The murine choroid plexus epithelium expresses the 2Cl-/H+ exchanger ClC-7 and Na+/H+ exchanger NHE6 in the luminal membrane domain.

The choroid plexus epithelium within the brain ventricles secretes the majority of the cerebrospinal fluid (CSF). The luminal Na+-K+-ATPase acts in concert with a host of other transport proteins to mediate efficient fluid secretion across the epithelium. The CSF contains little protein buffer, but the pH value seems nonetheless maintained within narrow limits, even when faced with acid-base challenges. The involvement of choroid plexus acid-base transporters in CSF pH regulation is highlighted by the expression of several acid-base transporters in the epithelium. The aim of the present study was to identify novel acid-base transporters expressed in the luminal membrane of the choroid plexus epithelium to pave the way for systematic investigations of each candidate transporter in the regulation of CSF pH. Mass spectrometry analysis of proteins from epithelial cells isolated by fluorescence-activated cell sorting identified the Cl-/H+ exchangers ClC-3, -4, -5, and -7 in addition to known choroid plexus acid-base transporters. RT-PCR on FACS isolated epithelial cells confirmed the expression of the corresponding mRNAs, as well as Na+/H+ exchanger NHE6 mRNA. Both NHE6 and ClC-7 were immunolocalized to the luminal plasma membrane domain of the choroid plexus epithelial cells. Dynamic imaging of intracellular pH and membrane potential changes in isolated choroid plexus epithelial cells demonstrated Cl- gradient-driven changes in intracellular pH and membrane potential that are consistent with Cl-/H+ exchange. In conclusion, we have detected for the first time NHE6 and ClC-7 in the choroid plexus, which are potentially involved in pH regulation of the CSF.

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.

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.

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, Alzheimer's 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.