Acidification Rate Of Cells Research Articles

K +/NH 4+ antiporter: a unique ammonium carrying transporter in the kidney inner medulla

The mechanism of NH 4 + transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH 4 +) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH 4 + and monitored for pH i changes by the use of the pH-sensitive dye BCECF. The rate of cell acidification following the initial cell alkalinization was measured as an index of NH 4 + transport. The rate of NH 4 + transport was the same in the presence or absence of sodium in the media (0.052±0.003 vs 0.048±0.004 pH/min, P>0.05), indicating that NH 4 + entry into the cells was independent of sodium. The presence of ouabain, bumetanide, amiloride, barium, or 4,4′-di-isothiocyanostilbene-2-2′-disulfonic acid (DIDS) did not block the NH 4 +-induced cell acidification, indicating lack of involvement of Na +:K +-ATPase, Na +:K +:2 Cl − transport, Na +:H + exchange, K + channel, or Cl −/base exchange, respectively, in NH 4 + transport. The NH 4 +-induced cell acidification was significantly inhibited in the presence of high external [K +] as compared to low external [K +] (0.018±0.001 vs. 0.049±0.003 pH/min for 140 mM K + vs. 1.8 mM K + in the media, respectively, P<0.001). Inducing K + efflux by imposing an outward K + gradient caused intracellular acidification by ∼0.3 pH unit in the presence but not the absence of NH 4 +. This K + efflux-induced NH 4 + entry increased by extracellular NH 4 + in a saturable manner with a Km of ∼5 mM, blocked by increasing extracellular K + and was not inhibited by barium. The K + efflux-coupled NH 4 + entry was electroneutral as monitored by the use of cell membrane potential probe 3,3′-dipropylthiadicarbocyanine. These results are consistent with the exchange of internal K + with external NH 4 + in a 1:1 ratio. The K +-NH 4 + antiporter was inhibited by verapamil and Schering 28080 in a dose-dependent manner, was able to work in reverse mode, and did not show any affinity for H + as a substrate, indicating that it is distinct from other NH 4 +-carrying transporters. We conclude that a unique transporter, a potassium-ammonium (K +/NH 4 +) antiport, is responsible for NH 4 + transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH 4 + and K + as substrates. The K +/NH 4 + antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.

Nerve growth factor stimulates rapid metabolic responses in PC12 cells.

Research into the effects of nerve growth factor (NGF) has involved study of either the signal transduction process or the morphological result of growth factor treatment (cell proliferation and/or differentiation). The Cytosensor Microphysiometer, a silicon-based biosensor system that allows the continuous and real-time monitoring of extracellular acidification rate changes of cells, was used to study the response of PC12 cells to NGF. Stimulation resulted in a rapid increase in the acidification rate of cells in a concentration-dependent fashion (0.1-200 ng/ml NGF; mean effective concentration value of 153 +/- 54 pM). Inhibition of the NGF receptor-linked protein tyrosine kinase by either genistein or K252a attenuated the acidification rate response to NGF. In addition, the acidification response to NGF could be modified by inhibiting Na+/H+ exchange and, separately, glycolysis. This implicates these processes in the metabolic response of PC12 cells to NGF stimulation.

Separation, identification, tissue culture, and physiological recording of ciliary body epithelial cells

The mechanism of NH4+ transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH4+) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH4+ and monitored for pHi changes by the use of the pH-sensitive dye BCECF. The rate of cell acidification following the initial cell alkalinization was measured as an index of NH4+ transport. The rate of NH4+ transport was the same in the presence or absence of sodium in the media (0.052±0.003 vs 0.048±0.004 pH/min, P>0.05), indicating that NH4+ entry into the cells was independent of sodium. The presence of ouabain, bumetanide, amiloride, barium, or 4,4′-di-isothiocyanostilbene-2-2′-disulfonic acid (DIDS) did not block the NH4+-induced cell acidification, indicating lack of involvement of Na+:K+-ATPase, Na+:K+:2 Cl− transport, Na+:H+ exchange, K+ channel, or Cl−/base exchange, respectively, in NH4+ transport. The NH4+-induced cell acidification was significantly inhibited in the presence of high external [K+] as compared to low external [K+] (0.018±0.001 vs. 0.049±0.003 pH/min for 140 mM K+ vs. 1.8 mM K+ in the media, respectively, P<0.001). Inducing K+ efflux by imposing an outward K+ gradient caused intracellular acidification by ∼0.3 pH unit in the presence but not the absence of NH4+. This K+ efflux-induced NH4+ entry increased by extracellular NH4+ in a saturable manner with a Km of ∼5 mM, blocked by increasing extracellular K+ and was not inhibited by barium. The K+ efflux-coupled NH4+ entry was electroneutral as monitored by the use of cell membrane potential probe 3,3′-dipropylthiadicarbocyanine. These results are consistent with the exchange of internal K+ with external NH4+ in a 1:1 ratio. The K+-NH4+ antiporter was inhibited by verapamil and Schering 28080 in a dose-dependent manner, was able to work in reverse mode, and did not show any affinity for H+ as a substrate, indicating that it is distinct from other NH4+-carrying transporters.We conclude that a unique transporter, a potassium-ammonium (K+/NH4+) antiport, is responsible for NH4+ transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH4+ and K+ as substrates. The K+/NH4+ antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.

Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process.

In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH-sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium-coupled H+/OH-/HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule.