chinese hamster ovary cells to study their role in cellular proliferation and their regulation by nucleotides and cell volume. Their ability to influence cell proliferation was assessed by measuring the growth of antiporter-deficient cells and of the different transfectants in media of varying pH. While antiporter-deficient cells were unable to grow at acidic pH levels, all three isoforms supported proliferation under these conditions. Therefore, while the epithelia-specific isoforms (NHE-2 and NHE-3) are thought to play primarily a role in transcellular ion transport, they can also contribute to intracellular pH (pHi) homeostasis and have a permissive role in cell growth. The activity of the three isoforms was markedly inhibited by depletion of cellular ATP. In the pHi 6.0-7.2 range, decreases in the affinity for internal H+ and/or the maximal rate of transport accounted for the inhibitory effect, depending on the isoform. The osmotic responsiveness of the three isoforms was also compared. As reported earlier, NHE-1 was stimulated by hypertonicity. Under similar conditions, NHE-2 was also stimulated to a comparable extent. Conversely, both isoforms were inhibited in hypotonic media. In contrast, NHE-3 was markedly inhibited by hypertonic cell shrinking but was unaffected by hypotonicity. Osmotic inhibition of NHE-3 was rapid, reversible, and apparent throughout the pH range studied. Osmotic inhibition of NHE-3 may play a role in the physiology and pathophysiology of epithelia.

Competitive inhibition by genistein and ATP dependence of daunorubicin transport in intact MRP overexpressing human small cell lung cancer cells

In several multidrug resistant tumor cell lines without overexpression of P-glycoprotein (non-Pgp MDR), a decreased accumulation of drugs has been shown to contribute to resistance. We have recently reported that daunorubicin (DNR) accumulation was decreased in the multidrug resistance-associated protein overexpressing GLC4/ADR non-Pgp MDR small cell lung cancer cell line due to an enhanced energy-dependent efflux which could be inhibited by the isoflavonoid genistein. The purpose of this work was 2-fold: (i) to investigate the mechanism by which genistein inhibits the DNR efflux in the GLC4/ADR cells; and (ii) to characterize the dependence of DNR transport on ATP concentration in intact GLC4/ADR cells. The active transport of DNR in GLC4/ADR cells appeared to be a saturable process with an apparent K m of DNR of 1.4 ± 0.4 μM. Genistein increased the apparent K m value of DNR, suggesting that this agent is a competitive inhibitor of DNR transport. These data provide additional evidence that energy-dependent DNR transport in GLC4/ADR cells is a protein-mediated process. In addition, genistein decreased cellular ATP concentration in a dose-dependent manner in sensitive as well as in resistant cells. Marked inhibition of DNR transport activity in intact GLC4/ADR cells was found when cellular ATP concentration was decreased below 2 mM by sodium azide or 2-deoxy- d-glucose. Thus, since DNR transport in intact GLC4/ADR is already inhibited at modest cellular ATP depletion, a limitation in ATP supply might open ways to make MDR cells more susceptible to drug toxicity.

Modulation of thyroxine uptake and efflux in vitro by temelastine and phenobarbital in cultured hepatocytes from different species, in relation to toxicological effects on the thyroid gland

Toxicological studies in the rat with phenobarbital and temelastine (SK&F 93944) are associated with thyroid lesions, characterized by thyroid stimulated hormone-mediated thyroid follicular cell hypertrophy and hyperplasia. It has previously been demonstrated that these compounds enhance the hepatocellular accumulation and clearance of thyroxine (T 4), in rat but not dog or mouse. In this study these events were further characterized in vitro using cultured hepatocytes from different species. Exposure of rat hepatocytes in vitro to phenobarbital and temelastine produced significant increases ( P < 0.05) in hepatocellular [ 125I] l-thyroxine accumulation, following 3 hr of exposure to either drug (at a concentration of 10 μ m), in the presence of [ 125I]T 4 (0.107 n m final concentration). At this concentration the accumulation of [ 125I]T 4 after xenobiotic exposure was 132.6 ± 1.5% (phenobarbital) and 135 ± 2.0% (temelastine) of control values. There was no apparent xenobiotic-induced cytotoxicity (as determined by the mitochondrial MTT index) up to 20 μ m temelastine and 50 μ m phenobarbital in rat hepatocytes. Experiments performed at 4°C [or under conditions of cellular ATP depletion, induced by 1 μ m carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) treatment] failed to show any such increase in hepatocellular thyroxine accumulation. Pretreatment of hepatocytes with either phenobarbital or temelastine for 3 hr before the addition of radiolabelled thyroxine produced increases in hepatocellular hormone accumulation similar to those observed when [ 125I]T 4 and drug were added to the cultures simultaneously. The earliest time at which any increase in [ 125I]T 4 accumulation was observed after drug exposure was approximately 90 min. Exposure of hepatocytes from guinea pig or beagle dog to phenobarbital or temelastine in vitro failed to produce similar increases in hepatocellular [ 125I]T 4 accumulation, demonstrating species specificity of the xenobiotic effect in vitro.

Systemic 3-nitropropionic acid: long-term effects on locomotor behavior

Systemic administration of 3-nitropropionic acid (3-NP) results in striatal atropy by irreversibly inhibiting the citric acid cycle, and thereby resulting in cellular ATP depletion. The neuropathological outcome following 3-NP injections is thought to resemble that seen in Huntington's disease (HD) [1]. The current study administered systemic injections in 6- and 10-week-old rats of low-dose 3-NP every other 4 days for a period of 28 days in order to investigate the effects on locomotor behavior and striatal D 1 dopamine receptor binding. Experimental and control animals received intraperitoneal injections of 3-NP (10 mg/kg in 0.9% saline) and 0.9% saline, respectively. Animals were tested behaviorally prior to the first and after the last 3-NP administration. Brains were then removed and striatal tissue samples were analyzed for D 1 dopamine receptor binding using [ 3H]SCH23390. Behaviorally, 6-week-old injected animals developed bradykinesia with no signs of stiffness or rigidity, while 10-week-old injected animals displayed an uncoordinated gait, stiffness and ventral recumbency with hind limbs extended in a rigid or fixed position. These visual observations of hypoactivity were supported by a significant decline in both experimental groups' locomotor activity as measured by Digiscan monitors. Furthermore, [ 3H]SCH23390 specific binding to D 1 dopamine receptors revealed a small but significant decrease in 10-week-old injected animals compared to controls. These results demonstrate that both 6- and 10-week-old rats do exhibit behavioral alterations after long-term 3-NP administration, however the former may not show accompanying gross D 1 receptor changes.

Protection of Hepatocytes Against Death Due to Mitochondrial Failure: Effect of Di-Calciphor on Antimycin A-Induced Toxicity

Di-Calciphor is a synthetic derivative of prostaglandin B 1 that protects against cerebral and cardiac ischemia apparently by preserving mitochondrial function. To determine whether di-Calciphor specifically protects against mitochondrial failure, we studied its effects on mitochondrial functions in hepatocytes treated with the specific mitochondrial poison, antimycin A. The results show that 1 μM di-Calciphor protects against cell death at concentrations of antimycin A that inhibited mitochondrial respiration and caused cellular ATP depletion. Di-Calciphor did not protect against loss of ATP but did protect against the loss of mitochondrial Δ ψ and ΔpH. In addition, di-Calciphor protected against antimycin A-induced loading of phosphate into mitochondria and an associated mitochondrial swelling. Thus, these results show that di-Calciphor protects against a specific mitochondrial poison and support the interpretation that di-Calciphor is a mitochondrial protective agent. In addition, the results suggest that the protection of the mitochondria involves preservation of mitochondrial ionic and osmotic stability and does not involve improved ATP supply.

Routes of delivery of muscarinic acetylcholine receptors to the plasma membrane in NG108-15 cells.

1. We have examined the routes of delivery of muscarinic acetylcholine receptors to the plasma membrane in unstimulated and agonist-stimulated NG108-15 cells. Delivery of receptors to the plasma membrane was measured by irreversible alkylating receptors already at the cell surface with propylbenzilylcholine mustard (PrBCM) and then following the recovery of binding of the polar radioligand [3H]-N-methylscopolamine ([3H]-NMS) in intact cells. 2. In unstimulated cells, recovery of [3H]-NMS binding after 2 h of incubation at 37 degrees C was 30% of binding in control cells. Binding affinity of [3H]-NMS was unchanged. In cells that had been pre-exposed to carbachol (0.5 mM) for 30 min, initial [3H]-NMS binding was reduced by 38% but recovery of binding was increased from 30% to 43% of control binding. 3. When the cells were pre-incubated for 1 h with the protein synthesis inhibitor cycloheximide (20 micrograms ml-1), recovery of [3H]-NMS binding was reduced by similar extents in unstimulated (30% to 9%) and carbachol-stimulated (43% to 19%) cells. Incubation of the cells at 20 degrees C instead of 37 degrees C reduced recovery of [3H]-NMS binding from 30% to 9% in unstimulated cells and from 43% to 23% in carbachol-stimulated cells. 4. Depletion of cellular ATP by addition of antimycin (50 nM) and deoxyglucose (50 mM), reduced recovery of binding to 12% in unstimulated cells and to 6% in carbachol-stimulated cells. 5. These results indicate that in unstimulated NG108-15 cells, delivery of muscarinic receptors to the plasma membrane is almost exclusively through the synthetic pathway. In agonist-stimulated cells,receptor sequestration into an intracellular compartment (probably endosomes) occurs. Under these circumstances, delivery of receptors to the plasma membrane along the synthetic route is unaffected but an additional route of delivery (recycling) now operates.