Interaction of organic ions with proteins.

In this study we have investigated how different proteins interact with big organic ions. Two ions that are similar in size and chemical structure (Ph4B- anion and Ph4As+ cation) were studied. The proteins chosen are the two major allergenic proteins of cow's milk, ß-lactoglobulin and ß-casein, and bovine serum albumin, BSA, as the reference protein. First, a quantitative study to determine the hydrophobic degree of the proteins was performed. Then, electrokinetic and stability measurements on protein-coated polystyrene (PS) microspheres as a function of the tetraphenyl ion concentration were carried out. Our results show that the affinity of the organic ions depends on the hydrophobicity of the interface. Big charge inversions and re-stabilization patterns were observed at very low concentrations of tetraphenyl ions for the most hydrophobic protein studied (with ß-casein). Besides, the ionic concentrations needed to destabilize these colloidal systems were roughly one order of magnitude lower for the anion than for the cation. In addition, we studied conformational changes of the adsorbed proteins with a quartz crystal microbalance. Proteins were adsorbed onto hydrophobic flat substrates and then exposed to the tetraphenyl ions. The protein films swelled or collapsed as a function of the accumulation of tetraphenyl ions. Similarly to the electrokinetic/stability studies, the ionic concentration necessary to trigger structural changes of the protein films was one order of magnitude larger for the cation than for the anion. All the results evidence that the accumulation of these organic ions on an interface depends directly on its degree of hydrophobicity. We attribute the different interactions of the anion and the cation with these interfaces to their dissimilar hydration, which makes the anion show a more hydrophobic behaviour than the cation.

Intramitochondrial accumulation of cationic Atto520-biotin proceeds via voltage-dependent slow permeation through lipid membrane.

Conjugation to penetrating cations is a general approach for intramitochondrial delivery of physiologically active compounds, supported by a high membrane potential of mitochondria having negative sign on the matrix side. By using fluorescence correlation spectroscopy, we found here that Atto520-biotin, a conjugate of a fluorescent cationic rhodamine-based dye with the membrane-impermeable vitamin biotin, accumulated in energized mitochondria in contrast to biotin-rhodamine 110. The energy-dependent uptake of Atto520-biotin by mitochondria, being slower than that of the conventional mitochondrial dye tetramethyl-rhodamine ethyl ester, was enhanced by the hydrophobic anion tetraphenylborate (TPB). Atto520-biotin also exhibited accumulation in liposomes driven by membrane potential resulting from potassium ion gradient in the presence valinomycin. The induction of electrical current across planar bilayer lipid membrane by Atto520-biotin proved the ability of the compound to permeate through lipid membrane in a cationic form. Atto520-biotin stained mitochondria in a culture of L929 cells, and the staining was enhanced in the presence of TPB. Therefore, the fluorescent Atto520 moiety can serve as a vehicle for intramitochondrial delivery of hydrophilic drugs. Of importance for biotin-streptavidin technology, binding of Atto520-biotin to streptavidin was found to cause quenching of its fluorescence similar to the case of fluorescein-4-biotin.

Effects of EDTA on routine and specialized coagulation testing and an easy method to distinguish EDTA-treated from citrated plasma samples.

Coagulation testing is performed with citrate-treated plasma. Samples submitted in other anticoagulants, such as EDTA, should not be tested. We aimed to evaluate the effects of EDTA on routine and specialized coagulation tests and to establish sodium tetraphenylborate testing as a quick and reliable method to identify EDTA-treated plasma samples. We performed the following measurements on citrateand EDTA-treated plasma samples from 10 healthy volunteers: sodium tetraphenylborate testing, prothrombin time (PT), partial thromboplastin time (PTT), potassium concentration, and functional assays for factors II, V, VII, VIII, IX, X, XI, XII, proteins C and S, and antithrombin. Mean values for citrate- and EDTA-treated plasma were most different for PT, PTT, factors V and VIII, and proteins C and S. Sodium tetraphenylborate testing correctly classified 100% of citratetreated and EDTA-treated samples. We confirm that EDTA has effects on coagulation assays. Sodium tetraphenylborate testing is a quick, simple, and inexpensive method for coagulation laboratories to identify samples erroneously submitted in EDTA.

Two reaction pathways for transformation of high potential cytochrome b559 of PS II into the intermediate potential form.

This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized ("O-type" effects) or when it is reduced ("R-type" effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the "O-type" mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an "R-type" mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q(C) which interacts with Cyt b559 and is clearly not the Q(B) site. Binding of compounds L to the Q(C) site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E(m) of HP Cyt b559 with increasing concentration of L (up to 10 K(ox)(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q(C) site when HP Cyt b559 is reduced (described by K(red)(L)) induce a conversion of HP Cyt b559 to lower potential redox forms ("R-type" transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q(C) site are discussed.

Use of lipophilic probes of membrane potential to assess human neutrophil activation. Abnormality in chronic granulomatous disease.

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.

Formation of electrical field accompanying temperature jump in isolated spinach chloroplasts.

Temperature-jump-induced absorbance changes of spinach chloroplasts in the dark were studied. After the temperature rise, a fast absorbance decrease and a succeeding slow absorbance increase were observed at the wavelength of 515 nm. The spectrum of the fast phase had positive maxima (increase in absorbance) at 430, 470 and 673 nm and a negative maxima (decrease in absorbance) at 525 nm. Permeant ions, tetraphenylboron-, tetraphenylarsonium+, and tetraphenylphosphonium+, decreased the extent of the fast absorbance change and increased the rate of slow recovery. Additions of inorganic potassium salts had a similar effect. Valinomycin, added in the presence of potassium ion, also increased the rate of slow recovery. These ions and ionophore had a parallel effect also on the recovery of flash-induced 515-nm absorbance change in chloroplasts. Electroneutral nigerericin did not affect the temperature-jump-induced absorbanc change. These results suggest the formation of electrical field across the thylakoid membrane in the dark accompanying the temperature rise. A possible involvement of the movement of water molecules (thermo-osmosis) in the observed absorbance changes is also discussed.

Effect of tetraphenylboron upon the uptake of the lipophilic cation dibenzyldimethylammonium by yeast cells.

The rate of uptake of the lipophilic cation dibenzyldimethylammonium by yeast cells is increased by tetraphenylboron. However, tetraphenylboron increases also the equilibrium partition of dibenzyldimethylammonium between cells and medium, probably because a complex between tetraphenylboron and dibenzyldimethylammonium is trapped inside the cells. Accumulation of dibenzyldimethylammonium in the presence of tetraphenylboron is not reversed by dinitrophenol, whereas accumulation of the lipophilic cation in the absence of tetraphenylboron appears to be almost completely reversible.

Enhancement of the uptake of 1-methyl-4-phenylpyridinium ion (MPP+) in mitochondria by tetraphenylboron.

The uptake of 1-methyl-4-phenylpyridinium (MPP+) by intact mitochondria was measured by an electrode sensitive to MPP+. The electrode was constructed with a polyvinyl chloride membrane that contained tetraphenylboron (TPB) as an ion-exchange. MPP+ was taken up by mitochondria in an energy-dependent process. TPB rapidly enhanced MPP+ uptake by mitochondria, and then induced release of MPP+ from mitochondria in medium containing glutamate and malate. No release of MPP+ from mitochondria after addition of TPB could be observed in medium containing succinate, the oxidation of which is not inhibited by MPP+. The release of MPP+ was caused by respiratory inhibition by MPP+ taken up in mitochondria. Since the release of MPP+ did not increased O2 uptake in mitochondria, the major part of MPP+ released from the matrix, where no respiratory enzyme inhibited by MPP+ exists. We concluded the following effect of TPB on MPP+ uptake from the results: (1) The increase of MPP+ concentration in matrix by addition of TPB increased the amount of bound to the inner membranes of mitochondria. (2) The increase of the amount of MPP+ in the inner membranes enhanced the respiratory inhibition. (3) The respiratory inhibition induced to release MPP+ from the matrix. The relation between MPP+ distribution in the membrane of mitochondria and the respiratory inhibition by MPP+ are discussed.

Calcium release from sarcoplasmic reticulum of normal and dystrophic mice.

Contraction of skeletal muscle is triggered by release of calcium from the sarcoplasmic reticulum. In this study, highly purified normal and dystrophic mouse sarcoplasmic reticulum vesicles were compared with respect to calcium release characteristics. Sarcoplasmic reticulum vesicles were actively loaded with calcium in the presence of an ATP-regenerating system. Calcium fluxes were followed by dual wavelength spectrophotometry using the metallochromic indicators antipyrylazo III and arsenazo III, and by isotopic techniques. Calcium release from sarcoplasmic reticulum vesicle was elicited by (a) changing the free calcium concentration of the assay medium (calcium-induced calcium release); (b) addition of a permeant anion to the assay medium, following calcium loading in the presence of a relatively impermeant anion (depolarization-induced calcium release); (c) addition of the lipophilic anion tetraphenylboron (TPB-) to the assay medium and (d) using specific experimental conditions, i.e. high phosphate levels and low magnesium (spontaneous calcium release). Drugs known to influence Ca2+ release were shown to differentially affect the various types of calcium release. Caffeine (10 mM) was found to enhance calcium-induced calcium release from isolated sarcoplasmic reticulum. Ruthenium red (20 microM) inhibited both calcium-induced calcium release and tetraphenylboron-induced calcium release, and partially inhibited spontaneous calcium release and depolarization-induced calcium release. Local anesthetics inhibited spontaneous calcium release in a time-dependent manner, and inhibited calcium-induced calcium release instantaneously, but did not inhibit depolarization-induced calcium release. Use of pharmacological agents indicates that several types of calcium release operate in vitro. No significant differences were found between normal and dystrophic sarcoplasmic reticulum in calcium release kinetics or drug sensitivities.

Hydrophobic ion interaction on Na+ activation and dephosphorylation of reconstituted Na+,K(+)-ATPase.

In liposomes with reconstituted shark Na+,K(+)-ATPase an uncoupled Na(+)-efflux and a Na+/Na+ exchange can be induced on inside-out oriented pumps by the addition of external (cytoplasmic) Na+ and MgATP to liposomes that either do not contain Na+ (and other alkali cations), or include 130 mM Na+ internally (extracellular). Both modes of exchange are electrogenic and accompanied by a net hydrolysis of ATP. The coupling ratio of positive net charges translocated per ATP split is found to be close to 3:1 and 1:1, respectively, for the two modes of exchange reactions at pH 7.0. By addition of the hydrophobic anion tetraphenylboron (TPB-), which imposes a negative electrostatic membrane potential inside the lipid bilayer, the ATP hydrolysis accompanying uncoupled Na+ efflux is increased with increasing TPB- concentrations. Cholesterol which increases the inner positive dipole potential of the bilayer counteracted this activation by TPB- of uncoupled Na+ efflux. Using the structural analog tetraphenylphosphonium (TPP+), which elicits an inside positive membrane potential, ATP hydrolysis accompanying uncoupled Na(+)-efflux is decreased. The rate of dephosphorylation in the absence of extracellular alkali cations was affected in a similar manner, whereas the dephosphorylation in the presence of extracellular Na+ inducing Na+/Na+ exchange was unaffected by the hydrophobic ions. In both modes of exchange the phosphorylation reaction was independent of the presence of hydrophobic ions. The hydrophobic ions affected the apparent affinity for cytoplasmic Na+, indicating that binding of cytoplasmic Na+ may involve the migration of cations to binding sites through a shallow cytoplasmic access channel. The results are in accordance with the simple electrostatic model for charge translocation in which two negative charges in the cytoplasmic binding domain of the Na+,K(+)-ATPase co-migrate during cation transport.

Effect of the calcium-channel blockers on calcium accumulation in sarcoplasmic reticulum of skeletal muscle.

Vesicular fragments of sarcoplasmic reticulum isolated from rabbit skeletal muscle were actively loaded with Ca2+ in the presence of ATP and an ATP-regenerating system using Arsenazo III as metallochromic indicator to monitor Ca2+ movements across the membrane. Once the Ca2+ release is triggered by the presence of tetraphenylboron in the reaction medium, the addition of verapamil or diltiazem gives rise to a net Ca2+ entry inside the vesicles. Preincubation in the presence of verapamil does not abolish the tetraphenylboron-induced Ca2+ release, the verapamil-induced Ca2+ accumulation being still observed. There appears to be a high-affinity site for verapamil titrated in the micromolar concentration range, whereas diltiazem demonstrates more complex behavior when its concentration is raised. This study suggests the existence of a Ca2+ pathway (putative channels) which is blocked by the drugs tested allowing Ca2+ accumulation inside the vesicles owing to the Ca2+-dependent ATPase activity.

Inhibition of intercellular junctional communication in human fibroblasts by triphenylmethane, triphenylmethylchloride, tetraphenylboron and related compounds.

Intercellular junctional communication was measured using [14C]citrulline incorporation in co-cultures of argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts. Triphenylmethane, triphenylmethylchloride and tetraphenylboron inhibited communication at concentrations at least 12-fold lower than cytotoxic concentrations. This inhibition was of rapid onset and was rapidly reversible by washing the co-cultures. Refractoriness to inhibition did not develop after prolonged exposure. Several other compounds also induced communication inhibition, but only at concentrations slightly below cytotoxic concentrations. Treatment of co-cultures with calcium ionophore A23187 or cycloheximide did not cause communication inhibition. It is suggested that triphenylmethane, triphenylchloride and tetraphenylboron may be useful inhibitors for studying the roles of intercellular junctional communication in some biological systems.

Mechanism of energization of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation [DMP+] into an acrA strain of Escherichia coli.

The mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) into cells and vesicles of the acrA strain AS-1 of Escherichia coli was examined. Uptake was energized by substrate oxidation and discharged by uncouplers. Uptake was enhanced by the presence of tetraphenylphosphonium cation, tetraphenylboron anion and tributyltin chloride, which may inhibit the efflux system for DMP+. Uptake was inhibited by 5-methoxyindole-2-carboxylic acid (MIC). By the use of ionophores with right-side-out vesicles loaded with monovalent cations it was shown that DMP+ uptake could be driven both by the establishment of a membrane potential across the vesicle membrane and by a H+/DMP+ antiport system. Attempts to demonstrate the latter mechanism in everted membrane vesicles were unsuccessful.

Exploring the potential of phyllosilicate minerals as potassium fertilizers using sodium tetraphenylboron and intensive cropping with perennial ryegrass.

In response to addressing potassium (K) deficiency in soil and decreasing agricultural production costs, the potential of K-bearing phyllosilicate minerals that can be directly used as an alternative K source has been investigated using sodium tetraphenylboron (NaTPB) extraction and an intensive cropping experiment. The results showed that the critical value of K-release rate and leaf K concentration was 3.30 g kg(-1) h(-1) and 30.64 g (kg dry matter)(-1), respectively under the experimental conditions. According to this critical value, the maximum amount of released K that could be utilized by a plant with no K deficiency symptoms was from biotite (27.80 g kg(-1)) and vermiculite (5.58 g kg(-1)), followed by illite, smectite and muscovite with 2.76, 0.88 and 0.49 g kg(-1), respectively. Ryegrass grown on phlogopite showed K deficiency symptoms during the overall growth period. It is concluded that biotite and vermiculite can be directly applied as a promising and sustainable alternative to the use of classical K fertilizers, illite can be utilized in combination with soluble K fertilizers, whereas muscovite, phlogopite and smectite may not be suitable for plant growth. Further field experiments are needed to assess the use of these phyllosilicate minerals as sources of K fertilizer.

Enhancement by tetraphenylborate of technetium-99m-MIBI uptake kinetics and accumulation in cultured chick myocardial cells.

Myocellular uptake and retention of technetium-99m-hexakis (2-methoxyisobutylisonitrile) (Tc-MIBI), a lipophilic cationic myocardial perfusion and viability imaging agent, is dependent on both mitochondrial and plasma-membrane potentials. To test for enhancement of uptake kinetics by lipophilic anions, cultured chick heart cells were exposed to tetraphenylborate (TPB), which produced a concentration-dependent maximal 15-fold increase in Tc-MIBI uptake kinetics (at 3 x 10(-5) M) and enhanced peak accumulation of Tc-MIBI from 165.4 +/- 26.3 to 705.6 +/- 61.3 fmoles/mg protein.nMo (P less than 0.001). Carbonyl cyanide-m-chloro phenylhydrazone (CCCP; 10(-5) M), a mitochondrial uncoupler, rapidly depleted cellular content of Tc-MIBI in the presence of TPB (10(-5) M) from 300.0 +/- 30.0 to 42.5 +/- 1.9 fmole/mg protein.nMo (p less than 0.001). TPB enhanced both uptake rates and net accumulation of Tc-MIBI at all buffer Ko concentrations between 130 mM and 0.54 mM. Tc-MIBI influx rates allowed estimation of plasma-membrane potential as a function of Ko in the presence of valinomycin with a slope of -67 mV/decade (r = -0.99). The results further support a potential-dependent mechanism for cell uptake of Tc-MIBI and suggest a rational approach for increasing tissue extraction fraction in vivo.

Antagonism by lipophilic quaternary ions of the K+ channel opener, levcromakalim, in vascular smooth muscle.

1. The aim of this study was to characterize the interaction between the K+ channel opener levcromakalim (LKM) and several quaternary ions, in vascular smooth muscle, in vitro. Segments of isolated, thoracic aorta of the rat were suspended in organ baths filled with Krebs solution at 37 degrees C. Cumulative concentration-response curves to LKM were obtained in the absence and in the presence of increasing concentrations of quaternary ions using a number of agents to pre-constrict the vessel. The ions tested were tetraphenylphosphonium (chloride TPP-Cl and bromide TPP-Br salts), tetrapentylammonium (TPeA), tetraethylammonium (TEA), tetraphenylarsonium (TPAs) and tetraphenylboron (TPB). 2. For the compounds which antagonized the vasorelaxation responses of LKM, 'apparent pKB' values were estimated on the basis of a single concentration of antagonist. These were then used to obtain the following order of potency: TPP-Br (7.22 +/- 0.25) = TPAs (7.12 +/- 0.04) = TPP-Cl (7.11 +/- 0.15) > TPeA (6.23 +/- 0.20). TEA and TPB were both found to be inactive at the maximum concentrations used. 3. The interaction between the cationic TPP and anionic TPB was also investigated. The shift in the LKM concentration-response curve constructed in the presence of both of these compounds was compared to that when each agent was present separately. We found that TPB, at concentrations greater than 1 microM, reversed the blockade of the LKM-mediated relaxation induced by TPP (3 microM). 4. Similar experiments were undertaken combining TPB with either alinidine or glibenclamide (both functional antagonists of K+ channel openers). It was found that TPB (10 microM) partially reversed the antagonism induced by alinidine (30 and 100 microM) but had no effect on the action of glibenclamide(3 microM).5. These studies show that lipophilic cations such as TPP and TPAs are potent antagonists of levcromakalim-mediated vasorelaxation responses in the rat thoracic aorta. The mechanism by which these compounds cause their antagonism is not known. However, given the lipophilicity of these compounds, it is possible they may act at a number of sites including the KATP channel itself or possibly via some other intracellular mechanism.

The effects of tetraphenylboron on neuromuscular transmission in the frog.

1 The effect of tetraphenylboron (TPB) were studied on the frog sciatic nerve-sartorius muscle preparation. 2 TPB (0.01-1 mM) blocked indirectly elicited twitches of the preparation. 3 TPB (0.01-0.1 mM) produced no depolarization but lowered membrane resistance. TPB increased miniature endplate potential (m.e.p.p.) frequency, the rate of rise of the endplate potential (e.p.p.), and slowed the rate of rise and rate of fall of the muscle action potential. 4 In Mg2+ solutions the quantal content of e.p.ps was initially increased by TPB (0.01 mM). This was followed by a decrease of e.p.p. and m.e.p.p. amplitudes, accompanied by a lack of e.p.p. failures. 5 Larger concentrations of TPB (0.1 mM) produced an increase in e.p.p. amplitude followed by the sudden abolition of e.p.ps. This effect was associated with abolition of the nerve terminal spike. 6 TPB (0.1 mM) exhibited no postjunctional blocking action. 7 The results indicate that TPB acts prejunctionally, initially causing an increased release of acetylcholine. Subsequently, transmitter output is reduced by a reduction of quantal size.

The effects of tetraphenylboron on spontaneous transmitter release at the frog neuromuscular junction.

1 The effects of tetraphenylboron (TPB) on spontaneous transmitter release were studied in the frog sartorius muscle preparation. 2 TPB (0.001-1 mM) produced a time-dependent increase in miniature endplate potential (m.e.p.p.) activity that was not sustained. TPB (0.1 mM) produced similar effects on m.e.p.p. frequency in normal Ringer solutions, in the absence of Ca2+ or Cl- and in the presence of excess Ca2+ and of tetrodotoxin. The effect of TPB (0.01 mM) was reduced but not abolished in the absence of Ca2+. 3 As m.e.p.p. frequency fell from its peak level in TPB (0.04 mM) m.e.p.p. amplitude was reduced. The reduction of m.e.p.p. amplitude was not prevented by choline (30-300 muM). 4 When m.e.p.p. activity fell below the noise level in the presence of TPB (0.1 mM), lanthanum (0.5 mM) was ineffective in promoting measurable m.e.p.p. activity. 5 The effects of TPB were slowly reversible by washing. 6 The results indicate that TPB acts to reduce the nerve terminal stores of acetylcholine, probably by a combination of rapid release and concomitant inhibition of transmitter storage.

Hepatic uptake and biliary excretion of organic cations--II. The influence of ion pair formation.

The influence of an anorganic anion iodide (I-) and an organic anion tetraphenylborate (TPB-) on the hepatic uptake and biliary excretion of three organic cations, triethylmethyl ammonium (TEMA), tripropylmethyl ammonium (TPMA) and tri-n-butylmethyl ammonium (TBuMA) was studied. The compounds were injected as a bolus (D = 1 mumole) and studied in isolated perfused livers. In the perfusion medium 25% of the amount of NaCl (3 mmole) was replaced by NaI, whereas in two other experiments TPB- was added to the medium in two concentrations (2 microM and 200 microM). NaI did not affect the biliary output of the three quaternary ammonium compounds (QACs) although an increased net rate of hepatic uptake was found for all compounds, most likely due to a decreased liver to plasma transport. Liver to plasma concentration ratios were increased, while the ratios between bile to liver and bile to plasma were not affected. TPB- in catalytic amounts added to the medium (2 microM) decreased the biliary output of TEMA and TBuMA, whereas the kinetic profile of TPMA was unchanged. The decreased biliary excretion rate of TEMA was explained by a decreased plasma level (due to the increased liver uptake) assuming that the small molecular weight compounds can enter the bile directly from plasma via the junctional complexes between the cells. The bile to plasma (B/P) ratio was not affected. In contrast, the bile to plasma (B/P) ratio and the bile to liver (B/L) ratio of TBuMA were decreased, compared with the control, probably due to an increased reabsorption from the bile, whereas the back transport from the liver into the plasma was also decreased. A large amount of TPB- (200 microM), added to the perfusion medium, dramatically changed the kinetic profile of the three QACs. Ion pair formation between the QACs and TPB- was supposed to be responsible for this effect. Plasma levels dropped more rapidly as a result of an increased rate of liver uptake. The biliary excretion of all compounds was greatly reduced (the excretion rates were 0.022, 0.19 and 0.18 nmole/min, compared with 0.047, 0.71 and 7.5 nmole/min for the controls). It is concluded that ion pair formation may play a role in the hepatobiliary transport. The rate of liver uptake of the QACs is enhanced in the presence of an anion, which is due to an increase in plasma to liver transport (k12) and a reduced liver to plasma transport (k21).(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative effects of neutral dipolar compounds and lipophilic anions on technetium 99m-hexakis (2-methoxyisobutyl isonitrile) accumulation in cultured chick ventricular myocytes.

RATIONALE AND OBJECTIVES: Non-flow-dependent myocellular accumulation and uptake kinetics of the myocardial perfusion and viability imaging agent, hexakis (2-methoxyisobutyl isonitrile) technetium 99m(I) (Tc-SESTAMIBI), are thermodynamically driven by large negative sarcolemmal and mitochondrial membrane potentials, and can be enhanced by addition of the lipophilic anion, tetraphenylborate (TPB). To further understand the general properties required of a co-administered compound for increasing the kinetic response of Tc-SESTAMIBI to membrane potential, a systematic appraisal of additional candidate lipid-soluble anions and neutral dipolar compounds was undertaken. METHODS: Each compound was biologically tested for its ability to enhance Tc-SESTAMIBI accumulation in a cultured heart cell model, and electronic dipole moments were evaluated using semi-empirical molecular orbital calculations. RESULTS: Of this series, phloretin (100 microM), TPB (10 microM), and to a lesser degree, 8-anilino-1-naphthalene sulfonate (100 microM) enhanced myocellular accumulation of Tc-SESTAMIBI. Phloretin enhancement was pH-dependent, showing maximal effect at pH 7.4, and was not additive to the augmentation induced by TPB. A series of additional lipid soluble anions and structural analogues of phloretin were without effect. CONCLUSION: Although selected compounds enhanced Tc-SESTAMIBI accumulation, overall, no direct relationship of dipole moment to biologic enhancement was demonstrated.