Ionic Milieu Research Articles

Contribution of cytosolic ionic and energetic milieu change to ischemia- and reperfusion-induced injury in guinea pig heart: fluorometry and nuclear magnetic resonance studies.

The contribution of cytosolic ion and energy milieu changes to ischemia/reperfusion injury was investigated in isolated guinea-pig hearts and mitochondria, with fluorometry and 31P nuclear magnetic resonance (NMR). The fura-2 Ca2+ signal during ischemia in the guinea-pig Langendorff heart changed triphasically (phases I, II, and III) and rapidly returned to the control level after the reperfusion. These triphasic changes during ischemia were affected by various agents that affect the cytosolic ion milieu: the combination of asebotoxin-III and dihydroouabain (which increase intracellular Na+) caused an increase in Ca2+ levels in the final stage (phase III) with a manifestation of contracture after the reperfusion of the heart. Inhibitors of the H+-Na+ exchange such as 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) produced a significant restorative effect on the contractility of the reperfused heart with increased proton and decreased Na+ and Ca2+ in the cytosol. The mitochondrial matrix Ca2+ ([Ca2+]m) preloaded with abnormally high Ca2+ levels was markedly increased by perfusion with either a physiologic concentration of Ca2+ or an acidified perfusate. These [Ca2+]m increases were reduced by the H+-Na+ and H+-K+ exchange inhibitor (EIPA; omeprazole), respectively. These findings will help to explain the Ca paradox at the mitochondria level (i.e., mitochondria for Ca2+ pumping play an essential role in the cellular homeostasis of Ca2+ for the maintenance of cell functions of the heart, acting like a Ca2+ scavenger in the cytosol). Factors that induce Ca2+ overload on mitochondria via sarcolemmal Ca2+ influx and any exchange mechanisms with Na+, K+, Ca2+, and H+ will lead to a loss of contractility, associated with the extremely reduced level of free energy change predicted from the reduced ATP x PCr/Pi ratio by 31P NMR.

Thermal stabilization of carboxypeptidase A as a function of pH and ionic milieu.

In this study we investigated the contribution of pH, phosphate anions and salt concentration to the catalytic and structural thermostability of the carboxypeptidase A (CPA). The concentration of 75-100 mM phosphate as well as neutral pH values were found to be optimal for stabilizing CPA at high temperatures. Although moderate concentrations of sodium chloride had no effect on thermal stability, high concentrations of the salt destabilized the enzyme. The experimental results and theoretical analysis suggested that the main contribution to heat stabilization of CPA is related to intramolecular electrostatic interactions and Arginine and/or Lysine are the putative groups able to bind phosphate and stabilize the enzyme molecule against thermal denaturation.

DNA binding specificity of MunI restriction endonuclease is controlled by pH and calcium ions: involvement of active site carboxylate residues.

Gel shift analysis reveals [Lagunavicius, A., & Siksnys, V. (1997) Biochemistry 36 (preceding paper in this issue)] that at pH 8.3 in the absence of Mg2+, MunI restriction endonuclease exhibits little DNA binding specificity, as compared with the D83A and E98A mutants of MunI. This suggests that charged carboxylate residue(s) influence the DNA binding specificity of MunI. In our efforts to establish the determinants of MunI binding specificity, we investigated the possible role of the ionic milieu, and we found that lowering pH or elevating Ca2+ levels per se induces specific DNA recognition by WT MunI. In contrast to the binding experiments at pH 8.3, gel shift analysis at pH 6.5 indicated tight sequence-specific binding of WT MunI in the absence of Mg2+, suggesting that protonation of active site carboxylate residue(s) which manifest anomalously high pKa value(s) control binding specificity. Interestingly, Ca2+ ion concentrations, which did not support DNA cleavage by MunI also induced DNA binding specificity in WT MunI at pH 8.3. To explore possible structural changes upon DNA binding, we then used a limited proteolysis technique. Trypsin cleavage of MunI-DNA complexes indicated that in the presence of cognate DNA the MunI restriction endonuclease became resistant to proteolytic cleavage, suggesting that binding of specific DNA induced a structural change. CD measurements confirmed this observation, suggesting minor secondary structural differences between complexes of MunI with cognate and noncognate DNA. These results therefore suggest that binding of MunI to its recognition sequence triggers a conformational transition that correctly juxtaposes active site carboxylate residues, which then chelate Mg2+ ions. In the absence of Mg2+ ions, at pH 8.3, conditions in which carboxylate groups would be expected to be completely ionized, electrostatic repulsion between charged carboxylates and phosphate oxygens is enhanced such as to interfere with specific DNA binding. Elimination of such repulsive constraints by replacement of carboxylate residues, by lowering pH, or by metal ion binding, then promotes MunI binding specificity.

Three-Dimensional Organization of Smooth Endoplasmic Reticulum in Hippocampal CA1 Dendrites and Dendritic Spines of the Immature and Mature Rat

Recent studies have shown high levels of calcium in activated dendritic spines, where the smooth endoplasmic reticulum (SER) is likely to be important for regulating calcium. Here, the dimensions and organization of the SER in hippocampal spines and dendrites were measured through serial electron microscopy and three-dimensional analysis. SER of some form was found in 58% of the immature spines and in 48% of the adult spines. Less than 50% of the small spines at either age contained SER, suggesting that other mechanisms, such as cytoplasmic buffers, regulate ion fluxes within their small volumes. In contrast, >80% of the large mushroom spines of the adult had a spine apparatus, an organelle containing stacks of SER and dense-staining plates. Reconstructed SER occupied 0.001-0.022 microm3, which was only 2-3.5% of the total spine volume; however, the convoluted SER membranes had surface areas of 0.12-2.19 microm2, which were 12 to 40% of the spine surface area. Coated vesicles and multivesicular bodies occurred in some spines, suggesting local endocytotic activity. Smooth vesicles and tubules of SER were found in continuity with the spine plasma membrane and margins of the postsynaptic density (PSD), respectively, suggesting a role for the SER in the addition and recycling of spine membranes and synapses. The amount of SER in the parent dendrites was proportional to the number of spines and synapses originating along their lengths. These measurements support the hypothesis that the SER regulates the ionic and structural milieu of some, but not all, hippocampal dendritic spines.

Evidence for rapid consumption of millimolar concentrations of cytoplasmic ATP during rigor-contracture of metabolically compromised single cardiomyocytes.

Cytoplasmic ATP can be measured continuously in single cardiac myocytes by monitoring the luminescence from microinjected firefly luciferase. We show here that the signals are markedly influenced by changes in cytoplasmic pH, and the calibration of the signals as ATP concentration is markedly affected by cytoplasmic protein. Measurements with a pH-sensitive fluorescent dye show that intracellular pH (pHi) can be clamped at pH 7.08 by perfusing cells with a modified bicarbonate-buffered Krebs saline containing 92 mM NaHCO3 and equilibrated with 20% CO2. Calibration of the firefly luciferase signal in vitro in the presence of high concentrations of BSA (180 mg/ml), to simulate the cytoplasmic protein concentration, revealed a shift in Km (ATP) to 2 mM, from approx. 400 microM in the absence of albumin in an identical ionic milieu. Luciferase measurements in pH-clamped cells show that metabolically poisoned isolated rat ventricle cardiomyocytes enter rigor at a cytoplasmic ATP concentration of between 1 and 2 mM. As the cells shorten in rigor, a process that is complete in 30-40 s, the cytoplasmic ATP concentration falls simultaneously to a level of typically 20 microM. When cyanide is removed 10 min later, to simulate reoxygenation, the signal recovers over a period of 2-3 min to a level approx. 70% of the original in the healthy cell. These studies indicate that rigor-mediated depletion of cytoplasmic ATP in metabolically poisoned cardiomyocytes is considerably more extreme than hitherto indicated.

Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O

Plasmodium falciparum is an intracellular parasite of human red blood cells (RBCs). Like many other intracellular parasites, P. falciparum resides and develops within a parasitophorous vacuole which is bound by a membrane that separates the host cell cytoplasm from the parasite surface. Some parasite proteins are secreted into the vacuolar space and others are secreted, by an as yet poorly defined pathway, into the RBC cytosol. The transport of proteins from the parasite has been followed mainly using morphological methods. In search of an experimental system that would allow (i) dissection of the individual steps involved in transport from the parasite surface into the RBC cytosol, and (ii) an assessment of the molecular requirements for the process at the erythrocytic side of the vacuolar membrane, we permeabilized infected RBCs with the pore-forming protein streptolysin O using conditions which left the vacuole intact. The distribution of two parasite proteins which served as markers for the vacuolar space and the RBC cytosol respectively was analysed morphologically and biochemically. In permeabilized RBCs the two marker proteins were sorted to the same compartments as in intact RBCs. The protein which was destined for the RBC cytosol traversed the vacuolar space before it was translocated across the vacuolar membrane. Protein transport could be arrested in the vacuole by removing the RBC cytosol. Translocation across the vacuolar membrane required ATP and a protein source at the erythrocytic face of the membrane, but it was independent of the intracellular ionic milieu of the RBC.

Evidence for a single non-arachidonic acid-specific fatty acyl-CoA synthetase in heart which is regulated by Mg2+

Previous reports indicated that arachidonic acid is incorporated into the isolated perfused rabbit heart in preference to other fatty acids, and that incorporation of arachidonic acid, but not other fatty acids, is inhibited during Mg2+ depletion. In this study, we have not been able to demonstrate an arachidonic acid-specific fatty acyl-CoA synthetase in rat or rabbit heart by hydroxyapatite chromatography. Kinetic evidence was consistent with a single enzyme, as the slopes of pseudo-Hill plots were not significantly different from -1. The single fatty acyl-CoA synthetase present appears to prefer C18:0 unsaturated fatty acids to arachidonate, and had about the same affinity for C10:0 -C14:0 saturated fatty acids as for arachidonate. At 35 microM arachidonate, enzyme velocity increased as the total Mg2+ was increased from 3 to 80 mM. Calculated [MgATP] indicated that the MgATP complex was not rate-limiting. At low concentrations, Mn2+ and Ni2+ supported activity, but Cu2+ and Zn2+ did not. Low Ca2+ concentrations activated only oleic acid conversion. Kinetic analysis indicated that the Vmax of the enzyme was increased with increasing concentrations of ionized Mg2+ for both oleic acid and arachidonic acid. The data are constant with the hypothesis that Mg2+ has a direct effect on fatty acyl-CoA synthetase activity, and suggest that preference for oleic acid and arachidonic acid can be influenced by the ionic milieu.

Mature Cathepsin L Is Substantially Active in the Ionic Milieu of the Extracellular Medium

The activity of cathepsin L is affected by ionic strength, resulting in the measured pH optimum being higher in acetate–4-morpholineethane sulfonic acid (MES)–Tris buffers of constant ionic strength than in phosphate buffers of constant molarity (and hence varying ionic strength). In acetate–MES–Tris and phosphate buffers of constant ionic strength across the pH range, the catalytic constant,kcat, generally peaked at ca. pH 6.5 and essentially independently of ionic strength.Kmvalues, of ca. 5 μM, manifested a slight rising trend with increasing ionic strength, with a sharp increase to 20–25 μM, specifically at pH 6.5 andI= 0.4. At physiological ionic strengths, the specific buffer ions present affected the activity of mature cathepsin L,kcat/Kmdeclining above pH 6.5 in phosphate buffer, but only above pH 7 in acetate–MES–Tris buffer. In Hanks’ balanced salt solution, a model of the extracellular fluid, measured values at pH 7.2 werekcat, 18.9 s−1;Km, 13.5 μM; andkcat/Km, 1.4 × 106M−1s−1. The stability of cathepsin L in the physiological pH range was also differentially affected by the specific buffer ions, generally in parallel with the enzyme activity. In Hanks’ balanced salt solution, mature cathepsin L was substantially active and stable, having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumor pH).

Electrophysiologic changes in ischemic ventricular myocardium: I. Influence of ionic, metabolic, and energetic changes.

Myocardial ischemia leads to significant changes in the intracellular and extracellular ionic milieu, high-energy phosphate compounds, and accumulation of metabolic by-products. Changes are measured in extracellular pH and K+, and intracellular pH, Ca2+, Na+, Mg2+, ATP, ADP, and inorganic phosphate. Alterations of membrane currents occur as a consequence of these ionic changes, adrenergic receptor stimulation, and accumulation of lactate, amphipathic compounds, and adenosine. Changes in the volume of the extracellular and intracellular spaces contribute further to the ultimate perturbations of active and passive membrane properties that underlie alterations in excitability, abnormal automaticity, refractoriness, and conduction. These characteristic changes of electrophysiologic properties culminate in loss of excitability and failure of impulse propagation and form the substrate for ventricular arrhythmias mediated through abnormal impulse formation and reentry. The ability to detail the changes in ions, metabolites, and high-energy phosphate compounds in both the extracellular and intracellular spaces and to correlate them directly with the simultaneously occurring electrophysiologic changes have greatly enhanced our understanding of the electrical events that characterize the ischemic process and hold promise for permitting studies aimed at developing interventions that may lessen the lethal consequences of ischemia.

Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.

Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.

P1-186 - Ischemic changes of ionic and energetic milieu in myocardium under drug action

P1-186 - Ischemic changes of ionic and energetic milieu in myocardium under drug action

Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells.

In order to improve calibration of firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells we have investigated why the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and why the decay of the intracellular signal is remarkably slow. We have shown that inorganic pyrophosphatase greatly depresses the signal in vitro and that micromolar concentrations of inorganic pyrophosphate, comparable with that in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations of pyrophosphate depress the signal by inhibiting ATP-binding to luciferase. Luciferase-injected cells exposed to extracellular luciferin concentrations above about 100 mumol/l (corresponding to a cytoplasmic level of c. 5-10 mumol/l because of a transplasmalemmal gradient) show a gradual, irreversible loss of signal. We attribute this phenomenon (which is not seen in vitro) to the gradual accumulation of a luminescently inactive, irreversible, luciferase-oxyluciferin complex. At low luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100 mumol/l extracellular luciferin, the pyrophosphate level in the cytoplasm fails to fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders of magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2-4 mumol/l.

Free insulin-like growth factors (IGF-I and IGF-II) in human serum

Using ultrafiltration by centrifugation we have isolated the free, unbound fractions of insulin-like growth factor I and II (free IGF-I and IGF-II) in human serum. In this way near in vivo conditions could be maintained before and during isolation. The recovery was 80 to 100% in the ultrafiltrates, which contained no detectable amounts of IGF-binding proteins (IGFBPs) as measured by Western ligand blotting and IGFBP-1 and IGFBP-3 immunoassays. The concentration of free peptides was measured in two ultrasensitive non-competitive IGF-I and IGF-II time-resolved fluoroimmunoassays. We found that (i) equilibrium between free and protein-complexed IGF was strongly dependent on re-establishment of in vivo conditions (temperature, pH, ionic milieu and dilution); (ii) metabolic events (glucose load and fasting) caused significant changes in free IGF-I and IGF-II levels without concomitant changes in total circulating levels of IGFs; (iii) in 49 healthy adult subjects (20 to above 60 years) free IGF-I was inversely related to age and ranged from 950 ± 150 ng/l (mean ± S.E.M.) (20–30 years) to 410 ± 70 ng/l (⪢ 60 years). The relative percentage was, however, unchanged, being 0.38 ± 0.02% of total IGF-I. In contrast, free IGF-II was independent of age, being 1,480 ± 80 ng/l (∼0.20 ± 0.01% of total IGF-II).

Aluminum-induced alterations in [3H]Ouabain binding and ATP hydrolysis catalyzed by the rat brain synaptosomal (Na+ + K+)-ATPase°

The (Na(+)+K+)-ATPase is responsible for maintenance of the ionic milieu of cells. The objective of this study is to investigate the effect of aluminum, an ion implicated in several neurological disorders, on ATP hydrolysis catalyzed by the rat brain synaptosomal (Na(+)+K+)-ATPase and on the binding of [3H]ouabain to this enzyme. AlCl3 (25-100 microM) inhibits the phosphatase activity of the (Na(+)+K+)-ATPase in a dose-dependent manner. AlCl3 appears to act as a reversible, noncompetitive inhibitor of (Na(+)+K+)-ATPase activity by decreasing the maximum velocity of the enzyme without significantly affecting the apparent dissociation constant with respect to ATP. AlCl3 may affect Mg2+ sites on the (Na(+)+K+)-ATPase but does not appear to interact with Na+ or K+ sites on the enzyme. In contrast to this inhibitory effect on the phosphatase function of the enzyme, AlCl3 (1-100 microM) stimulates the binding of [3H]ouabain to the (Na(+)+K+)-ATPase. This effect is due to an increase in the maximum [3H]ouabain binding capacity of the enzyme with no change in the [3H]ouabain binding affinity. These data support the hypothesis that AlCl3 may stabilize the phosphorylated form of the synaptosomal (Na(+)+K+)-ATPase which increases [3H]ouabain binding while inhibiting the phosphatase activity of the enzyme.

Modulation of the ionic milieu of the airway in health and disease.

Airway surface liquid (ASL) is an integral part of lung defense mechanisms. Ion transport by airway epithelia regulates the volume and composition of this fluid. A better understanding of the mechanisms of ion transport will enable the development of new therapies for airway diseases associated with defects in these mechanisms. A useful model of a disease with abnormal airway epithelial ion transport is cystic fibrosis (CF), a distinct genetic syndrome of altered lung defense mechanisms characterized by chronic bacterial infection and a steady decline in lung function. Traditional therapies for CF include antibacterial drugs and augmentation of clearance of secretions, but investigators are now studying pharmacological approaches to target the more basic defect of the disease, i.e. abnormal sodium and chloride ion transport. Early treatment in childhood, prior to lung damage, might prevent or at least retard the decline in pulmonary function that remains the hallmark of CF. Ion transport dysfunction may also contribute to other airway diseases such as asthma and chronic bronchitis. Pharmacological intervention at this level may prove beneficial in these common lung diseases as well.

Carbon tetrachloride-induced cell death in perfused livers from phenobarbital-pretreated rats under hypoxic conditions and various ionic milieu: Further evidence for calcium-dependent irreversible changes

The role of Ca 2+ in the initiation of carbon tetrachloride (CCl 4) hepatotoxicity was studied using perfused livers isolated from phenobarbital-pretreated rats in a single-pass system. Krebs-Henseleit bicarbonate buffer containing 1.3 mM CaCl 2 (KHB) was the regular ionic milieu. In the liver perfused with fructose-supplemented regular KHB equilibrated with 95% N 2−5% CO 2, infusion of 0.5 mM CCl 4 caused an early uptake of Ca 2+ coupled with K + leakage and Na + uptake within the infusion time of 30 min, which was followed by a marked lactic dehydrogenase (LDH) leakage into the effluent perfusate and further Ca 2+ uptake by the liver. With Ca 2+-free medium, the prenecrotic K + leakage and the successive LDH leakage were suppressed markedly. However, a perfusate exchange from regular to Ca 2+-free KHB at the end of the prenecrotic stage did not protect against the LDH leakage, and the perfusate exchange conversely did not produce LDH leakage. Perfusion of the liver with high K +(Cl −) medium under 20% O 2 markedly suppressed CCl 4-induced LDH leakage even in the presence of Ca 2+, whereas once CCl 4 had acted under regular KHB perfusion, changing the medium to high K + did not further prevent the LDH leakage. High K +-lactobionic acid medium containing Ca 2+ and supplemented with fructose also suppressed LDH leakage under 95% N 2 without the accompanying prenecrotic Ca 2+ uptake. However, a change of the medium after CCl 4 infusion to regular KHB containing Ca 2+ caused LDH leakage and K + leakage, with Ca 2+ uptake. The prevention of LDH leakage in a different ionic milieu may not be due to suppression of CCl 4 bioactivation, since the liver cytochrome P450 content decreased to a similar extent. These findings suggest that entry of extracellular Ca 2+ into hepatocytes coupled with K + leakage and Na + entry is a prerequisite for CCl 4-induced hepatocyte death and that association of Ca 2+ with a CCl 4-derived radical-mediated process may be necessary for early and irreversible plasma membrane damage.

Aldose reductase gene expression and osmotic dysregulation in cultured human retinal pigment epithelial cells

A "compatible osmolyte hypothesis" proposes that intracellular nonionic organic osmolytes such as sorbitol, myo-inositol, taurine, betaine, and glycerophosphorylcholine respond coordinately to changes in external osmolality, thereby maintaining the intracellular ionic milieu. Osmoregulation may be the primary physiological function of aldose reductase, which catalyzes the conversion of glucose to sorbitol. Glucose-induced sorbitol accumulation in isosmotic hyperglycemic states is associated with compensatory depletion of myo-inositol and taurine. Because such depletion may predispose to chronic diabetic complications, the relationship between osmolyte shifts and aldose reductase gene expression was studied in two human retinal pigment epithelial cell lines, one exhibiting osmoregulated and the other high basal aldose reductase gene expression. High basal expression of the aldose reductase gene was associated with rapid sorbitol accumulation and myo-inositol depletion in response to hyperglycemic (20 mM) concentrations of glucose. Myo-inositol and sorbitol behaved as compensating intracellular osmolytes by accumulating markedly in response to hyperosmolality (300 mM mannitol). Thus the pattern of response of myo-inositol to hyperglycemic and hyperosmotic levels of glucose and mannitol was related to the degree of basal aldose reductase gene expression, which may therefore influence the development of diabetic complications.

Control of the Na+,K(+)-ATPase under normal and pathological conditions.

The Na+,K(+)-ATPase is an important enzyme in determining the ionic milieu of the cerebromicrovasculature and neurons. The effect of hypertension or aging on this enzyme, as well as its susceptibility to regulation by fatty acids or aluminum, is the focus of this study. A significant increase (34%) in the apparent affinity constant (KD) but no change in the maximum binding capacity (Bmax) for [3H]ouabain binding to the cerebromicrovascular Na+,K(+)-ATPase occurs after induction of acute hypertension. In addition, long chain unsaturated fatty acids stimulate the binding of [3H]ouabain to the enzyme in microvessels from normotensive and hypertensive rats. The synaptosomal Na+,K(+)-ATPase is sensitive to aluminum. AlCl3 (1-100 microM) inhibits the K(+)-dependent-p-nitrophenylphosphatase (K(+)-NPPase) activity of the Na+,K(+)-ATPase in a dose-dependent manner. AlCl3 (100 microM) decreases the Vmax by 14% but does not alter the KM, suggestive of non-competitive inhibition. The enzyme from aged brain displays a greater Vmax, but shows the same susceptibility to AlCl3 as the enzyme from younger brain. In summary, disruption of the Na+,K(+)-ATPase may underlie, at least in part, abnormalities of nerve and vascular cell function in disorders where elevated concentrations of fatty acids or metal ions are involved.

Cellular changes in HeLa cells and cervix cells after treatment with cyclic nucleotides.

Determination of tumor cell maturity and induction of cell differentiation are significant issues in oncology. We studied here in vitro the effects of cyclic AMP and cyclic GMP on human cervix carcinoma cells (HeLa cells), using normal human cervix cells as controls. Relative plasma membrane fluidity (which corresponds with the adhesive power of the cell, membrane permeability, and the ability to maintain the ionic milieu), cell cycle characteristics, and protein kinase C activity (a regulator of the cell cycle) were determined. In the untreated HeLa cells, membrane fluidity and protein kinase C activity were increased versus the controls. Administration of dbcAMP decreased the membrane fluidity and the protein kinase C activity, prolonged the G0/G1 phase of the cell cycle and shortened the S + G2 + M phase; with dbcGMP, the opposite changes were recorded (all findings, p < 0.05). Findings from HeLa cells treated with dbcAMP approached those from the normal controls. Each of the parameter studied reflected the differentiation of the HeLa cells during dbcAMP treatment. They may be of potential use for the determination of tumor cell maturity in clinical oncology.

P-634 - Effect of ionic milieu on CCl4-hepatotoxicity in perfused rat liver.

P-634 - Effect of ionic milieu on CCl4-hepatotoxicity in perfused rat liver.