Increase In Rate Of Efflux Research Articles

Abstract 172: Adipocytes decrease daunorubicin concentration in acute lymphoblastic leukemia cells

Abstract Obesity is associated with increased cancer incidence and mortality. Adipose tissue has been shown to play a role in several types of cancer, including acute lymphoblastic leukemia (ALL). We have demonstrated that murine ALL cells actively migrate into adipose tissue in mice, and that adipocytes protect ALL cells from daunorubicin (DNR) in vitro. We have further shown that media cultured by both adipocytes and ALL cells together (ALCM) can protect ALL cells from DNR. We hypothesize that adipocytes protect ALL cells from DNR by decreasing ALL cell intracellular drug accumulation. Human pre B ALL cells (BV173) were co-cultured with 3T3-L1 adipocytes using a transwell system. Intracellular DNR was visualized using con-focal fluorescence microscopy at various time points in leukemia cells during DNR treatment. Drug uptake in leukemia cells treated with DNR and co-cultured with adipocytes was measured by flow cytometry. By preloading cells with DNR, drug efflux assays were also performed with ALL cells. Gene expression of ABCB1a and cell surface expression of MDR1 were quantified using qPCR and flow cytometry, respectively, in ALL cells co-cultured with adipocytes. After 72 hours of DNR treatment, there were significantly more viable BV173 cells in the transwells placed over adipocytes than those over fibroblasts (290,000± 60,500 vs. 108,000± 13,000, p=0.008, n=4). Co-culture with adipocytes was associated with a lower intracellular DNR in BV173 cells. After 4 hours, DNR was 30% lower, and by 24 hours intracellular DNR was 80% less (n=1). However, co-culturing ALL cells with adipocytes was not associated with a significant increase in efflux rate of DNR from preloaded BV173 (half-life = 36±13 vs. 31±9 min., p=0.23, n=3). The mRNA expression of ABCB1a, the gene encoding MDR1, was increased in BV173 cells (130% increase, n=1) co-cultured with adipocytes for 24 hours. However, we did not detect an increase in surface MDR1 expression in BV713 co-cultured with adipocytes. We then tested whether adipocyte-secreted factors would cause efflux of DNR from ALL cells. There was a tendency for DNR efflux rate to be higher in BV173 cells cultured in ALCM (half-life = 36±13 vs. 27±10 min., p=0.08, n=3). Further investigation is needed to confirm this effect. These findings demonstrate that adipocytes may protect ALL cells from daunorubicin treatment in part by decreasing intracellular drug accumulation. However, it is not clear whether this effect is due to altered uptake, efflux, or intracellular drug action. Citation Format: Xia Sheng, Jonathan Tucci, James Behan, Steven D. Mittelman. Adipocytes decrease daunorubicin concentration in acute lymphoblastic leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 172. doi:10.1158/1538-7445.AM2014-172

Protons activate a cation conductance in a sub‐population of rat dorsal root ganglion neurones.

1. The responses of adult and neonatal rat dorsal root ganglion (DRG) neurones to buffered acidic solutions were studied with both voltage clamp and radioactive ion flux techniques. Electrophysiological experiments were made on acutely isolated neurones and ion flux experiments were made on cells that had been in culture for 3-6 days. 2. Acid solutions of pH < 6.2 evoked a sustained, slowly inactivating inward current in neurones voltage clamped at negative holding potentials. The size of the current increased with increasing proton concentrations. This response was restricted to a sub-population (approximately 45%) of adult and neonatal rat DRG neurones and was distinct from a rapidly activating and inactivating proton-induced inward sodium current that was also found in DRG neurones. 3. The proton-activated sustained current was due to an increase in cation conductance that allowed K+, Cs+ and Na+ to pass with PK/PNa = 1.32 and PCs/PNa = 1.12. 4. Radioactive ion efflux experiments made on neonatal rat cultured DRG neurones showed that protons also increased the permeability to both [14C]guanidinium and 86Rb+ ions. The half-maximal increase in efflux rate for 86Rb+ occurred at pH 5.8. Acid solution also stimulated the efflux of 86Rb+ in cultures of adult rat neurones. 5. Cells that showed a late, sustained proton-activated current also responded to capsaicin. In addition, no proton-activated fluxes of either [14C]guanidinium or 86Rb+ ions were observed in cultures of DRG neurones that had been treated with high concentrations of capsaicin (10 microM) to kill the capsaicin-sensitive neurones. Thus this proton-activated current is restricted largely, if not exclusively, to capsaicin-sensitive peripheral sensory neurones.

Potassium (86Rb+) efflux from the rat submandibular gland under sodium-free conditions in vitro.

1. Fragments of rat submandibular gland were pre-loaded with 86Rb+, an isotopic marker of potassium transport, and rate constants for 86Rb+ efflux were determined during superfusion with a physiological salt solution. 2. In sodium-containing solutions acetylcholine evoked a rapid and immediate increase in efflux rate. After reaching a peak value, the efflux rate initially declined rapidly, but a second, slowly declining phase to the response was also evident. The response could be resolved into Ca2(+)-independent and Ca2(+)-dependent phases. 3. The basal efflux rate was elevated during superfusion with solutions in which sodium had been replaced with either lithium or N-methyl-D-glucammonium (NMDG+). Although lithium had a greater effect, which was absent under calcium-free conditions, addition of calcium to initially calcium-free, lithium-containing solutions did not affect the rate of efflux. 4. In the presence of calcium the response to acetylcholine was augmented during exposure to lithium-containing, sodium-free solutions but, in contrast, slightly inhibited when NMDG+ was used as a sodium substituent. 5. The transient, calcium-independent component of the response to acetylcholine was unaffected by exposure to lithium, whereas the calcium-dependent phase of the response was inhibited. 6. Responsiveness to acetylcholine was reduced during superfusion with a calcium-free, NMDG+-containing solution. The response normally observed when extracellular Ca2+ was subsequently elevated, in the continued presence of acetylcholine, was also inhibited. Sensitivity to acetylcholine was retained, however, when the tissue was initially exposed to a solution containing approximately 20 mumol l-1 Ca2+. The response was smaller than that evoked in sodium-containing solutions. 7. The use of lithium as a sodium substituent presents special problems, possibly related to the effects of this ion on the metabolic cycling of phosphatidylinositol-4,5-bisphosphate metabolites.

Membrane permeability characteristics of perfused human senile cataractous lenses

Human cataractous lenses were removed by the cryoprobe technique and were maintained for up to 24 hr in a solution of similar ionic composition to human aqueous humour. The bimodal distribution of internal sodium concentrations was similar to that previously reported for unincubated human lenses. Lenses with lower total and free sodium contents had relatively higher membrane potentials and they lost 86Rb at a slower rate than lenses with high internal sodium. The 86Rb efflux in these lenses was stimulated four-fold by removing external calcium. The efflux was reduced by inereasing external calcium, but was increased during a small (60 mosmol) hyperosmotic shock. A similar hyperosmotic shock also surprisingly increased 86Rb efflux. Lenses with increasing internal sodium (and calcium) levels showed an inereasing rate of loss of 86Rb and the stimulation by calcium removal was progressively diminished. The efflux from lenses with disturbed ion levels was also relatively to changes in external osmolarity and to increasing external potassium. Lenses with raised sodium concentrations also had an increased inulin space. Frog, rat and rabbit lenses were also exposed to the same range of stimuli and only frog lenses responded to the low calcium solution with more than a four-fold increase in efflux rate. Although only a two- to four-fold increase in efflux rate was obtained from rabbit lenses exposed to Ca-free conditions, this was the only type of animal lens so far tested that, like the human lens, responded to both hyperosmotic and isosmotic shocks with an increase in efflux rate. All three species of mammalian lenses responded with an increase in efflux rate when exposed to a hyperosmotic test solution while in the frog, the efflux rate from the lens decreased. The glucose efflux from human cataractous lenses was inhibited by cytochalasin B in a similar manner to the efflux from rat and frog lenses. It was concluded, therefore, that the cryoprobed human lens can be kept for a limited period in a relative simple artificial aqueous humour solution. The potassium permeability characteristics of low sodium cataracts remained relatively intact and showed a unique response (relative to lenses from other animals) when exposed to various stimuli that are known to be potentially cataractogenic.

The effect of pH on 86Rubidium efflux from pancreatic islet cells

The influence of pH on the K + permeability of pancreatic islet cells was investigated by measuring 86Rb + fluxes in isolated rat islets perifused or incubated in the absence of glucose. Acidification of the medium (to pH 6.5), by decreasing the concentration of HCO − 3 or by increasing pCO 2 at constant HCO − 3 reversibly reduced the rate of 86Rb + efflux from perifused islets. Alkalinization of the medium (to pH 7.8), by decreasing the pCO 2, reversibly increased 86Rb + efflux. Similar changes were recorded upon alteration of the pH in a bicarbonate-free, Hepesbuffered medium with or without calcium. Alteration of the CO 2 level at constant external pH — in order to modify internal pH — produced only a small and transient increase in efflux rate when CO 2 was lowered, and a decrease in efflux rate when CO 2 was raised. NH 4Cl reversibly augmented 86Rb + efflux in the presence and in the absence of HCO − 3. At low pH (6.5), 86Rb + uptake by islet cells was reduced after 10 min (25%) and 30 min (11%), but not after 60 min, of incubation; it was not significantly affected by high pH (7.8). Calcium uptake and insulin release were reduced at low pH and increased at high pH. These results show that the K + permeability of islet cells is affected by changes in extracellular but not in intracellular pH. They suggest that the endogenous production of protons that accompanies glucose stimulation of islet cells is not the mediator of the decrease in K + permeability induced by the sugar.

Effect of exogenous ATP on the permeability properties of transformed cultures of mouse cell lines.

HEPPEL,~ AND ILAN FRIEDBERG§ From the Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, England External ATP produces an extensive change in the mem- brane permeability of transformed cultured mouse cells, leading to a depletion of intracellular pools labeled with 13Hluridine, L3H1adenosine, ““Rb, or Z-deoxy[“HIglucose. For example, when 3T6 (a cell line derived from 3T3 by spontaneous transformation), SV3T3 (3T3 cells transformed by simian vacuolating virus 40), and PY3T3 (3T3 cells trans- formed by polyoma virus) cells are exposed to as little as 0.2 mM ATP in buffered saline medium for several minutes, there results a 20- to 30-fold increase in rate of efflux of the acid-soluble uridine nucleotide pool. A much smaller stimu- lation (from 0- to 3-fold) is seen with resting or growing 3T3 cells or with secondary cultures of mouse embryo fibro- blasts. The increase in permeability produced by ATP is highly specific; it was not obtained with GTP, CTP, UTP, ADP, inorganic phosphate, and various chelating agents. The effect of ATP in increasing permeability is pa-depend- ent and occurs much faster at 37.5” than at 20”. Stimulation of efflux persists after removal of ATP and the enhanced rate of efflux is the same at 20” as at 37.5”. The effect of ATP is seen in various buffered saline media. When the solutions are removed and replaced with Dul- becco’s modified Eagle’s serum-free medium (Smith, J. D., Freeman, G., Vogt, D., and Dulbecco, R. (1960) Virology 12, 185-196), the increased rate of efflux is quickly suppressed. The presence of bicarbonate, divalent cations, and a more neutral pH all contribute to restore a normal permeability barrier. Upon addition of serum, the acid-soluble pools are restored in 1 h and the process can be repeated. This treat- ment with ATP does not affect the subsequent appearance of the cultured cells or their rate of growth.

The effects of extracellular pH and buffer concentration on the efflux of lactate from frog sartorius muscle

1. The rate of efflux of lactate from isolated frog sartorius muscles is measured with a superfusion technique. Efflux curves are followed after raising the internal lactate level of the muscles by repetitive electrical stimulation over a 200 sec period.2. With an external pH of 7.0 or below the measured efflux rates following stimulation reach 100-150 n-mole/g.min. Increasing the pH of the superfusion fluid to 8.0 results in a two or threefold increase in the peak efflux rate. The effect is independent of the buffer system used and occurs fairly rapidly when the pH of the superfusion fluid is changed. This suggests that the effect of pH on lactate efflux is extracellular.3. The increase in efflux rate due to an increase in pH is dependent on buffer concentration. This fact together with measurements of surface pH changes in muscles following arrest of superfusion indicates that a pH gradient exists through the muscle thickness during lactate efflux.4. The low lactate efflux rate seen at a low buffer concentration (1 mM) is reduced to an even lower level by depolarization with potassium sulphate suggesting a membrane potential dependent component. At pH 8.0 with a high buffer concentration (25 mM) potassium sulphate only reduces efflux rate slightly.The observations are interpreted as indicating that a fraction of lactate lost is in the form of undissociated acid and that this fraction increases with increasing external pH.5. Conditions which favour loss of hydrogen ions and lactate from muscle are also associated with improved recovery of twitch tension.

Ion fluxes in photoreception in Limulus polyphemus ventral eye. I. The response of potassium efflux to light

1. 1. Radioactive tracers were used to study the effect of light on the efflux of K + from Limulus polyphemus ventral eye. Eyes were labeled in artificial sea water containing 42KCl and then perfused with non-radioactive artificial sea water. 2. 2. After the first 2 min of perfusion, 42K in the eye decreased with time as the sum of two exponential terms of half-times 0.3 and 2 h. 3. 3. Illumination of an eye labeled with 42K caused an abrupt increase in the rate of release of 42K. The additional 42K released in response to light comes from the photoreceptor cell bodies rather than from the axons. 4. 4. The proportional increase in efflux rate upon illumination did not vary with the length of time that an eye was perfused. This suggests that the two exponential terms above reflect two classes of photoreceptor cells rather than photoreceptor cells and glial cells. 5. 5. By collection of perfusate at intervals of less than one second, it was shown that the instantaneous rate of 42K release increases up to 20-fold at the onset of illumination. With continuous illumination, the rate remains above the dark rate, but by less than a factor of 2. Thus, the response of potassium efflux to light has transient and steady phases like the receptor potential. The increased driving force on internal potassium resulting from the receptor potential is not sufficient to explain the flux changes, if one makes the constant field assumption for the photoreceptor membrane. 6. 6. A double label method with 42K and 42K was used to show that the average rate of influx over a 10-min period is not affected by illumination. 7. 7. In eyes cooled in the dark to 2–3 °C, light still elicited an increase in the rate of 42K release and large, prolonged receptor potentials. Thus, the photoreception mechanism of the ventral eye is not, as has been previously suggested, inactive at low temperatures.