Na+-independent uptake of nutrients inTetrahymena

Summary Cell multiplication rates in cultures ofTetrahymena pyriformis andT. thermophila were independent of the external Na⁺ concentrations at the levels of 0.5, 10 and 22 mM. The Na_i/Na₀ and Cl_i/Cl₀ ratios were determined at the low and high Na⁺ concentrations, and assuming that Cl^– is distributed passively, the electrochemical Na⁺ gradients for the two situations were calculated. The energy in these gradients allows a net co-transport of nutrients only in the high Na⁺ medium. Since the doubling times are independent of the Na⁺ concentrations we conclude thatTetrahymena can obtain its energy for nutrient uptake from other sources than the electrochemical Na⁺ gradient.     

The basic asymmetry of Na+-dependent glycine transport in Ehrlich cells

Influx and efflux of glycine have been examined as a function of external and internal Na⁺ concentrations, respectively, when $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$ it was found that at comparable external and cellular Na⁺ levels, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for efflux was larger by an order of magnitude than the value for influx and the $\text{V}$ for efflux was several times greater than the $\text{V}$ for influx. For both fluxes the major effect of Na⁺ was to decrease the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value. The observations are consistent with the conclusion that the Na⁺-dependent transport system is asymmetric per se. Influx and efflux of glycine were increased in a near linear manner by increasing the Na⁺ concentration from 13 to 100 mM, the half-time for glycine equilibration being a function of the Na⁺ concentration in absence of an electrochemical potential difference for Na⁺. In Na⁺-free media ($\text{[}\text{Na}{}^{\mathrm{+}}\text{] < 5}\text{mM}$) equilibration of glycine between cells and medium was not achieved after 60 min at 25°C. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$, efflux (or uptake) of glycine was not affected by internal (or external) K⁺ between 20 and 120 mM suggesting that K⁺ plays no direct role in Na⁺-dependent transport of glycine in Ehrlich cells.     

Carrier-mediated transfer of d-glucose in brush border vesicles derived rom rabbit renal tubules. Na+-dependent versus Na+-independent transfer

A brush border preparation from rabbit renal tubules containing a high yield of vesicles has been used to study the transfer of d-glucose through the brush border membrane. In the presence of an Na⁺ gradient across the vesicular membrane, the vesicles could concentrate d-glucose to a factor of 1.5, whereas in the absence of an Na⁺ gradient, only equilibrium with the medium was achieved. Two types of transfer could be distinguished by their requirement of Na⁺, their sensitivity to phlorizin and their pH optimum. The Na⁺-independent transfer was about 100 times less sensitive to phlorizin than the Na⁺-dependent path and exhibited a pH optimum between 7 and 8, whereas the Na⁺-dependent transfer was highest at a pH between 8 and 9.The brush border preparation could be freed of most of the contaminating material derived from the basal and lateral tubular cell membrane by a discontinuous density gradient centrifugation. It still showed both forms of transfer to a similar extent, indicating that both are located in the brush border membrane.A study of the sensitivity of d-glucose transfer to phlorizin, in the presence and absence of Na⁺ at different temperature, suggests a single carrier species functioning in two interchangeable conformational states with different affinities for phlorizin rather than two transfer systems working independently.     

Na+-dependence of 45Ca2+ uptake in adult rat isolated cardiac cells

We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca²⁺-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed ⁴⁵Ca²⁺ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca²⁺ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na⁺ by Li⁺ or Tris increased ⁴⁵Ca²⁺ uptake by the fast compartment; high [K⁺]_o increased this uptake further. These increases appeared to be related also to internal concentrations of Na⁺. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na⁺-dependent ⁴⁵Ca²⁺ uptake is affected by [Na⁺]_o, [Na⁺]_i and [K⁺]_o is compatible with the Na⁺-Ca²⁺ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca²⁺ transport in cardiac muscles.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent K_m value of 46 μM and a V_max of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na⁺ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na⁺ was related to the transmembraneous Na⁺-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent Km value of 46 microM and a Vmax of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na+ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na+ was related to the transmembraneous Na+-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

The characterisation of two partially purified systems for Na+-dependent amino acid transport

Two systems mediating the transport of amino acids were studied in vesicles derived from protein-depleted membranes of pigeon erythrocytes. One system (ASC system) catalysed the Na⁺-dependent exchange of small neutral amino acids, such as alanine, serine and cysteine. The other system, also Na⁺-dependent, mediated the active transport of glycine. The ASC and glycine systems were distinguished by the sensitivity of the latter to the anion present, by the former's requirement for an exchangeable amino acid and by the inability of alanine to inhibit the transport of glycine. Preliminary results indicated that the influx of glycine was electrically silent. The only major integral protein retained in the vesicles was the band 3 protein, but that could not be unequivocally identified as the transporter.     

The properties of the preferential uptake of l-leucine by isolated intestinal epithelial cells

1.

1. Epithelial cells isolated from rat intestine have shown the ability to preferentially take up 1 mM l-leucine as compared to the d-isomer. The uptake was found to be concentrative. l-Leucine uptake was inhibited by neutral l-amino acids and basic l-amino acids but was not inhibited by d-valine or d-isoleucine. Galactose and α-methyl-d-glucose were inhibitory; glucose was significantly less inhibitory; and fructose activated uptake. Inhibitors of energy metabolism, sulfhydryl inhibitors, ouabain, and procedures which damaged the morphology of the cell all decreased l-leucine uptake. l-Leucine uptake was decreased in the absence of either Na⁺, K⁺, Ca²⁺, or Mg²⁺ and exhibited a broad ph optimum between 4 and 8. d-Leucine uptake was a linear function of time during the first 5 min of incubation. The apparent K_m for l-leucine uptake was 3.2 mM, and l-valine was competitive inhibitor of l-leucine uptake. Inhibitors of protein biosynthesis did not reduce l-leucine uptake. The efflux of l-leucine from the cells was inhibited by the cold.     

Bimodal effects of cellular amino acids on Na+-dependent amino acid transport in ehrlich cells

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K⁺] ($\text{10 mM ± 5 mM}$).With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of infiux is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells.Knowing that increases and decreases in the membrane potential are achieved by using varying external [K⁺] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the $\text{V}$ for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased $\text{V}$. Changes in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ also occur when the membrane potential is varied.     

Activation of Rb+ and Na+ uptake into yeast by monovalent cations

⁸⁶Rb⁺ uptake by yeast was not only stimulated by Rb⁺ or K⁺ but also by Na⁺. The uptake of ²²Na⁺ was enhanced by both Rb⁺ and K⁺, but not by Na⁺, which was inhibitory at all concentrations applied. Inhibition of ²²Na⁺ uptake by inactive Na⁺ occurred in two phases: one phase refers to inhibition at low Na⁺ concentrations and the other to inhibition at high Na⁺ concentrations. Our results can be qualitatively described by a two-site transport mechanism, having two cation binding sites, which must be occupied with monovalent cations before transport can occur.     

Immunohistochemical localization of Na+-dependent glucose transporter in the rat digestive tract

Summary Glucose is actively absorbed in the intestine by the action of the Na⁺-dependent glucose transporter. Using an antibody against the rabbit intestinal Na⁺-dependent glucose transporter (SGLT1), we examined the localization of SGLT1 immunohistochemically along the rat digestive tract (oesophagus, stomach, duodenum, jejunum, ileum, colon and rectum). SGLT1 was detected in the small intestine (duodenum, jejunum and ileum), but not in the oesophagus, stomach, colon or rectum. SGLT1 was localized at the brush border of the absorptive epithelium cells in the small intestine. Electron microscopical examination showed that SGLT1 was localized at the apical plasma membrane of the absorptive epithelial cells. SGLT1 was not detected at the basolateral plasma membrane. Along the crypt-villus axis, all the absorptive epithelial cells in the villus were positive for SGLT1, whose amount increased from the bottom of the villus to its tip. On the other hand, cells in the crypts exhibited little or no staining for SGLT1. Goblet cells scattered throughout the intestinal epithelium were negative for SGLT1. These observations show that SGLT1 is specific to the apical plasma membrane of differentiated absorptive epithelial cells in the small intestine, and suggest that active uptake of glucose occurs mainly in the absorptive epithelial cells in the small intestine.     

Active Na+ uptake in the isolated midgut of larval Sarcophaga bullata

The isolated midgut of larval Sarcophaga bullata actively accumulates Na⁺ from the gut lumen into the haemolymph. The active transport of Na⁺ out of the gut lumen is responsible for the transepithelial potential difference measured across the midgut epithelium, such that the midgut lumen is negative in respect to the haemolymph side. Both the net movement of Na⁺ out of the midgut lumen and the transepithelial potential are inhibited by CN^? and, in addition, the potential in blocked by ouabain.