carbocyanines and triphenylmethylphosphonium

Four indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3'dipentyloxacarbocyanine [di-O-C5(3)], 3,3'dipentylindocarbocyanine [di-I-C5(3)], and 3,3'dipropylthiodicarbocyanine [di-S-C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di-S-C3(5) not only exhibits a different fluorescence response mechanism from di-O-C5(3) [and di-I-C5(3)] but also that the fluorescence of di-S-C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB-, to shorten the TPMP+ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB- are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di-O-C5(3) and di-I-C5(3) but cannot be avoided with di-S-C3(5) or TPMP+/TPB-. Using di-O-C5(3) with the fluorescence-activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe-specific problems or response heterogeneity.

Topics: Carbocyanines; Cells, Cultured; Chemotactic Factors; Chemotaxis, Leukocyte; Fluorescent Dyes; Humans; Membrane Potentials; Neutrophils; Onium Compounds; Quinolines; Superoxides; Trityl Compounds

The membrane potential of the human platelet was investigated using the membrane potential probes 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide and tritiated triphenylmethylphosphonium bromide. The membrane potential in physiologic buffer was estimated to be 52-60 mV inside negative. The membrane was depolarized when extracellular potassium or hydrogen ion concentrations were increased. Changes in extracellular sodium, chloride, or calcium ion concentration had no measurable effect on membrane potential. Elevated extracellular potassium has been shown to increase platelet sensitivity to the aggregating agent, adenosine diphosphate. Our results show that changes in extracellular ion concentrations that depolarize platelets increase platelet sensitivity to aggregating agents. These results suggest that membrane potential changes may play a role in modulating the response of platelets to aggregating agents.

Topics: Benzothiazoles; Blood Platelets; Carbocyanines; Electrolytes; Humans; Inulin; Membrane Potentials; Onium Compounds; Platelet Aggregation; Spectrometry, Fluorescence; Trityl Compounds; Valinomycin

The accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma N1E 115 cells in the presence of tetraphenylboron is reduced by 3,3'-diethylthiadicarbocyanine iodide and by 3,3'-dipropylthiadicarbocyanine iodide. This reduction in uptake of the lipophilic cation is not due to the carbocyanine dyes depolarizing the plasma membrane of these cells but due to an interaction between the carbocyanine dyes and tetraphenylboron leaving less of the lipophilic anion free in solution to assist uptake of the lipophilic cation. This interaction is shown to have a 1:1 stoicheiometry.

Topics: Animals; Benzothiazoles; Biological Transport; Carbocyanines; Cell Line; Fluorescent Dyes; Indicators and Reagents; Kinetics; Membrane Potentials; Mice; Neoplasms, Experimental; Neuroblastoma; Onium Compounds; Quinolines; Thiazoles; Tritium; Trityl Compounds

Methods for the measurement of membrane potentials in cells not easily penetrated by microelectrodes were assessed for use in epithelia. The lipophilic cation triphenylmethylphosphonium appeared to distribute in a Nernstian fashion in the epithelial cells of Necturus gallbladder as judged by parallel microelectrode measurements. In the toad urinary bladder, the distribution of this cation gave a value for the membrane potential of epithelial cells under short-circuit conditions of -62 mV in normal Ringer's solution and -51 mV after 2 h treatment with ouabain. In our laboratory the dye 3,3'-dipropylthiadicarbocyanine iodide, when used with cell suspensions, yielded results comparable to those of other workers, but we were unable to record a redistributional signal from epithelial sheets or scraped cells. The dye appeared to enter cells and become irreversibly bound. The membrane-bound merocyanine dyes have not been used on epithelial cells. They appear to hold the greatest promise for dynamic experiments on epithelial membrane potentials.

Topics: Animals; Anions; Anura; Benzothiazoles; Carbocyanines; Cations; Epithelium; Membrane Potentials; Necturus; Onium Compounds; Trityl Compounds; Urinary Bladder; Urodela

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.

Topics: Adolescent; Adult; Calcimycin; Carbocyanines; Chediak-Higashi Syndrome; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Female; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Membrane Potentials; Neutrophils; Onium Compounds; Superoxides; Tetradecanoylphorbol Acetate; Tetraphenylborate; Trityl Compounds; Valinomycin