TRANSMERIDIAN JET LAG AND ITS PHARMACOLOGICAL CORRECTION.

The article deals with the development and correction of acute jet lag in a flight across several time zones. The investigation had the purpose to study dynamics of subjective and objective psychophysiological parameters and demonstrate methods of prophylaxis and correction of acute jet lag due to transmerdian flights. Subjects were 8 normal volunteers (experimental group) at the age of 26-55 years flying eastward over 7 times zones. The investigation included 3 stages: baseline, preparatory (21-d course of Euricoma longifolia extraction) and main (intake of donormil, cirkadin and artificial sleep during the flight). Functional diagnostics was performed on the baseline stage, on completion of the preparatory stage and on the next day after the flight (21-22 days from the beginning of the preparatory stage). Objective and subjective methods were used to evaluate the autonomic and cardiovascular systems and mental performance. In the control group (n = 4) functional diagnostics was performed on the same days. The investigations showed the benefit of preparation for transmeridian air travel and experimentally demonstrated positive effects of the proposed pharmacological correction of acute transmeridian jet lag.

Glycosphingolipids modulate renal phosphate transport in potassium deficiency.

BACKGROUND: Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism. The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. METHODS: K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days, which resulted in a marked decrease in serum and tissue K content. RESULTS: K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V(max) of brush-border membrane (BBM) Na/Pi cotransport activity (1943 +/- 95 in control vs. 1184 +/- 99 pmol/5 sec/mg BBM protein in K deficiency, P < 0.02). Surprisingly, the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1), and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin, glucosylceramide, and ganglioside GM3 content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficient rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 1152 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. CONCLUSION: K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.

Dietary sulfate regulates the expression of the renal brush border Na/Si cotransporter NaSi-1.

Dietary inorganic sulfate (Si) intake is an important factor in the regulation of renal proximal tubular sodium-dependent Si transport (Na/Si cotransport). The purpose of the present study was to determine whether modulation of Na/Si cotransport activity by dietary Si is mediated through regulation of the renal expression of the recently cloned NaSi-1 protein located in the apical brush border membrane (BBM) of the proximal tubule. It was found that rats fed a high Si diet had a marked increase in the renal excretion of Si and a concomitant decrease in BBM Na/Si cotransport activity when compared with rats on a control Si diet. The 43% decrease in BBM Na/Si cotransport activity was associated with a 33% decrease in BBM NaSi-1 protein abundance, as determined by Western blotting, and a 2.7-fold decrease in cortical NaSi-1 mRNA abundance, as determined by Northern blotting. Furthermore, cortical mRNA from rats fed a high Si diet when injected into Xenopus laevis oocytes led to a 2.2-fold decrease in Na/Si cotransport activity compared with mRNA isolated from control Si diet rats. This study indicates that adaptation to a high Si diet is accompanied by a decrease in renal cortical NaSi-1 mRNA abundance, which results in reduced expression of the NaSi-1 protein at the level of the proximal tubular BBM.

Renal Na-Si cotransporter NaSi-1 is inhibited by heavy metals.

Heavy metal intoxication leads to a number of reabsorptive and secretory defects in renal transport systems. We have studied the effects of several heavy metals on the expression of the renal Na-Si cotransporter NaSi-1. NaSi-1 cRNA was injected into Xenopus oocytes, and Na-Si cotransport activity was measured in the presence of mercury, lead, cadmium, or chromium. Mercury strongly inhibited NaSi-1 transport irreversibly by reducing both maximal velocity (Vmax) and Michaelis constant (Km) for inorganic sulfate (Si). Lead inhibited NaSi-1 transport reversibly by decreasing Vmax but not Km for Si. Cadmium showed weak reversible inhibition of NaSi-1 transport by decreasing only NaSi-1 Vmax. Chromium strongly inhibited NaSi-1 cotransport reversibly by reducing Km for Si by sevenfold, most probably by binding to the Si site, due to the strong structural similarity between the CrO4(2-) and SO4(2-) substrates. In conclusion, this study presents an initial report demonstrating heavy metals inhibit renal brush border Na-Si cotransport via the NaSi-1 protein through various mechanisms and that this blockade may be responsible for sulfaturia following heavy metal intoxication.

Identification of a cDNA/protein leading to an increased Pi-uptake in Xenopus laevis oocytes.

In a previous report we documented an increased Na(+)-dependent transport of inorganic phosphate (P(i)) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211-216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P(i)-uptake stimulator) lead to a 3-4-fold stimulation of Na(+)-dependent P(i)-uptake (10ng cRNA injected, 3-5 days of expression). Na(+)-independent uptake of P(i) was also affected but transport of sulphate and L-arginine (in the presence or absence of sodium) remained unchanged. The apparent K(m)-values for the induced Na(+)-dependent uptake were 0.26 +/- 0.04 mM for P(i) and 14.8 +/- 3.0 mM for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of approximately 2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P(i)-uptake into Xenopus laevis oocytes, but which is not a P(i)-transporter itself.

Transport characteristics of a murine renal Na/Pi-cotransporter.

A complementary deoxyribonucleic acid (cDNA) corresponding to a murine renal cortical Na/phosphate-(Na/Pi-) cotransporter was isolated and its transport properties characterized by electrophysiological techniques after expression in Xenopus laevis oocytes. A Na-dependent inward movement of positive charges ("short-circuit current") was observed upon superfusion with Pi (and with arsenate). Increasing the Na concentration led to a sigmoidal elevation in Pi-induced short-circuit current; the apparent Michaelis constant, Km, (around 40 mM Na) was increased by lowering the pH of the superfusate but was not influenced by altering the Pi concentration. Increasing the Pi (and arsenate) concentration led to a hyperbolic elevation in Na-dependent short-circuit current (apparent Km for Pi at 100 mM Na was around 0.1 mM; apparent Km for arsenate was around 1 mM); lowering the Na concentration decreased the apparent affinity for Pi. The Pi-induced short-circuit current was lower at more acidic pH values (at pH 6.3 it was about 50% of the value at pH 7.8); this pH dependence was similar if the Pi concentration was calculated in total, or if distinction was made between its mono- and divalent forms. Thus, the pH dependence of Na-dependent Pi transport (total Pi) may not be related primarily to a pH-dependent alteration in the availability of divalent Pi, but includes also a competitive interaction of Na with protons. The effect of Pi and Na concentration on the apparent Km values for Na or Pi, respectively, provides evidence for an ordered interaction of "cosubstrate" (Na first) and "substrate" (Pi or arsenate second).

Regulation of opossum kidney (OK) cell Na/Pi cotransport by Pi deprivation involves mRNA stability.

Renal proximal tubular Na-dependent phosphate transport (Na/Pi cotransport) has been studied extensively in the opossum kidney (OK) cell line. Recently, we cloned a complementary deoxyribonucleic acid (cDNA) (NaPi-4) from OK cells encoding an apical NaPi cotransport system. OK cells exposed to a low-Pi medium, as compared to high-Pi media, responded with an increase in Na/Pi cotransport, which was followed by an increase in NaPi-4 messenger ribonucleic acid (mRNA) abundance; maximal stimulation of Na/Pi cotransport was reached in 2 h, with no further increase for up to 16 h. NAPi-4 mRNA abundance was unaltered for 2 h, then increased to a maximum after 6-16 h in cells treated with low Pi medium. NaPi-4 mRNA decay rate was lowered by low-Pi media when compared to high-Pi media, with no increase in the NaPi-4 mRNA transcription rate. These data suggest that the upregulation of Na/Pi cotransport in OK cells by low-Pi media involves two regulatory mechanisms: an immediate (early) increase (after 2 h) in the expression of Na/Pi cotransport, independent of mRNA synthesis or stability, and a delayed (late) effect (after 4-6 h), resulting in an increase in NaPi-4 mRNA abundance, due to an increased stability.

Expression of sodium-dependent phosphate (NadPi) transport in Xenopus laevis oocytes induced by mRNA from 1 alpha, 25-dihydroxyvitamin D3-treated rat osteoblast-like cells.

Injection of messenger ribonucleic acid (mRNA) isolated from 1 alpha, 25-dihydroxyvitamin D3-treated osteoblast-like (PyMS) cells leads to an enhanced sodium-dependent phosphate (NadPi) transport in Xenopus laevis oocytes, when compared to untreated cells. After mRNA size fractionation, mRNA with an average size of 2.2-3.8 kilobases showed up to a 1.8-fold stimulation of NadPi transport encoding either directly a NadPi transporter(s) or proteins controlling their activity. No hybridization was observed in Northern blots with RNA from rat bone or PyMS cells with the recently cloned rat renal brush border NadPi transporter NaPi-2; hybrid depletion with a NaPi-2 antisense oligonucleotide did not abolish the PyMS mRNA-induced NadPi transport in oocytes. We present the first evidence for functional expression in Xenopus laevis oocytes of a new type of NadPi transport system in bone cells, which is different from the renal type.

Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate.

Previously, we isolated a cDNA (NaPi-1) related to a rabbit renal proximal tubular Na-Pi cotransporter (A. Werner, M.L. Moore, N. Mantei, J. Biber, G. Semenza, and H. Murer. Proc. Natl. Acad. Sci. USA 88:9608-9612, 1991.). In this study, we isolated an additional (rabbit renal) cDNA (NaPi-6), which induces Na-dependent Pi uptake in Xenopus laevis oocytes. Substrate specificity and kinetic properties corresponded to those known for rabbit renal brush-border membrane (BBM) Na-Pi cotransport. NaPi-6 was cloned by homology using NaPi-2 cDNA, a rat renal BBM Na-Pi cotransporter (S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, and H. Murer. Proc. Natl. Acad. Sci. USA 90: 5979-5983, 1993). NaPi-6 encodes a protein of 642 amino acids, exhibiting at least eight transmembrane domains. NaPi-6 mRNA and protein in kidneys of rabbits fed a low-Pi diet (LPD; 0.11% Pi) for 1 wk were increased by 1.5- and 4-fold, respectively, compared with those of rabbits fed a high-Pi diet (HPD; 1.20% Pi). This effect was correlated with an increase in Na-Pi cotransport of BBM vesicles isolated from animals adapted to LPD (2.5-fold with respect to HPD). In contrast, NaPi-1 mRNA and protein were not altered in response to LPD. Thus rabbit proximal tubular BBMs contain two different Na-Pi cotransport systems: NaPi-1 (type I) and NaPi-6 (type II). Only the type II transport system seems to be under regulatory control in response to low-Pi dietary intake.

Electrophysiological analysis of Na+/Pi cotransport mediated by a transporter cloned from rat kidney and expressed in Xenopus oocytes.

Phosphate (Pi) reabsorption in renal proximal tubules involves Na+/Pi cotransport across the brush border membrane; its transport rate is influenced by the Na(+)-coupled transport of other solutes as well as by pH. In the present study, we have expressed a cloned rat renal brush border membrane Na+/Pi cotransporter (NaPi-2) in Xenopus laevis oocytes and have analyzed its electrophysiologic properties in voltage- and current-clamp studies. Addition of Pi to Na(+)-containing superfusates resulted in a depolarization of the membrane potential and, in voltage-clamped oocytes, in an inward current (IP). An analysis of the Na+ and/or Pi concentration dependence of IP suggested a Na+/Pi stoichiometry of 3:1. IP was increased by increasing the pH of the superfusate; this phenomenon seems to be mainly related to a lowering of the affinity for Na+ interaction by increasing H+ concentration. The present data suggest that known properties of Pi handling at the tubular/membrane level are "directly" related to specific characteristics of the transport molecule (NaPi-2) involved.

Expression of rat ileal Na(+)-sulphate cotransport in Xenopus laevis oocytes: functional characterization.

Small-intestinal sulphate absorption is a Na(+)-dependent process having its highest rate in the ileum; it involves brush-border membrane Na(+)-sulphate cotransport. Injection of rat ileal mRNA into Xenopus laevis oocytes induced Na(+)-dependent sulphate uptake in a dose-dependent manner, with no apparent effect on Na(+)-independent sulphate uptake. For mRNA-induced transport, the apparent Km value for sulphate interaction was 0.6 +/- 0.2 mM and that for sodium interaction was 25 +/- 2 mM (Hill coefficient: 2.3 +/- 0.3). mRNA-induced transport, was inhibited by thiosulphate, but not by phosphate or 4,4,'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Using a rat renal Na(+)-sulphate cotransporter cDNA as a probe [NaSi-1; Markovich et al. (1993) Proc Natl Acad Sci USA 90:8073-8077], the highest hybridization signals (2.3 kb and 2.9 kb) were obtained in size fractions showing the highest expression of Na(+)-dependent sulphate transport in oocytes. Hybrid depletion experiments using antisense oligonucleotides (from the NaSi-1 cDNA sequence), provided further evidence that rat small-intestinal (ileal) Na(+)-sulphate cotransport is closely related to rat proximal-tubular brush-border membrane Na(+)-sulphate cotransport.

Cloning of a Na/Pi cotransporter from opossum kidney cells.

Opossum kidney (OK) cells have been extensively used to study cellular mechanisms of renal proximal tubular Na/P(i) cotransport. We have cloned a cDNA (NaPi-4) most likely encoding an apical Na/P(i) cotransporter from OK cells. The cloning strategy was based on homology to the recently cloned human renal (NaPi-3) Na/P(i) cotransporter (Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5979-5983). Kinetic characterization (P(i) interaction, sodium interaction, and pH dependence) of NaPi-4-induced Na/P(i) uptake showed high similarity to apical Pi transport in OK cell monolayers. The NaPi-4 cDNA is 2548 base pairs long and encodes a protein of 70.5 kDa, containing at least 8 predicted transmembrane domains. Northern blot analysis with OK cell mRNA shows a NaPi-4-related signal (2.5 kilobases) in cells grown on impermeant and permeant supports. Hybrid depletion with NaPi-4 antisense oligonucleotides abolished the mRNA-induced Na/P(i) cotransport in oocytes. Similarly, NaPi-4 antisense oligonucleotides inhibited (up to 70%) Na/P(i) cotransport in OK cell monolayers. We presume that NaPi-4 is closely related to the OK cell apical Na/P(i) cotransporter.

Stimulation of system y(+)-like amino acid transport by the heavy chain of human 4F2 surface antigen in Xenopus laevis oocytes.

A kidney cortex cDNA clone (rBAT) has recently been isolated, which upon in vitro transcription and capping complementary RNA (cRNA) and injection into Xenopus laevis oocytes induces a system b0,(+)-like amino acid transport activity. This cDNA encodes a type II membrane glycoprotein that shows significant homology to another type II membrane glycoprotein, the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Here we demonstrate that injection of human 4F2hc cRNA into oocytes results in the activation of a cation-preferring amino acid transport system that appears to be identical to the y(+)-like transport already present in the oocyte. This is based on the following results: (i) Injection of in vitro transcripts from 4F2hc cDNA (4F2hc cRNA) into oocytes stimulates up to 10-fold the sodium-independent uptake of L-arginine and up to 4.1-fold the sodium-dependent uptake of L-leucine. In contrast, 4F2hc cRNA does not increase the basal sodium-independent uptake of L-leucine. (ii) Basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine is completely inhibited by L-leucine in the presence of sodium. Similarly, the basal and 4F2hc cRNA-stimulated sodium-dependent uptake of L-leucine is entirely inhibited by L-arginine. (iii) The stimulation of sodium-independent uptake of L-arginine and the stimulation of sodium-dependent uptake of L-leucine induced by injection of 4F2hc cRNA are both completely inhibited by dibasic L amino acids and to a lesser extent by D-ornithine. (iv) Both basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine show two additional characteristics of the system y+ transport activity: inhibition of L-arginine uptake by L-homoserine only in the presence of sodium and an increase in the inhibition exerted by L-histidine as the extracellular pH decreased. Our results allow us to propose that an additional family of type II membrane glycoproteins (composed by rBAT and 4F2hc) is involved in amino acid transport, either as specific activators or as components of amino acid transport systems.