Tracey Speake

Molecular mechanisms of cerebrospinal fluid production

The epithelial cells of the choroid plexuses secrete cerebrospinal fluid (CSF), by a process which involves the transport of Na(+), Cl(-) and HCO(3)(-) from the blood to the ventricles of the brain. The unidirectional transport of ions is achieved due to the polarity of the epithelium, i.e. the ion transport proteins in the blood-facing (basolateral) membrane are different to those in the ventricular (apical) membrane. The movement of ions creates an osmotic gradient which drives the secretion of H(2)O. A variety of methods (e.g. isotope flux studies, electrophysiological, RT-PCR, in situ hybridization and immunocytochemistry) have been used to determine the expression of ion transporters and channels in the choroid plexus epithelium. Most of these transporters have now been localized to specific membranes. For example, Na(+)-K(+)ATPase, K(+) channels and Na(+)-2Cl(-)-K(+) cotransporters are expressed in the apical membrane. By contrast the basolateral membrane contains Cl(-)- HCO(3) exchangers, a variety of Na(+) coupled HCO(3)(-) transporters and K(+)-Cl(-) cotransporters. Aquaporin 1 mediates water transport at the apical membrane, but the route across the basolateral membrane is unknown. A model of CSF secretion by the mammalian choroid plexus is proposed which accommodates these proteins. The model also explains the mechanisms by which K(+) is transported from the CSF to the blood.

Mechanisms of CSF secretion by the choroid plexus

The epithelial cells of the choroid plexus secrete cerebrospinal fluid (CSF), by a process that involves the movement of Na+, Cl− and HCO3− from the blood to the ventricles of the brain. This creates the osmotic gradient, which drives the secretion of H2O. The unidirectional movement of the ions is achieved due to the polarity of the epithelium, i.e., the ion transport proteins in the blood‐facing (basolateral) are different to those in the ventricular (apical) membranes. Saito and Wright (1983) proposed a model for secretion by the amphibian choroid plexus, in which secretion was dependent on activity of HCO3− channels in the apical membrane. The patch clamp method has now been used to study the ion channels expressed in rat choroid plexus. Two potassium channels have been observed that have a role in maintaining the membrane potential of the epithelial cell, and in regulating the transport of K+ across the epithelium. An inward‐rectifying anion channel has also been identified, which is closely related to ClC‐2 channels, and has a significant HCO3− permeability. This channel is expressed in the apical membrane of the epithelium where it may play an important role in CSF secretion. A model of CSF secretion by the mammalian choroid plexus is proposed that accommodates these channels and other data on the expression of transport proteins in the choroid plexus. Microsc. Res. Tech. 52:49–59, 2001.

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na(+)/K(+)-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.

Modulation of calcium signals by intracellular pH in isolated rat pancreatic acinar cells

1 We have investigated the interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in isolated rat pancreatic acinar cells. The fluorescent dyes fura‐2 and BCECF were used to measure [Ca2+]i and pHi, respectively. 2 Sodium acetate and ammonium chloride (NH4Cl) were used to acidify and alkalinize pHi, respectively. Cytosolic acidification had no effect on [Ca2+]i in resting pancreatic acinar cells, whereas cytosolic alkalinization released Ca2+ from intracellular stores. 3 Cytosolic acidification using either acetate or a CO2‐HCO3−‐buffered medium enhanced Ca2+ signals evoked by acetylcholine (ACh) and cholecystokinin (CCK). In contrast, both NH4Cl and trimethylamine (TMA) inhibited Ca2+ signals during stimulation with either ACh or CCK. This inhibitory effect was also observed in the absence of extracellular Ca2+, and was therefore not due to changes in Ca2+ entry. 4 Calcium oscillations evoked by physiological concentrations of CCK were enhanced by cytosolic acidification and inhibited by cytosolic alkalinization. 5 In order to determine the effects of pHi upon Ca2+ handling by intracellular Ca2+ stores, intraorganellar [Ca2+] was monitored using the low affinity Ca2+ indicator mag‐fura‐2 in permeabilized cells. Addition of NH4Cl, which is expected to alkalinize intraorganellar pH, did not alter intraorganellar [Ca2+] in permeabilized cells, suggesting that changing intraorganellar pH does not release Ca2+ from intracellular stores. Addition of NH4Cl or acetate also did not affect the rate of Ca2+ release induced by inositol 1,4,5‐trisphosphate (InsP3). 6 Modification of extraorganellar (‘cytosolic’) pH did not affect the rate of ATP‐dependent Ca2+ uptake into stores, but did modify the rate of Ca2+ release evoked by submaximal concentrations of InsP3. The rate of Ca2+ release was increased at more alkaline extraorganellar pHs. These results would suggest that manipulation of intraorganellar pH does not affect Ca2+ handling by the intracellular stores. In contrast, extraorganellar (‘cytosolic’) pH does affect InsP3‐induced Ca2+ release from the stores. 7 In conclusion, changes in intracellular pH in pancreatic acinar cells can profoundly alter cytosolic [Ca2+]. This may shed light on earlier observations whereby cell‐permeant weak acids and bases can modulate fluid secretion in epithelia.

Expression of the Na+/K+/2CI-cotransporter in alpha and beta cells isolated from the rat pancreas.

Abstract. The expression of the Na+-K+-2Cl– cotransporter (NKCC1) in α cells and β cells from the rat pancreas was examined. Isolated α cells and β cells in a mixed islet cell preparation were identified by volume using video-imaging methods, and by the expression of glucagon or insulin. The expression of mRNA for NKCC1 in pancreatic islets was demonstrated by RT-PCR. Immunocytochemical studies showed that the NKCC1 protein was expressed in rat β cells, but not α cells. The activity of Na+-K+-2Cl– cotransporter was also examined, by studying cell volume regulation in response to HEPES-buffered, hypertonic solutions. A regulatory volume increase was observed in the β cells but not the α cells. It is concluded that the NKCC1 is expressed in rat pancreatic β cells but not α cells. This is consistent with the hypothesis that Cl– is accumulated above the expected equilibrium distribution in β cells, but is below equilibrium in α cells.

Inward-rectifying anion channels are expressed in the epithelial cells of choroid plexus isolated from ClC-2 'knock-out' mice

Choroid plexus epithelial cells express inward‐rectifying anion channels which have a high HCO3− permeability. These channels are thought to have an important role in the secretion of cerebrospinal fluid. The possible relationship between these channels and the ClC‐2 Cl− channel was investigated in the present study. RT‐PCR, using specific ClC‐2 primers, amplified a 238 bp fragment of mRNA from rat choroid plexus, which was 99 % identical to the 5′ sequence of rat ClC‐2. A 2005 bp clone was isolated from a rat choroid plexus cDNA library using a probe for ClC‐2. The clone showed greater than 99 % identity with the sequence of rat ClC‐2. Inward‐rectifying anion channels were observed in whole‐cell recordings of choroid plexus epithelial cells isolated from ClC‐2 knock‐out mice. The mean inward conductance was 19.6 ± 3.6 nS (n= 8) in controls (3 heterozygote animals), and 22.5 ± 3.1 nS (n= 10) in three knock‐out animals. The relative permeability of the conductances to I− and Cl− (PI : PCl) was determined. I− was more permeant than Cl− in both heterozygotes (PI:PCl= 4.0 ± 0.9, n= 3) and knock‐out animals (PI : PCl= 4.1 ± 1.4, n= 3). These results indicate that rat choroid plexus expresses the ClC‐2 variant that was originally reported in other tissues. ClC‐2 does not contribute significantly to inward‐rectifying anion conductance in mouse choroid plexus, which must therefore express a novel inward‐rectifying anion channel.

The role of calcium in the volume regulation of rat lacrimal acinar cells

Abstract. Earlier studies have suggested a role for Ca2+ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca2+-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca2+ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd3+ inhibited RVD, suggesting that Ca2+ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca2+]i during exposure to hypotonic solutions. The absence of any changes in [Ca2+]i resulted from the buffering of cytosolic Ca2+ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca2+ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca2+]i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca2+-activated K+ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca2+ in controlling cell volume in lacrimal acinar cells.

Ion channels in epithelial cells of the choroid plexus isolated from the lateral ventricle of rat brain

Whole-cell patch clamp methods were used to determine the expression of ion channels in the epithelial cells of choroid plexus isolated from the lateral ventricle of the rat brain. A single population of cells with a mean capacitance of 61.5+/-1.7 pF was identified in 103 recordings. This value is significantly greater than that measured for cells from the fourth ventricle (P<0.01 by unpaired t-test), indicating that cells from the lateral ventricle have a greater surface area. Voltage-dependent, outward currents were recorded using a K(+)-rich electrode solution. These currents were partially inhibited by 10 nM margatoxin or 10 nM dendrotoxin-K and blocked by 5 mM TEA(+). An inward-rectifying chloride conductance was observed in K(+)-free solutions. The relative permeability of this conductance to anions was P(I)>P(Cl)>P(aspartate). A volume-sensitive anion conductance was observed when cell swelling was induced using a hypertonic electrode solution. The properties of each conductance were similar to conductances previously identified in fourth ventricle choroid plexus cells. Furthermore, there were no significant differences between the magnitudes of any of the conductances in cells from the lateral and fourth ventricle choroid plexus. Thus, the ionic conductances expressed in rat lateral and fourth ventricle choroid plexus are very similar.

Functional Characterisation of the Volume-Sensitive Anion Channel in Rat Pancreatic β-Cells

The whole‐cell and perforated patch configurations of the patch‐clamp technique were used to characterise the volume‐sensitive anion channel in rat pancreatic β‐cells. The channel showed high permeability (P) relative to Cl− to extracellular monovalent organic anions (PSCN/PCll= 1.73, Pacetate/PCll= 0.39, Plactate/PCll= 0.38, Pacetoacetate/PCll= 0.32, Pglutamate/PCll= 0.28) but was less permeable to the divalent anion malate (Pmalate/PCll= 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9‐dideoxyforskolin (100 μM) and 4‐OH tamoxifen (10 μM). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume‐sensitive anion channel in non‐swollen cells. Furthermore, addition of 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13‐dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume‐sensitive anion channel in pancreatic β‐cells can be activated by protein kinase A. Furthermore, the β‐cell channel does not appear to be subject to regulation via protein kinase C.