Characterization of solute/proton cotransport in plasma membrane vesicles from Ricinus cotyledons,and a comparison with other tissues

Plasma membrane vesicles, purified by aqueous two-phase partitioning, were used to investigate the presence of sugar and amino acid carriers in cotyledons and roots of Ricinus communis L. and in roots of red beet (Beta vulgaris L.). Artificial pH and electrical gradients were generated across the plasma membrane, and [¹⁴C]acetate and [¹⁴C]tetraphenylphosphonium were used to demonstrate the presence of an internal alkaline pH gradient and an internal negative membrane potential, respectively. In Ricinus cotyledons, uptake of sucrose was more strongly inhibited than that of glutamine by p-chloromercuribenzenesulphonic acid, phlorizin and phenylglyoxal. The sucrose transport system showed a high degree of substrate specificity with only the presence of maltose and phenyl--glucoside significantly affecting sucrose uptake; in contrast, the glutamine transport system was inhibited by a number of other amino acids. pH+gD-driven glutamine uptake showed saturation kinetics with a K _m of 0.35 mol · m^–3. Sucrose and glutamine -driven uptake was pH dependent with an optimum in the acidic range (pH 6.25) and a decrease at higher pH values. Vesicles obtained from cotyledons and roots of Ricinus showed different transport properties. In the cotyledons, gDH+gD-driven transport for both sucrose and glutamine were observed at similar levels; however, in the root tissue, pH--driven glutamine transport was the dominant uptake process. Uptake rates for glucose and fructose were low in the cotyledons whereas, in the roots, glucose and sucrose transport were slightly higher than that of fructose. In vesicles from red beet tissue there was a different uptake profile, with evidence of proton-coupled cotransport systems for sucrose and glucose, but lower uptake of glutamine and fructose. The results are discussed in relation to the reported different pathways for loading and unloading of solutes in these tissues.Abbreviations CCCP carbonyl cyanide-m-chlorophyenyl hydrazone - DEPC diethyl pyrocarbonate - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid - TPP tetraphenylphosphonium ion - gDH⁺ proton electrochemical potential gradient - membrane potential We would like to thank the SERC(UK) and the Royal Society for financial support.     

Metabolic state and cell cycle as determinants of facilitated uptake of genetic information by yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The transformation efficiency of yeast cells during exponential growth might be characterised as undulatory. The aim of the study was to investigate the reason for the fluctuation in transformation efficiency of yeast Saccharomyces cerevisiae p63-DC5 cells during exponential growth. The heightened response to exogenous DNA was observed with the growing yeast culture when budded cells were predominant. To confirm this phenomenon we carried out synchronization of yeast cells with 10 mM hydroxyurea. Results showed that synchronous yeast cells in the S-phase of cell cycle have enhanced transformation efficiency. Furthermore, S. cerevisiae p63-DC5 cells in the S-phase were successfully transformed with plasmid pl13 in the absence of lithium acetate. We indicated that the permeability of yeast cells in the S-phase to tetraphenylphosphonium (TPP) cations was significantly higher than in asynchronous culture. The results of our study showed that the fluctuation in transformation efficiency was strictly dependent on the metabolic state of yeast cells and the capacity of the yeast cells to become competent was related to the S-phase of cell cycle.     

Age and growth-related changes in cyclopiazonic acid-potentiated lipophilic cation accumulation by cultured cells and binding to freeze-thaw lysed cells

In a previous study (1) we demonstrated that increased tetraphenylphosphonium (TPP) uptake by renal epithelial cells (LLC-PK₁) exposed to the fungal metabolite cyclopiazonic acid (CPA) was not a result of hyperpolarization across the plasma membrane even though CPA-potentiated TPP uptake could be totally inhibited by the depolarizing agent carbonylcyanide-m-chlorophenylhydrazone (CCCP). We now demonstrate that CPA potentiates TPP accumulation by proliferating skeletal muscle (L6) and LLC-PK₁ cells but not by nonproliferating primary rat hepatocytes. In LLC-PK₁ cells, CPA-potentiated TPP accumulation is observed in cells at all ages. In s cells, CPA-potentiated TPP accumulation is maximal soon after subculturing, and as the cells age they become less sensitive to CPA until TPP accumulation by CPA-treated cells approaches that of untreated cells. The temporal change in sensitivity of L6 cells to CPA may be related to biochemical and/or metabolic changes which occur as the cells age in culture. Hepatocytes, LLC-PK₁ cells, and L6 cells permeabilized by freeze-thaw lysis, all exhibit CPA-potentiated TPP partitioning, even in the presence of CCCP. This result indicates that both TPP and CPA must have access to the intracellular space in order for potentiated TPP partitioning to be observed. We hypothesize that the site of interaction between CPA and TPP is intracellular and probably associated with the cytoplasmic side of the plasma membrane and possibly the mitochondria.     

Membrane Potential and Catecholamine Secretion by Bovine Adrenal Chromaffin Cells: Use of Tetraphenylphosphonium Distribution and Carbocyanine Dye Fluorescence

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.     

Interference of calmidazolium with measurement of mitochondrial membrane potential using the tetraphenylphosphonium electrode or the fluorescent probe rhodamine 123

Calmidazolium (CMZ) is a positively charged, hydrophobic compound used as a calmodulin antagonist. It may cause unspecific effects in mitochondria, e.g., a decrease in membrane potential (deltapsi), swelling, and uncoupling. Several groups have advised against use of CMZ in studying signal transduction in mitochondria. We report here that it interferes with measurement of deltapsi in rat liver mitochondria (RLM) when using the tetraphenyl phosphonium (TPP+) electrode. We also found that CMZ reduces the signal, indicating an apparent drop in deltapsi. CMZ itself gave a signal with the TPP+ electrode in the absence of RLM. At high concentrations, > 10 microM, it also reduced the fluorescence quenching of the probe rhodamine 123. This may be due to an interference with mitochondrial uptake and binding of this positively charged probe or to an uncoupling effect. It is concluded that CMZ and similar positively charged calmodulin antagonists such as trifluoperazine may be used in mitochondria if these interferences are controlled and calibration is carried out under the experimental conditions used.     

Effects of Chronic Nicotine Treatment on the Accumulation of [3H]Tetraphenylphosphonium by Cerebral Cortical Synaptosomes

Abstract: Chronic exposure of rats to nicotine increases the number of [³H]nicotine binding sites in the brain; however, it is not clear whether nicotinic cholinergic receptor function is altered as well. In this study, we have used [³H]tetraphenylphosphonium as a probe of synaptosomal membrane potential to investigate whether exposure to nicotine in vivo alters the ability of cerebral cortical synaptosomes to maintain a potential difference and to depolarize in response to in vitro nicotine. Treatment of rats for 14 days with 0.475 mg of nicotine base/day via subcutaneously implanted minipumps resulted in a decrease in the synaptosomal accumulation of [³H]tetraphenylphosphonium in physiological buffer, corresponding to a decrease in estimated membrane potential from –55 mV to –50 mV. The onset of the decrease in membrane potential occurred after 7 days of in vivo nicotine treatment and was significantly correlated with an increase in [³H]nicotine binding to cerebral cortical synaptosomal (P₂) membranes. Nicotine, at in vitro concentrations of 3–1,000 μ M , decreased [³H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes from control animals. When compared to accumulation in buffer alone, in vitro nicotine and other nicotinic agonists did not significantly decrease [³H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes prepared from rats treated with nicotine in vivo. These studies provide evidence that chronic treatment with nicotine results in an average lower membrane potential in cerebral cortical synaptosomes and in functional down-regulation of the depolarization response to nicotinic cholinergic receptor stimulation.     

31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE

The binding of tetraphenylphosphonium (TPP⁺) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by ³¹P cross-polarisation–magic-angle spinning nuclear magnetic resonance spectroscopy (CP–MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP–MAS could selectively distinguish binding of TPP⁺ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.     

Pitfalls in the measurement of membrane potential in yeast cells using tetraphenylphosphonium

The uptake of the lipophilic cation tetraphenylphosphonium (Ph₄P⁺) by Saccharomyces cerevisiae was measured using yeast grown on glucose and harvested either at the logarithmic or at the stationary phase of growth. When yeast was collected at the stationary phase, Ph₄P⁺ uptake proceeded steadily during several hours until an equilibrium was reached. When yeast was collected in the logarithmic phase of growth, a biphasic uptake was observed. The second phase of uptake began when the glucose of the incubation medium had been exhausted. From experiments in the presence of cycloheximide or chloramphenicol it is concluded that the second phase of Ph₄P⁺ uptake is dependent on the synthesis of some protein(s) repressed by glucose but unrelated with the existence of functional mitochondria. The addition of compounds which collapse the membrane potential provokes an efflux from the yeast cells of the Ph₄P⁺ accumulated both during the first phase and the second phase of uptake. It is concluded that accumulation of Ph₄P⁺ in yeast cells is a complex process and that Ph₄P⁺ cannot be used to give a quantitative measure of the yeast plasma membrane potential.     

The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

Membrane potential in yeast cells measured by direct and indirect methods

The membrane potential, ΔΨ, of various yeasts estimated from the distribution of tetraphenylphosphonium cations ranged from ?50 to ?120 mV, depending on species, incubation conditions and technique of measurement. Values obtained directly with a microelectrode in Endomyces magnusii were consistently lower than those determined indirectly.