Dipropylthiodicarbocyanine Iodide Research Articles

The effect of the fluorescent probe, 3,3′-dipropylthiodicarbocyanine iodide, on the membrane potential of Ehrlich ascites tumor cells

The effects of the potential-sensitive fluorescent dye, 3,3′dipropylthiodicarbocyanine iodide, on factors establishing the membrane potential of Ehrlich ascites tumor cells have been tested. The dye itself induces membrane hyperpolarization as monitored by electrophysiological methods. In addition, the dye inhibits active (Na ++K +-transport and increases cell membrane permeability to K + by about 65% in these cells.

Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping, deplete cellular ATP and inhibit respiration of isolated mitochondria

3,3′-Dipropylthiodicarbocyanine iodide, a widely used fluorescent probe of membrane potential, was found to inhibit anti-Ig antibody, induced capping of mouse lymphocytes. The dye also lowered the cell ATP content. Experiments with isolated mitochondria revealed that the probe had a potent inhibitory action at site I of the respiratory chain. This mitochondrial blockade helps to explain the ATP depletion and blockade of capping, and gives cause for caution in the use of this dye as a probe of cell membrane potential. Three related dicarbocyanine dyes had similar toxic effects, but two cyanine dyes with much longer alkyl side chains, which have been used as probes of membrane fluidity, did not.

Membrane potential-sensitive fluorescence changes during Na+-dependent D-glucose transport in renal brush border membrane vesicles.

When D-glucose was added to a suspension of renal brush border membrane vesicles equilibrated in a Na+-containing medium, there was a rapid transient increase in the fluorescence of the probe, 3,3'-dipropyl thiodicarbocyanine iodide (DiS-C3-(5)). This sugar-induced response was stereospecific for the D isomer, dependent on Na+, inhibited by phlorizin, and blocked by ionophores, valinomycin plus nigericin, which dissipate ionic gradients. The enhancement in fluorescence suggests the entrance into the vesicle of Na+, cotransported with the sugar. This would lead to the interior of the membrane vesicle becoming more positive, resulting in depolarization of the membrane potential. That the sugar induced the transport of Na+ was confirmed by direct measurement of 22Na+ uptake. Thus, the Na+-sugar co-transport system provides a mechanism for D-glucose to stimulate the flux of Na+ as well as for the Na+ electrochemical gradient to enhance the transport of D-glucose.

The influence of cellular amino acids and the Na +: K + pump on the membrane potential of the Ehrlich ascites tumor cell

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na +: K + pump. The membrane potential (monitored by the fluorescent dye, 3,3′-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na + but not cellular ATP. Na + efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na + and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na + (approx. 30 mM), the Na +: K + pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na + was raised to 60 mM, the activity of the pump changed the membrane potential from the range −25 to −30 mV to −44 to −63 mV. This hyperpolarization required external K + and was inhibited by ouabain.

Effect of Concanavalin A on Membrane Potential of Polymorphonuclear Leukocyte Monitored by Fluorescent Dye

Changes in fluorescence intensity of the dye 3, 3'-dipropylthiodicarbocyanine iodide were monitored in guinea pig polymorphonuclear leukocytes (PMN) in the presence of reagents that modulate plasma membrane dynamics. Valinomycin, a specific K+ ionophore, increased fluorescence intensity as a function of log K+ concentration in incubation medium. The fluorescence was intensified by treating cells with concanavalin A, and 100μg/ml of the lectin was required for a half maximum change. The change was temperature dependent. It was inhibited by potent depressants of membrane fluidity, such as cholesterol ester and cepharanthine, or by colchicine which degrades microtubules. Cytochalasin B which degrades microfilament assembly stimulated fluorescence change. The change was also dependent on the ionic environment and energy state of PMN. These data suggest that the fluidity of the surface membrane and cytoskeletal system are closely related to changes in PMN membrane potential. The data further suggest that the changes might be involved in the concanavalin A-related transmembrane control mechanism.

Evidence for the electrogenic nature of the ATP-ADP exchange system in rat liver mitochondria

A transient decrease in the fluorescent intensity of the dye, 3,3′-dipropylthiodicarbocyanine iodide was seen upon the addition of ATP to rat liver mitochondria which had been pre-treated with 2 · 10 −6 M rotenone and 3.3 μg oligomycin/ml. This decrease which is indicative of a hyperpolarization (internal more negative) was half maximal at 2 · 10 −5 M ATP and was not seen with either ADP or AMP. Atractyloside, bongkrekic acid, ADP and to a lesser extent AMP inhibited the decrease observed with ATP. The characteristics of inhibition by these compounds were similar to those observed in experiments where either transport or binding of adenine nucleotides was measured. The addition of ADP after ATP led to a transient increase in fluorescent intensity indicative of depolarization. This increase was also blocked by atractyloside and bongkrekic acid. The evidence presented supports the hypothesis that the exchange of ADP and ATP via an adenine nucleotide exchange mechanism is electrogenic.

Monitoring membrane potentials in Erhlich ascites tumor cells by means of a fluorescent dye

1. 1. The fluorescent intensity of the dye 3,3′-dipropylthiodicarbocyanine iodide was measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potentials under a variety of different ionic and metabolic conditions. 2. 2. In the presence of valinomycin, fluorescent intensity is dependent on log [K +] medium (the fluorescent intensity increased with increasing [K +] medium) where K + replaced Na + in the medium. Cellular K + content also influenced fluorescent intensity in the presence of valinomycin. With lower cellular K +, fluorescent intensity in the presence of valinomycin for any given concentration was increased. 3. 3. In the presence of gramicidin fluorescent intensity was highest in Krebs-Ringer and decreased with the substitution of choline + for Na +. 4. 4. The observations with ionophores are consistent with the hypothesis that the dye monitors membrane potential in these cells with an increase in fluorescence indicating membrane depolarization (internal becomes more positive). 5. 5. The estimated membrane potential were influenced by the way in which the cells were treated. Upon dilution of the cells from 1 in 20 to 1 in 300 the initial estimations were between −50 and −60 mV. With incubation at 1 in 300 dilution for 1 h at room temperature or a 37 °C, the membrane potentials ranged from −18 to −42 mV. 6. 6. Estimations of membrane potential on the basis of chloride distribution (Cl − cell/Cl − medium) in equilibrated cells ranged from −13 to −32 mV. 7. 7. Addition of glucose to cells equilibrated at 37 °C for 30 min in the presence of rotenone led to a decrease in fluorescent intensity indicating hyperpolarization. Addition of ouabain in turn led to a 70 to 100% reversal of fluorescent intensity. This hyperpolarization is therefore probably due to the electrogenic activity of the sodium pump. 8. 8. The addition of amino acids known to require external Na + for transport increased fluorescent intensity (depolarization) reaching a maximum at higher concentrations of amino acids. Plots of 1/Δfluorescence vs. 1/[glycine] were linear with an apparent K m of 2–3 mM. The increase in fluorescence with amino acids always required external Na +. Plots of 1/fluorescence vs. 1/[Na +] medium were also linear with an apparent K m of 29 mM. These apparent K m values compare favorably with those derived from amino acid transport studies using tracers. These data indicate that the Na +-dependent transport of amino acids in these cells is electrogenic.

Membrane potentials in mitochondrial preparations as measured by means of a cyanine dye

Changes in the fluorescent intensity of the dye 3,3′-dipropylthiodicarbocyanine iodide were measured in suspensions of hamster liver mitochondria upon the development of a K + diffusion potential by the addition of valinomycin and upon the development of the energized state by the addition of succinate or ATP. The changes (large decreases) seen with the addition of succinate or ATP (inhibitable by NaCN and oligomycin respectively) were comparable to those recorded upon the addition of valinomycin to mitochondria suspended in media containing low concentrations of K +. The change observed with succinate was partially reversed by the addition of either 2,4-dinitrophenol or ADP. Oligomycin prevented the reversal seen with ADP. Decreases in fluorescent intensity were also recorded when succinate was added to suspensions of inner membranes (prepared from rat liver mitochondria) containing the dye. With submitochondrial particles (also from rat liver mitochondria), however, increases in fluorescent intensity were seen upon the addition of succinate or ATP. These observations are consistent with the idea that a large negative (internal) potential develops across the inner membrane of the mitochondrion during energization and with other aspects of the chemiosmotic hypothesis.