Choline uptake across the ventricular membrane of neonate rat choroid plexus.

The uptake of [3H]choline from the cerebrospinal fluid (CSF) side of the rat neonatal choroid plexus was characterized in primary cultures of the choroidal epithelium grown on solid supports. Cell-to-medium concentration ratios were approximately 5 at 1 min and as high as 70 at 30 min. Apical choline uptake was facilitated; the Km was approximately 50 microM. Several organic cations (e.g., hemicholinium-3 and N1-methylnicotinamide) inhibited uptake. The reduction or removal of external Na+ or the addition of 5 mM LiCl had no effect on uptake. However, increasing external K+ concentration from 3 to 30 mM depolarized ventricular membrane potential (-70 to -15 mV) and reduced uptake to 45% of that for the control. Treatment with 1 mM ouabain or 2 mM BaCl2 reduced uptake 45%, and intracellular acidification reduced uptake to approximately 90% of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled to Na+ influx but is sensitive to plasma membrane electrical potential.

Functional characterization of choroid plexus epithelial cells in primary culture.

The objective of this study was to develop and evaluate a primary culture system for choroid plexus epithelial cells as an in vitro model for studying organic cation transport. Cells were dispersed from choroid plexus of neonatal rats by enzymatic digestion and grew as differentiated monolayers when plated on solid or permeable support. Electron microscopy showed that cultured cells were morphologically similar to intact choroid plexus epithelium, having apical tight junctions between cells, numerous mitochondria, basal nuclei and apical microvilli and cilia. As previously demonstrated for intact choroid plexus, immunocytochemistry showed that Na+,K+-ATPase was localized to the apical membrane, and GLUT-1, the facilitative glucose transporter, was localized to the basolateral membrane of cultured cells. Apical transport of L-proline by cultured cells was mediated by a sodium-dependent, electrogenic process, as in whole tissue. 14C-Tetraethylammonium (TEA), a prototypic organic cation, was accumulated by isolated choroid plexus in a time-dependent manner; uptake was inhibited by tetrapentylammonium (TePA). In cultured cells, apical TEA transport was mediated by a saturable process coupled to cellular metabolism. Unlabeled TEA and other organic cations (TePA, N1-methylnicotinamide and mepiperphenidol) inhibited TEA transport; the organic anion, p-aminohippurate, had no effect. Finally, TePA-sensitive transport of 14C-TEA was stimulated after preloading the cells with unlabeled TEA. Based on the morphological, biochemical and functional properties of these cultured cells, we conclude that this primary culture system should be an excellent in vitro model for experimental characterization of choroid plexus function.

Hydrogen peroxide stimulates sodium-potassium pump activity in cultured pulmonary arterial endothelial cells.

Oxidant injury to pulmonary vascular endothelium is an important factor in the pathogenesis of acute lung injury. Oxidant injury to other cell types has been reported to alter the function of Na-K-adenosinetriphophatase (ATPase) an enzyme important in maintenance of cellular ionic homeostasis and in transport of ions across biological membranes. We investigated the effect of H2O2 (0.001-10 mM) or xanthine (X) (15.2 micrograms/ml) plus xanthine oxidase (XO) (0.0153 U/ml) on the Na-K pump activity of cultured bovine pulmonary arterial endothelial cells (PAECs). We used a functional assay, using 86RbCl as a tracer for K+ and expressing Na-K pump activity as ouabain-inhibitable K+ uptake. Our results demonstrate that H2O2 and X/XO stimulate Na-K pump activity of bovine PAECs, an effect prevented by catalase. In addition, we assessed the affinity, number, and turnover of [3H]ouabain binding sites on intact endothelial monolayers and found that H2O2 increased affinity to [3H]ouabain, decreased the number of binding sites, and increased the rate of pump turnover. Influx of 22Na increased in response to a nonlytic concentration of H2O2. Cell injury, as assessed by 51Cr release, adherent cell number, and phase-microscopic morphology, was not observed after 30-min incubations with the lowest dose (1 mM) of H2O2 effective in stimulating Na-K pump activity, or after incubation with X/XO. Na-K pump inhibition by ouabain significantly increased the 51Cr release caused by H2O2 or by X/XO, suggesting that the increase in Na-K pump activity may be a compensatory response to the cellular alterations produced by H2O2. Incubation with H2O2 decreased cell ATP content, an effect which was not prevented by coincubation with ouabain. In summary, these results show that H2O2 increases Na-K pump activity of PAECs, an effect mediated, at least in part, by increased intracellular [Na] and by an increased rate of pump turnover. It is possible that the increased pump activity may be an early marker of endothelial cell perturbation.

Immunofluorescent cytometry and electron microscopic immunolocalization of insulin-like growth factor (IGF)-II receptors in infant rat choroid plexus.

The choroid plexus contains receptors for insulin-like growth factor (IGF)-II, it synthesizes IGF-II, and it secretes soluble IGF binding proteins with high affinity for IGF-II. Although the actions of IGFs in the choroid plexus are unknown, IGFs may participate in CNS growth and differentiation, CNS injury/repair mechanisms, regulation of satiety, and growth hormone secretion. Because the choroid plexus is a heterogeneous tissue containing several cell types, to study the role of IGF-II in the choroid plexus we need to know precisely which cells contain IGF-II receptors. Therefore, we studied the distribution of IGF-II receptors in infant rat choroid plexus by electron microscopic immunohistochemistry and immunofluorescent cytometry of dispersed choroid plexus cells using a well-characterized, highly specific rabbit anti-rat IGF-II receptor antibody. These studies show that IGF-II receptors are detectable in large quantities in both choroid plexus epithelial cells and in the fenestrated capillary endothelial cells. Results were confirmed by immunofluorescent cell sorting using the anti-IGF-II receptor antiserum. Other cell types in the choroid plexus contained lower levels of immunoreactivity, including red blood cells. Physiological studies of IGF-II effects on intact choroid plexus may, therefore, reflect effects on both of these cell populations. These data provide a compelling rationale for the development of choroid plexus epithelial cell lines or pure culture techniques to study the physiology of IGF-II in individual cell types.

Response of infant and adult rat choroid plexus potassium transporters to increased extracellular potassium.

The choroid plexus (CP) has an important role in regulating ion concentrations in the cerebrospinal fluid (CSF). Maintenance of potassium concentration [( K]) in brain extracellular fluids lower than in serum is critical to proper neuronal function. We studied K (86Rb) transport by the isolated, lateral CP from infant and mature rats to determine the nature and development of CSF [K] regulation. Comparison of transport characteristics in media with 3-11 mM [K] showed that the adult CP increased uptake in a stepwise fashion with each 2 mM increase in [K], up to a 90% increase with 9 mM [K] over control (3 mM [K]). In contrast, 3-day CP increased uptake in 5 mM, but could not increase K uptake further with higher [K]. One-week tissues showed an intermediate response. Kinetic analysis of the ouabain-inhibitable component suggested that the immature tissues may express a different isoform of the catalytic (alpha) subunit of the Na,K-ATPase. These data may explain the previously established inability of neonatal rats to regulate CSF [K] when serum [K] is elevated, and they indicate that active K transport by the CP cells plays an integral role in CSF [K] regulation.

Potassium cotransport with sodium and chloride in the choroid plexus.

The effects of loop diuretics and ion substitution on the 2-min uptake of K (86Rb as marker) were analyzed to obtain evidence for K cotransport with Na and Cl in the choroid plexus epithelium. The isolated plexuses, which were excised from lateral ventricles of adult rats, were bathed in artificial cerebrospinal fluid (aCSF). At concentrations of 10(-6) to 10(-4) M, the specific cotransport inhibitors, bumetanide and piretanide, suppressed uptake of K in a dose-dependent manner. Ouabain-insensitive K uptake was stimulated by preincubating the choroid plexus in aCSF very low in [Na] and [K], then incubating it in much higher concentrations of these cations; bumetanide (10(-4)M) blocked this stimulated uptake by 52%. Moreover, tissue preincubation in Na- or Cl-free medium, followed by incubation with normal concentrations of both ions, stimulated the ouabain-insensitive uptake of K from 15 (baseline) to 35 nmol/mg dry weight. This stimulation of K transport depended on the simultaneous presence of both Na and Cl in aCSF, and replacing either ion alone did not stimulate the ouabain-insensitive K uptake. Collectively, these findings, together with those from a previous pharmacological study of 22Na and 36Cl transport, constitute strong evidence for the cotransport of Na, K, and Cl in rat choroid plexus.

Expression of insulin and insulin-like growth factor receptors and binding proteins by retinal pigment epithelium.

Insulin-like growth factors (IGFs) I and II are mitogenic polypeptides structurally homologous to insulin, which are thought to mediate important neurobiologic actions in the CNS. The purpose of this study was to determine if cultured bovine retinal pigment epithelial cells (RPE) express IGF receptors and secrete soluble IGF binding proteins, and to characterize these receptors and binding proteins. We also characterized the soluble IGF binding proteins present in juvenile and adult rat vitreous and serum, as well as those in fetal bovine vitreous and serum, in order to facilitate identification of the RPE IGF binding protein, and to determine potential destinations for this protein once produced. Affinity labeling was used to characterize insulin, IGF-I and IGF-II receptors. Western radioligand blotting and immunoprecipitation were used to characterize IGF binding proteins. We found that RPE cells in culture express virtually no insulin receptors, and only modest amounts of IGF-I receptors. IGF-II receptors were abundantly expressed. Additionally, RPE cells secrete a soluble IGF binding protein which is immunologically related to IGFBP-2, the primary IGF binding protein produced in the central nervous system. Bovine vitreous was found to contain a mixture of IGF binding proteins (IGFBP). The most prominent IGFBP in this mixture is immunologically related to IGFBP-2. Likewise, juvenile and adult rat vitreous contained only one IGF binding protein that was shown to be immunologically related to IGFBP-2. Juvenile rat vitreous contained more binding activity corresponding to IGFBP-2 than did adult vitreous, suggesting developmental regulation. These data suggest that IGF's and their binding proteins may have important, and as yet undefined, roles in retinal neurophysiology.

Characterization of insulin-like growth factor binding proteins produced in the rat central nervous system.

Insulin-like growth factor binding proteins (IGFBP) are thought to modulate the biological actions of the insulin-like growth factors (IGF), including possible regulatory roles in the growth and differentiation of the central nervous system. Extracellular fluids usually contain a mixture of IGFBPs, three of which have been cloned, sequenced, and designated IGFBP-1, -2, and -3. We used Western ligand blotting, immunoprecipitation, and competitive binding analysis to characterize IGFBPs found in fetal and adult rat cerebrospinal fluid (CSF) and IGFBPs produced by cultures of neonatal rat choroid plexus, astrocytes, and C6 glial cells. Pooled rat CSF contains primarily IGFBP-2 (a narrow band at Mr = 29,000), lesser quantities of IGFBP-3 (a multicomponent broad band at Mr = 37,500-43,000), and trace amounts of low mol wt IGFBPs. Conditioned medium from cultures of choroid plexus cells contained a single binding protein corresponding to IGFBP-2, whereas C6 cells made predominately an IGFBP corresponding to IGFBP-3. Astrocytes secreted two IGFBPs corresponding to IGFBP-2 and -3, primarily IGFBP-3. Neonatal CSF contained substantially more binding activity corresponding to IGFBP-2 than did adult CSF. In all samples showing Western ligand binding profiles corresponding to IGFBP-2, identification was established by immunoprecipitation. Competitive binding analysis performed on choroid plexus IGFBP showed preferential high affinity binding for IGF-II compared with that for IGF-I. In conclusion, CSF contains a mixture of distinct IGFBPs, primarily IGFBP-2. The other IGFBPs found in CSF are capable of being synthesized locally within the central nervous system by glial cells and neurons, suggesting that they are not derived from plasma by transport across the blood-brain barrier.

Volume regulation in lens epithelial cells and differentiating lens fiber cells.

Previous studies from our laboratory have led us to conclude that lens cell elongation is caused by an increase in cell volume. This volume increase results from an increase in the potassium content of the cells due to decreased potassium efflux. In contrast, an increase in the volume of most cells triggers a regulatory volume decrease (RVD) that is usually mediated by increased potassium efflux. For this reason, chicken embryo lens epithelial cells were tested to see whether they were capable of typical cell volume regulation. Changes in cell volume during lens fiber differentiation were first estimated by 3H2O water uptake. Cell water increased in proportion to cell length in elongating lens cells. Treatment of epithelial cells cultured in basal medium with dilute or concentrated medium, or with medium containing 50 mM sucrose, resulted in typical volume regulatory responses. Cells lost or gained volume in response to osmotic stress, then returned to their previous volume. In addition, the elongation and increase in cell volume that accompanies lens fiber cell differentiation occurred normally in either hypo- or hypertonic media. This observation showed that the activation of mechanisms to compensate for osmotic stress did not interfere with the increase in volume that accompanies elongation. The ability of elongating cells to volume regulate was also tested. Lens epithelial cells were stimulated to elongate by exposure to embryonic vitreous humor, then challenged with hypotonic medium. These elongating cells regulated their volume as effectively as unstimulated cells. Therefore, cells that were increasing their volume due to reduced potassium efflux could adjust their volume in response to osmotic stress, presumably by increasing potassium efflux. This suggests that the changes in potassium efflux that occur during differentiation and RVD are regulated by distinct mechanisms.

Cotransport of sodium and chloride by the adult mammalian choroid plexus.

Cerebrospinal fluid formation stems primarily from the transport of Na and Cl in choroid plexus (CP). To characterize properties and modulation of choroidal transporters, we tested diuretics and other agents for ability to alter ion transport in vitro. Adult Sprague-Dawley rats were the source of CPs preincubated with drug for 20 min and then transferred to cerebrospinal fluid (CSF) medium containing 22Na or 36Cl with [3H]mannitol (extracellular correction). Complete base-line curves were established for cellular uptake of Na and Cl at 37 degrees C. The half-maximal uptake occurred at 12 s, so it was used to assess drug effects on rate of transport (nmol Na or Cl/mg CP). Bumetanide (10(-5) and 10(-4) M) decreased uptake of Na and Cl with maximal inhibition (up to 45%) at 10(-5) M. Another cotransport inhibitor, furosemide (10(-4) M), reduced transport of Na by 25% and Cl by 33%. However, acetazolamide (10(-4) M) and atriopeptin III (10(-7) M) significantly lowered uptake of Na (but not Cl), suggesting effect(s) other than on cotransport. The disulfonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 10(-4) M), known to inhibit Cl-HCO3 exchange, substantially reduced the transport of 36Cl. Bumetanide plus DIDS (both 10(-4) M) caused additive inhibition of 90% of Cl uptake, which provides strong evidence for the existence of both cotransport and antiport Cl carriers. Overall, this in vitro analysis, uncomplicated by variables of blood flow and neural tone, indicates the presence in rat CP of the cotransport of Na and Cl in addition to the established Na-H and Cl-HCO3 exchangers.

Development of potassium transport capability by choroid plexus of infant rats.

Developmental maturation of K transport and Na+-K+-adenosinetriphosphatase (ATPase) function in the choroid plexus (CP) was investigated in infant rats. K fluxes (86Rb), Na+-K+-ATPase enzyme activities, and [3H]ouabain binding were measured in isolated, lateral CPs removed from anesthetized Sprague-Dawley rats of several ages ranging from 1 day to 6 wk. Morphological integrity of incubated tissue was confirmed by electron microscopy. CP epithelia accumulated K at a lower level during the 1st wk after birth (40 nmol/mg wet wt) compared with CP from rats aged 11-28 days (56 nmol/mg wet wt). However, efflux curves and efflux rate coefficients among the age groups were similar. Both Na+-K+-ATPase activity in CP tissue homogenates and [3H]ouabain binding to isolated CP began to increase between age 10 and 14 days. It is concluded that CP Na+-K+-ATPase has limited capacity in the lateral CP of neonatal rats but that this important transport function matures rapidly in the 1st 2-3 wk after birth.

Decreased membrane permeability to potassium is responsible for the cell volume increase that drives lens fiber cell elongation.

Differentiation of lens epithelial cells into fiber cells involves an increase in cell volume which previously was proposed to be the direct cause of the extensive cell elongation which accompanies fiber formation. In this study we have continued to investigate the mechanism underlying cell elongation by defining the minimum nutrient and ion requirements of elongating cells, measuring potassium and sodium fluxes in stimulated and unstimulated lens epithelia, and determining the effects of several pharmacological agents on elongation and ion transport. We have shown that elongation will occur in a basic salt/glucose solution with Insulin-like growth factor I/somatomedin-C stimulation. Neither sodium nor any metabolite appears to be the osmotically active species which drives the increase in cell volume. However, potassium efflux rate coefficient was 47% lower in differentiating cells than in unstimulated cells, whereas potassium uptake rates and ouabain effects were similar. Cells did not elongate in potassium-free medium nor in the presence of several drugs which prevent the accumulation of intracellular potassium or hinder osmotic water flux. Unstimulated cells elongated, however, with the application of quinidine, a drug known to block potassium channels. We propose that stimulation of lens epithelial cells with an insulin-like growth factor signals the closure of a certain population of potassium channels. As a result, potassium efflux from the differentiating cells slows while active potassium uptake continues at a constant rate. Potassium then accumulates within the cell causing water influx, an increase in cell volume, and cell elongation.

Measurement of steady currents around the frog lens.

Steady electrical currents were measured and mapped around the frog lens using a vibrating probe. Ion substitutions and ouabain were used to help identify the ionic basis of the observed currents. In physiological frog Ringer solution, current densities at the equator averaged 26 microA cm-2 and were directed outward. At both optical poles, inward currents were measured, with those at the posterior pole about 30% greater than those at the anterior pole. Currents at all loci decreased when external potassium concentration was increased at the expense of sodium, and when Na and K concentrations approached those of lens cytosol, all currents approached zero. Current direction reversed in a solution containing 105 mM K and 2.5 mM Na. In an Na-free medium, where sodium was replaced with choline, currents were reduced significantly at the poles and equator, but they changed direction only at the anterior pole. This effect was fully reversible on return to solution containing sodium. Ouabain (0.1 mM) caused a significant decrease in currents only at the equator, and this effect had two components. A rapid 10% decline in current was complete in 2 min, followed by a steady, slower decrease which stabilized 8-12 min later at a new level approximately 30% lower than the initial current.

Effects of calcium on the steady outward currents at the equator of the rat lens.

The relationships between calcium and the steady outward currents at the equator of the rat lens were studied using the vibrating probe technique. In a calcium-free medium, the current was greatly increased and it returned to its original level when calcium was restored to the medium. The Ca-free effect was not observed in Na-free medium. Iodoacetate (IAA) inhibited the initial current, but a current then returned which is referred to as a secondary current. The secondary current was not observed in a Ca-free medium and, therefore, it is thought to be a calcium-dependent potassium current. These responses are consistent with effects on potassium efflux measured by others and lend support to the interpretation that the outward currents observed at the equator of the rat lens are potassium currents. The currents are partially inhibited but not abolished in Na-free bathing medium. This is consistent with the view that the inward currents at the optical poles may be related to the influx of sodium.

Esophageal desalination of seawater in flounder: role of active sodium transport.

The esophagus of the flounder, Pseudopleuronectes americanus, was studied to determine how salinity of ingested seawater (SW) is decreased before fluid absorption in the intestine. Drinking rate was 2.5 ml X h-1 X kg-1. Stomach fluid osmolality was 45% that of seawater, and intestinal fluid was isosmotic to plasma. Esophagus and stomach were nearly impermeable to 28Mg; thus Mg concentrations were accurate indicators of fluid addition and NaCl removal between pharynx and stomach. Measurements of water and ion fluxes across isolated esophageal epithelium mounted in Ussing chambers and bathed by Ringer solution showed that the tritiated water flux was lower in esophagus than in intestine and that 22Na flux ratio was 1.4 (Jm leads to s/Js leads to m) regardless of acclimation medium (100 or 10% SW). Potential difference was zero, and electrical resistance averaged 90 omega X cm2. Mucosal-to-serosal Na transport was inhibited by 0.1 mM amiloride, 0.1 mM ouabain, and Cl-free medium, whereas 1.0 mM furosemide had no effect. Net esophageal Na absorption (mucosal-to-serosal) averaged 10.0 mumol X h-1 X cm-2 with mucosa exposed to SW and was inhibited 46% by 0.1 mM ouabain. Taken together the above observations suggest a role for both passive and active esophageal Na transport in SW desalination.