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Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.

In this study, the short component of the 23Na T2 (T2f) and the 23Na and 2H quadrupolar interactions (νQ) were measured in bone-cartilage samples of osteoarthritic (OA) and osteoporotic (OP) patients. 23Na νQ was found to increase in osteoarthritic articular cartilage relative to controls. Similar results were found in bovine cartilage following proteoglycan (PG) depletion, a condition that prevails in osteoarthritis. 23Na νQ and 1/T2f for articular cartilage obtained from osteoporotic patients were significantly larger than for control and osteoarthritic cartilage. Decalcification of both human and bovine articular cartilage resulted in an increase of 23Na νQ and 1/T2f, showing the same trend as the osteoporotic samples. Differences in the ratio of the intensity of the large 2H splitting to that of the small one in the calcified zone were also observed. In osteoporosis, this ratio was twice as large as that obtained for both control and osteoarthritic samples. The 2H and 23Na results can be interpreted as due to sodium ions and water molecules filling the void created by the calcium depletion and to calcium ions being located in close association with the collagen fibers. To the best of our knowledge, this is the first study reporting differences of NMR parameters in cartilage of osteoporotic patients. Magn Reson Med, 2010. © 2010 Wiley-Liss, Inc.

Purpose To examine the effects of the alkylating anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on 23Na MRI and the water apparent diffusion coefficient (ADC) in subcutaneously- (sc-) implanted 9L glioma in rats. Materials and Methods 23Na MRI and 1H water ADC measurements were performed on sham-treated control (N = 6) and BCNU-treated (N = 15) Fisher rats one day before BCNU injection and then one, three, and five days after BCNU injection. Results The BCNU-treated tumors were divided into BCNU-responsive (RBCNU) and BCNU-nonresponsive (NRBCNU) groups depending on the tumor volume changes that occurred after therapy. The pretreatment 23Na MRI signal intensity (SI) and water ADC values were higher in RBCNU tumors compared to NRBCNU tumors. 23Na MRI SI and water ADC increased with tumor growth in control and NRBCNU groups, but these changes were interrupted by BCNU therapy in RBCNU group. Conclusion 23Na MRI and water ADC measurements may be useful for predicting and monitoring response to chemotherapy in some tumors. However, the changes that occurred in 23Na MRI SI and water ADC in sc-implanted 9L tumors are in contrast to previously published results for BCNU therapy of orthotopic 9L tumors. This may have important implications for monitoring therapy response in tumors. J. Magn. Reson. Imaging 2006. © 2006 Wiley-Liss, Inc.

Unilateral nephrectomy results in a rapid and specific stimulation of growth and function of the remaining kidney (Ogden, 1967; Fine, 1986). In experimental animals, the bulk of such renal growth is accounted for by hypertrophy of the proximal tubule, which is detectable within 24-48 h and is preceded by an increase in proximal tubule sodium reabsorption via activated apical sodium-hydrogen exchange (NHE) (Fine, 1986; Pollock & Field, 1993). Although the existence of a kidney-specific humoral growth factor, which incites and/or regulates compensatory renal growth, has been long established by parabiotic experiments (Van Vroonhoven, Soler-Montesinos & Malt, 1972; Austin, Goldin & Preuss, 1981; Malt, 1983), serum injections in live animals (Lowenstein & Stern, 1963; Austin et al. 1981; Malt, 1983; Pollock, Nobes, Gyory, Heng & Field, 1996) and in vitro assays (Austin et al. 1981; Malt, 1983; Yamada, Kanetake, Saito, Kondo & Yamamoto, 1983; Yamamoto, Kanetake & Yamada, 1983; Yun, Areas, Yamamoto & Preuss, 1988; Esbrit, Garcia Ocana, Garcia Canero, Manzano & Jiminez Clavero, 1991; Garcia Ocana & Esbrit, 1994; Nobe, Pollock, Heng & Field, 1995), its precise identity remains elusive. Attempts at characterization have so far suggested that this factor is species specific (Yamamoto et al. 1983; Fine, 1986; Yun et al. 1988), unaffected by dialysis or heating (Fine, 1986), and possibly synthesized by the liver and activated by the remnant kidney in a time-dependent fashion following nephrectomy (Dicker, Morris & Shipolini, 1977; Fine, 1986; Garcia Ocana & Esbrit, 1992). Insulin-like growth factor-I (IGF-I), a 7.5 kDa peptide produced in the liver and kidney, exerts renotropic effects in proximal tubule cells (PTCs) and enhances Na+ reabsorption in in vitro and in vivo studies (Zumkeller & Schofield, 1992; Hammerman & Miller, 1993; Nobes et al. 1995; Feld & Hirschberg, 1996; Johnson et al. 1997b). A humoral role in the development of renal hypertrophy has been postulated since binding of IGF-I to PTCs is enhanced following reductions in renal mass (Polychronakos, Guyda & Posner, 1985; Hise, Lahn, Shao, Mantzouris & Fontana, 1993), endogenous renal cortical IGF-I accumulation precedes compensatory renal growth (Stiles, Sosenko, D'Ercole & Smith, 1985; Fagin & Melmed, 1987; Flyvbjerg, Thorlacius Ussing, Naeraa, Ingerslev & Orskov, 1988; Lajara et al. 1989; Zumkeller & Schofield, 1992; Feld & Hirschberg, 1996; Gronboek, Nielsen, Flyvbjerg & Orskov, 1997) and administration of a somatostatin analogue prevents compensatory renal growth and increased renal IGF-I content (Flyvbjerg, Frystyk, Thoracius Ussing & Orskov, 1989; Hammerman & Miller, 1993). Although serum IGF-I levels are reported to be either unchanged (Stiles et al. 1985; Fagin & Melmed, 1987; Lajara et al. 1989; Hammerman & Miller, 1993; Gronboek et al. 1997) or decreased (Flyvbjerg et al. 1988) and renal IGF-I mRNA levels are either unchanged (Lajara et al. 1989) or increased (Fagin & Melmed, 1987) following reductions in renal mass, the accumulation and enhanced action of IGF-I at the proximal tubule following uninephrectomy might be explained by alterations in circulating levels of IGF binding proteins (IGFBPs), which tightly regulate the delivery of serum IGF-I to tissues (Jones & Clemmons, 1995; Feld & Hirschberg, 1996). Circulating IGF-I may then be trapped in the proximal tubule by post-nephrectomy increases in IGF receptor number/affinity or in cell-associated IGFBPs, which potentiate IGF-I action on PTCs (Johnson et al. 1997c). However, the circulating levels of IGFBPs following unilateral nephrectomy and their effects on IGF-I binding to PTCs have not been studied to date. Therefore, the aim of the present study was to determine whether alterations in the IGF-I/IGFBP axis and its direct interaction with human PTCs account for the renotropic effect of serum following a reduction of renal mass.

1. We have used highly purified right-side-out luminal and basolateral membrane vesicles (LMVs and BLMVs) isolated from rat medullary thick ascending limb (MTAL) to study directly the possible roles of the LMV and BLMV Na(+)-H+ exchangers in the transport of NH4+. 2. Extravesicular NH4+ ((NH4+)o) inhibited outward H+ gradient-stimulated 22Na+ uptake in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pHi). 3. Conversely, in both plasma membrane preparations, the imposition of outward NH4+ gradients stimulated 22Na+ uptake at the acidic pHi (6.60) of MTAL cells, under conditions in which possible alterations in pHi were prevented. All NH4+ gradient-stimulated Na+ uptake was sensitive to 0.5 mM 5-(N,N-dimethyl)-amiloride. 4. The BLMV and LMV Na(+)-H+ exchangers had a similar apparent affinity for internal H+ (Hi+), with pK (-log of dissociation constant) values of 6.58 and 6.52, respectively. 5. These findings indicate that NH4+ interacts with the external and internal transport sites of the LMV and BLMV Na(+)-H+ antiporters, and that both of these exchangers can mediate the exchange of internal NH4+ ((NH4+)i) for external Na+ (Na+o) at the prevailing pHi of MTAL cells. 6. We conclude that operation of the BLMV Na(+)-H+ exchanger on the NH4(+)-Na+ mode may represent an important pathway for mediating the final step of NH4+ absorption, whereas transport of NH4+ on the apical antiporter may provide negative feedback regulation of NH4+ absorption.

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na+-translocating Escherichia coli/Propionigenium modestum ATPase hybrid with a Na+-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel becomes impermeable for Na+, N side Na+ ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na+ ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na+ ions on H+ transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22NaCl and ATP, 1 mol 22Na+/mol enzyme was occluded. With the parent hybrid ATPase, 22Na+ occlusion was not observed. The occluded 22Na+ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li+ but not Na+ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na+ ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na+, Li+, or H+-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.

A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na+ at elevated pH, was deficient in energy-dependent Na+ extrusion. The capacity of the mutant JC901 for Na(+)-dependent pH homeostasis was unaffected relative to the wild-type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3'-terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane-spanning regions at its C-terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP-binding proteins that are components of transport systems of the ATP-binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p-chlorophenylhydrazone (CCCP), and more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na(+)-sensitive phenotype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol-resistance does not result from NatAB-dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+ efflux, which was further stimulated by CCCP. In the absence of CCCP, NatAB-mediated Na+ efflux was stimulated by K+. Concomitant NatAB-dependent K+ uptake occurred, as monitored by 86Rb+ uptake; this uptake was inhibited by CCCP and is thus secondary to the primary, electrogenic Na+ efflux. A B. subtilis mutant strain (BsAJ96) in which most of natA and all of natB was replaced by a spectinomycin-resistance-gene cassette exhibited phenotypic properties identical to JC901 Under anaerobic conditions, using a strain of B. subtilis deleted in atp genes encoding the F1F0-ATPase (BD99-A), glucose energized Na+ exclusion in an arsenate-sensitive manner; this exclusion capacity was absent in a strain deleted both in atp and natAB genes (BsAJ96-A). We conclude that NatAB is an inducible, ABC transport system that catalyses ATP-dependent electrogenic Na+ extrusion without mechanistically coupled proton or K+ uptake. This is a novel mode of Na+ extrusion that is hypothesized to play an inducible role in exclusion of cytotoxic Na+ and in the secondary stimulation of K+ uptake, especially when the function of the membrane as an ion-permeability barrier is compromised by agents such as alcohols or uncouplers.

A quantitative in vivo method for obtaining maps of tissue sodium concentration (TSC) by MRI is compared to the invasive, global 22 Na radionuclide dilutional technique in the normal rat brain. The MR method uses a three-dimensional projectional acquisition scheme to minimize signal losses from transverse relaxation. Internal calibration standards are used to convert the signal intensity into TSC after correction for B 1 inhomogeneities by using the ratio of 23 Na and 1 H images obtained with identical B 1 distributions and sensitivities at the two frequencies. Over the biological range of concentrations, the TSC, measured as the ratio of MR signals of 23 Na and 1 H, gives a linear response with concentration. In the normal rat brain, the mean TSC measured using the MRI method (TSC = 45 ± 4 mM, animals = 5) is not significantly different from the global 22 Na radionuclide method (TSC = 49 ± 6 mM, animals = 7).

1. Capillary permeability-surface area (PS) products for the low molecular weight radioactive tracers, 22Na, 51Cr-EDTA (relative molecular mass 357) and 57Co-cyanocobalamin (relative molecular mass 1353) were measured in the fetal circulation of isolated dually perfused lobules of normal term human placentae using the single circulation, multiple-tracer dilution technique. 2. In lobules perfused with M199 medium, containing dextran and 5 g l-1 bovine albumin, the extractions of all three tracers decreased as the flow was increased over the range of 2-8 ml min-1, and PS products for 51Cr-EDTA and 57Co-cyanocobalamin, but not for 22Na, reached constant values at flows above 0.1 ml min-1 g-1. 3. Flow-independent PS products in the presence of albumin were 0.025 +/- 0.002 ml min-1 g-1 (mean +/- S.E.M., n = 25) for 57Co-cyanocobalamin and 0.057 +/- 0.003 ml min-1 g-1 (n = 25) for 51Cr-EDTA. The ratio of PS values (51Cr-EDTA/57Co-cyanocobalamin) was 2.28, while the ratio of the corresponding free diffusion coefficients was 1.79, indicating substantial restriction to the diffusion of the 57Co-cyanocobalamin. 4. In another series of lobules perfused in the absence of albumin, extraction values for all three test tracers were constant over the same flow range. Values at high flow rates were therefore about twice those measured in the presence of albumin, and PS products for all three tracers failed to reach diffusion-limited values. 6. Lobules perfused with and without albumin were fixed using a glutaraldehyde fixative containing 1% Alcian Blue dye. An ultrastructural examination of the endothelium showed no significant changes in cell or cleft morphology, or in the glycocalyx, in the absence of albumin which might account for the observed permeability change. 7. These data are the first physiological measurements specifically characterizing fetal microvascular permeability in the human placenta. The results suggest that permeability resembles that found in skeletal muscle and, as such, the endothelium presents a significant barrier to the diffusion of large solutes. The observed 'protein effect' indicates that albumin can interact with elements of the solute pathway to increase its restrictiveness.

The effects of forskolin, dibutyryl cyclic AMP, and 5′-N-ethylcarboxamide adenosine on specific 22Na uptake by synaptosomes stimulated by veratridine were investigated. All substances inhibited 22Na uptake, with forskolin more potent than 5′-N-ethylcarboxamide and this latter one more potent than dibutyryl cyclic AMP. In the absence of prein-cubation with forskolin, this substance caused little or no effect on 22Na uptake by synaptosomes. In the presence of the adenosine antagonist dipropylsulfophenylxanthine, the inhibitory effect of 5′-N-ethylcarboxamide adenosine on 22Na uptake was consistently antagonized. The results were interpreted as forskolin and 5′-N-ethylcarboxamide adenosine increasing cyclic AMP accumulation, and dibutyryl cyclic AMP mimicking it, and by these mechanisms decreasing sodium uptake through the sodium channels.

1. Na+ influx into cultured vascular smooth muscle cells (VSMC) obtained from spontaneously hypertensive rats (SHR) and from Wistar-Kyoto rats (WKY) was measured. Na+ influx via the Na+/H+ exchange system was measured as the rate of 22Na+ influx into cultured VSMC sensitive to ethylisopropylamiloride (EIPA), a specific inhibitor of the exchange system. 2. The total 22Na+ influx rate in SHR was significantly higher than in WKY (6.08 +/- 0.16 vs 4.13 +/- 0.09 nmol/min per mg protein; P less than 0.001; n = 14). The EIPA (1 X 10(-4) mol/L)-sensitive 22Na+ influx rate in SHR was significantly higher than that in WKY (4.32 +/- 0.27 vs 2.17 +/- 0.14 nmol/min per mg protein; P less than 0.001; n = 14). There was no difference in EIPA-insensitive 22Na+ influx between SHR and WKY. The EIPA-sensitive 22Na+ influx rate into VSMC was significantly decreased in SHR but not in WKY by the addition of 1 X 10(-4) mol/L 1-(5-isoquinoline-sulfonyl)-methylpiperazine (H-7), an inhibitor of protein kinase C (PK-C). 3. These results suggest that the increase in Na+ influx in SHR may be due to elevation of the Na+/H+ exchange activity, and possible involvement of PK-C in the increased Na+/H+ exchange activity in VSMC from SHR.

1. To explore its effect on blood pressure (BP) in relation to body sodium (Na), felodipine was given to New Zealand genetically hypertensive (GH) and normotensive (N) rats and to Japanese SHR and WKY rats, prepared for repeated measurements of body sodium (Na) by a whole body counting technique (22Na) using an Na-free pelleted diet and 22Na-NaCl fluid as the sole source of Na, with water also available. The drug was added to the food before pelleting pellets, 0.5 mg/g food. 2. Felodipine reduced BP in each strain; this effect was as great on normal Na intake as when on a zero Na intake. Felodipine caused an increase in plasma renin activity in each strain but the increase reached significant levels only in GH, SHR and WKY. Plasma aldosterone was decreased by treatment in GH and N and increased in SHR and WKY. 3. In rats on a zero Na intake (water sole drinking fluid), felodipine caused no fall in body Na (i.e. no natriuretic effect was detectable) except in WKY. When saline was freely available, felodipine tended to stimulate saline intake and was associated with an increase in body Na in all strains except SHR; body Na was high in SHR even in the absence of felodipine and did not increase further with felodipine treatment. Felodipine slightly speeded up the excretion of an Na load in rats on an ample NaCl intake (choice of 0.5% 22Na-NaCl and water) but this may have been due, in part, to the treated rats having taken, by choice, a greater intake of saline in the pre-load period. 4. In this study, felodipine reduced BP and stimulated PRA and salt appetite. Its effects on Na handling varied to some extent between the strains; there was no consistent natriuretic effect, but rather some evidence of Na retention.

1. The diffusional permeability of collecting ducts to 22Na+ and 36Cl- was measured in rat papillae in vitro. 2. The permeability of the collecting duct to 36Cl- was 0.72 (s.e.m. = 0.01; n = 356) microns/sec which was significantly higher than the value of 0.51 (s.e.m. = 0.01; n = 356) microns/sec measured for 22Na+. 3. Collecting ducts in papillae taken from rats on a high sodium intake had a 22Na+ permeability of 0.63 (s.e.m. = 0.04; n = 53) microns/sec which was significantly higher than the value on a normal salt intake (0.50, s.e.m. = 0.04; n = 46 microns/sec). 4. When papillae from normal rats were studied in plasma taken from salt loaded rats, the 22Na+ permeability of 0.59 (s.e.m. = 0.04; n = 18) microns/sec was significantly higher than when incubated in plasma from normal rats (0.44, s.e.m. = 0.05; n = 12) microns/sec. 5. An extract of urine with natriuretic activity had no effect on 22Na+ permeability when tested in this system. 6. Adrenalectomy, PGE2, indomethacin and antidiuretic hormone had no significant effect on 22Na+ and 36Cl- permeability. 7. A substance exists in plasma from salt loaded animals that increases the permeability of collecting ducts to sodium. This effect could explain the component of the natriuresis that follows saline infusion which is independent of changes in glomerular filtration rate, aldosterone, or proximal tubule reabsorption.

1. The action of four volatile anaesthetics, ethrane, halothane, isoflurane and methoxyflurane on stimulus-secretion coupling has been studied in isolated bovine adrenal medullary cells. All four agents inhibited the secretion of adrenaline and noradrenaline evoked by 500 microM carbachol at concentrations within the anaesthetic range. Total catecholamine secretion induced by stimulation with 77 mM potassium was also inhibited but at higher concentrations. All four agents inhibited the 45Ca influx evoked by stimulation with 500 microM carbachol and the 45Ca influx in response to K+-depolarization. 2. When total catecholamine secretion in response to potassium or carbachol was modulated by varying extracellular calcium or by adding halothane or methoxyflurane to the incubation medium, the amount of catecholamine secretion for a given Ca2+ entry was the same. 3. The action of methoxyflurane on the relationship between intracellular free Ca and exocytosis was examined using electropermeabilised cells, which were suspended in solutions containing a range of concentrations of ionised calcium between 10(-8) and 10(-4)M. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. 4. Halothane and methoxyflurane inhibited the carbachol-induced secretion of catecholamines in a non-competitive manner. 5. Halothane and methoxyflurane inhibited the increase in 22Na influx evoked by carbachol. For halothane and methoxyflurane this inhibition of Na influx appears to be sufficient to account for the inhibition of the evoked catecholamine secretion. 6. We conclude that the volatile anaesthetics ethrane, halothane, isoflurane and methoxyflurane inhibit the secretion of adrenaline and noradrenaline induced by carbachol at concentrations that lie within the range encountered during general anaesthesia. In addition all four also inhibit the secretion of catecholamines induced by depolarization with 77 mM K+ but at much higher concentrations. The decrease in Ca influx caused by methoxyflurane accounts fully for the decrease in secretion in response to depolarization with potassium. Similar actions at synapses within the CNS may underlie the general anaesthetic effects of these agents.

Sprague-Dawley rats were given treatments, known to decrease 22Na movement into choroid plexus and CSF, to investigate their effect on 22Na transfer across the cerebral capillaries. Acidic salts, acetazolamide, or amiloride was injected intraperitoneally into bilaterally nephrectomized rats, and the rate of 22Na uptake into parietal cortex, pons-medulla, and CSF was determined at 12, 18, and 24 min. Severe acidosis (arterial pH 7.2), produced by HCl injection, decreased the rate of 22Na entry into both brain regions and CSF by 25%, whereas mild acidosis (pH 7.3) from NH4Cl injection reduced brain entry by 18%, but CSF entry by only 10%. Like HCl acidosis, amiloride reduced transport into both brain and CSF by 22%. Penetration of 22Na into parietal cortex was unchanged by acetazolamide, but that into CSF was slowed 30%. Since uptake of 22Na into cortical regions is primarily movement of tracer across the cerebral capillaries when tracer uptake time is less than 30 min, the results indicate that both metabolic acidosis and amiloride decrease Na+ permeativity at the cerebral capillaries as well as at the choroid plexus. Acetazolamide, on the other hand, alters Na+ movement only across the choroidal epithelium.

A {sup 23}Na NMR assay for measurement of erythrocyte Na+/K+ ATPase activity is presented. Using the nonpermeant shift reagent dysprosium tripolyphosphate the signals of intra- and extracellular sodium are separated, enabling measurement of sodium fluxes nondestructively, without the need to physically separate the cells from their environment. By increasing membrane permeability with nystatin we have shown that the assay allows the detection of differences in membrane permeability. With low doses of nystatin the ouabain-sensitive sodium flux increased more than twofold. With high doses of nystatin the Na+/K+ pump could not prevent an almost total equilibration of intra- and extracellular sodium. All sodium that entered the cells remained NMR visible, proving that sodium influx can be measured quantitatively. {sup 31}P NMR spectra taken before and after the assay revealed a slight acidification of the cells and no significant change in ATP concentration. No evidence of Dy3+ entering the cell was observed.

A new method is described for the quantification of leakage between etched enamel and restorative resin. A significant correlation was demonstrated between the plane of enamel section prior to etching and subsequent leakage. It is suggested that these findings will be of value in the design of cavities for use with the acid-etch technique; when possible, enamel should be prepared to expose prisms transversely prior to etching and bonding.

We have studied the in vitro short-term effects of aldosterone (1.0-1000 nmol l-1), cortisol (0.5-5.0 mumol l-1), fludrocortisone (1.0-10 nmol l-1) and carbenoxolone (0.5-3 mmol l-1) on 86rubidium influx (a model for potassium), 22sodium efflux, and [3H]-ouabain binding capacity in intact human leucocytes. No effect of aldosterone (at concentrations present in Conn's syndrome) or fludrocortisone could be demonstrated on cation fluxes or [3H]-ouabain binding compared to controls. No significant effect of cortisol, at concentrations either physiological or present in Cushing's syndrome, could be demonstrated on cation fluxes or [3H]-ouabain binding compared to controls. Carbenoxolone significantly increased 86Rb influx and 22Na efflux at concentrations known to cause hypokalaemia in man. The effect was not blocked by propranolol. No effect could be demonstrated for [3H]-ouabain binding.

Transcapillary movements of 22Na, 51Cr-EDTA and 125I-myoglobin were studied in the cat choroid by means of the single injection technique, using labelled albumin as the reference tracer. By an external shunting of blood, recirculation of tracer was delayed and complete venous outflow curves became available for analysis. The initial extractions observed were 0.66, 0.35 and 0.08 respectively. The extractions decreased with time. Within 20--25 s the extracted fractions of the tracers had almost completely returned to the blood. The results indicate high permeability coefficients and small extravascular volumes of distribution. The retinal pigment epithelium seems to have a very low permeability to all three substances. Extraction of tritiated water at 20--25 s was about 0.30 probably due to rapid penetration of labelled water into the retina. Evidence for a stereo-specific transport system for glucose in the retinal pigment epithelium was found in experiments with labelled D-glucose and 3-O-methyl-D-glucose. The total amount of glucose delivered by the choroid was calculated from measurements of the a-v differences for unlabelled glucose.