Helle Hasager Damkier

Cerebrospinal Fluid Secretion by the Choroid Plexus

The choroid plexus epithelium is a cuboidal cell monolayer, which produces the majority of the cerebrospinal fluid. The concerted action of a variety of integral membrane proteins mediates the transepithelial movement of solutes and water across the epithelium. Secretion by the choroid plexus is characterized by an extremely high rate and by the unusual cellular polarization of well-known epithelial transport proteins. This review focuses on the specific ion and water transport by the choroid plexus cells, and then attempts to integrate the action of specific transport proteins to formulate a model of cerebrospinal fluid secretion. Significant emphasis is placed on the concept of isotonic fluid transport across epithelia, as there is still surprisingly little consensus on the basic biophysics of this phenomenon. The role of the choroid plexus in the regulation of fluid and electrolyte balance in the central nervous system is discussed, and choroid plexus dysfunctions are described in a very diverse set of clinical conditions such as aging, Alzheimers disease, brain edema, neoplasms, and hydrocephalus. Although the choroid plexus may only have an indirect influence on the pathogenesis of these conditions, the ability to modify epithelial function may be an important component of future therapies.

Mice with targeted Slc4a10 gene disruption have small brain ventricles and show reduced neuronal excitability

Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Slc4a10 gene, which encodes the Na+-coupled Cl−–HCO3− exchanger Slc4a10 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4a10 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4a10 also was expressed in neurons. Within the hippocampus, the Slc4a10 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridine-induced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure.

Epithelial Pathways in Choroid Plexus Electrolyte Transport

A stable intraventricular milieu is crucial for maintaining normal neuronal function. The choroid plexus epithelium produces the cerebrospinal fluid and in doing so influences the chemical composition of the interstitial fluid of the brain. Here, we review the molecular pathways involved in transport of the electrolytes Na+, K+, Cl-, and HCO3(-)across the choroid plexus epithelium.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na+-dependent HCO3− importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pHi) recordings were performed upon acidification in the presence of CO2/HCO3− by 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na+-dependent pHi recovery, which was accompanied by an increase in the intracellular Na+ concentration evaluated by use of the Na+-sensitive fluorophore CoroNa Green. The estimated Na+:HCO3− stoichiometry was 1:2. Cl− is most likely the counterion maintaining electroneutrality because (i) Na+-dependent pHi recovery was eliminated in Cl−-depleted cells; (ii) acute extracellular Cl− removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS)-sensitive and Na+- and HCO3−-dependent 36Cl−-efflux during pHi recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na+ and are expected to move more HCO3− molecules/turnover were targeted by site-directed mutagenesis. Na+-dependent pHi recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na+-dependent Cl−/HCO3− exchanger in which four specific charged amino acids seem necessary for ion transport.

17β-Estradiol induces nongenomic effects in renal intercalated cells through G protein-coupled estrogen receptor 1

Steroid hormones such as 17β-estradiol (E2) are known to modulate ion transporter expression in the kidney through classic intracellular receptors. Steroid hormones are also known to cause rapid nongenomic responses in a variety of nonrenal tissues. However, little is known about renal short-term effects of steroid hormones. Here, we studied the acute actions of E2 on intracellular Ca(2+) signaling in isolated distal convoluted tubules (DCT2), connecting tubules (CNT), and initial cortical collecting ducts (iCCD) by fluo 4 fluorometry. Physiological concentrations of E2 induced transient increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in a subpopulation of cells. The [Ca(2+)](i) increases required extracellular Ca(2+) and were inhibited by Gd(3+). Strikingly, the classic E2 receptor antagonist ICI 182,780 also increased [Ca(2+)](i), which is inconsistent with the activation of classic E2 receptors. G protein-coupled estrogen receptor 1 (GPER1 or GPR30) was detected in microdissected DCT2/CNT/iCCD by RT-PCR. Stimulation with the specific GPER1 agonist G-1 induced similar [Ca(2+)](i) increases as E2, and in tubules from GPER1 knockout mice, E2, G-1, and ICI 182,780 failed to induce [Ca(2+)](i) elevations. The intercalated cells showed both E2-induced concanamycin-sensitive H(+)-ATPase activity by BCECF fluorometry and the E2-mediated [Ca(2+)](i) increment. We propose that E2 via GPER1 evokes [Ca(2+)](i) transients and increases H(+)-ATPase activity in intercalated cells in mouse DCT2/CNT/iCCD.

NHE1 knockout reduces blood pressure and arterial media/lumen ratio with no effect on resting pHi in the vascular wall

Key points  •  Small arteries are important for regulation of blood pressure and local blood flow. •  Changes in intracellular pH alter artery tone although the mechanistic background has been unclear. •  Using knockout mice for Na+/H+ exchanger NHE1 with reduced acid extrusion from cells in the arterial wall and low intracellular pH in the absence of CO2/HCO3‐, we show that intracellular acidification alters enzymatic activity and consequently artery dilatation and contraction. •  Although lack of NHE1 does not affect intracellular pH in the arterial wall when CO2/HCO3− is present, arteries from NHE1 knockout mice have thinner walls and produce less active force due to reduced volume and cross‐sectional area of individual smooth muscle cells. •  These results highlight the interplay between intracellular pH and artery function, provide new targets to consider for modulating artery structure, and underscore the need to evaluate acid–base transport in conditions of vascular disease and blood pressure disturbances.

Genetic ablation of Slc4a10 alters the expression pattern of transporters involved in solute movement in the mouse choroid plexus

Mutational changes of one transporter can have deleterious effects on epithelial function leaving the cells with the options of either compensating for the loss of function or dedifferentiating. Previous studies have shown that the choroid plexus epithelium (CPE) from mice lacking the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger (NCBE) encoded by Slc4a10 leads to retargeting of the Na(+)/H(+) exchanger 1 (NHE1) from the luminal to the basolateral plasma membrane. We hypothesized that disruption of NCBE, the main basolateral Na(+) importer in the CPE, would lead to a compensatory increase in the abundance of other important transport proteins in this tissue. Aquaporin-1 (AQP1) abundance was 42.7% lower and Na,K-ATPase 36.4% lower in the CPE of Slc4a10 knockout mice, respectively. The NHE1 binding ezrin cytoskeleton appeared disrupted in Slc4a10 knockout mice, whereas no changes were observed in cellular polarization with respect to claudin-2 and appearance of luminal surface microvilli. The renal proximal tubule constitutes a leaky epithelium with high transport rate similar to CPE. Here, Slc4a10 knockout did not affect Na,K-ATPase or AQP1 expression. CPE from AQP1 knockout mice has a secretory defect similar to Slc4a10 mice. However, neither NCBE nor Na,K-ATPase expression was affected in CPE from AQP1 knockout mice. By contrast, the abundance of Na,K-ATPase and NBCe1 was decreased by 23 and 31.7%, respectively, in AQP1 knockout proximal tubules, while the NHE3 abundance was unchanged. In conclusion, CPE lacking NCBE seems to spare the molecular machinery involved in CSF secretion rather than compensate for the loss of the Na(+) loader. Slc4a10 knockout seems to be more deleterious to CPE than AQP1 knockout.

Fluctuations in surface pH of maturing rat incisor enamel are a result of cycles of H+-secretion by ameloblasts and variations in enamel buffer characteristics

It is disputed if ameloblasts in the maturation zone of the enamel organ mainly buffer protons released by hydroxyapatite (HA) crystal growth or if they periodically secrete protons to create alternating acidic and alkaline conditions. The latter hypothesis predicts alternating pH regimes in maturing enamel, which would be affected by pharmacological interference with ameloblast H(+)-secretion. This study tests these predictions. Colorimetric pH-indicators and ratiometric fluorometry were used to measure surface pH in maturation zone enamel of rat incisors. Alternating acidic (down to pH6.24±0.06) and alkaline zones (up to pH7.34±0.08) were found along the tooth coinciding with ameloblast morphological cycles. Underlying the cyclic pattern, a gradual decrease in pH towards the incisal edge was seen. Vinblastine or FR167356 (H(+)-ATPase-inhibitor) disturbed ameloblast acid-secretion, especially in the early parts of acidic zones. Enamel surface pH reflects the titration state of surface PO4(3-)-ions. At the pH-values observed, PO4(3-) would be protonated (pKa>12) and HA dissolved. However, by molecular dynamics simulations we estimate the pKa of HPO4(2-) at an ideal HA surface to be 4.3. The acidic pH measured at the enamel surface may thus only dissolve non-perfect domains of HA crystals in which PO4(3-) is less electrostatically shielded. During repeated alkaline/acidic cycles, near-perfect HA-domains may therefore gradually replace less perfect HA-domains resulting in near-perfect HA-crystals. In conclusion, cyclic changes in ameloblast H(+)-secretion and the degree of enamel maturation determine enamel surface pH. This is in accordance with a hypothesis implicating H(+)-ATPase mediated acid-secretion by ameloblasts.

Na + dependent acid-base transporters in the choroid plexus; insights from slc4 and slc9 gene deletion studies

The choroid plexus epithelium (CPE) is located in the ventricular system of the brain, where it secretes the majority of the cerebrospinal fluid (CSF) that fills the ventricular system and surrounds the central nervous system. The CPE is a highly vascularized single layer of cuboidal cells with an unsurpassed transepithelial water and solute transport rate. Several members of the slc4a family of bicarbonate transporters are expressed in the CPE. In the basolateral membrane the electroneutral Na+ dependent Cl−/HCO3− exchanger, NCBE (slc4a10) is expressed. In the luminal membrane, the electrogenic Na+:HCO3− cotransporter, NBCe2 (slc4a5) is expressed. The electroneutral Na+:HCO3− cotransporter, NBCn1 (slc4a7), has been located in both membranes. In addition to the bicarbonate transporters, the Na+/H+ exchanger, NHE1 (slc9a1), is located in the luminal membrane of the CPE. Genetically modified mice targeting slc4a2, slc4a5, slc4a7, slc4a10, and slc9a1 have been generated. Deletion of slc4a5, 7 or 10, or slc9a1 has numerous impacts on CP function and structure in these mice. Removal of the transporters affects brain ventricle size (slc4a5 and slc4a10) and intracellular pH regulation (slc4a7 and slc4a10). In some instances, removal of the proteins from the CPE (slc4a5, 7, and 10) causes changes in abundance and localization of non-target transporters known to be involved in pH regulation and CSF secretion. The focus of this review is to combine the insights gathered from these knockout mice to highlight the impact of slc4 gene deletion on the CSF production and intracellular pH regulation resulting from the deletion of slc4a5, 7 and 10, and slc9a1. Furthermore, the review contains a comparison of the described human mutations of these genes to the findings in the knockout studies. Finally, the future perspective of utilizing these proteins as potential targets for the treatment of CSF disorders will be discussed.

Transport across the choroid plexus epithelium

The choroid plexus epithelium is a secretory epithelium par excellence. However, this is perhaps not the most prominent reason for the massive interest in this modest-sized tissue residing inside the brain ventricles. Most likely, the dominant reason for extensive studies of the choroid plexus is the identification of this epithelium as the source of the majority of intraventricular cerebrospinal fluid. This finding has direct relevance for studies of diseases and conditions with deranged central fluid volume or ionic balance. While the concept is supported by the vast majority of the literature, the implication of the choroid plexus in secretion of the cerebrospinal fluid was recently challenged once again. Three newer and promising areas of current choroid plexus-related investigations are as follows: 1) the choroid plexus epithelium as the source of mediators necessary for central nervous system development, 2) the choroid plexus as a route for microorganisms and immune cells into the central nervous system, and 3) the choroid plexus as a potential route for drug delivery into the central nervous system, bypassing the blood-brain barrier. Thus, the purpose of this review is to highlight current active areas of research in the choroid plexus physiology and a few matters of continuous controversy.