A comparative study of specificity of the intestinal Na+/sugar cotransport among vertebrates

Kt values for various monosaccharides were determined from sugar-induced increments of the transmural potentials in isolated small intestines of the goldfish, bullfrog, turtle, quail, guinea pig, rat and rabbit, and specificity patterns of the Na+/sugar cotransporters were compared among these animal species. Absolute requirement of the D-pyranose ring structure was seen in all animals. Requirements of C2-OH and C6 were strong, but not absolute, and OH groups on C3, C4, C6 and the O-atom of the pyranose ring were also suggested to participate, in some degree, in the interaction with the carrier. Comparison of the disaccharide-evoked potentials revealed that there were considerable species differences in activities of trehalase, sucrase and lactase among animals examined, but the differences were relatively small for maltase activity.     

Temperature sensitivity and substrate specificity of two distinct Na+-activated D-glucose transport systems in guinea pig jejunal brush border membrane vesicles

D-Glucose transport was studied with isolated brush border membrane vesicles from guinea pig jejunum. Saturation curves were carried out at either 25 or 35 degrees C in buffers containing Na+, Li+, K+ (100 mM chloride salt), or sorbitol (200 mM). Uncorrected uptake rates were fitted by nonlinear regression analysis to an equation involving one diffusional and two saturable terms. In the presence of Na+ at 35 degrees C, two saturable systems (Km = 0.4 and 24 mM, respectively) were evident, as well as a diffusion component quantitatively identical with that measured with L-glucose in separate experiments. In contrast, at 25 degrees C only one saturable system was apparent (Km = 1.2 mM): the second exhibited diffusion-like kinetics. In the presence of Na+ at 35 degrees C, D-glucose uptake was fully inhibited by both D-glucose and D-galactose, whereas alpha-methylglucoside gave kinetics of partial inhibition. We conclude that in the presence of Na+ there are at least two distinct D-glucose transport systems: 1) System I, a low temperature-sensitive system, fully inhibited by D-glucose, D-galactose, and alpha-methylglucoside; we identify it as the "classical" D-glucose/Na+ cotransport system, insensitive to inhibition by cytochalasin B and obligatorily dependent on Na+; and 2) System II, a high temperature-sensitive system where D-glucose and D-galactose inhibit but alpha-methylglucoside is inert. Its cation specificity is unclear but it appears to be sensitive to cytochalasin B inhibition. When Li+ or K+ substituted for Na+, only one transport system was apparent. The Li+-activated transport was: independent of the incubation temperature; inhibited by D-glucose and D-galactose but not by alpha-methylglucoside, 2-deoxy-D-glucose, D-mannose, and D-xylose; and sensitive to cytochalasin B inhibition. The exact nature of the system (or systems) involved in D-glucose transport in the absence of sodium remains to be established.     

1,25-Dihydroxycholecalciferol-related Na+/D-glucose transport in brush-border membrane vesicles from embryonic chick jejunum. Modulation by triiodothyronine

1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na(+)-gradient-driven D-glucose uptake by brush-border membrane vesicles, but had no effect on Na(+)-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ([Na+]o = 100 mM; [Na+]i = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na(+)-dependent D-glucose transport through changes in the electrochemical Na+ gradient. D-[U-14C]Glucose tracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane. 10 nM triiodothyronine, which by itself has no effect on Na(+)-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecalciferol such that in the presence of both hormones, Na+/D-glucose-carrier activity was increased fourfold above control levels.     

Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells

A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.     

Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts.     

Na+/K+/Cl- cotransport is stimulated by a Ca(++)-calmodulin-mediated pathway in BALB/c 3T3 fibroblasts.

In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl- cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl- cotransport was stimulated by the growth factors EGF, TGF-alpha, IGF-1, and IGF-2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++. In addition, direct activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) did not affect the Na+/K+/Cl- cotransport activity of quiescent cells. The Na+/K+/Cl- cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl- cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl- cotransport is stimulated predominantly by a protein kinase C-independent mechanism in BALB/c 3T3 fibroblasts. Both the intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine (CP), blocked serum- and mitogen-stimulated Na+/K+/Cl- cotransport. These results suggest that the Na+/K+/Cl- cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca(++)-calmodulin-mediated pathway in the synchronized BALB/c 3T3 fibroblasts.     

The effect of cellular calcium on Na+/K+ cotransport in human red blood cells

The increase in Ca²⁺ permeability by addition of ionophore A23187 in the presence of external Ca²⁺ did not alter the bumetanide-sensitive Na⁺/K⁺ effluxes in human red blood cells. An inhibition of this pathway by cellular Ca²⁺ could be observed only under conditions in which the cellular ATP content was drastically depleted.     

Na+/K+/Cl- cotransport in resealed ghosts from erythrocytes of the Milan hypertensive rats.

The erythrocytes (RBC) of the Milan hypertensive rats (MHS) have a smaller volume and faster Na+/K+/Cl- cotransport than RBC from normotensive controls (MNS). The difference in Na+/K+/Cl- cotransport is no longer present in inside-out Vesicles (IOV) of RBC membrane. To differentiate between cytoplasmic or membrane skeleton abnormalities as possible causes of these differences. Resealed ghosts (RG) were used to measure ion transport systems. The following results have been obtained: (1) RG from MHS have a smaller volume than MNS (mean +/- S.E. 20.7 +/- 0.45 vs. 22.09 +/- 0.42 fl, P < 0.05). (2) RG showed a bumetanide-sensitive Na efflux that retains the characteristics of the Na+/K+/Cl- cotransport of the original RBC: it is K(+)- and Cl(-)-sensitive and dependent on the intracellular Na+ concentration. (3) The Na+/K+/Cl- cotransport was faster in RG from MHS than in those from MNS (mean +/- S.E. 0.095 +/- 0.01 vs. 0.066 +/- 0.01 rate constant h-1, P < 0.01). These results, together with those of IOV, support the hypothesis that an abnormality in the membrane skeletal proteins may play a role in the different Na+/K+/Cl- cotransport modulation between MHS and MNS erythrocytes.     

Relative contributions of Na+/H+ exchange and Na+/HCO3- cotransport to ischemic Nai+ overload in isolated rat hearts

The Na(+)/H(+) exchanger (NHE) and/or the Na(+)/HCO(3)(-) cotransporter (NBC) were blocked during ischemia in isolated rat hearts. Intracellular Na(+) concentration ([Na(+)](i)), intracellular pH (pH(i)), and energy-related phosphates were measured by using simultaneous (23)Na and (31)P NMR spectroscopy. Hearts were subjected to 30 min of global ischemia and 30 min of reperfusion. Cariporide (3 microM) or HCO(3)(-)-free HEPES buffer was used, respectively, to block NHE, NBC, or both. End-ischemic [Na(+)](i) was 320 +/- 18% of baseline in HCO(3)(-)-perfused, untreated hearts, 184 +/- 6% of baseline when NHE was blocked, 253 +/- 19% of baseline when NBC was blocked, and 154 +/- 6% of baseline when both NHE and NBC were blocked. End-ischemic pH(i) was 6.09 +/- 0.06 in HCO(3)(-)-perfused, untreated hearts, 5.85 +/- 0.02 when NHE was blocked, 5.81 +/- 0.05 when NBC was blocked, and 5.70 +/- 0.01 when both NHE and NBC were blocked. NHE blockade was cardioprotective, but NBC blockade and combined blockade were not, the latter likely due to a reduction in coronary flow, because omission of HCO(3)(-) under conditions of NHE blockade severely impaired coronary flow. Combined blockade of NHE and NBC conserved intracellular H(+) load during reperfusion and led to massive Na(+) influx when blockades were lifted. Without blockade, both NHE and NBC mediate acid-equivalent efflux in exchange for Na(+) influx during ischemia, NHE much more than NBC. Blockade of either one does not affect the other.     

Modulation of Na+/alanine cotransport in liver sinusoidal membrane vesicles by internal divalent cations

Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane.     

Inhibition of the Na+/K+ cotransport system by cyclic AMP and intracellular Ca2+ in human red cells

Human erythrocytes are able to incorporate cyclic AMP (cAMP) in amounts larger than those required to saturate cAMP-dependent protein kinase. In contrast to previous observations in avian red blood cells in which cAMP stimulates the Na+/K+ cotransport system, we demonstrate that cAMP inhibits this system in human erythrocytes. The cotransport inhibition is enhanced by addition of phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine to the incubation medium. The cAMP concentration giving half-maximal cotransport inhibition showed a wide variation among different individuals (from 0.1 to 5 mM external cAMP concentration). In contrast to cAMP, cyclic GMP showed little effect on the cotransport system. Ca2+ introduced into the cell interior was an inhibitor of the Na+/K+ cotransport system. These results suggest that in human cells in which endogeneous levels of cAMP and Ca2+ are modulated by hormones, the Na+/K+ cotransport system may be under hormonal regulation.     

Activation of the Na+/K+/Cl- cotransport system by reorganization of the actin filaments in Ehrlich ascites tumor cells.

Reorganization (disassembly) of the actin filaments in Ehrlich ascites tumor cells, either by hypotonic treatment in the presence of Ca2+ or by addition of cytochalasin B, results in activation of the Na+/K+/Cl- cotransport system. However, other regulatory processes, some of which may be dependent on an intact filament system, are responsible for the activation of the Na+/K+/Cl- cotransport system after cell shrinkage.     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

Polarized distribution of Na+/H+ antiport and Na+/HCO3- cotransport in primary cultures of renal inner medullary collecting duct cells.

Primary cultures of rat renal inner medullary collecting duct cells were grown to confluence on glass coverslips and treated permeant supports, and the pH-sensitive fluorescent probe 2,7-biscarboxyethyl-5,6-carboxyfluorescein was employed to delineate the nature of the transport pathways that allowed for recovery from an imposed acid load in a HCO3-/CO2-buffered solution. The H+ efflux rate of acid-loaded cells was 13.44 +/- 0.94 mM/min. Addition of amiloride, 10(-4) M, to the recovery solution reduced the H+ efflux rate to 4.06 +/- 0.63 mM/min. The amiloride-resistant pHi recovery mechanism displayed an absolute requirement for Na+ but was Cl(-)-independent. Studies performed on permeable supports demonstrated that the latter pathway was located primarily on the basolateral-equivalent (BE) cell surface and was inhibited by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In a Na(+)-replete solution containing DIDS (50 microM) and amiloride (10(-4) M), acid-loaded cells failed to return to basal pHi. To delineate further the amiloride-inhibitable component of pHi recovery, monolayers were studied in the nominal absence of HCO3-/CO2. In 70% of monolayers studied, Na(+)-dependent, amiloride-inhibitable H+ efflux was the sole mechanism whereby acid-loaded cells returned to basal pHi. A Na(+)-independent pathway was observed in 30% of monolayers examined and represented only a minor component of the pHi recovery process. In studies performed on permeable supports, the Na(+)-dependent amiloride-inhibitable pathway was found to be confined exclusively to the BE cell surface. In summary, confluent monolayers of rat renal inner medullary collecting duct cells in primary culture possess two major mechanisms that contribute toward recovery from an imposed acid load, namely, Na+/H+ antiport and Na+/HCO3- cotransport. Na(+)-independent pHi recovery mechanisms represent a minor component of the pHi recovery process in the cultured cell. Both the Na+/H+ antiporter and Na+/HCO3- cotransporter are located primarily on the BE cell surface.     

Inhibition of 3-O-methylglucose transport in human erythrocytes by forskolin

The effect of forskolin, an activator of adenylate cyclase, was investigated on glucose transport in human erythrocytes. Forskolin was found to be a potent inhibitor of 3-O-methylglucose (3-O-MG) influx in human erythrocytes. The inhibition of 3-O-MG transport was instantaneous and reversible. The inhibitory effect of forskolin was concentration-dependent, having an IC50 value of 7.5 microM. Forskolin caused a decrease in Vmax of carrier-mediated 3-O-MG transport from 35.32 to 1.56 mumol/ml of cell X min in the presence of 50 microM forskolin. Inhibition of influx was not reversed at high concentrations of 3-O-MG. In addition, forskolin inhibited the influx of other carbohydrates including galactose, ribose, and fructose. In contrast, forskolin was without effect on adenosine transport. To unravel the underlying mechanism responsible for the inhibitory action of forskolin, the possible involvement of cyclic AMP in controlling glucose transport was examined. Erythrocytes treated with 50 microM forskolin exhibited an increase in cyclic AMP content from the basal levels of 258 fmol/ml of cell to 334 fmol/ml of cell within 10 s after forskolin exposure. However, erythrocytes in which cyclic AMP was allowed to accumulate in excess of 10,000 times the basal level, by means of preincubation with exogenous cyclic AMP, displayed 3-O-MG transport indistinguishable from that of cyclic AMP-poor control cells. In view of the finding that cyclic AMP plays no discernible role in the erythrocyte 3-O-MG transport, it is suggested that the forskolin inhibition is mediated by a mechanism other than by stimulating adenylate cyclase activity. Moreover, forskolin appears to directly inactivate the 3-O-MG transport system since glucose-sensitive cytochalasin B binding to erythrocyte membranes is virtually abolished by 50 microM forskolin.     

Conserved glutamate residues are critically involved in Na+/nucleoside cotransport by human concentrative nucleoside transporter 1 (hCNT1)

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.     

Structural state of the Na+/D-glucose cotransporter in calf kidney brush-border membranes. Target size analysis of Na+-dependent phlorizin binding and Na+-dependent D-glucose transport

Target sizes of the renal sodium-D-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at -50 degrees C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na+-dependent D-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4-4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na+-dependent phlorizin binding unit was estimated to be 230 000 +/- 38 000. From the tracer exchange experiments a molecular weight of 345 000 +/- 24 500 was calculated for the D-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and gamma-glutamyltransferase, whose target sizes were found to be 68 570 +/- 2670 and 73 500 +/- 2270, respectively. These findings provide further evidence for the assumption that the sodium-D-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and D-glucose translocation.     

Kinetic dissection of two distinct proton binding sites in Na+/H+ exchangers by measurement of reverse mode reaction

We examined the effect of intracellular acidification on the reverse mode of Na+/H+ exchange by measuring 22Na+ efflux from 22Na+-loaded PS120 cells expressing the Na+/H+ exchanger (NHE) isoforms NHE1, NHE2, and NHE3. The 5-(N-ethyl-N-isopropyl)amiloride (EIPA)- or amiloride-sensitive fraction of 22Na+ efflux was dramatically accelerated by cytosolic acidification as opposed to thermodynamic prediction, supporting the concept that these NHE isoforms are activated by protonation of an internal binding site(s) distinct from the H+ transport site. Intracellular pH (pHi) dependence of 22 Na+ efflux roughly exhibited a bell-shaped profile; mild acidification from pHi 7.5 to 7 dramatically accelerated 22Na+ efflux, whereas acidification from pHi 6.6 gradually decreased it. Alkalinization above pHi 7.5 completely suppressed EIPA-sensitive 22Na+ efflux. Cell ATP depletion and mutation of NHE1 at Arg440 (R440D) caused a large acidic shift of the pHi profile for 22Na+ efflux, whereas mutation at Gly455 (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na+/H+ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.