Characteristics of the thyroid iodide translocator and of iodide-accumulating phospholipid vesicles

Some biochemical characteristics of the thyroid I- translocator and of I- accumulating phospholipid vesicles (P-vesicles) were studied. P-vesicles were made from thyroid plasma membranes (PM) and soybean phospholipids by sonication. The optimal incubation temperature for Na+-dependent I- accumulation in P-vesicles was from 18-26 C. Only a small amount of Na+-independent I- accumulation was observed at various incubation temperatures, but it increased in proportion with the temperature up to 36 C. The optimal incubation pH (7.0-7.5) was near the physiological extracellular pH. When PM were heated at 55 C for 30 min before preparation of P-vesicles, Na+-dependent I- accumulation in the vesicles decreased by 35%. When they were heated at 65 C for 30 min, the I- -accumulating activity was almost completely lost. The translocator was also inactivated when PM were sonicated at 37 C in the presence of trypsin. The internal and external administration of ouabain to the vesicles did not affect the activity of Na+-dependent I- accumulation. When PM were treated with sodium dodecyl sulfate at a final concentration of 0.2-0.6 mg/ml, the I- translocator was inactivated or detached from PM, whereas the ouabain-sensitive Na+, K+-ATPase activity was preserved in the PM fragments. These observations suggest that the thyroid I- translocator consists of a protein component that is bound to PM at a site separate from Na+,K+-ATPase.     

Inhibition of iodide accumulation by perchlorate and thiocyanate in a model of the thyroid iodide transport system

The manner of inhibition of thyroid I- accumulation by perchlorate (CIO4-) and thiocyanate (SCN-) was studied using a newly developed biological model of the I- transport system. CIO4- inhibited I- accumulation in phospholipid vesicles made from thyroid plasma membrane and soybean phospholipids by decreasing Na+-dependent I- influx. The anion did not at all induce I- leakage from the vesicles. On the basis of Lineweaver-Burk plot analysis, it did not change Vmax for I- concentration. These results suggest that CIO4- is a competitive inhibitor of thyroid I- transport. In contrast, SCN- did increase I- leakage from the phospholipid vesicles to diminish I- accumulation. This anion might cause only slight depression of Na+-dependent I- entry, if any. The results do not support the idea that SCN- may be a competitive inhibitor, in spite of the fact that the anion did not change Vmax for I- transport on the basis of Lineweaver-Burk plot analysis.     

Sodium-calcium ion exchange in cardiac membrane vesicles.

Membrane vesicles isolated from rabbit ventricular tissue rapidly accumulated Ca2+ when an outwardly directed Na+ gradient was formed across the vesicle membrane. Vesicles loaded internally with K+ showed only 10% of the Ca2+ uptake activity observed with Na+-loaded vesicles. Dissipation of the Na+ gradient with the monovalent cation exchange ionophores nigericin or narasin caused a rapid decline in Ca2+ uptake activity. The Ca2+-ionophore A23187 inhibited Ca2+ uptake by Na+-loaded vesicles and enhanced the rate of Ca2+ loss from the vesicles after uptake. Efflux of preaccumulated Ca2+ from the vesicles was stimulated 30-fold by the presence of 50 mM Na+ in the external medium. Na+-dependent uptake and efflux of Ca2+ were both inhibited by La3+. The results indicate that cardiac membrane vesicles exhibit Na+-Ca2+ exchange activity. Fractionation of the vesicles by density gradient centrifugation revealed a close correspondence between Na+-Ca2+ exchange activity and specific ouabain-binding activity among the various fractions. This relationship suggests that the observed Na+-Ca2+ exchange activity derives from the sarcolemmal membranes within the vesicle preparation.     

Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.

Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and from those transformed by simian virus 40 catalyzed carrier-mediated uptake of L-alpha-aminoisobutyric acid. Concentrative uptake required the presence of a Na+ gradient (external Na+ greater than internal Na+) and occurred independently of endogenous (Na+ + K+) ATPase activity. This process is electrogenic, since uptake was stimulated by a K+ diffusion gradient (internal greater external) in the presence of valinomycin or by the addition of the Na+ salt of a permeant ion, conditions expected to create an interior-negative membrane potential. Both the initial rate of concentrative uptake of L-alpha-aminoisobutyric acid and its maximal accumulation, driven by a standard Na+ gradient, were decreased in vesicles from density-inhibited, untransformed cells and increased in those from cells transformed by simian virus 40 compared with vesicles from proliferating untransformed cells. An increased maximal velocity (Vmax) of uptake stimulated by Na+ gradient was observed in vesicles from transformed cells compared with those from untransformed cells, suggesting an increase in the number of carriers or in their mobility. Since the relative extent of accumulation of this model amino acid driven by a standard Na+ gradient also differed with growth or transformed status, an additional possibility for cellular regulation of this process could be alteration of membrane Na+ permeability or carrier response to Na+.     

Proton accumulation and ATPase activity in Golgi apparatus-enriched vesicles from rat liver.

We have studied the mechanism by which liver Golgi apparatus maintains the acidity of its contents, using a subcellular fraction from rat liver highly enriched in Golgi marker enzymes. Proton accumulation (measured by quenching of acridine-orange fluorescence) and anion-dependent ATPase were characterized and compared. Maximal ATPase and proton accumulation required ATP; GTP and other nucleotides gave 10% to 30% of maximal activity. Among anions, Cl- and Br- approximately doubled the activities; others were much less effective. Half-maximal increase of ATPase and H+ uptake required 55 mmol/L and 27 mmol/L Cl-, respectively. In predominantly chloride media, SCN- and NO3- markedly inhibited H+ uptake. Nitrate competitively inhibited both the chloride-dependent ATPase (apparent Ki 6 mmol/L) and proton uptake (apparent Ki 2 mmol/L). Nitrate and SCN- also inhibited uptake of 36Cl. Replacing K+ with Na+ had no effect on the initial rate of proton uptake but somewhat reduced the steady state attained. Replacement of K+ with NH4+ and choline reduced proton uptake without affecting ATPase. The ATPase and H+ uptake were supported equally well by Mg2+ or Mn2+. The ATPase was competitively inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (apparent Ki 39 mumol/L). Other agents inhibiting both H+ uptake and ATPase were N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn2+, Co2+ and Cu2+. In the Cl- medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin greater than valinomycin greater than carbonyl cyanide mchlorophenylhydrazone; the last of these also reduced ATPase activity. In the absence of Cl-, monensin and valinomycin both stimulated the ATPase. These results show a close association between ATPase activity and acidification of liver Golgi vesicles. They support a role for Cl- that depends on its uptake as a counter ion for H+ and suggest that it may also stimulate proton transport by a more direct effect on a component of the transport system.     

Sodium/proton exchange in the maintenance of intracellular pH in FRTL-5 thyroid cells

In the present study we describe a Na+/H+ exchange system in FRTL-5 rat thyroid cells and its role in the regulation of intracellular pH (pHi). The pHi of these cells was measured in acidic medium (pH 6.5) using the equilibrium distribution of 14C-labeled 5,5'-dimethyl-oxazolidine-2,4-dione. The estimated pHi in 10 separate experiments was 7.30 +/- 0.01 (mean +/- SE). The pHi declined to 6.87 +/- 0.02 when equimolar concentrations of choline+ replaced Na+ in the incubation medium, but was restored when cells were placed in a medium containing 50 mM Na+. The necessity for external Na+ to maintain pHi, when cells are exposed to acidic solutions, is consistent with a Na+/H+ antiport system in these cells. This is further supported by the observation that pHi decreased to 6.70 +/- 0.01 when amiloride, an inhibitor of Na+/H+ exchange, was added, and that this agent prevented the recovery of pHi in Na+-depleted cells after addition of Na+ to the medium. This report also provides additional information concerning the relationship between H+ and I- uptake and suggests that there are two I- transport systems: one that is Na+-dependent, 4,4'-diisothiocyano 2,2'-disulfonic acid stilbene-sensitive, and not associated with intracellular acidification, and the other operative at an external pH of 6.5 and associated with intracellular acidification.     

H+ disposal by rabbit gastric mucosal surface cells

Exposure of isolated gastric mucosal surface cells to NH4+ results in acidification of cells as determined by a fluorescent dye technique using acridine orange. The resulting intracellular pH gradient is maintained when cells are suspended in either buffered HCO3- -free Ringer's or choline chloride solution. Cells suspended in a Na+-containing but K+-free solution exhibit dissipation of the proton gradient. When Na+ is added to cells suspended in Na+, K+-free solution, the gradient rapidly dissipates with a half-maximal response occurring at 56 mM Na+. The effect of Na+ is amiloride sensitive with half-maximal inhibition occurring at 38 microM at a Na+ concentration of 50 mM. The K+ does not cause dissipation of the gradient and neither ouabain nor valinomycin have an effect. Yet, K+ has a modulating influence on Na+/H+ exchange by the isolated surface cells. The addition of K+ to acid-loaded cells resuspended in Na+-free solution decreases the ability of subsequent Na+ addition to evoke gradient dissipation. The data suggest that Na+/H+ exchange appears to be at least one mechanism whereby gastric mucosal surface cells could protect themselves against diffusing acid. This ion exchange mechanism is amiloride sensitive and appears to be unrelated to Na+, K+ adenosine triphosphatase activity, but is affected by the external K+ concentration.     

Relation between pH regulation and iodide transport in turtle thyroid glands

Mechanisms of pH recovery after alkalinization and acidification by exposing or prepulsing turtle thyroid slices with a Hanks' balanced salt solution (HBSS) containing NH4Cl or CO2 were studied by examining the effects of amiloride, 4-acetamido-4'-isocyanostilbene-2,2'-disulphonic acid (SITS), frusemide and acetazolamide, and of reducing the concentration of Na+ or Cl- in the incubation medium. When alkalinization was produced either during exposure to NH4Cl or after a CO2 pulse, the pH in thyroid slices rose rapidly and then recovered gradually. Addition of SITS (0.1 mmol/l) or reduction of the Cl- concentration markedly inhibited pH recovery. However, amiloride (0.1 mmol/l) and low Na+ in the medium had no significant effect on recovery from alkalinization induced by NH4Cl exposure or by a CO2 pulse. These data suggest that pH recovery from alkalinization in turtle thyroid gland is achieved by an exchange of internal HCO3- for external Cl-. When acidification was accomplished by either exposure to CO2 or removal of NH4Cl, the pH of thyroid slices fell rapidly and then recovered gradually. If amiloride was added or the Na+ concentration in the medium was reduced, the pH recovery was greatly attenuated. However, SITS and low Cl- in the medium did not affect the recovery from an acid load in turtle thyroid slices. These results suggest that pH recovery from acidification in turtle thyroid gland is achieved by an exchange of internal H+ for external Na+. Both frusemide and acetazolamide prevented the pH recovery in turtle thyroid slices during exposure to and withdrawal from NH4Cl. These results suggest that besides the Na(+)-H+ and Cl(-)-HCO3- exchange processes, other mechanisms may also be involved in pH regulation in turtle thyroid glands. Simultaneous uptakes into turtle thyroid slices of 125I- and 22Na+ and of 125I- and 36Cl- were studied during and following exposure to NH4Cl in the absence and presence of different transport inhibitors, such as frusemide, amiloride, SITS and acetazolamide. When the thyroid slices were exposed to HBSS containing 30 mmol/l NH4Cl (alkalinization phase), the tissue/medium (T/M) ratios of 125I- increased gradually, reached the highest point in 10 min, and were maintained at this level for the next 20 min. The T/M ratios of 22Na+ and 36Cl- of thyroid slices also slowly increased after exposure to NH4Cl.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thyroid hormones increase Na+-H+ exchange activity in renal brush border membranes.

Na+-H+ exchange activity, i.e., amiloride-sensitive Na+ and H+ flux, in renal proximal tubule brush border (luminal) membrane vesicles was increased in the hyperthyroid rat and decreased in the hypothyroid rat, relative to the euthyroid animal. A positive correlation was found between Na+-H+ exchange activity and serum concentrations of thyroxine (T4) and triiodothyronine (T3). The thyroid status of the animal did not alter amiloride-insensitive Na+ uptake. The rate of passive pH gradient dissipation was higher in membrane vesicles from hyperthyroid rats compared to the rate in vesicles from hypothyroid animals, a result which would tend to limit the increase in Na+ uptake in vesicles from hyperthyroid animals. Na+-dependent phosphate uptake was increased in membrane vesicles from hyperthyroid rats; Na+-dependent D-glucose and L-proline uptakes were not changed by the thyroid status of the animal. The effect of thyroid hormones in increasing the uptake of Na+ in the brush border membrane vesicle is consistent with the action of the hormones in enhancing renal Na+ reabsorption. Further, the regulation of transtubular Na+ flux has now been shown to be concomitant with modulation of the entry of Na+ into the tubular cell across its luminal membrane, mediated by the exchange reaction, and with the previously reported control of the pumping of Na+ out of the cell across its basolateral membrane, mediated by the Na+,K+-ATPase.     

Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana.

Potassium is a major nutrient in higher plants, where it plays a role in turgor regulation, charge balance, leaf movement, and protein synthesis. Terrestrial plants are able to sustain growth at micromolar external K+ concentrations, at which K+ uptake across the plasma membrane of root cells must be energized despite the presence of a highly negative membrane potential. However, the mechanism of energization has long remained obscure. Therefore, whole-cell mode patch clamping has been applied to root protoplasts from Arabidopsis thaliana to characterize membrane currents resulting from the application of micromolar K+. Analysis of whole cell current/voltage relationships in the presence and absence of micromolar K+ enabled direct testing of K+ transport for possible energization by cytoplasmic ATP and the respective trans-membrane gradients of Na+, Ca2+, and H+. Subtracted current/voltage relations for K(+)-dependent membrane currents are independent of ATP and reverse at potentials that imply H(+)-coupled K+ transport with a ratio of 1 H+:K+. Furthermore, the reversal potential of the K+ current shifts negative as external H+ activity is decreased. K(+)-dependent currents saturate in the micromolar concentration range with an apparent Km of 30 microM, a value in close agreement with previously reported Km values for high-affinity K+ uptake. We conclude that our results are consistent with the view that high-affinity K+ uptake in higher plants is mediated by a H+:K+ symport mechanism, competent in driving K+ accumulation to equilibrium ratios in excess of 10(6)-fold.     

Effects of 4,4'-di-isothiocyano-2,2'-stilbene disulphonate on iodide uptake by primary cultures of turtle thyroid follicular cells

Iodide uptake by primary cultures of turtle thyroid follicular cells is directly proportional to the Na+ concentration and is inversely proportional to the HCO3- concentration in culture medium, but is not affected by the Cl- concentration. Addition of 4,4'-di-isothiocyano-2,2'-stilbene disulphonate (DIDS; 10 mumol/l and higher doses) to medium containing different concentrations of Na+ (5-140 mmol/l), HCO3- (0-40 mmol/l) and Cl- (120 mmol/l) generally enhanced iodide uptake by the cultured cells; however, there was no significant effect in Na+-free and in low Cl- (90 mmol/l and less) medium. The inhibitory effects on iodide uptake of ouabain, frusemide and perchlorate were attenuated by DIDS which also antagonized the stimulatory effects on iodide uptake of TSH, although both DIDS and TSH increased the 125I- cell/medium ratio when they were given alone. At doses of 100 mumol/l and higher, DIDS lowered the intracellular pH of cultured cells when the pH of the medium was maintained at a constant level. It also increased the intracellular Cl- concentration, but had no effect on intracellular Na+ or K+. The input and specific resistances of cell membranes in cultured thyroid cells and in isolated thyroid slices increased (decreased conductance) after adding DIDS to the perfusion fluids. Both Na+/K+- and HCO3(-)-ATPase activities in homogenates of turtle thyroid tissue were inhibited by DIDS. Results from this investigation demonstrate (1) that in addition to preventing the leak of iodide from thyroid cells, DIDS may act to increase the sensitivity of the Na+-anion carrier to I- and thereby increases iodide uptake, and (2) that a HCO3(-)-Cl- exchange system is present in the thyroid cell membrane and appears to be linked to the transport of iodide into thyroid cells.     

Canine cardiac sarcolemmal vesicles demonstrate rapid initial Na(+)-Ca2+ exchange activity

To identify a rapid, uninhibited rate of exchange activity, we investigated in canine sarcolemmal vesicles the rapid kinetics of Na(+)-Ca2+ exchange. Sarcolemmal vesicles were incubated in 160 mM NaCl and 20 mM HEPES at 25 degrees C (pH 7.4) and actively loaded with 45Ca2+ for 2 minutes by Na(+)-Ca2+ exchange. After further uptake was inhibited by dilution into 0.15 mM Na(+)-free EGTA, sarcolemmal vesicles were immobilized on a rapid filtration apparatus that allowed millisecond resolution of 45Ca2+ fluxes. In the presence of external NaCl (Na+o) but not other monovalent cations (i.e., K+, Li+), a biphasic pattern of Ca2+ release was observed--an initial brief and rapid rate of Ca2+ release followed by a second slower, prolonged phase of Ca2+ release. Semilogarithmic plots of sarcolemmal Ca2+ content versus time were not linear but were consistent with a biexponential rate of Na+o-induced Ca2+ release during the first several seconds of the exchange reaction. The fast phase of Na+o-stimulated Ca2+ release was several thousand-fold more rapid than that in the absence of Na+o. Both phases of Ca2+ release showed a similar Na+o dependence (Km, approximately 12 mM) with evidence of a positive cooperative effect of Na+. Vmax of the fast and slow phases were approximately 37.0 and approximately 0.76 nmol/mg/sec, respectively. Using rapid-reaction techniques, we demonstrated in the present study that the initial velocity of sarcolemmal Na(+)-Ca2+ exchange activity is greater than previously reported in sarcolemmal vesicles and that this exchange process exhibits complex rate behavior with a biphasic pre-steady state kinetic pattern.     

Serum stimulates the Na+,K+ pump in quiescent fibroblasts by increasing Na+ entry.

Two ionophores (monensin and gramicidin) that carry Na+ into 3T3 cells markedly enhance the rate of 86Rb+ uptake. Ouabain prevents both ionophores from increasing 86Rb+ uptake, indicating that the ionophores activate the Na+,K+ pump. Measurements of 86Rb+ uptake and cell Na+ and K+ over a range of monensin concentrations show that the activity of the Na+,K+ pump in 3T3 cells is limited by the supply of internal Na+ and is extremely sensitive to small changes in internal Na+. Serum rapidly enhances the rate of 22Na+ uptake and net Na+ entry when Na+ exit is inhibited by ouabain. At 0.3 microgram/ml, monensin increases the rate of net Na+ entry and activates the Na+,K+ pump by the same degree as serum. The stimulation of 86Rb+ uptake by serum or the ionophores has an absolute requirement for external Na+. Thus, serum appears to stimulate the Na+,K+ pump in quiescent 3T3 cells by increasing its supply of Na+.     

Na(+)-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I(-)-transporting cultured thyroid cells.

The active accumulation of I- in the thyroid gland is mediated by the Na(+)-I- symporter and driven by the Na+ gradient generated by the Na+/K(+)-ATPase. Thyrotropin (TSH) stimulates thyroidal I- accumulation. Rat thyroid-derived FRTL-5 cells require TSH to accumulate I-. TSH withdrawal for over 7 days results in complete loss of Na(+)-I-symport activity in these cells [Weiss, S. J., Philp, N. J. and Grollman, E. F. (1984) Endocrinology 114, 1090-1098]. Surprisingly, membrane vesicles prepared from FRTL-5 cells maintained in TSH-free medium [TSH(-)cells]accumulate I-, suggesting that the absence of Na(+)-I- symport activity in TSH(-) cells cannot be due solely to a decrease in the biosynthesis of either the symporter or a putative activating factor. This finding indicates that the Na(+)-I- symporter is present, probably in an inactive state, in TSH(-) cells despite their lack of Na(+)-I- symport activity. Na(+)-I- symport activity in thyroid membrane vesicles is enhanced when conditions for vesicle preparation favor proteolysis. Subcellular fractionation studies in both TSH(+) and TSH(-) cells show that Na(+)-I- symport activity is mostly associated with fractions enriched in plasma membrane rather than in intracellular membranes, suggesting that the Na(+)-I- symporter may constitutively reside in the plasma membrane and may be activated by TSH.     

Charge movements during the Na+-Ca2+ exchange in heart sarcolemmal vesicles.

The Na+-Ca2+ exchange was studied in a highly purified vesicular preparation derived from heart sarcolemma. The initial velocity of the Na+-driven Ca2+ influx, which was monitored continuously with a specific electrode, was 15 nmol/mg of protein per sec; the total Ca2+-accumulation capacity was 80 nmol/mg of protein. The Na+-Ca2+ exchange generated a current that was compensated for by the uptake of tetraphenylphosphonium in (Ph4P+) (when the latter was present in the medium), the influx of K+, and the efflux of Cl-. The movements of Ph4P were followed with a specific electrode. Ca2+ in the concentration range 3-50 microM induced an increase in the permeability of the sarcolemmal membrane to K+. Under conditions of optimal charge neutralization by K+ (i.e., in the presence of valinomycin), the Km (Ca2+) of the exchanger was 1.5 microM. The Na+-Ca2+ exchange was inhibited by chlorpromazine and was not inhibited by vanadate.     

Sodium-induced calcium release from mitochondria in brown adipose tissue.

Coupled mitochondria of brown adipose tissue can accumulate Ca2+ if a substrate is present. The Ca2+ is released by addition of 20 mM Na+, but not by addition of K+ or choline +. Energy-dissipating Na+-induced Ca2+ cycling occurs maximally with 20 mM Na+ and 10 microM Ca2+. In brown adipocytes, the Ca2+ ionophore A23187 and the Na+ ionophore monensin increase respiration if substrate is added, and incubation in a low-Na+ buffer decreases norepinephrine-induced respiration. Thus Na+-induced Ca2+ release takes place in brown adipose tissue; released Ca2+ could have a regulatory or thermogenic role or both.     

Stimulatory action of endothelin-1 on membrane Na+ transport in vascular smooth muscle cells in culture

Endothelin-1 was able to induce an immediate and transient increase in cytosolic free Ca2+ concentrations in the A10 cell line of vascular smooth muscle. This was associated with a strong stimulation of the Na+:H+ exchange, the Na+, K+ pump and the [Na+,K+,Cl-]-cotransport system. Pump stimulation appeared to be secondary to sodium entry through Na+:H+ exchange because it was absent in Na+ loaded cells and in the presence of ethyl-isopropyl-amiloride. Cotransport stimulation was blocked by indomethacin, suggesting the involvement of a cyclooxygenase product. In conclusion, the monovalent ionic perturbations associated to the vasoconstrictor and mitogenic actions of endothelin-1 are counterbalanced by activation of the Na+,K+ pump and the [Na+,K+,Cl-]-cotransport system.     

Water and electrolyte contents, cell pH and membrane potentials of cultured turtle thyroid cells

Water and electrolyte contents, cell pH, membrane potential and 125I- uptake were determined in cultured follicular cells of turtle thyroid. The Na+, K+ and Cl- concentrations in the cultured thyroid cells were 59.2, 119.0 and 50.9 mmol/l cell water respectively. Treatment with TSH (10 mu./ml for 24 h) increased the K+ and Cl- and decreased the Na+ concentrations in cells. The water and protein contents of these cells were 81.6 and 8.7 g/100 g cells respectively. The cell pH was 6.91. With glass micro-electrodes, the resting membrane potential of thyroid cells cultured in Medium 199 averaged 33.9 +/- 0.63 mV which is slightly higher than 29.8 +/- 1.6 mV as calculated from the data on the uptakes of [14C]methyltriphenylphosphonium and 3H2O by the cells. The potential varied linearly with the log of external K+ concentration (between 15 and 120 mmol/l) with a slope of about 24 mV per tenfold change in K+ concentration. Both TSH and cyclic AMP depolarized the cell membrane. Calculations based on the values for the electrolyte concentrations in cells and in culture medium indicated that Na+, K+ and Cl- were not distributed according to their electrochemical gradients across the cell membrane. Na+ was actively transported out of the cells and K+ and Cl- into the cells. Follicular cells of turtle thyroid cultured in the medium without addition of TSH formed a monolayer. Their iodide-concentrating ability was low and they did not respond to TSH with an increase in iodide uptake. In contrast, cells cultured in medium containing TSH tended to aggregate and organize to form follicles. They had higher ability to concentrate iodide and respond to TSH.     

Phorbol ester inhibits furosemide-sensitive potassium transport in BALB/c 3T3 preadipose cells.

The tumor promoter phorbol 12-myristate 13-acetate (PMA) rapidly decreased the rate of 86Rb+ uptake into BALB/c 3T3 preadipose cells. The component of total 86Rb+ influx affected by PMA is insensitive to ouabain but sensitive to the diuretic furosemide. Experiments designed to investigate the characteristics of the K+ transport system sensitive to PMA revealed that: (i) 86Rb+ uptake is highly dependent on external Na+, (ii) 86Rb+ uptake is highly dependent on external Cl-, (iii) 22Na+ uptake is dependent on external K+, and (iv) a major component of 86Rb+ efflux that is sensitive to PMA and furosemide is not dependent on extracellular K+. These features strongly implicate a Na+K+/Cl- cotransport system as the target of PMA and furosemide in these experiments. PMA caused a net intracellular accumulation of K+ within 15 min in these cells, presumably via its inhibitory effect on furosemide-sensitive K+ transport. Within 30 min after PMA treatment, the mean cell volume was significantly reduced in treated compared to control cells, with a maximum decrease of 21% attained at 4 hr after PMA. The significance of these findings for biologic changes induced by PMA is discussed.     

Sodium ion-coupled uptake of taurocholate by rat-liver plasma membrane vesicles

ABSTRACT— Uptake of taurocholate into plasma membrane vesicles isolated from rat liver was investigated. In the presence of an extra- to intravesicular gradient of Na+ ions, a typical “overshoot” phenomenon in the accumulation pattern was observed. Osmotic manipulation of the incubation medium indicated that the transport of this bile acid occurs into an osmotically active intravesicular space. Uptake of taurocholate as measured after 1 min was specifically stimulated by Na+ ions: NaNO₃ and NaCl were capable of supporting accumulation, whereas KNO₃ was not. Na+-coupled uptake of taurocholate showed saturation kinetics and was inhibited by other bile acids or by preloading the vesicles with Na+. Our observations support the idea of a carrier-mediated bile-acid uptake system, as suggested previously for the intact rat liver and isolated rat hepatocytes. When the electrical potential difference across the vesicle membrane was changed by inducing different diffusion potentials (anion replacement), a more negative potential inside stimulated Na+-dependent taurocholate transport. The results demonstrate that rat-liver plasma membrane vesicles possess an electrogenic Na+-coupled transport system for taurocholate.