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Differential Effects of Diabetes on Rat Choroid Plexus Ion Transporter Expression Richard D

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Differential Effects of Diabetes on Rat Choroid Plexus Ion Transporter Expression Richard D Differential Effects of Diabetes on Rat Choroid Plexus Ion Transporter Expression Richard D. Egleton, Christopher C. Campos, Jason D. Huber, Rachel C. Brown, and Thomas P. Davis Though diabetes is a disease with vascular complica- alus (6,7). Hydrocephalus is caused by excessive retention tions, little is known about its effects on the blood-brain or production of cerebrospinal fluid (CSF) within the barrier or the blood–cerebrospinal fluid barrier central nervous system. CSF is produced by the choroidal (BCSFB). The BCSFB is situated at choroid plexuses epithelial cells of the choroid plexus (8). The choroid located in the lateral, third, and fourth ventricles. Cho- plexuses are also the site of the BCSFB and are located in roid plexuses are the primary site of cerebrospinal fluid the lateral, third, and fourth ventricles. Capillaries of the (CSF) production and express numerous ion transport- choroid plexus are fenestrated, allowing extracellular fluid ers. Previous studies have shown a perturbation of ion formation (unlike other brain capillary beds). CSF produc- transport in the periphery and brain during diabetes. In tion involves the combination of a number of transport this study, we investigated the effect of diabetes on ion systems in the choroid plexus (9) (Fig. 1). transporters in the choroid plexuses of streptozotocin ϩ ϩ (STZ)-induced diabetic rats. Diabetes was induced in Animal models of diabetes have shown that the Na -H exchanger (10–12), Naϩ-Kϩ-2ClϪ cotransporter (13), and male Sprague-Dawley rats by intraperitoneal injection ϩ ϩ of STZ (60 mg/kg in citrate buffer, confirmed by glucose Na -K -ATPase (14–17) have altered expression and ac- -analysis: 601 ؎ 22 mg/dl diabetic rats, 181 ؎ 46 mg/dl tivity in a number of vascular beds. These three transport age-matched controls); and at 28 days, rats were killed, ers are critical for CSF production and pH maintenance choroid plexuses harvested, and protein extracted. (9). In this study, we have investigated the effects of the Western blot analyses were carried out using antibodies -؉ ؉ ؊ streptozotocin (STZ) model of type 1 diabetes on expres for ion transporters, including Na -K -2Cl cotrans- ϩ ϩ ϩ ϩ Ϫ -؉ ؉ sion of the Na -H exchanger, Na -K -2Cl cotrans porter and the Na -K -ATPase ␣1-subunit. The efflux of ϩ ϩ .؉ ؉ porter, and Na -K -ATPase in the rat choroid plexus the K analog 86Rb from choroid plexus was also studied. Diabetic rats showed an increase in expression ؉ ؉ ؊ ؉ ؉ of the Na -K -2Cl cotransporter and the Na -K - RESEARCH DESIGN AND METHODS ATPase ␣1-subunit, as compared with age-matched con- ؉ ؉ Induction of diabetes. All protocols used in this study were approved by the trols, a decrease in Na -H exchanger expression, and University of Arizona Institutional Animal Care and Use Committee and abide ؉ ؉ no change in Na -K -ATPase ␤1- or ␤2-subunit. The net by National Institutes of Health guidelines. Diabetes was induced in male 300- effect of these changes was a 66% increase in 86Rb؉ to 325-g Sprague-Dawley rats (Harlan, Indianapolis, IN) via an intraperitoneal efflux from diabetic choroid plexus compared with con- injection of 60 mg/kg STZ (Sigma, St. Louis, MO) in sterile phosphate buffered trols. These changes in expression may affect choroid saline. Control animals were injected intraperitoneally with phosphate buff- plexus ion balance and thus significantly affect CSF ered saline. The animals were housed under standard 12-h light-dark condi- Diabetes tions and received food and water ad libitum for 28 days. Induction of diabetes production in diabetic rats. 52:1496–1501, was assessed by weight changes and blood glucose levels. 2003 Blood and CSF chemistry. Heparinized blood samples were collected and analyzed using an ABL 505 blood gas analyzer (Radiometer Copenhagen). CSF samples were taken from the cisterna magna before decapitation and analyzed as for blood. iabetes is a disease with numerous vascular Protein isolation. Rats were anesthetized with sodium pentobarbital (64.8 mg/kg) and decapitated. The brains were rapidly removed, and the choroid complications. Previous studies have shown plexuses of the lateral ventricles were removed. Protein was isolated from the perturbations in ion transport in both the pe- choroid plexuses by incubating overnight in 6 mol/l urea buffer (6 mol/l urea, riphery and the brain during diabetes (1–3). 10 mmol/l Tris, 1 mmol/l dithiothreitol, 5 mmol/l MgCl2, 5 mmol/l EGTA, 150 D mmol/l NaCl, pH 8.0, complete mini EDTA free protease inhibitor [one Though diabetes is a vascular disease, little is known regarding the effects of diabetes on the blood-brain barrier tablet/10 ml of buffer; Roche, Mannheim, Germany]). Protein was quantified using the bicinchoninic acid method (Pierce, Indianapolis, IN). (BBB) or the blood–cerebrospinal fluid barrier (BCSFB). Western blot analyses. Protein samples (20 ␮g) were separated on Novex It has been reported that diabetes is a risk factor not only 4–12% Tris-glycine gels using an electrophoretic field at 125 V for 75–90 min. for stroke (4,5), but also for normal-pressure hydroceph- Proteins were transferred to polyvinylidene fluoride membranes using 240 mA at 4°C for 30 min. The membranes were then blocked using 5% nonfat milk/Tris-buffered saline (20 mmol/l Tris, 137 mmol/l NaCl, pH 7.6) with 0.1% From the Department of Pharmacology, College of Medicine, The University Tween-20. Membranes were incubated overnight at 4°C with primary antibod- of Arizona, Tucson, Arizona. ies (anti–Naϩ-Hϩ exchanger [1:250; Transduction Laborotories]; anti–Naϩ-Kϩ- Address correspondence and reprint requests to Dr. Thomas P. Davis, 2ClϪ cotransporter [1:2,000; The University of Iowa Hybridoma Bank]; anti-rat Department of Pharmacology, P.O. Box 245050, The University of Arizona, Naϩ-Kϩ-ATPase ␣1-, ␤1-, and ␤2-subunits [1:2,000; Research Diagnostics]). Tucson, AZ 85724. E-mail: [email protected]. Primary antibodies were chosen based on their ability to bind to rat trans- Received for publication 14 July 2002 and accepted in revised form 24 porters. Following incubation with primary antibodies, the membranes were February 2003. aCSF, artificial cerebrospinal fluid; BBB, blood-brain barrier; BCSFB, washed with 5% nonfat milk/TBS buffer before incubation with respective blood–cerebrospinal fluid barrier; CSF, cerebrospinal fluid; PKC, protein secondary antibodies (anti-mouse and anti-rabbit [Amersham, Springfield, IL] kinase C; STZ, streptozotocin. at 1:2,000 and 1:3,000 dilutions, respectively, in PBS/0.5% BSA) for 30 min at © 2003 by the American Diabetes Association. room temperature. Membranes were developed using the enzyme chemilumi- 1496 DIABETES, VOL. 52, JUNE 2003 R.D. EGLETON AND ASSOCIATES adding 1 ml TS2 tissue solubilizer (Research Products International, Mt. Prospect, IL) and incubated overnight. Before counting on a Beckman LS5000 counter (Beckman Instruments Fullerton, CA), 100 ␮l 30% acetic acid and 4 ml Budget Solve scintillation cocktail (Research Products International, Mt. Prospect, IL) were added to each sample. Efflux rate constants were calculated for 86Rbϩ from the slope of the logarithmic plot of 86Rbϩ remaining in the choroid plexus against time. Total tissue 86Rbϩ was taken as 100% at time 0. The amount of 86Rbϩ remaining at each time point was based on sequential subtractions of the amount effluxed for each 20-s time point from the total radioactivity. Data analysis. All data are presented as mean Ϯ SE of measurements from 4–24 separate animals (numbers for individual experiments noted in text and figures). Statistical significance was calculated using Student’s t tests between control and diabetic values, using the Pharmacological Calculation System software package (19). RESULTS Blood and CSF chemistry. Diabetes was assessed in this study by monitoring weight changes and blood glucose levels of both PBS- and STZ-injected rats (Table 1). Control rats gained 101 Ϯ 26 g over the 28-day period, while diabetic rats lost 68 Ϯ 9 g. Weight loss was paralleled by a significant increase (P Ͻ 0.01) in blood glucose levels, from 181 Ϯ 46 mg/dl in control to 601 Ϯ 22 mg/ml in diabetic rats. Levels of electrolytes within the CSF and plasma were similar, with a small increase in plasma Kϩ levels in diabetic rats compared with controls (Table 2). FIG. 1. Schematic showing location of transporters and direction of ion Western blot analyses of transporter expression. flow during CSF production by choroidal epithelial cells, based on Figure 2A– C shows the expression of the ␣1-, ␤1-, and Speake et al. (9). Carbonic anhydrase (C.A.) catalyzes the production ϩ ϩ -؊ ␤2-subunits of the Na -K -ATPase transporter. A signifi of HCO3- ions from H2O and CO2.Cl accumulation in the choroidal ؊ Ͻ ϳ epithelial cell is driven by the basolateral HCO3-/Cl exchanger. HCO3- cant increase (P 0.05) of 60% was seen in expression can also move down an electrochemical gradient across the apical of the ␣1 catalytic subunit in diabetic rats as compared membrane via various channels. Cl؊ enters the CSF via either anion ␤ ؉ ؉ ؊ ؉ channels or the Na -K -2Cl cotransporter. Na enters the choroid with controls (Fig. 2A), with no significant change in 1- or .(plexus from the blood down a chemical gradient via the Na؉-H؉ ␤2-subunit expression (Fig. 2B and C exchanger on the basolateral membrane (H؉ supplied from the produc- ϩ ϩ Ϫ ؉ Figure 2D shows the expression of the Na -K -2Cl tion of HCO3-). Na crosses the apical membrane to the CSF via the ,Na؉-K؉-ATPase and the Na؉-K؉-2Cl؊ cotransporter. Secretion of Na؉, cotransporter, which showed a significant increase (40% ؊ Cl , and HCO3- into the ventricles leads to an osmotic gradient, which P Ͻ 0.05) in expression in diabetic choroid plexuses as drives H2O movement into the CSF via aquaporins.
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