Interaction of the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane with the beta-receptor adenylate cyclase system in Chang liver cell membranes

Interaction of the insecticide 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane (DDT) with beta-receptor binding and adenylate cyclase activity of biological membranes has been studied. Following exposure of cultured Chang liver cells to DDT, maximal binding of the catecholamine antagonist [125I]-iodohydroxybenzylpindolol (HYP) to isolated cell membranes was decreased by 30% whereas the dissociation constant remained unchanged. Both basal activity and maximal isoproterenol-stimulated activity of adenylate cyclase were not altered. The isoproterenol concentration required for half-maximal stimulation of the enzyme was increased about 2-fold as was the agonist concentration required for half-maximal displacement of the antagonist HYP at the beta-receptor binding site. Thus, coupling efficiency of hormone-stimulated adenylate cyclase activity was not influenced by the presence of DDT in these membranes. The data show that interaction of DDT with the beta-receptor adenylate cyclase complex is restricted to the receptor component. Enzyme activity is directly linked to changes of agonist binding at the beta-receptor. Interference of DDT with signal transduction via 'fluidization' of membrane lipids has not been detected.     

Membrane potential of Plasmodium-infected erythrocytes

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.     

Transmembrane potential and intracellular potassium ion activity in fetal and maternal liver

We have compared transmembrane potentials (Em) of maternal liver with Em of fetal liver, and as an initial step to account for differences in Em, we have measured intracellular potassium ion activities (aiK) in both tissues. Paired segments of maternal and fetal (day 17) mouse liver were suffused (15 ml/min) with Krebs' physiologic salt solution equilibrated with 95% 02-5% CO2 (pH 7.3-7.4) at 37 degrees C. To measure Em, cells were impaled with open-tip microelectrodes filled with 0.5 M KCl. Intracellular voltage recordings that were stable +/- 2 mV for at least 10 s were considered valid impalements. Maternal liver mean Em = -41 +/- 1 (SEM) mV, n = V 10 animals. In contrast, fetal liver mean Em = -23 +/- 1 (SEM) mV, n = 10 animals. In the same segments we measured aiK with potassium-selective liquid ion-exchanger microelectrodes. Maternal liver mean aik = 95 +/- 7 (SEM) mM and fetal liver mean aiK = 62 +/- 4 (SEM) mM. in addition, Em and aiK of fetal liver increased to values comparable to those of maternal liver during the first 8 days of neonatal life. The differences of Em and aik between fetal and maternal liver, and the changes in these values that occur in the neonate, may result from activity of a membrane Na-K exchange pump that increases with tissue development.     

Plasma membrane biophysical properties linked to the antiproliferative effect of interferon-alpha

The relationship of plasma membrane biophysical properties to the anti-proliferative effect of interferon-alpha (IFN-alpha) was investigated in Daudi lymphoblasts cell lines with sensitivity to growth inhibition, parallel clonal variants selected for resistance, and one revertant subclone. Lateral mobility of surface differentiation antigens (I2, CD19, CD20, and sIgM-kappa) were measured by fluorescence recovery after photobleaching (FRAP). The mean diffusion coefficients, D, values for two clones of IFN-alpha resistant Daudi cells were significantly higher (D = 8.1-11 x 10(-10) cm2/sec) than for parental sensitive cells (D = 4.9-7.4 x 10(-10) cm2/sec). Microviscosity of the plasma membranes were probed by electron spin resonance (ESR) spectrometry. These results also indicate a greater degree of molecular motional freedom in resistant cells. Treatment of sensitive lymphoblasts with IFN-alpha (100-400 U/10(6) cells) for 5-30 min consistently increased mean values of D and the degree of spin-probe motional freedom, whereas no significant differences were detected in resistant cells. The effect of IFN-alpha on the membrane potential (Em) of Daudi cells was quantitated by flow cytometry using a voltage-sensitive oxonol dye. Membrane potential of all clones was similar (-50 to -56 mV). Treatment with IFN-alpha for 8-10 min caused hyperpolarization in the sensitive cells (deltaEm up to 45 mV), but only minimal hyperpolarization in the resistant ones (deltaEm up to 7 mV). We concluded that sensitivity to IFN-alpha and treatment with IFN-alpha are related to the biophysical status of plasma membranes.     

Role of lipids in the Neurospora crassa membrane. II. Membrane potential and resistance studies; the effect of altered fatty acid composition on the electrical properties of the cell membrane.

The effect of doubling the saturated fatty acid content on the electrophysiology of Neurospora crassa membranes was studied. Intracellular membrane input resistance (Rm) and potential (Em) were measured for wild-type (w/t) and cel- (Tween 40) organisms as a function of temperature. Over the 0 to 40 degrees C temperature range studied, mean Em values of both w/t and cel- (Tw 40) organisms increased from -160 to -210 mV. This difference is greater than that expected from Nernst potential considerations, indicating an active component of Em. This active component is insensitive to a doubling of the saturated fatty acid content. Rm exhibits a temperature dependence and hysteresis. Averaged data indicate an increase in Rm with decreased temperature. The slope of the temperature dependence varies among individual hyphae. Above 17.5 degrees C cel- (Tw 40) hyphae averaged greater than 70% higher values of Rm than w/t. Below 17.5 degrees C w/t Rm data divided into low and high temperature dependence groups, while cel- data exhibited a low temperature dependence. The results are discussed in relation to gel-liquid crystal phase transitions, membrane fluidity, and the contribution of fatty acid structure to membrane electrical properties.     

Estrogen receptors and effects of estrogen on membrane electrical properties of coronary vascular smooth muscle

The effect of estrogen stimulation in vitro on the electrical properties of vascular smooth muscle (VSM), and the concentration of estrogen receptors in VSM were measured in isolated coronary arteries. Microelectrode measurements of the dog coronary artery membrane potential (Em) showed quiescent values of -51 millivolts (mV) and an input resistance (rin) of 10 megohms. Addition by diethylstilbestrol (DES) at 10(-6) M hyperpolarized the membrane to -64 mV and reduced input resistance (rin) to 5 megohms within 15 minutes. Extrapolation of the Em vs. log [K]o curve to zero potential gave similar values of [K]i of around 170 mM in both normal and DES treated muscles suggesting that the DES induced hyperpolarization is not due to increased Na-K pump activity. The 0.5% ethanol vehicle alone had no effect on the membrane potentials. Tetraethylammonium ion (TEA) induced action potentials in the previously quiescent tissue. When DES was applied in the presence of TEA, the membrane potential increased and the action potentials were abolished. Scatchard analysis of the estrogen receptor binding demonstrated both a high and a low affinity receptor for estrogen in the VSM. These data indicate that DES hyperpolarizes the VSM cells by a mechanism other than an increased Na-K pump activity. The mechanism of this increased Em may be due to factors which increase K+ conductance either mediated directly through estrogen interaction with its cytosolic receptors or through some unidentified second mechanism.     

Electrophysiological studies on the cultured cells obtained from transplantable pancreatic carcinoma in Syrian golden hamsters

A pancreatic ductal carcinoma was established as a transplantable tumor line in an inbred strain of Syrian golden hamsters. Intracellular recordings of membrane potentials and input resistance were made from cultured cells obtained from the transplanted tumors using indwelling glass microelectrode. The mean value of the resting membrane potential was -46.5 +/- 1.8 mV (S.E.) (n = 13), while the mean resting input resistance was 21.2 +/- 4.3 M omega (S.E.) (N = 13). Dibutyryl cyclic AMP (2 X 10(-3)M) caused a marked hyperpolarization of about 30 mV accompanied by a reduction of input resistance. The transplantable tumor and its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use in pancreatic cancer research.     

Intracellular microelectrode measurements in small cells evaluated with the patch clamp technique.

Microelectrode penetration of small cells leads to a sustained depolarization of the resting membrane potential due to a transmembrane shunt resistance (Rs) introduced by the microelectrode. This has led to underestimation of the resting membrane potential of various cell types. However, measurement of the fast potential transient occurring within the first few milliseconds after microelectrode penetration can provide information about pre-impalement membrane electrophysiological properties. We have analyzed an equivalent circuit of a microelectrode measurement to establish the conditions under which the peak of the impalement transients (Ep) approaches the pre-impalement resting membrane potential (Em) of small cells most closely. The simulation studies showed that this is the case when the capacitance of the microelectrode is low and the membrane capacitance of the cell high. In experiments performed to assess the reliability of Ep as a measure of Em, whole-cell patch clamp measurements were performed in the current clamp mode to monitor, free from the effects of Rs, Em in cultured human monocytes. Microelectrode impalement of such patch clamped cells and measurement of Ep made it possible to detect correlation between Ep and Em and showed that for small cells such as human monocytes Ep is on average 6 mV less negative than the resting membrane potential.     

Membrane and junctional properties of dissociated frog lens epithelial cells

Summary Individual cells and cell pairs were isolated from frog lens epithelium. Individual cells were whole cell voltage clamped and the current-voltage relationship was determined. The cells had a mean resting voltage of –54.3 mV and a mean input resistance of 1.4 G. The current-voltage relationship was linear near the cell resting voltage, but showed decreased resistance with large depolarization or hyperpolarization. Junctional currents between pairs of cells were recorded using the dual whole cell voltage-clamp technique. The corrected junctional resistance was 15.5 M (64.5 nS). The junctional current-voltage relationship was linear. A combination of ATP and cAMP, in the electodes, stabilized junctional resistance. Currents recorded when uncoupling was nearly complete, showed evidence of single connexon gating events. A single-channel conductance of about 100 pS was prominent. Dye spread between isolated cell pairs was demonstrated using Lucifer Yellow CH in a whole cell configuration. Photodamage to the cells due to the dye was apparent. Dye loaded cells, in the presence of exciting light, showed decreased resting voltages, decreased input resistances and morphological changes. Glutathione (20mm) delayed this damage.     

Redox properties of membrane-bound b-type cytochromes and a soluble c-type cytochrome of nitrate reductase in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.     

Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase

Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase.     

Low intensity microwave radiation effects on the ultrastructure of Chang liver cells

Chang liver cells (CCL-13 ATCC) exposed to 2450 MHz microwaves of field intensities ranging from 5 to 20 mW/cm2 for different periods up to 2 h show distinct alterations in the cytomembrane ultrastructure. A 30-min exposure of 10 mW/cm2 produces well-defined cytoplasmic lesions which appear as clear areas of degenerated rough endoplasmic reticulum (RER). Extensive degeneration of RER along with fragmentation and vacuolation, disorganization of mitochondrial membranes and matrix, increased lysosomal activity, and in some cases disruptions of nuclear membrane are seen in longer exposures. Radiation at 20 mW/cm2 produces significant damage to cell membranes in short exposures and treatments of 30 min and longer exposures lead to total disruption of organized cell ultrastructure. The identity of many organelles is lost as the cells become highly heteropycnotic with numerous cytoplasmic projections. Short exposures of 5 mW/cm2 produce very few noticeable differences in ultrastructure. These results confirm earlier observations that membranes may be the primary targets of microwave radiation in cells.     

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.     

Hormonal imprinting in cell culture I. Impact of a single exposure to insulin on cellular insulin binding capacity in permanent cell lines

Three permanent cell lines showed a durable change in cellular insulin binding capacity in response to a single exposure to insulin. Initial increase in binding of FITC-labeled insulin to both cytoplasmic and nuclear membrane was followed by a lasting decrease in the case of fibroblasts (NCTC) and return to approximately the control level in epithelial (Chang liver) and tumour (HeLa) cell cultures. While the amplifying effect of a single 4-h exposure was measurable as soon as after 24 h, that of a single 24-h exposure came into display only after 72 h in the epithelial and tumour cell cultures. The latter two lines showed return of the binding capacity to the control value and a drop of it to a minimum 20 days after exposure for 4 and 24 h, respectively. These observations support the implication that although the degree and manner of insulin binding differs between cell lines, the impact of a single exposure to insulin is durably "felt" by all.     

Modulation of resting membrane potential of human and rat hepatocytes by sex steroids

The effect of sex steroids on the regulation of hepatocyte resting membrane potential (Em) was investigated. In adult rat liver snips, Em was significantly lower in males than females. In prepubertal animals no sex related difference was observed and the Em was comparable to that of adult females. Exposure of the human liver cell line, HepG2 cells, to 10 microM testosterone resulted in a significant hyperpolarization. These data indicate that the more negative Em found in male animals is specifically due to the influence of testosterone. In addition, they also suggest that sex hormone regulation of Em is maintained by HepG2 cells. This cell line may prove to be a good model for the study of liver cell function.     

Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst.

The oxidation-reduction midpoint potential of the cytochrome b found in the plasma membrane of human neutrophils has been determined at pH 7.0 (Em,7.0) from measurements of absorption spectra at fixed potentials. In both unstimulated and phorbol myristate acetate-stimulated cells Em,7.0 was -245 mV. Changes in pH affected the Em of the cytochrome b, with a slope of approx. 25 mV/pH unit change. The Em,7.0 of the haem group(s) of the membrane-bound myeloperoxidase of human neutrophils was found to be +34 mV. The plasma membranes contained no detectable ubiquinone, and no iron-sulphur compounds were detected by e.p.r. spectroscopy at 5-20 K. No flavins were detected by e.p.r. spectroscopy. The cytochrome b-245 was not reduced by added NADH or NADPH. Dithionite-reduced cytochrome b-245 formed a complex with CO, supplied as a saturated solution, which was dissociated with 26 microseconds illumination from a xenon flash lamp, and the recombination with CO had a half-time of approx. 6 ms. Partly (80%) reduced cytochrome b-245 was oxidized by added air-saturated buffer with a half-time faster than 1 s at 20 degrees C, a resolution limited by mixing time. These results are compatible with cytochrome b-245 acting as an oxidase.     

Electrical properties of normal and transformed mammalian cells.

The transmembrane potential difference, Em, and DC membrane resistance were measured in 3T3 and polyoma virus-transformed 3T3 cells. Em was a function of cell density and was -12 and -25 mV for the normal and transformed cells, respectively. The external concentrations of K+, Na+, and Cl were varied in order to study the nature of the differences between the two cell types. The relative permeability of ions was calculated to be: PNa/PK, 1.0; PCl/PK, 1.88; PNa/PCl, 0.53 for 3T3 cells, and 0.27, 1.75, and 0.15 for the transformed cells. In contrast to the normal cells, PNa/PK varied as a function of the external K+ concentration for the transformed cells. It was emphasized that the manipulation of variables directly affecting the electrical properties of cells also involves the indirect manipulation of a network of interconnected physiological determinants.     

ATP-binding cassette transporter enhances tolerance to DDT in Tetrahymena

The reuse of dichlorodiphenyltrichloroethane(DDT) as an indoor residual spray was permitted by the World Health Organization in 2007, and approximately 14 countries still use DDT to control disease vectors. The extensive exposure of insects to DDT has resulted in the emergence of DDT resistance, especially in mosquitoes, and the mechanism for this resistance in mosquitoes has been widely reported. Spraying can also introduce DDT directly into surface water, and DDT can subsequently accumulate in microorganisms, but the mechanism for the resistance to DDT degradation in microorganisms is unclear. Using whole-genome microarray analysis, we detected an abcb15 gene that was up-regulated in a specific manner by DDT treatment in T. thermophile. The deduced ABCB15 peptide sequence had two transmembrane domains(TMDs) and two nucleotide-binding domains(NBDs) to form the structure TMD-NBD-TMD-NBD, and each NBD contained three conserved motifs: Walker-A, C-loop, and Walker-B, which indicated the T. thermophila abcb15 was a typical ABC transporter gene. The expression of ABCB15 fused with a C-terminal green fluorescent protein was found to be on the periphery of the cell, suggesting that ABCB15 was a membrane pump protein. In addition, cells with abcb15 partially knocked down(abcb15-KD) grew slower than wild-type cells in the presence of 256 mg L-1 DDT, indicating the tolerance of abcb15-KD strain to DDT exposure was decreased. Thus, we suggest that in Tetrahymena, the membrane pump protein encoded by ABCT gene abcb15 can enhance the tolerance to DDT and protect cells from this exogenous toxin by efficiently pumping it to the extracellular space.     

Effects of carbamylcholine on membrane potential and Na-K pump activity of cultured rat skeletal myotubes

1. We measured changes in resting membrane potential (Em) and Na-K pump activity, assayed by ouabain-sensitive 86Rb uptake, in response to carbamylcholine (CCh) and its continued presence in single rat skeletal myotubes in culture. 2. CCh caused immediate depolarization from control Em (-80 to -85 mV) to near 0 followed by repolarization of varying degrees depending on the age of the culture and temperature of the recording medium; repolarization of Em was most apparent by culture age 8-9 days in vitro (DIV), Em reaching values as high as -60 mV by 5-10 min after peak depolarization at 37 degrees C. 3. Input resistance, which decreased during CCh depolarization, increased only slightly during the initial phase of repolarization and then remained essentially unchanged during the major component of membrane repolarization in the presence of CCh. 4. Ouabain, given before CCh, prevented repolarization of Em and, when given after repolarization had begun, reversed it and caused Em to return to about -7 mV. 5. Na-K pump activity was decreased in myotubes in which Em did not repolarize or did so only slightly, and was increased by over 40-50% in myotubes whose Em repolarized by 40-60 mV, even though CCh was still present in the medium. Inhibition of pump activity in non repolarizing myotubes was related to Na influx, inhibition being reversed to stimulation when CCh was administered to myotubes in Na-free medium. 6. Repeated (three or four times) or prolonged (up to 60-min) administration of CCh to myotubes in which repolarization was hardly expressed (age 6-7 DIV) caused increases both in the amount of repolarization and in 86Rb uptake, both being related to the number or duration of CCh exposures. 7. We conclude that repolarization of Em following CCh-induced depolarization of cultured rat skeletal myotubes depends to a large extent on an increase in activity of the electrogenic Na-K pump.     

Primary photochemistry in the facultative green photosynthetic bacterium Chloroflexus aurantiacus

The mechanism of primary photochemistry has been investigated in purified cytoplasmic membranes and isolated reaction centers of Chloroflexus aurantiacus. Redox titrations on the cytoplasmic membranes indicate that the midpoint redox potential of P870, the primary electron donor bacteriochlorophyll, is +362 mV. An early electron acceptor, presumably menaquinone has Em 8.1 = -50 mV, and a tightly bound photooxidizable cytochrome c554 has Em 8.1 = +245 mV. The isolated reaction center has a bacteriochlorophyll to bacteriopheophytin ratio of 0.94:1. A two-quinone acceptor system is present, and is inhibited by o-phenanthroline. Picosecond transient absorption and kinetic measurements indicate the bacteriopheophytin and bacteriochlorophyll form an earlier electron acceptor complex.