Ventricular Choline Transport

To determine whether organic cation transporter (OCT) family members might mediate choline transport in choroid plexus (CP), the handling of choline by cloned transporters and by intact CP isolated from the adult rat was investigated. Expression of OCT1 and OCT2 in Xenopus oocytes increased hemicholinium-3-sensitive choline uptake. In contrast, OCT3 did not mediate choline transport. Estimated Km values for choline in rOCT1-, rOCT2-, and hOCT2-expressing oocytes were 346 ± 50, 441 ± 67, and 102 ± 80 μm, respectively. Membrane potential was the major driving force for choline uptake in rat and human OCT2-expressing oocytes and in intact CP in vitro. Lowering of medium pH (6 versus 7.4) was equally effective at inhibiting choline uptake in CP, suggesting that there might be a non-OCT component of choline uptake that is responsive to an H+ gradient. However, choline efflux from CP was not stimulated by a trans-applied H+ gradient. Choline uptake by CP was Na+-independent with an estimated Km of 183 μm. Reverse transcriptase-polymerase chain reaction detected OCT2 and OCT3, but not OCT1, mRNA expression in CP. Transfection of intact CP with a rOCT2/green fluorescent protein fusion construct resulted in strong apical membrane fluorescence with no detectable signal in the basal and lateral plasma membranes. These data indicate that OCT2 mediates choline transport across the ventricular membrane of CP. To determine whether organic cation transporter (OCT) family members might mediate choline transport in choroid plexus (CP), the handling of choline by cloned transporters and by intact CP isolated from the adult rat was investigated. Expression of OCT1 and OCT2 in Xenopus oocytes increased hemicholinium-3-sensitive choline uptake. In contrast, OCT3 did not mediate choline transport. Estimated Km values for choline in rOCT1-, rOCT2-, and hOCT2-expressing oocytes were 346 ± 50, 441 ± 67, and 102 ± 80 μm, respectively. Membrane potential was the major driving force for choline uptake in rat and human OCT2-expressing oocytes and in intact CP in vitro. Lowering of medium pH (6 versus 7.4) was equally effective at inhibiting choline uptake in CP, suggesting that there might be a non-OCT component of choline uptake that is responsive to an H+ gradient. However, choline efflux from CP was not stimulated by a trans-applied H+ gradient. Choline uptake by CP was Na+-independent with an estimated Km of 183 μm. Reverse transcriptase-polymerase chain reaction detected OCT2 and OCT3, but not OCT1, mRNA expression in CP. Transfection of intact CP with a rOCT2/green fluorescent protein fusion construct resulted in strong apical membrane fluorescence with no detectable signal in the basal and lateral plasma membranes. These data indicate that OCT2 mediates choline transport across the ventricular membrane of CP. cerebrospinal fluid artificial cerebrospinal fluid choroid plexus organic cation organic cation transporter (prefix r, m, or h denotes rat, mouse, or human, respectively) green fluorescent protein rat organic cation transporter 2-green fluorescent protein fusion construct endoplasmic reticulum tetraethylammonium tetramethylammonium N 1-methylnicotinamide N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyri dinium dibromide reverse transcriptase-polymerase chain reaction base pair hemicholinium-3 In the brain, free choline is essential for the synthesis of membrane phospholipids and the neurotransmitter acetylcholine. However, there is very little de novo synthesis of choline in the brain, and a constant supply from plasma is required (1Crews F.T. Hirata F. Axelrod J. J. Neurochem. 1980; 34: 1491-1498Crossref PubMed Scopus (168) Google Scholar). Movement of choline across the brain capillary endothelium changes with its concentration in plasma (physiological range of 10–50 μm), whereas the choline concentration in cerebrospinal fluid (CSF)1 is tightly controlled (5–7 μm) (2Cornford E.M. Braun L.D. Oldendorf W.H. J. Neurochem. 1978; 30: 299-308Crossref PubMed Scopus (169) Google Scholar, 3Klein J. Gonzalez R. Koppen A. Loffelholz K. Neurochem. Int. 1993; 22: 293-300Crossref PubMed Scopus (106) Google Scholar, 4Erb C. Seidel A. Frank H. Platt K.L. Oesch F. Klein J. Biochem. Pharmacol. 1999; 57: 681-684Crossref PubMed Scopus (9) Google Scholar, 5Vargas H.M. Jenden D.J. Life Sci. 1996; 58: 1995-2002Crossref PubMed Scopus (14) Google Scholar). Thus, the balance between plasma choline levels and the net flux of brain choline is an important mechanism for maintaining brain choline homeostasis. It has been postulated that perturbations in this balance may have a role in central cholinergic dysfunctions and that impeding the transepithelial movement of choline from CSF to blood may prove to be an effective therapy (6Koppen A. Klein J. Holler T. Loffelholz K. J. Pharmacol. Exp. Ther. 1993; 266: 720-725PubMed Google Scholar, 7Jenden D.J. Rice K.M. Roch M. Booth R.A. Lauretz S.D. Adv. Neurol. 1990; 51: 131-138PubMed Google Scholar). Ventriculocisternal perfusion studies and in vitroexperiments on isolated choroid plexus (CP) show carrier-mediated uptake of choline, suggesting that the CP is actively involved in the maintenance of brain choline homeostasis through removal of excess choline from CSF (6Koppen A. Klein J. Holler T. Loffelholz K. J. Pharmacol. Exp. Ther. 1993; 266: 720-725PubMed Google Scholar, 7Jenden D.J. Rice K.M. Roch M. Booth R.A. Lauretz S.D. Adv. Neurol. 1990; 51: 131-138PubMed Google Scholar, 8Aquilonius S.M. Winbladh B. Acta Physiol. Scand. 1972; 85: 78-90Crossref PubMed Scopus (37) Google Scholar, 9Lanman R.C. Schanker L.S. J. Pharmacol. Exp. Ther. 1980; 215: 563-568PubMed Google Scholar, 10Ehrlich B.E. Wright E.M. Brain Res. 1982; 250: 245-249Crossref PubMed Scopus (18) Google Scholar). Recently, Villalobos et al. (11Villalobos A.R. Parmelee J.T. Renfro J.L. Am. J. Physiol. 1999; 276: C1288-C1296Crossref PubMed Google Scholar), using primary cultures of the choroidal epithelium, found that the characteristics of apical choline uptake in CP cells are strikingly similar to those identified for the renal basolateral organic cation (OC) transport system. Such a reversal of functional polarity is conceivable because the movement of OCs from CSF to blood requires the membrane potential-sensitive entry step to take place across the apical membrane of the cells of the CP. Therefore, we propose that the molecular basis for CP function in the regulation of free choline concentration in CSF might be the expression of organic cation transporter (OCT) family members. Three candidate transporters, OCT1, OCT2, and OCT3, have been cloned recently from a variety of species including rat, mouse, pig, rabbit, and man (12Schweifer N. Barlow D.P. Mamm. Genome. 1996; 7: 735-740Crossref PubMed Scopus (73) Google Scholar, 13Wu X. Ganapathy M.E. Leibach F.H. Ganapathy V. FASEB Abstr. 1998; 12: A1042Google Scholar, 14Kekuda R. Prasad P.D. Wu X. Wang H. Fei Y. Leibach F.H. Ganapathy V. J. Biol. Chem. 1998; 273: 15971-15979Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, V. M. H. PubMed Scopus Google Scholar, J. F. N. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, V. A. C. F. H. Biol. PubMed Scopus Google Scholar, M. H. Y. M. K. Biochem. Res. 1996; PubMed Scopus Google Scholar, K.M. 1998; PubMed Scopus Google Scholar, R.C. FASEB J. 1996; Scholar, K.M. Pharmacol. 51: PubMed Scopus Google Scholar). transporters mediate the entry of OCs the a potential mechanism X. Ganapathy M.E. Leibach F.H. Ganapathy V. FASEB Abstr. 1998; 12: A1042Google Scholar, 14Kekuda R. Prasad P.D. Wu X. Wang H. Fei Y. Leibach F.H. Ganapathy V. J. Biol. Chem. 1998; 273: 15971-15979Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, V. M. H. PubMed Scopus Google V. A. C. F. H. Biol. PubMed Scopus Google Scholar, Am. J. Physiol. 1999; Google Scholar). are in that transport are by an of that and tetramethylammonium 1-methylnicotinamide and in between OCT1 and OCT2 have been identified H. V. J. Biol. 1999; PubMed Scopus Google and K.M. Pharmacol. 1998; PubMed Scopus Google Scholar), but on OCT3 is R. Prasad P.D. Wu X. Wang H. Fei Y. Leibach F.H. Ganapathy V. J. Biol. Chem. 1998; 273: 15971-15979Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, X. R. Fei Leibach F.H. J. Ganapathy V. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), between transporters to is by the that there are species in the expression and of transporters in in oocytes versus cells versus intact in oocytes the of rat OCT1 for that of μm, but in cells are versus μm, respectively) V. M. H. PubMed Scopus Google Scholar, Am. J. Physiol. 1999; Google Scholar, Y. M. H. K. J. Pharmacol. Exp. Ther. 1998; Google Scholar). Thus, increased of the transporters at the molecular and of the functional between the are in oocytes the rat for with the μm, μm, and R. Prasad P.D. Wu X. Wang H. Fei Y. Leibach F.H. Ganapathy V. J. Biol. Chem. 1998; 273: 15971-15979Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, V. M. H. PubMed Scopus Google Am. J. Physiol. 1999; Google Scholar). It has been that and are by but is not R. Prasad P.D. Wu X. Wang H. Fei Y. Leibach F.H. Ganapathy V. J. Biol. Chem. 1998; 273: 15971-15979Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, V. M. H. PubMed Scopus Google Scholar, Am. J. Physiol. 1999; Google Scholar). prove to be a with for ± μm), ± μm), and the Km for was in cells and not Xenopus oocytes R. Prasad P.D. Wu X. Wang H. Fei Y. Leibach F.H. Ganapathy V. J. Biol. Chem. 1998; 273: 15971-15979Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, C. V. T. C. A. H. Am. J. Physiol. PubMed Google Scholar). In of the that in CP function in the regulation of free choline in we and V. A. C. F. H. Biol. PubMed Scopus Google Scholar, X. R. Fei Leibach F.H. J. Ganapathy V. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R.C. FASEB J. Scholar, V. F. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google have found that choline uptake by and and that choline is by and However, was that choline is a for but is not a for or N. Pharmacol. 1999; PubMed Scopus Google Scholar). there are to whether OCT1, OCT2, and OCT3 are in brain V. M. H. PubMed Scopus Google Scholar, J. F. N. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, V. A. C. F. H. Biol. PubMed Scopus Google Scholar, X. R. Fei Leibach F.H. J. Ganapathy V. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). we that OCT2 and OCT3 are in rat CP and that choline uptake by intact rat choroid plexus has the characteristics of suggesting that this an important role in central choline homeostasis. and transport of the rOCT2/green fluorescent protein fusion and the endoplasmic reticulum construct have been Am. J. Physiol. PubMed Google Scholar, PubMed Scopus Google Scholar). and were the from Ganapathy R. Prasad P.D. Wu X. Wang H. Fei Y. Leibach F.H. Ganapathy V. J. Biol. Chem. 1998; 273: 15971-15979Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, X. Ganapathy M.E. Wang H. R. Leibach F.H. Ganapathy V. Am. J. Physiol. PubMed Google and from K. J. Pharmacol. PubMed Scopus Google Scholar). and uptake were Am. J. Physiol. 1999; Google Scholar, T. Pharmacol. 1999; PubMed Scopus Google Scholar, Am. J. Physiol. 1999; 276: Google Scholar). adult Xenopus were with and and oocytes were isolated by with and at in and were to for was from using in Three with oocytes were of and at for or in or in the or of uptake studies concentration to a to the membrane potential V. M. H. PubMed Scopus Google there was a in concentration from to to the of the was in in a with was isolated from lateral CP of adult using the to the of was for h at with of reverse in a reaction of and and of the reverse reaction was for chain reaction with the rOCT1-, rOCT2-, and and and and were reaction the but no reaction reaction using adult rat and reaction with CP are at for by of for for and for were on a with lateral CP from adult were in and at mRNA was isolated using mRNA of CP mRNA was for h at with of reverse in a reaction of and of the reverse reaction was for plexus and uptake were R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). adult were with CP were and to artificial cerebrospinal fluid and pH with of was in of CP in of in the or of and at for uptake studies were It has been that an in from to ventricular membrane potential to in primary cultures of rat CP (11Villalobos A.R. Parmelee J.T. Renfro J.L. Am. J. Physiol. 1999; 276: C1288-C1296Crossref PubMed Google Scholar). the efflux CP were isolated and by in for at CP were with and at in of or to pH medium were at the CP was by of the by medium the and the in the CP. In the in the was with and CP were in for CP were with of using in with a of of essential medium and was at with in for the of the CP were by fluorescence and h CP and cells were using a with a were by with an at was in the and a was in of the were or and to a were with and were from choline, and were from fluorescent plasma was from were from and were of the and choline uptake that was by the organic cation transport and by the choline transport hemicholinium-3 In contrast, increased choline uptake was not in oocytes the of functional transporters, by a in uptake not OCT3 to transport choline not choline uptake by and was to an effective of brain choline levels for and that choline uptake was for at in and OCT2-expressing Xenopus oocytes not uptake was to the for oocytes no transport. oocytes were in and uptake was in the of was at and choline a of the component of uptake component was from uptake to the of the component a for choline of 346 ± for and 441 ± for choline Km for of 102 ± 80 was found not These values are similar to values for the transporters V. A. C. F. H. Biol. PubMed Scopus Google Scholar, C. V. T. C. A. H. Am. J. Physiol. PubMed Google V. C. A. H. Pharmacol. 1999; PubMed Scopus Google Scholar). by was to determine whether its driving force and transport characteristics were those for Am. J. Physiol. 1999; Google Scholar). uptake was by and for Am. J. Physiol. 1999; Google Scholar). H. and J. B. Choline uptake a similar with choline an choline uptake by the rat and human OCT2 was by the membrane potential for transport by Am. J. Physiol. 1999; Google of membrane on choline uptake. uptake with was on oocytes with or with a of choline uptake that was the membrane potential was was no on were in and the data are values ± from To determine whether OCT1, OCT2, OCT3 are in CP, mRNA or was in from plexus and for reverse were with rOCT1-, rOCT2-, and using of the were detected for and that are in CP and may a role in choline from CSF was detected for in CP. were for CP with adult rat the for choroid plexus was and found to be to cloned from rat M. H. Y. M. K. Biochem. Res. 1996; PubMed Scopus Google Scholar, R.C. FASEB J. 1996; Scholar). whether the driving for choline uptake by intact CP in the those by family members CP choline uptake that was by uptake was potential-sensitive by the in the from to the concentration of in the transport was to CP choline transport was to by pH from to To determine whether this was to we efflux from CP H+ to choline at increased H+ versus pH a the of on choline efflux was because is to efflux Am. J. Physiol. 1999; Google Scholar). Choline efflux was by whereas excess H+ to have an of choline by intact CP was by uptake of and of on choline efflux from isolated adult rat choroid isolated plexus was by of at in plexus was and at in pH or to pH medium were at the Choline efflux from CP was by excess stimulated by choline transport across the apical membrane not an are of and values are ± the the was with a for pH and for denotes of on choline uptake in intact choroid isolated plexus was for at in or to or Choline uptake was the in and uptake. that apical CP choline uptake is not to the is not by a was and the data are values ± from a denotes and denotes uptake by intact CP was for at not and were to CP were in choline, and uptake was not component was estimated by the of the by uptake at and transport was by the for at concentration from the uptake of the component of uptake an estimated Km for choline of 183 fluorescent plasma membrane was to the between the plasma of the CP and the capillary show the of plexus and is of capillary by a of cells that the cerebrospinal of the is that the apical membrane of the cells is by the and the basal membrane is the fluorescence show a with the cells of the choroidal were by a of fluorescence and apical membrane a in with the and the basal membrane is in and is with the capillary and the capillary were To the of intact adult rat CP were isolated in and with CP with a fluorescence that the and the CP with the membrane a to the and the of endoplasmic reticulum that with there is no of the plasma membrane and no signal in the In contrast, was to the apical plasma membrane and was from the and basal of CP cells and capillary and the of the choroid and the brain from the to the and Scholar, in and Scholar). between the cells the of and the the of the fluid of the brain fluid efflux from the brain, are required to of including and Therefore, of the transporters in and the that function is to the maintenance of brain homeostasis and the basis for its that we the mechanism of transport and of OCT2 in the CP of the adult of choline transport by OCT2, of OCT2 mRNA in CP, of the mechanism of choline uptake in intact CP, and of OCT2 in the apical membrane of CP OCT2 may a role in the maintenance of brain choline homeostasis and of the brain capillary endothelium have that the mechanism the entry of choline, and the brain has functional similar to those by OCT1 and OCT2, OCT2 the net flux of brain choline in its N. H. H. M. T. Y. J. Pharmacol. 1999; 51: PubMed Scopus Google Scholar, H. N. N. H. Y. Pharmacol. Res. Scopus Google Scholar). To the movement of choline, the brain capillary and CP polarity of and In cells basolateral uptake of organic is by the potential across the membrane and apical a the transport responsive to driving for uptake and of Physiol. 1993; PubMed Scopus Google and Biochem. 1999; PubMed Scopus Google Scholar). Choline transport across the apical membrane of primary cultures of choroidal from was by membrane (11Villalobos A.R. Parmelee J.T. Renfro J.L. Am. J. Physiol. 1999; 276: C1288-C1296Crossref PubMed Google Scholar). that choline uptake across the apical membrane of intact rat CP in is membrane These data are with the polarity of the choroidal for its role in the of OCs from CSF to that the potential-sensitive entry step across the ventricular of the similar reversal of function between and CP has been R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google for organic transport. we have that the major driving force for and choline uptake is the membrane potential CP choline uptake was to be suggesting that a mechanism may be However, H+ to choline whereas trans-applied stimulated that an not mediate choline transport across the apical membrane of adult rat CP It was that the and choline transporters in cholinergic a role in apical CP choline transport. R.C. Schanker L.S. J. Pharmacol. Exp. Ther. 1980; 215: 563-568PubMed Google Scholar, 10Ehrlich B.E. Wright E.M. Brain Res. 1982; 250: 245-249Crossref PubMed Scopus (18) Google Scholar, C. Brain Res. 1990; PubMed Scopus Google Scholar, M. C. Exp. Brain Res. PubMed Scopus Google on intact adult CP rat, rabbit, and that apical CP choline transport is to the at through a to the for the choline transporter in cholinergic Brain Res. PubMed Scopus Google Scholar, J. Neurochem. 1978; 30: PubMed Scopus Google Scholar, J. Neurochem. 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PubMed Scopus Google and that of to increased choline levels in the brain D.J. Rice K.M. Roch M. Booth R.A. Lauretz S.D. Adv. Neurol. 1990; 51: 131-138PubMed Google Scholar). was to be to the of choline transport by the of to D.J. Rice K.M. Roch M. Booth R.A. Lauretz S.D. Adv. Neurol. 1990; 51: 131-138PubMed Google Scholar). In of this the of from by an in rat brain has been H.M. Jenden D.J. Life Sci. 1996; 58: 1995-2002Crossref PubMed Scopus (14) Google Scholar). Thus, the of of on choline transport and the of this transporter by Am. J. Physiol. 1999; Google with on CP choline that the of with choline to choline levels in the to increased brain choline levels the that of choline transport be an effective therapy for central cholinergic (6Koppen A. Klein J. Holler T. Loffelholz K. J. Pharmacol. Exp. Ther. 1993; 266: 720-725PubMed Google Scholar). In we have that the of apical choline uptake in CP with those for we have detected expression of OCT2 in CP and apical membrane of in intact CP. that OCT2 mediates apical CP choline uptake from the the and of and