Effect of norepinephrine on intracellular pH in kidney proximal tubule: role of Na+-(HCO-3)n cotransport.

We examined the effect of norepinephrine (NE) on intracellular pH (pHi) and activity of Na+ (aNai) in the isolated perfused kidney proximal tubule of Ambystoma, using single-barreled voltage and ion-selective microelectrodes. In control HCO-3 Ringer, addition of 10(-6) M NE to the bath reversibly depolarized the basolateral membrane potential (V1), the luminal membrane potential (V2), and the transepithelial potential difference (V3) and increased pHi by 0. 14 +/- 0.02. These effects were mimicked by isoproterenol but were abolished after pretreatment with SITS or in the absence of CO2/HCO-3. Removal of bath Na+ depolarized V1 and V2, hyperpolarized V3, and decreased pHi. These effects are largely mediated by the electrogenic Na+-(HCO-3)n cotransporter. In the presence of NE, the effects of Na+ removal on membrane potential differences and the rate of change of pHi were significantly smaller. Reducing bath HCO-3 concentration from 10 to 2 mM at constant CO2 (pH 6.8) depolarized V1 and V2, decreased pHi, and lowered aNai. These changes are also due to Na+-(HCO-3)n. In the presence of NE, reducing bath [HCO-3] caused a smaller depolarizations of V1 and V2, and the rate of pHi decrease was significantly reduced. Our results indicate: 1) NE causes an increase in pHi; 2) the NE-induced alkalinization is mediated by a SITS-sensitive and HCO-3-dependent transporter on the basolateral membrane; and 3) in the presence of NE, the reduced effects caused by basolateral HCO-3 changes or Na+ removal are indicative of an inhibitory effect of NE on Na+-(HCO-3)n cotransport.

Effect of norepinephrine on cellular sodium transport in Ambystoma kidney proximal tubule.

The effect of norepinephrine (NE) on mechanisms of cellular Na+ transport in the isolated, perfused proximal tubule of Ambystoma tigrinum was examined. Single-barreled voltage and ion-selective microelectrodes were used to determine basolateral (V1), luminal (V2), and transepithelial (V3) membrane potentials and intracellular Na+ activity (alpha Nai). In CO2/HCO3- control solution, addition of NE (10(-6) M) to the bath caused depolarizations of V1, V2, and V3 are decreased alpha Nai. These effects were mimicked by isoproterenol and inhibited by propranolol. Addition of NE in the absence of luminal Na+ and substrates did not cause any changes in V1, V2, V3, or alpha Nai. NE did not affect the changes in membrane potential difference (PD) or alpha Nai caused by removal and readdition of luminal substrates and/or Na+. To study the effect of NE on Na-K-adenosinetriphosphatase (Na-K-ATPase), the pump was inhibited by external K+ removal and then reactivated by readdition of 12 mM K+ to the bath in the presence and absence of NE. Reactivation of the pump caused hyperpolarization of membrane PDs, and alpha Nai recovered monotonically in 3-5 min. The peak hyperpolarizations of V1 and V2 (approximately 1 min) were significantly larger in the presence of NE. During the first 3 min, and also at the same alpha Nai, the rate of decrease of alpha Nai was significantly faster in the presence of NE. In conclusion, these results show a direct effect of NE on cell membrane PDs and alpha Nai in the kidney proximal tubule. Most likely, beta-receptors are involved in mediating the action of NE. Neither Na/H exchange nor Na-substrate cotransport at the luminal membrane are affected by NE. On the other hand, NE activates Na-K-ATPase.

Intracellular pH regulation in rat Schwann cells.

We examined H+ and HCO3- transport mechanisms that are involved in the regulation of intracellular pH of Schwann cells. Primary cultures of Schwann cells were prepared from the sciatic nerves of 1-3-day-old rats. pHi of single cells attached to cover slips was continuously monitored by measuring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein incorporated intracellularly. The average pHi of neonatal Schwann cells bathed in HEPES mammalian solution was 7.17 +/- 0.02 (n = 32). In the nominal absence of HCO3-, pHi spontaneously recovered from an acute acid load induced by exposing the Schwann cells to 20 mM NH4+ (NH4+ prepulse). This pHi recovery from the acute acid load was totally inhibited in the absence of external Na+ or in the presence of 1 mM amiloride. In both cases, the pHi recovery was readily restored upon readdition of external Na+ or removal of amiloride. In the steady-state, addition of amiloride caused a small and slow decrease in pHi which was readily reversed upon removal of amiloride. In the presence of HCO3-, removal of external Cl- caused pHi to rapidly and reversibly increase by 0.23 +/- 0.03 (n = 15) and the initial rate of alkalinization was 20.6 +/- 2.7 x 10(-4) pH/sec. In the absence of external Na+, removal of bath Cl- still caused pHi to increase by 0.15 +/- 0.02 and the initial rate of pHi increase was not significantly altered. In the nominal absence of HCO3-, removal of bath Cl- caused pHi to increase very slightly (0.05 +/- 0.01) with an initial dpHi/dt of only 4.4 +/- 0.2 x 10(-4) pH/sec (n = 4). Addition of 100 microM DIDS did not inhibit the pHi increase caused by removal of bath Cl-. These data indicate that 1) Rat Schwann cells regulate their pHi via an Na-H exchange mechanism which is moderately active at steady-state pHi. 2) In the presence of HCO3-, there is a Na-independent Cl-HCO3 (base) exchanger which also contributes to regulation of intracellular pH in Schwann cells.

Electrochemical potentials of potassium in skeletal muscle under different metabolic states.

Intracellular potassium and membrane potential were measured simultaneously by means of double-barrelled liquid ion-exchange microelectrodes in single fibers of rat thigh muscle in vivo in rats maintained in seven different metabolic states. The K+ equilibrium potential (EK) was more negative than the simultaneously measured membrane potential (Em) in the normal state by 18.4 mV. K+ loading, acute and chronic, resulted in depolarization of Em due to increased serum K+ (hyperkalemia) with no increase in intracellular K+. K+ depletion resulted in hyperpolarization of Em as plasma K+ decreased proportionately more than intracellular K+. Low Na+ diet had no effect. Intracellular K+ was decreased in acute acidosis but not in the chronic state. Thus K+ depletion and acute acidosis are associated with intracellular K+ decrease. The fact that hyperpolarization exists in the former and not the latter is a reflection that hypokalemia accompanies the former condition. The hyperpolarizing states of K+ depletion and chronic acidosis are accompanied by decreased excitability and muscle weakness.

Transport of prostaglandins through normal and diabetic rat hepatocytes.

Transport of alprostadil (prostaglandin E1) and dinoprost (prostaglandin F2 alpha) was studied in enzymatically dispersed normal and streptozocin-treated rat hepatocytes prepared by collagenase perfusion. Cell suspensions incubated at 37 degrees were sampled at time intervals for a period of 5 min and the supernatant analyzed for prostaglandins after centrifugation. The data analysis employed a theory and a model for solute transfer at the cell membrane-water interphase. Biophysical parameters such as the effective partition and the apparent permeability constants were used to define the transport mechanism. The apparent permeability coefficient of alprostadil and dinoprost transfer through normal hepatocytes was calculated to be 5 X 10(-3) and 3 X 10(-3) cm/sec with a mean partition coefficient of 1345 and 764 for both solutes, respectively. The permeability coefficient of alprostadil and dinoprost transfer through diabetic hepatocytes were 3 X 10(-3) and 2 X 10(-3) cm/sec with partition coefficient of 572 and 206, respectively. The results showed differences in prostaglandin transport between normal and diabetic hepatocytes, resulting from morphological and lipid alteration in the cytoplasmic membrane.

Factors regulating prostaglandin uptake by rat small intestine.

The uptake of prostaglandin E1 by rat intestine mucosal strips was investigated. The results indicate that: (1) PGE1 did not accumulate in the epithelial cells of rat intestine. The intracellular to extracellular distribution ratio did not reach unity in spite of the extended periods of incubation. (2) A decrease in the sodium gradient across the brush-border membrane of intestinal cells significantly inhibited PGE1 uptake by the mucosal strips. (3) PGE1 uptake increases with increasing concentration of potassium in the incubation medium with a tendency for saturation at high K+ levels. (4) Calcium and magnesium ions had no effect on the steady-state uptake of PGE1 by the epithelial cells of rat intestine. (5) Addition of sodium carbonate or phosphate to the incubation medium significantly decreased PGE1 uptake by the intestinal cells. (6) Further studies of PGE1 uptake reveal a temperature-dependent process with a Q10 of 2.8.

Alanine influx across serosal border of Testudo graeca intestine.

The influx of alanine across the serosal membrane of Testudo graeca intestinal cells with preserved epithelial orientation was examined. Our results suggest that: 1. The mechanism of alanine influx across the serosal membrane of turtle intesintal cells is a carrier-mediated process that has the characteristics of facilitated diffusion. 2. Alanine influx mechanism is independent of intra- and extra-cellular changes in Na+ and K+ concentrations, and is not altered by reversal of Na+ and K+ gradients across the serosal membrane. 3. In Na+-free media the mechanism of transport of alanine at the mucosal membrane has the same pattern of competitive inhibition by amino acids as the serosal.

Intracellular bicarbonate of skeletal muscle under different metabolic states.

Intracellular bicarbonate of single muscle fibers in vivo was measured by a direct electrometric method simultaneously with the membrane PD in rats under seven different metabolic states. From the measured intracellular bicarbonate values and the PCO2, the bicarbonate equilibrium potential and the intracellular pH were calculated. The mean intracellular [HCO3-] under normal control conditions was 10.3 +/- 0.7 mM (SE). The intracellular bicarbonate fell significantly in both chronic metabolic acidosis and chronic K+ depletion. In contrast, intracellular bicarbonate was elevated in chronic metabolic alkalosis, K+ loading, and Na+ depletion. Taking intracellular pH as an index of the acid-base status of cells, we find that whereas the calculated cell pH decreased along with the cell bicarbonate in both chronic metabolic acidosis and K+ depletion, cell pH increased along with the bicarbonate only in chronic metabolic alkalosis. Cell pH was unchanged in both chronic K+ loading and Na+ depletion.

Effect on Mn2+ on permeability properties of frog skin.

Mn2+ added to the inner bathing solution of frog skin caused a transient increase in potential difference (PD) and a decrease in total skin conductance and mannitol influx. Net Na flux and short-circuit current (Is. c.) were also reduced, the isotopic net flux being reduced more than Is. c. This observed discrepancy appears to be the result of Cl- retention in the outer medium since it was not observed when the skin was bathed in a sulfate-substituted chloride-free solution. The effect of Mn2+ on the inner side of the frog skin appears to be due to a reduced permeation of Na+ and Cl- through the outer barrier of the skin. Addition of Mn2+ to the outer solution bathing the frog skin caused an increase in PD and a smaller increase in Is. c. These changes were not associated with alterations in the fluxes of Na+ or mannitol and were observed only when chloride was present in the bathing solutions. The effect of Mn2+ on this side of the frog skin may therefore be due to a net retention of Cl- in the outer solution.

Effect of bile salts on amino acid transport by rabbit intestine.

The effect of bile salts on alanine absorption across four regional sites of rabbit intestine was examined using an in vivo single-pass perfusion technique. Na-deoxycholate at a concentration of 3 mM reduced alanine absorption across all levels of the intestine, and a higher concentration (10 mM) of Na-taurodeoxycholate (TDC) caused only a minimal reduction of alanine absorption in the jejunum. TDC, however, was more effective in in vitro experiments, causing an incrase in transmural serosal-to-mucosal flux of alanine and phenylalanine, particularly when present in both the mucosal and serosal media. It also reduced the mucosal-to-serosal alanine flux rate when present only in the mucosal medium. The influx of these amino acids across the mucosal brush border membrane was also decreased by TDC. These amino acid transport changes correlated fairly well with some observed histological changes of the intestinal epithelium. This suggests that bile salt inhibition of amino acid absorption is nonspecific in type and can be mainly explained as being the result of an injurious action of these surface-active agents on the rabbit intestine.

Effects of flow rate and potassium intake on distal tubular potassium transfer.

Potassium transport was studied across proximal and distal tubular epithelium in rats on a normal, low- and high-potassium intake during progressive loading with isotonic saline (150 mM) or a moderately hypersomotic urea (200 mM) sodium chloride (100 mM) solution. Free-flow micropuncture and recollection techniques were used during the development of diruesis and tubular fluid (TF) analyzed for inulin-14C, potassium (K) and sodium (Na). Tubular puncture sites were localized by neoprene filling and microdissection. During the large increase in tubular flow rates (10 times): 1) fractional potassium reabsorption fell along the proximal tubule, 2) TFk along the distal tubule remained constant and independent of flow rate in control and high-k rats; thus, net potassium secretion increased in proportion to and was limited by flow rate. 3) In low-K rats TF k fell; with increasing flow rates distal K secretion was not effectively stimulated. 4) Distal tubular sodium reabsorption increased in all animals with flow rate, but tubular Na-K exchange ratios varied greatly. It is suggested that whenever sodium delivery stimulates distal tubular potassium secretion it does so by 1) increasing volume distal tubular potasssium secretion and by 2) augmenting the transepithelial electrical potential difference (lumen negative).

Effects of graded solute diuresis on renal tubular sodium transport in the rat.

Sodium transport was studied across proximal and distal tubules of rats undergoing progressive intravenous loading with either isomotic saline or urea (200 mosmol)-saline (100 mosmol) solutions. Free-flow as well as recollection micropuncture techniques were used, and tubular fluid (TF) samples were analyzed for inulin-14C and sodium (Na). With administration of progressively larger intravenous saline loads, the delivery of fluid and sodium into the distal tubule rose. Concomitantly, the normally observed decline of tubular sodium concentrations along the distal tubule became attenuated until it was abolished at the highest infusion rates of saline solutions. Absolute reabsorption rates of Na across the distal tubule increased in proportion to tubular flow rate, and no tubular maximum (Tm) was observed. It is suggested that the delivery of increased amounts of sodium to the normally unsaturated later parts of the distal tubule and the elevated tubular sodium concentration after saline loading account for the observed stimulation of distal tubular net sodium transport. The extent of transport stimulation is also subject to control by the amount of urea accumulation along the distal tubule. As the latter declines, sodium reabsorption is proportionately enhanced.