Search PubMed⌕ Search

Biomedical subjects

L M Loew

Publications and source records attributed to L M Loew.

At least 55 records · Page 3Linked to original sources
PubMed1989

Simultaneous imaging of cell and mitochondrial membrane potentials.

The distribution of charged membrane-permeable molecular probes between intracellular organelles, the cytoplasm, and the outside medium is governed by the relative membrane electrical potentials of these regions through coupled equilibria described by the Nernst equation. A series of highly fluorescent cationic dyes of low membrane binding and toxicity (Ehrenberg, B., V. Montana, M.-D. Wei, J. P. Wuskell, and L. M. Loew, 1988. Biophys. J. 53:785-794) allows the monitoring of these equilibria through digital imaging video microscopy. We employ this combination of technologies to assess, simultaneously, the membrane potentials of cells and of their organelles in situ. We describe the methodology and optimal conditions for such measurements, and apply the technique to concomitantly follow, with good time resolution, the mitochondrial and plasma membrane potentials in several cultured cell lines. The time course of variations induced by chemical agents (ionophores, uncouplers, electron transport, and energy transfer inhibitors) in either or both these potentials is easily quantitated, and in accordance with mechanistic expectations. The methodology should therefore be applicable to the study of more subtle and specific, biologically induced potential changes in cells.

Adenosine Triphosphate↗
PubMed1988

Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.

Animals↗
PubMed1987

Insertion of amphiphilic molecules into membranes is catalyzed by a high molecular weight non-ionic surfactant.

We have employed an amphiphilic fluorescent probe to elucidate the mechanism by which a class of oxyethylene-oxypropylene copolymers catalyzes the insertion of hydrophobic or amphiphilic molecules into membranes. The rate of binding can be accelerated by over two orders of magnitude in the presence of the catalyst which does not itself disrupt the lipid bilayer. The rate of probe binding to lipid vesicles does not depend on the lipid concentration in the presence or absence of catalyst but is linearly related to the concentration of the catalyst. Probe binding to the polyol surfactant appears to be a component of the catalytic mechanism and equilibrium binding parameters can be determined; these are used to indirectly establish quantitative binding parameters for the probe to the vesicle membrane. The polyol surfactant is also shown to catalyze insertion of the probe into the outer leaflet of a hemispherical lipid bilayer and the plasma membrane of HeLa cells. The latter were also stained by catalyzed transfer of a fluorescent lipid from lipid vesicles. The permeability of the cell membrane is not significantly altered under any of the catalytic conditions. These data, taken together, suggest that the polyol surfactant extracts a monomeric substrate molecule from its aggregate or microcrystal and passes it to the membrane via a loose and transient contact.

Cell Membrane↗
PubMed1987

Membrane potential induced by external electric field pulses can be followed with a potentiometric dye.

A potential-sensitive dye was recently used to measure the spatial variation in the membrane potential induced by an externally applied electric field. In this work, we demonstrate that the time course of these induced potentials can also be followed. Two experimental systems were explored. Dye fluorescence from HeLa cells could be modulated by a train of field pulses; the relative fluorescence change measured with a lock-in amplifier was linear with the field and similar to the fluorescence responses obtained in the static measurements. A model membrane system consisting of a hemispherical bilayer allowed convenient measurement of the dye absorbance change as a function of the bathing solution conductivity. The charging time of the membrane was inversely related to the aqueous conductance as predicted by the theoretical solution to Laplace's equation.

Cell Membrane↗
PubMed1986

Purification and pore-forming activity of two hydrophobic polypeptides from the secretion of the Red Sea Moses sole (Pardachirus marmoratus).

A new column chromatography procedure, based on ion exchange, chromatofocusing, and reverse phase high pressure liquid chromatography was employed to isolate the two main proteinaceous, toxic, cytolytic, pore-forming factors from the secretion of the Red Sea Moses sole Pardachirus marmoratus. Pardaxin I, comprising 10% of the gland secretion proteins, was shown to be 5-10 times more toxic, cytolytic, and active in membrane pore formation than pardaxin II (8% of gland secretion proteins). Gel electrophoresis, amino acid analysis, and NH2-terminal amino acid sequence reveals a high degree of homogeneity and resemblance between the two toxins. They are rich in aspartic acid, serine, glycine, and alanine and devoid of arginine, tyrosine, and tryptophan. Their NH2-terminal residue sequence was found to be NH2-Gly-Phe-Phe. Their hydrophobicity is evident from chromatographic behavior on a hydrophobic matrix, presence of 9 successive hydrophobic residues at the NH2 terminus, and a decrease in drop size during elution of active fractions during chromatographic purification. The minimal molecular weight of pardaxin I is about 3500 as determined by sodium dodecyl sulfate gel electrophoresis and amino acid analyses. It is composed of 35 amino acids and is free of carbohydrate and sialic acid residues. Mass spectrometry of the ethyl acetate extract of the gland secretion and purified toxin reveals the presence of sterols in the secretion but their absence in the purified toxins. Pardaxin I was iodinated without affecting its chemical and pore-forming properties. It binds to liposomes of different phospholipid compositions. In hyperpolarized unilamellar liposomes, pardaxin I produced a fast, nonspecific permeabilization and in multilamellar liposomes, a slow, cation-specific pore. It is suggested that pardaxins exert their effects due to their hydrophobic and pore-formation properties.

Amino Acid Sequence↗
PubMed1986

Optical imaging of cell membrane potential changes induced by applied electric fields.

We report the first imaging of the spatial distributions of transmembrane potential changes induced in nonexcitable cells by applied external electric fields. These changes are indicated by the fluorescence intensity of a charge-shift potentiometric dye incorporated in the cell plasma membrane and measured by digital intensified video microscopy.

Basidiomycota↗
PubMed1985

Spectra, membrane binding, and potentiometric responses of new charge shift probes.

The properties of a series of new potentiometric membrane probes have been explored. The probes all contain an (aminostyryl)pyridinium chromophore or a more highly conjugated analogue. The spectral properties of the dyes are discussed in terms of the excitation-induced charge shift from the pyridine to the aniline; this charge shift also provides the basis for the voltage dependence of the spectra according to an electrochromic mechanism. The spectral responses to a membrane potential on a hemispherical bilayer have been obtained and, grossly, are quite similar for all probes tested. The more subtle variations from dye to dye can be partially rationalized by consideration of binding parameters, the depth within the membrane, and structural factors. The most potential sensitive dye in this collection has been designated di-4-ANEPPS and has a 6-amino-2-naphthyl group in place of the p-anilino on the parent chromophore. Both the relative fluorescence emission and excitation responses have maxima of 8% per 100 mV, and these two spectra display a striking symmetry.

Chemical Phenomena↗
PubMed1985

Fluorometric analysis of transferable membrane pores.

When pore-forming factors insert into the hyperpolarized membranes of lipid vesicles, ion gradients are rapidly equilibrated, effecting complete depolarization. This process can be conveniently followed with a potentiometric cyanine dye. The generality of the method is demonstrated by applications to three diverse materials. The well-studied gramicidin channel is used to demonstrate that the method is sensitive down to concentrations of 10(-12)M. An extract from the shark repellent skin secretion of the Red Sea flatfish displays activity in the assay and is used to demonstrate the potential of the method to elucidate some of the characteristics of the pore, including its molecularity. That membrane-active factors can be detected and assayed in crude preparations is demonstrated with an impure extract of "amoebapore" from Entamoeba histolytica. In addition, variation of the buffer composition surrounding the vesicles can provide information about the ion selectivity of the pore under investigation.

Diffusion↗
PubMed1985

Aggregation, lipid exchange, and metastable phases of dimyristoylphosphatidylethanolamine vesicles.

A new fluorescent lipid analogue, bimanephosphatidylcholine, has been synthesized for use in lipid bilayers. This probe is well suited as an energy-transfer donor with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine as the acceptor. Dimyristoylphosphatidylethanolamine vesicles are prepared by sonication at pH 9 and characterized by electron microscopy and other methods. Resonance energy transfer between separately labeled donor and acceptor vesicles is monitored during HCl-induced aggregation to determine the kinetics of lipid randomization. Light scattering is also monitored to measure the kinetics of aggregation. The light scattering shows a marked reversal with NaOH while the energy transfer does not, indicating lipid exchange during a reversibly aggregated state; the extent of energy transfer suggests that only lipids in the outer monolayers exchange. The gel to liquid-crystalline phase transition temperature in HCl-treated vesicles is found to be 47 degrees C with diphenylhexatriene. The initial sonicated dispersion does not show a sharp phase transition. In vesicles labeled with both donor and acceptor probes, a small, irreversible increase in energy transfer is obtained upon lowering and then restoring the pH. These results suggest a metastable phase in the sonicated vesicles containing a randomized distribution of lipid and probes within the bilayers; the thermodynamically favored phase, whose formation is triggered by the pH shock, contains domains within which the probe lipids are more highly concentrated.

Energy Transfer↗
PubMed1985

Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon.

The characteristics of transmittance and fluorescence changes of 4-(p-aminostyryl)-1-pyridinium dyes in response to voltage-clamp pulses on the squid giant axon were examined. A zwitterionic styryl dye displays transmittance and excitation spectra on the voltage-clamped squid axon with shapes similar to those previously measured on a model membrane system and consistent with a postulated electrochromic mechanism. The speed of the transmittance response is faster than 1.2 microseconds. The size of the fluorescence change is a factor of 40 lower than on the model membrane; this diminution can be rationalized in terms of the background fluorescence from Schwann cells and the nonoptimal geometric arrangement of the axon membrane. When the emission spectrum is dissected from the excitation response, a nonelectrochromic component is found. This component might result from molecular motion during the excited state lifetime. A positively charged dye permeates the axon membrane and displays complex response waveforms dependent on the method of application and the axon holding potential. This contrasts markedly with model membrane results where the behavior of the cationic and zwitterionic dyes were indistinguishable.

Animals↗
PubMed1983

Diffusion potential cascade. Convenient detection of transferable membrane pores.

A valinomycin-mediated K+ diffusion potential across the membrane of multilamellar liposomes is stable for longer than 30 min and can be collapsed by a nonselective channel such as gramicidin. The kinetics of the potential collapse are complex but can be qualitatively broken down into a series of processes involving (1) binding of the gramicidin to the outer membrane, (2) dimerization to form a functional channel, (3) the flow of ions through the channel, (4) the establishment of a new diffusion potential on the next bilayer within the multilamellar liposome, and (5) the dissociation of gramicidin from the outer bilayer into the adjacent internal aqueous space. These processes are then repeated, in turn, for all the internal bilayers until the K+ concentration gradient (and membrane potential) is completely dissipated. Process 5 appears to be rate limiting at high gramicidin concentrations, but ion flux, process 3, becomes slower at low gramicidin concentrations where the collapse of the K+ gradient displays voltage dependence. Of course the rates of these processes can also be manipulated by changing the composition or size of the liposome and by varying the ion concentrations. Since the diffusion potential can be conveniently monitored with a voltage-sensitive fluorescent dye, 3,3'-diethylthiodicarbocyanine iodide [diS-C2-(5)], a simple method for the detection and partial characterization of membrane pores emerges from this investigation.

Dimyristoylphosphatidylcholine↗
PubMed1982

Design and characterization of electrochromic membrane probes.

Electrochronic membrane probes display a spectroscopic response to membrane potential by a direct electronic mechanism. This allows such probes to be designed a priori via quantum-chemical techniques. The detailed behavior of potentiometric optical probes can be elucidated with an apparatus based on phase-sensitive detection from a hemispherical lipid bilayer; several different types of response spectra can be obtained with this apparatus allowing distinction between the electrochromic mechanism and the more common molecular-motion based mechanisms. The development of 'fast' potentiometric dyes has now reached a stage where practical and exciting applications are rapidly appearing. It is anticipated that the emergence of a complementary set of electrochromic probes will lead to new applications; in particular, it may be possible to elucidate the molecular events which underlie biological or physiological phenomena.

Animals↗
PubMed1981

Charge-shift probes of membrane potential: a probable electrochromic mechanism for p-aminostyrylpyridinium probes on a hemispherical lipid bilayer.

The characteristics of the spectroscopic responses to membrane potential are examined for a series of dyes based on the 4-(p-aminostyryl)-1-pyridinium chromophore. An apparatus using an oxidized cholesterol hemispherical bilayer and phase-sensitive detection provides response spectra in either transmission or fluorescence excitation modes. All the probes with good binding properties display biphasic response spectra that are similar in both shape and magnitude. Detailed analysis of the response spectra allows all the previously discovered mechanisms for extrinsic potential sensitive molecular probes, which require a change in the probe's chemical environment, to be ruled out. The data are consistent with an electrochromic mechanism. Polarized fluorescence intensities from the membrane-bound probes indicate that the chromophore is optimally oriented for an electrochromic response.

Cholesterol↗
PubMed1979

Evidence for a charge-shift electrochromic mechanism in a probe of membrane potential.

Extrinsic optical probes have become important tools for monitoring membrane potential, with probes now available for many tissue or cell suspension systems. In each case that has been studied in detail, it seems that the mechanism involves a shift in the equilibrium population of the probe from one chemical environment to another in response to the transmembrane potential; the environments perturb the probe's spectrum differently. As this indirect mechanism involves a redistribution of dye between chemical environments that are likely to vary if a given probe is transferred from one membrane to another, a potential probe that is effective and calibrated for all membrane systems has not been realised. We present here evidence for a direct response of a probe chromophore to the electric field across membrane systems. The results suggest it might be possible to develop a universal set of membrane probes.

Cholesterol↗