Clinical significance of sodium-dependent lithium transport in affective psychoses

The steady state red blood cell/plasma lithium (Li) ratios were determined simultaneously with the in vitro rates of sodium-dependent Li transport of erythrocytes during prophylactic Li therapy in 30 unipolar, 52 bipolar forms of manic-depressive patients and in 58 nonmanic-depressive psychiatric patients as a control group. A reciprocal correlation was found between the Li ratios and the values of Na-dependent Li transport. These transport rates were in positive correlation with the steady state Li contents of red blood cells and that of the plasma. Significant differences were revealed between the different nosologic subgroups in the respect of in vitro rates of Na-dependent Li countertransport. Results are discussed in the light of clinical and pharmacogenetic aspects.     

Cadmium binding and sodium-dependent solute transport in renal brush-border membrane vesicles

Exposure to cadmium (Cd) impairs renal transport systems for glucose, amino acids, phosphate, and dicarboxylates. To investigate if these changes are directly related to a Cd binding to the renal brush-border membrane, Cd binding and the Na+-dependent uptakes of d-glucose, l-alanine, phosphate, and succinate were determined in rat renal brush-border membrane vesicles (BBMV) exposed to CdCl2. Cd uptake by BBMV showed time and concentration dependence. Changes in medium osmolality had no effect on Cd uptake, indicating that the process primarily involves binding of Cd to the membrane. Scatchard analysis indicated the presence of two types of Cd binding sites, differing in affinity and number. Increasing the medium Cd concentration from 50 to 200 microM resulted in a progressive increase in Cd binding to the membrane and decrease in Na+-dependent transport of d-glucose, l-alanine, inorganic phosphate, and succinate. In all cases, the inhibition of transport was directly proportional to the total amount of Cd binding to the membrane. These results suggest that, during chronic exposure to Cd, free Cd ions liberated in renal tubular cells may directly interact with brush-border membranes and impair Na+-dependent solute transports.     

Heterogeneous distribution of the sodium-dependent alanine transport activity in the rat hepatocyte plasma membrane

The localization of the sodium-dependent alanine uptake activity in rat liver cells was studied. Fractions representative of the canalicular, the contiguous (lateral) and the blood-sinusoidal surface of the hepatocyte were isolated by means of centrifugal fractionation and density gradient centrifugation. The distribution of various marker-enzyme activities in conjunction with the occurrence of alanine transport activity was studied both in fractions obtained after zonal density gradient centrifugation, and in the subcellular fractions mentioned above. It is concluded that the sodium-dependent alanine transport activity is primarily located in the blood-sinusoidal plasma membrane of the hepatocyte.     

Subcellular distribution of rat brain cortex high-affinity, sodium-dependent, glycine transport sites

The subcellular distribution of the membrane components, present in rat brain cortex homogenates, that interact with glycine in the presence of sodium ions was studied. The distribution in the primary fractions, as per cent of total binding in the homogenate, was: P1 ('nuclear'), 58%; P2 (large granule), 39%; P3 (microsomal), 2%9 Of the subfractions obtained by centrifuging P1 in a linear 0.32--1.5 M sucrose gradient, only the lighter fraction (P1-III) formed by large myelin fragments was enriched in specific binding activity with respect to P1. The pellet formed by purified nuclei had negligible binding, and fractions of intermediate density had a lower activity than P1. Transient exposure of P1-III to 1.5 M sucrose did not diminish its binding ability. Similarly, in the subfractions obtained by centrifuging P1 in a discontinuous sucrose gradient, only the least dense one, P1-A, that is formed exclusively by large myelin fragments, was enriched with respect to P1. The electron microscopy of these fractions is presented. The P2 subfractions, obtained in a linear 2--18% Ficoll gradient, had the following sodium-dependent activity (counts/min/mg protein, fractions being in the order of decreasing density): pellet, 0; P2-I, O; P2-II, 450; P2-III, 1770; P2-IV, 4130; unfractionated P2, 880; P2-IV, the least dense fraction being composed mainly of myelin. With P2 subfractions obtained in a discontinuous sucrose gradient (0.32, 0.8 and 1.2 M sucrose layers), it was also found that sodium-dependent glycine binding was only enriched, with respect to P2, in the myelin fraction P2-A. Glycine binding to purified brain cortex myelin was also found to be very high, while binding to non-myelin membranes, obtained during the purification procedure, was only 0--7% of that seen with myelin. These results suggest that high-affinity glycine binding is located in myelin proper, and possibly also in some other glial plasma membranes, but not in nuclei, mitochondria, endoplasmic reticulum or synaptosomes. The relevance of these findings for interpreting previous reports on high-affinity glycine transport in the central nervous system is analyzed.     

Regulation of intestinal sugar transport

The recent surge in knowledge of cellular and molecular mechanisms of intestinal sugar transport has fueled an enormous interest in adaptive mechanisms regulating sugar transport. We first review several functional considerations that help us interpret the different patterns of adaptation for different nutrients. We then distinguish nonspecific adaptive mechanisms leading to parallel changes in transport of different nutrients from specific adaptive mechanisms only affecting the transport of a single nutrient. Nonspecific adaptive mechanisms include changes in mucosal surface area and in the ratio of transporting to nontransporting cells; specific mechanisms include changes in site density of transporters and in affinity constants. We also enumerate the patterns of regulation and describe how sugar transport is affected by changes in diet, energy budgets, and environmental salinity as well as by intestinal resection, starvation, stress, and age. We relate the various signals linking these stimuli to adaptive mechanisms and make predictions about the nature of these signals. Finally, we describe the significance of the interactions among sugar, fluid, and electrolyte transport mechanisms and of the paracellular pathway to transepithelial transport of sugars. We close by drawing attention to promising directions for future research.     

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.     

Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands

The extracellular concentration of inorganic phosphate (Pi) is an important determinant of parathyroid cell function. The effects of Pi may be mediated through specific molecules in the parathyroid cell membrane, one candidate molecule for which would be a Na+-dependent Pi cotransporter. A complementary DNA encoding a Na+-Pi cotransporter, termed rat PiT-1, has now been isolated from rat parathyroid. The 2890-bp complementary DNA encodes a protein of 681 amino acids that shows sequence identities of 97% and 93% with the type III Na+-Pi cotransporters mouse PiT-1 and human PiT-1, respectively. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and is most abundant in brain, bone, and small intestine. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.     

Identification of a membrane protein involved in mitochondrial phosphate transport

A specific labeling by radioactive N-ethylmaleimide of a protein involved in phosphate transport was obtained by protecting one of the two-SH-groups of the transport system with low concentrations of mersalyl. Subsequently, the other free-SH groups were blocked with excess N-ethylmaleimide. Removal of mersalyl by cysteine and subsequent inbucation with labeled N-ethylmaleimide results in a "specific" binding of N-ethylmaleimide to one-SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled mitochondria was subjected to dodecylsulfate gel electrophoresis. The followin results were obtained. 1. The difference of the radioactivity pattern on the dodecylsulfate-polyacrylamide gel of inner membrane proteins, labeled with N-[14C]ethylmaleimide in the absence and with N-[3H-A1-ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only one main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32 degrees C and at 0 degrees C. 2. The position of the labeled peak on the dodecylsulfate-polyacrylamide gel corresponds to a protein of molecular weight of 26500 +/- 800. 3. The amount of one of the two-SH groups, involved in phosphate transport, was estimated to be 30 nmol per g of mitochondrial protein.     

Expression of sodium-dependent purine nucleoside carrier (SPNT) mRNA correlates with nucleoside transport activity in rat liver

Co-ligation of antigen receptor and complement receptor 2 (CD21) in the B cell membrane is important in the immune response to T-dependent antigens. Four CD21 ligands have so far been identified, but only the activated products of the third component of complement (C3) are known to augment the immune response to specific antigens. The most recently discovered ligand for CD21 is CD23. We have generated a CD32+ CD23+ fibroblast cell line which presents a surrogate antigen (anti-IgM) to human tonsil B cells in vitro. Incubation with these cells causes a 10- to 100-fold reduction in the threshold concentration of anti-IgM required for B cell proliferation. Anti-CD19 further enhances the response to antigen and induces proliferation in the absence of anti-IgM. Addition of soluble CD21 totally inhibits the effect of CD23, suggesting that CD21 mediates synergistic signaling by CD23.     

Characterization of two phosphate transport systems in Acinetobacter johnsonii 210A

The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.     

Regulation of intestinal nutrient transport is impaired in aged mice

To determine the effect of age on the regulation of intestinal nutrient absorption, we fed young (7.6-mo-old) and aged (24.8-mo-old) C57BL mice diets designed to stimulate in vitro sugar or amino acid uptake in the isolated small intestine. In each age group, diet had no effect on feeding rates and body weights. D-Glucose and D-fructose uptakes by the small intestine each increased by about two times in young and 1.5 times in aged mice fed high carbohydrate diets as compared with those fed no carbohydrate. Adaptive increases in uptake by the aged group were not only reduced but also restricted to more proximal regions of the small intestine. In both age groups, diet-stimulated increases in D-glucose transport were accompanied by parallel increases in number of Na(+)-D-glucose cotransporters as estimated by specific phlorizin binding. Diet had no effect on transporter Kd for phlorizin, turnover rate of each transporter, mucosal mass or mucosal permeability. A high protein diet stimulated the uptake of L-aspartate and L-proline in young mice and of only L-aspartate in aged mice. Uptake of essential amino acids and of nonessential amino acids sharing transporters with essential ones were not regulated. Although aged mice possess adaptive mechanisms to diet that are similar to those in young mice, the effectiveness of these mechanisms may be impaired with age and may result in malabsorption symptoms so prevalent in the elderly.     

Fimbrin in podosomes of monocyte-derived osteoclasts

Fimbrin, an actin-bundling protein, is a component of the osteoclast adhesion complexes called podosomes. In this study, we (1) determined the localization of fimbrin in the mature rabbit osteoclast as well as in differentiating osteoclasts using the avian monocyte-derived osteoclast differentiation model, (2) characterized the distribution and accumulation of three fimbrin isotypes (T, L, and I) in avian monocytes as they fused to form multinucleate osteoclast-like cells, and (3) report for the first time, a close spatial relationship between podosomes and microtubules using fimbrin as a marker of the podosome. Immunofluorescence using anti-T-fimbrin, anti-L-fimbrin, and pan-isotype-anti-fimbrin antibodies, showed that fimbrin is an integral component of the podosome core in the mature rabbit osteoclast and in the monocyte-derived osteoclast throughout differentiation. Anti-I-fimbrin, however, did not show immunoreactivity in these cultures. These studies also show that in the avian model of monocyte-derived osteoclast differentiation, day 2 cells (D2) are predominantly mononucleate and have few podosomes. By days 4 and 6 in culture (D4 and D6), many cells have fused and punctate rows of podosomes are commonly observed at cell margins. Analysis by Western blot of protein accumulation showed that after an initial small rise from D2 to D4, L-fimbrin levels remained relatively constant from D4 to D6. However, T-fimbrin protein levels increase steadily from D2 to D6, suggesting that it may be related to the increase in podosome formation as monocytes fuse to form osteoclasts. Finally, we examined the distribution of podosomes relative to other cytoskeletal elements such as microtubules and intermediate filaments. Double immunofluorescence labeling using anti-fimbrin and anti-tubulin showed podosomes lying adjacent to microtubules at cell margins. When osteoclasts were treated with nocodazole (1 X 10(-6) M) to disrupt microtubules, the distribution of podosomes became more random and was no longer confined to the cell periphery. These results suggest that microtubule-podosome interactions may play a role in osteoclast adhesion.     

Upregulation of c-fos protooncogene expression in pagetic osteoclasts

Using in situ hybridization we show that expression of the c-fos oncogene, a gene normally associated with osteosarcomas, is greatly elevated in osteoclasts of patients with Paget's disease. Immunohistochemical staining with c-fos antibodies also shows increased protein in pagetic osteoclasts. In light of transgenic mouse experiments showing a key role for c-fos in bone resorption, we propose that elevated c-fos gene expression in pagetic osteoclasts is an important component in producing the pagetic phenotype. Levels of c-fos gene and protein expression in pagetic osteoblasts are lower than those detected in osteoclasts but still higher than in nonpagetic osteoblasts. This may provide an explanation for the increased incidence of osteosarcomas in patients with Paget's disease because overexpression of c-fos in osteoblasts of transgenic mice induces osteosarcoma formation.