ORIGINAL PAPER

Regional Characteristics of Histamine Uptake into Neonatal RatAstrocytes

Katja Perdan-Pirkmajer • Sergej Pirkmajer •

Andreja Raztresen • Mojca Krzan

Received: 16 January 2013 / Revised: 20 March 2013 / Accepted: 23 March 2013

� Springer Science+Business Media New York 2013

Abstract Histaminergic signalling constitutes an attrac-

tive target for treatment of neuropsychiatric disorders. One

obstacle to developing new pharmacological options has

been failure to identify putative specific histamine trans-

porter responsible for histamine clearance. Although high-

affinity histamine uptake was detected in neonatal cortical

astrocytes, its existence in other brain regions remains lar-

gely unexplored. We investigated whether cerebellar and

striatal astrocytes participate in histamine clearance and

evaluated the role of organic cation transporters (OCT) in

astroglial histamine transport. Kinetic and pharmacological

characteristics of histamine transport were determined in

cultured astrocytes derived from neonatal rat cerebellum,

striatum and cerebral cortex. As well as astrocytes of cortical

origin, cultured striatal and cerebellar astrocytes displayed

temperature-sensitive, high-affinity histamine uptake.

Exposure to ouabain or Na?-free medium, supplemented

with choline chloride markedly depressed histamine trans-

port in cortical astrocytes. Conversely, histamine uptake in

striatal and cortical astrocytes was ouabain-resistant and was

only partially diminished during incubation in the absence of

Na?. Also, histamine uptake remained unaltered upon

exposure to OCT inhibitor corticosterone, although OCTs

were expressed in cultured astrocytes. Finally, histamine

transport in cerebellar and striatal astrocytes was not sensi-

tive to antidepressants. Despite common characteristics,

cerebellar astrocytes had lower affinity, but markedly higher

transport capacity for histamine compared to striatal astro-

cytes. Collectively, we provide evidence to suggest that

cerebellar, striatal as well as cortical astrocytes possess sat-

urable histamine uptake systems, which are not operated by

OCTs. In addition, our data indicate that Na?-independent

histamine carrier predominates in cerebellar and striatal

astrocytes, whereas Na?-dependent transporter underlies

histamine uptake in cortical astrocytes. Our findings impli-

cate a role for histamine transporters in regulation of extra-

cellular histamine concentration in cerebellum and striatum.

Inhibition of histamine uptake might represent a viable

option to modulate histaminergic neurotransmission.

Keywords Rat � Astrocytes � Histamine uptake �Regional characteristics

Introduction

Histamine is a monoamine neurotransmitter in the central

nervous system, where it participates in regulation of sleep-

wake cycle, fluid balance and feeding as well as higher brain

functions, such as mood, memory and locomotion [1]. Tar-

geting histamine signalling in the brain is the focus of efforts

to expand pharmacopoeia against neuropsychiatric disorders

[2], although exact role of the histaminergic system in the

pathogenesis of specific diseases remains poorly defined [1].

Several pharmacological approaches have been undertaken

to modulate neurotransmission in other monoaminergic

systems, including inhibition of monoamine uptake through

the specific, high-affinity Na?/Cl--dependent transporters

(uptake1) for serotonin (SERT), noradrenaline (NET) and

dopamine (DAT). Monoamine uptake inhibitors include

several pharmacotherapeutic agents as well as recreational

K. Perdan-Pirkmajer � A. Raztresen � M. Krzan (&)

Department of Pharmacology and Experimental Toxicology,

Faculty of Medicine, University of Ljubljana, Korytkova 2,

1001 Ljubljana, Slovenia

e-mail: mojca.limpel@mf.uni-lj.si

S. Pirkmajer

Faculty of Medicine, Institute of Pathophysiology, University

of Ljubljana, Zaloska 4, 1001 Ljubljana, Slovenia

123

Neurochem Res

DOI 10.1007/s11064-013-1028-x

substances, underscoring essential role of monoamine

transporters in regulation of the noradrenergic, dopaminer-

gic and serotoninergic systems and their physiological

functions [3]. In contrast, the role of histamine uptake in

histaminergic neurotransmission is incompletely character-

ized, not least because histamine remains the only mono-

amine neurotransmitter, whose putative specific transporter

has not been identified.

Neurons essentially lack plasmalemmal high-affinity

uptake system for histamine [4, 5], although histamine

shares the same vesicular transporter (VMAT2) with other

monoamines [6]. Indeed, synaptosomal studies have either

detected only low-efficiency histamine transport [7] or have

even failed to convincingly demonstrate saturable histamine

uptake [8], a unique exception among monoamine neuro-

transmitters [4]. In direct contrast, astrocyte-enriched brain

tissue fractions from adult rat cortex displayed high-affinity

histamine transport [9]. Moreover, high-affinity as well as

low-affinity histamine uptake was repeatedly demonstrated

in cultured astrocytes [10–14]. In addition, evidence sug-

gests glial cells participate in histamine uptake and/or

inactivation in vivo [15–17]. Taken together these findings

indicate that astrocytes may represent an important site of

histamine uptake. Yet carriers involved in astrocyte hista-

mine transport have been incompletely characterized.

Although selective uptake1 transporters, which mediate

high-affinity monoamine uptake into presynaptic terminals

[3], are expressed in astrocytes [18, 19] failure to observe

significant histamine uptake in neuronal terminals strongly

suggests histamine is not transported by SERT, DAT and/or

NET. Consistent with this notion, uptake studies have

demonstrated that exposure to histamine has either very low

or no inhibitory effect on serotonin and dopamine uptake by

SERT and DAT, respectively [20–22]. In fact, histamine is

not considered a substrate for SERT, NET or DAT and

histamine uptake assays have often been used to detect

and assess monoamine transport not dependent on uptake1

[17, 23, 24].

On the other hand, histamine is a prominent substrate for

the non-specific, corticosterone-sensitive organic cation

transporters (OCT) 2 (Slc22A2) and OCT 3 (extraneuronal

monoamine transporter (EMT) or Slc22A3) from the

Slc22A family of solute carriers [25, 26]. Although OCT2

and 3 mediate low-affinity, high-capacity transport of a

range of organic cations, they show relative preference

towards histamine compared to other monoamines [27, 28]

and may play an important role in histamine clearance in

the central nervous system. Importantly, OCT expression

has been detected in neurons as well as astrocytes [13, 19,

29, 30]. Furthermore, OCT inhibitors corticosterone and

decynium-22 (D22) reduce histamine uptake in periven-

tricular hypothalamic minces [23]. Similarly, D22 reduces

histamine clearance in murine hippocampus in vivo [17].

Thus, OCT2 and OCT3 are thought to represent a major

mechanism for low-affinity, high-capacity histamine

clearance in the brain. However, whereas neuronal OCT

expression is compatible with failure to identify efficient

high-affinity histamine uptake in synaptosomes [8], kinetic

parameters of OCTs [27, 28] markedly differ from those

observed for high-affinity histamine uptake in astrocytes

[10–12, 14]. These data, when taken together, suggest

histamine uptake in the central nervous system is not

exclusively mediated by OCTs.

Indeed, a significant fraction of total histamine clearance

in periventricular region of hypothalamus was not inhibited

by OCT inhibitors corticosterone and D22 [23], indicating

OCT-mediated histamine uptake is probably complemented

by other, unidentified transporters. In agreement with this

notion, striatal histamine levels during post-ischemic

reperfusion were unaltered between OCT3-deficient mice

and their wild type controls [31], although prominent OCT3

expression is normally detected in murine striatum [29]. On

the other hand, accentuated increase in histamine concen-

tration in reperfused cortex of OCT3 knock-out mice [31]

suggests impaired cortical histamine clearance and, by

extension, a role for OCT3 in histamine elimination in brain

cortex. Similarly, basal histamine concentration was affec-

ted only in cortex and thalamus/hypothalamus of OCT3-

deficient mice, while remaining unaltered in most other brain

regions, including striatum and cerebellum [32]. Available

evidence therefore indicates that relative contribution of

OCTs and/or other transporters to total histamine uptake may

substantially vary between brain regions.

Nevertheless, previous studies have focused almost

exclusively on histamine uptake in astrocytes from cerebral

cortex. Indeed, the potential role of astrocytes in subserv-

ing histamine elimination in most other brain regions,

including striatum and cerebellum, remains largely unex-

plored despite functional importance of histaminergic

neurotransmission in these areas [1]. We determined

whether cerebellar and striatal astrocytes could participate

in histamine clearance and evaluated the role of OCTs in

astroglial histamine transport. Our findings implicate a role

for histamine transporters in regulation of extracellular

histamine concentration in cerebellum and striatum.

Methods

Materials and Reagents

All cell culture reagents, except fetal bovine serum (FBS),

Cambrex IEP GmbH (Wiesbaden, Germany) were obtained

from Gibco, Invitrogen (Paisley, Scotland, UK). Cell cul-

ture flasks and plates were from TPP (Trasandingen,

Switzerland). [3H]-histamine (525.4 GBq/mmol) was

Neurochem Res

123

purchased from Perkin Elmer (Massachusetts, USA),

RNeasy Kit from Qiagen (Valencia, Canada), High

Capacity cDNA Reverse Transcription Kit, TaqMan Gene

Expression Assays and TaqMan Gene Expression Master

Mix from Applied Biosystems (Carlsbad, CA, USA).

Bradford total protein assay was obtained from Bio-Rad

(Hercules, CA, USA). Decynium 22 (D22), ouabain, flu-

oxetine and corticosterone were from Sigma-Aldrich (St.

Louis, MO, USA), while desipramine and amitriptyline

were purchased from Sandoz (Cham, Switzerland). All

other reagents, unless otherwise specified, were of analyt-

ical grade and from Sigma-Aldrich.

Animal Housing and Care

Experiments were approved by Veterinary Administration

of Republic of Slovenia (34401-1/2010/8). Wistar rats were

maintained on 12-h day/12-h night cycle, received standard

rodent chow and were cared for in accordance with the

national guidelines for the care and use of laboratory ani-

mals. Three-day old neonatal rats were sacrificed for

removal of cerebellum, striatum or cerebral cortex.

Cultured Astrocytes

Astrocyte cultures were prepared, as described [12, 33, 34].

Briefly, primary cultures derived from cerebellum, striatum

or cerebral cortex, were grown in high-glucose Dulbecco’s

modified Eagle’s medium (DMEM), containing 10 % FBS,

1 mM pyruvate, 2 mM glutamine, and 25 lg/ml gentamy-

cin at 37 �C in humidified 95 % air/5 % CO2. To reduce the

number of contaminating cells, confluent cultures were

exposed to overnight shaking at 225 rpm. Medium con-

taining detached cells was removed the next morning and

fresh growth medium was added. The whole procedure was

repeated three times. After the third overnight shaking,

adherent cells were trypsinized and subsequently cultured

in the presence of 10 lM cytosine arabinoside for 24 h.

Upon reaching confluence, the cells were seeded into

12-well or 6-well plates and were grown for additional

3 weeks before being used for histamine uptake or real-time

PCR experiments, respectively. Cultures used for all

experiments contained 93–100 % type 1 astrocytes [33, 34].

Histamine Uptake in Cultured Astrocytes

Monolayer cultures in 12-well plates were preincubated for

30 min in the uptake buffer (25 mM HEPES, 125 mM

NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4,

1.4 mM CaCl2, and 5.6 mM glucose, pH 7.4) at 37 �C

(total uptake) or at 4 �C (non-specific uptake). Cultured

astrocytes were incubated with 125 nM [3H]-histamine for

different time intervals to determine time dependence of

histamine uptake. Concentration dependence of histamine

uptake was determined by exposing cultured astrocytes to

different histamine concentrations, as indicated in the

Results, for 20 min. Experiments at 37 �C were terminated

by placing the plates on ice. [3H]-histamine was quickly

removed and the plates were washed twice with ice-cold

uptake buffer. The cells were subsequently lyzed in 300 ll

0.5 M NaOH. An aliquot (250 ll) of each sample was

transferred to a scintillation vial to measure the radioac-

tivity. The amount of transported histamine is normalized

to total protein content, which was determined in the

remaining aliquots (50 ll) of each sample with the Brad-

ford protein assay.

The Specific Histamine Uptake Rate and its Kinetics

The specific (temperature-sensitive) histamine uptake was

calculated as the difference between the total (37 �C) and

the non-specific histamine uptake (4 �C). Specific hista-

mine uptake rate data were linearly transformed and fitted

using linear regression to obtain Woolf, Eadie-Hofstee and

Lineweaver–Burk plots, which were used to determine the

apparent Michaelis–Menten constant (Km), the maximal

uptake rate (Vmax) and transport efficiency (Vmax/Km).

Kinetic parameters are reported as means with 95 % con-

fidence intervals.

Cell Treatments

To determine sensitivity of histamine transport to different

uptake (corticosterone, D22, antidepressants) or Na?/K?-

ATPase (ouabain) inhibitors, cultured astrocytes were first

preincubated in the uptake buffer for 30 min, followed by

incubation in the presence or absence of an inhibitor for

20 min at 37 �C. Astrocytes were then exposed to 125 nM

[3H]-histamine in the absence or presence of uptake or

Na?/K?-ATPase inhibitors for 20 min. To assess whether

histamine uptake is Na?-dependent, cultured astrocytes

were preincubated in the normal (Na?-containing) or Na?-

free uptake buffer, containing 125 mM choline chloride

((CH3)3N(Cl)CH2CH2OH) for 30 min. Astrocytes were

subsequently incubated with 125 nM [3H]-histamine in the

presence or absence of Na? for 20 min. Samples were

harvested and processed for radioactivity and protein

measurements as described above.

OCT mRNA Expression

The total RNA from rat cerebral cortex, neonatal kidney,

and cultured astrocytes was extracted using RNeasy kit

(Qiagen, Valencia, CA, U.S.A.) according to the manu-

facturer’s instructions. The RNA quantity and purity of

isolated total RNA was estimated by measuring the

Neurochem Res

123

absorbance at 260 and 280 nm with the Nanodrop 2000c

spectrophotometer, Thermo Fischer Scientific (Waltham,

MA, USA). cDNA was synthesized from 2 lg of total

RNA using High Capacity cDNA Reverse Transcription

Kit, Applied Biosystems (Carlsbad, CA, USA). Quantita-

tive real-time PCR was performed in the 96-well format,

using an ABI PRISM SDS 7500 sequence detection system

from the Applied Biosystems (Carlsbad, CA, USA). Taq-

Man gene expression assays were used to determine

mRNA expression of rat OCT1 (Rn00562250_m1), rat

OCT2 (Rn00580893_m1), rat OCT3 (Rn00570264_m1)

and rat beta actin (rACTB, 4352931E), which was used as

the endogenous control (reference gene). Samples were

analyzed in duplicates. 20 ll reaction mixture contained

10 ll TaqMan Gene Expression Master Mix, Applied

Biosystems (Carlsbad, CA, USA), 1 ll of the appropriate

Taqman Gene Expression Assay, 7 ll of diethylpyr-

ocarbonate-treated water and 2 ll of cDNA. Amplification

was carried out as follows: 2 min at 55 �C, denaturation for

10 min at 95 �C followed by 40 cycles at 95 �C and 40

cycles at 60 �C. Exponential phase of the amplification

reaction was used to determine the threshold cycle (Ct) for

target genes (OCT mRNA) and endogenous control

(ACTB). OCT mRNA levels were normalized to ACTB

mRNA and gene expression ratios are reported [35]. Brain

cortex and kidney from neonatal rats were used to validate

OCT gene expression assays.

Statistics

Results are presented as mean ± SEM. The kinetic

parameters Km, Vmax and transport efficiency (Vmax/Km)

were determined from Woolf, Eadie-Hofstee and Linewe-

aver–Burk plots using software Prism4 version 4.00

(GraphPad Software Inc., San Diego, USA). Comparisons

between groups were performed using Student’s t test or

one-way ANOVA, followed by Dunnett’s or Bonferroni’s

post hoc test, when several groups were compared. Sta-

tistical significance was established at P \ 0.05.

Results

Cerebellar and Striatal Astrocytes Display

Time-Dependent and Temperature-Sensitive

Histamine Uptake

First of all, we determined whether carrier-mediated hista-

mine uptake exists in cultured astrocytes from neonatal rat

cerebellum and striatum. Astrocytes were exposed to 125 nM

[3H]-histamine for 2–30 min at 4 or 37 �C and the non-spe-

cific and total histamine uptake, respectively, were measured

(Fig. 1). Histamine accumulated in a time-dependent manner

and was clearly temperature-sensitive, as histamine uptake

decreased 70–85 % during exposure to 4 �C. Overall, these

data indicate that cultured cerebellar and striatal astrocytes

possess temperature-sensitive, carrier-mediated histamine

uptake.

Cerebellar, Striatal and Cortical Astrocytes Display

Differences in Histamine Uptake Characteristics

Next we evaluated concentration-dependence and kinetics of

histamine transport in striatal and cerebellar astrocytes. To

investigate whether regional differences in basic characteris-

tics of histamine transport exist, histamine uptake in cortical

astrocytes was assessed in parallel. We have previously

reported some basic properties of histamine transport in

neonatal cortical astrocytes [12, 14], here we provide further

and more detailed kinetic data. Astrocytes were incubated

with 0.125–90 lM histamine for 20 min and the specific

histamine uptake rate (Fig. 2) was determined from the spe-

cific, temperature-sensitive histamine uptake which was cal-

culated as the difference between the total (37 �C) and the

non-specific (4 �C) uptake. Uptake rate data were linearly

transformed and Woolf, Eadie-Hofstee (Fig. 2) and Linewe-

aver–Burk (not shown) plots were used to determine kinetic

parameters (Table 1). Saturable, concentration-dependent

histamine uptake was detected in cerebellar, striatal and, as

previously reported [14], cortical astrocytes. In comparison

with striatum or cortex, histamine uptake in cerebellar

astrocytes had the highest capacity (Vmax = 8.42-13.5 pmol/

mg protein/min) but somewhat lower affinity (Km =

18.5–43.1 lM). Transport efficiency (Vmax/Km) in cerebellar

astrocytes (0.31–0.46 ll/mg protein/min) was comparable to

histamine uptake in astrocytes from cerebral cortex. In con-

trast, striatal astrocytes were characterized by the highest

affinity for histamine (Km = 6.35–8.23 lM), but markedly

lower uptake efficiency (0.06–0.07 ll/mg protein/min). In

sum, these data demonstrate that cultured astrocytes from

neonatal cerebellum possess histamine uptake with moderate

affinity and comparatively high capacity, whereas striatal

astrocytes display high-affinity, low-capacity histamine

transport.

Histamine Uptake is Ouabain-Sensitive in Cortical,

but Not in Cerebellar and Striatal Astrocytes

Monoamine uptake is mediated by the specific, high-affinity/

low-capacity, Na?/Cl--dependent transporters (uptake1) [3]

as well as by the non-specific, low-affinity/high-capacity,

Na?/Cl--independent carriers (uptake2), which are thought to

represent a major histamine uptake system [17, 23, 27, 28]. To

determine, whether histamine uptake is Na?-dependent,

sodium chloride in the uptake buffer was substituted by

125 mM choline chloride. Astrocytes were preincubated in

Neurochem Res

123

Na?-free medium for 30 min, which was followed by 20-min

exposure to 125 nM [3H]-histamine at 4 and 37 �C (Fig. 3).

Incubation in the presence of Na?-free, choline-containing

medium reduced total histamine uptake 30 % (P \ 0.05) and

21 % (P \ 0.05) in cerebellar and striatal astrocytes (Fig. 3a,

b), respectively. This was paralleled by 65 % decrease

(P \ 0.05) in total histamine uptake in cortical astrocytes

(Fig. 3c). These results suggest that Na?-independent com-

ponent of the specific histamine transport accounts for at least

55–64 % in cerebellar and striatal astrocytes, whereas only

22 % of the specific transport was Na?-independent in corti-

cal astrocytes.

However, choline is a quaternary amine and could act as

a competitive inhibitor of histamine uptake in cultured

astrocytes [23]. Inhibition of Na?/K?-ATPase dissipates

Na? gradient in astrocytes [36] and thereby diminishes

Na?-coupled transport. Na?-dependence of histamine

transport was therefore further tested by subjecting astro-

cytes to histamine uptake assay in the absence or presence

of 1 mM ouabain, a Na?/K?-ATPase inhibitor. Concen-

tration used is sufficient to exert total inhibition of all

rodent a-isoforms of Na?/K?-ATPase, including the oua-

bain-resistant a2-isoform [37]. Although choline decreased

total histamine accumulation 27–30 %, histamine uptake in

striatal and cerebellar astrocytes was not inhibited by

ouabain (Fig. 3). This indicates choline-sensitive fraction

may not represent Na?-coupled histamine transport. In

contrast, exposure to 1 mM ouabain decreased total hista-

mine uptake more than 58 % in cortical astrocytes

(P \ 0.05). Consistent with choline data, ouabain-insensi-

tive fraction accounted for approximately 22 % of the

specific histamine transport. Taken together these findings

suggest that Na?-independent carrier is responsible

for histamine transport in cerebellar and striatal astro-

cytes, whereas Na?-dependent transport seems to represent

a major uptake mechanism in cortical astrocytes.

Organic Cation Transporters (OCT) 1, 2 and 3 are

Expressed in Cerebellar, Striatal and Cortical

Astrocytes

Na?-independent organic cation transporters (OCT) 2 and3 are

currently the only known plasmalemmal transporters for his-

tamine [25–27] expressed in astrocytes [29, 38, 39]. In partic-

ular, since OCT2 also transports choline [26], we hypothesized

that OCT2-mediated transport could, at least in part, represent

choline-sensitive fraction of the histamine uptake in cerebellar

and striatal astrocytes. To determine OCT expression profile in

cultured neonatal astrocytes from different brain regions, OCT

mRNA expression was measured by real-time PCR. Cerebral

cortex and kidney from neonatal rats were used to validate

OCT gene expression assays. As expected, OCT3 mRNA was

the predominant OCT subtype in rat cerebral cortex (Fig. 4a),

while rat kidney highly expressed OCT1 and OCT2 mRNA

(Fig. 4b) [40]. Subsequently, OCT mRNA expression was

measured in cultured astrocytes (Fig. 4c–e). All three members

of OCT family were expressed in cerebellar, striatal as well as

cortical astrocytes. However, unlike in cortical tissue, OCT3

mRNA in cultured astrocytes was only lowly expressed com-

pared to OCT1 and OCT2 mRNA. Indeed, prominent OCT2

mRNA expression was consistently observed, which was most

pronounced in cortical astrocytes. Whereas histamine is not a

substrate for OCT1 according to most [25–27], although not all

[41] studies, our data were consistent with OCT2-mediated

histamine uptake in cultured astrocytes. Furthermore, although

low expression level did not support a major role for OCT3, its

possible involvement in mediating histamine uptake could not

be disregarded.

Fig. 1 Cultured cerebellar

(a) and striatal (b) astrocytes

display time-dependent and

temperature-sensitive histamine

uptake. Total (37 �C) and non-

specific (4 �C) histamine uptake

was determined after exposure

to 125 nM [3H]-histamine. Data

are mean ± SEM (n = 6–9)

Neurochem Res

123

Effect of OCT and Uptake1 Inhibitors on Histamine

Uptake

Na?-independent histamine transport in conjunction with

OCT2 and OCT3 mRNA expression in cultured astrocytes,

suggested a role for OCTs in histamine transport. To

establish the involvement of OCTs, astrocytes were

exposed to decynium-22 (D22) and corticosterone (Fig. 5a),

which are both potent and well-characterized OCT inhibi-

tors [28, 42]. While histamine uptake remained unaltered

upon exposure to D22 in cerebellar and striatal astrocytes

histamine accumulation was decreased 62 % (P \ 0.05) in

D22-treated cortical astrocytes. Corticosterone failed to

exert dose-dependent inhibition of histamine uptake in

cerebellar and striatal astrocytes, similar to what we pre-

viously determined in astrocytes from neonatal [43] as well

as adult rat [13] cerebral cortex. Taken together these data

indicate OCTs do not mediate high-affinity histamine

uptake in neonatal rat astrocytes.

Histamine shares vesicular transporter VMAT2 with

other monoamines [6], suggesting putative specific hista-

mine transporter could be inhibited by uptake1 inhibitors due

to common structural properties. Moreover, we have previ-

ously established that histamine uptake in cultured cortical

astrocytes from neonatal as well as adult rats was sensitive to

uptake1 inhibitors amitriptyline and/or desipramine [12, 13].

Fig. 2 Kinetic characteristics of specific histamine uptake in cultured

cerebellar (a–c), striatal (d–f) and cortical (g–i) astrocytes. Specific

histamine uptake rate (a, d, g) was determined after exposure to

0.125–90 lM histamine. Data are mean ± SEM (n = 6). Kinetic data

were plotted as specific uptake rate/histamine concentration ratio

versus histamine concentration or specific uptake rate versus hista-

mine concentration/specific uptake rate ratio and fitted by using linear

regression to obtain Woolf (b, e, h) or Eadie-Hofstee (c, f, i) plots,

respectively

Neurochem Res

123

Histamine uptake in cerebellar and striatal astrocytes was

therefore measured in the absence or presence of amitripty-

line (non-selective monoamine reuptake inhibitor), fluoxe-

tine (selective serotonin reuptake inhibitor) and desipramine

(selective noradrenaline reuptake inhibitor) (Fig. 5b).

Whereas desipramine showed some inhibitory activity in

striatal astrocytes, amitriptyline and fluoxetine did not alter

histamine uptake neither in cerebellar nor striatal astrocytes.

Overall these results demonstrate that histamine transport

in striatal and cerebellar astrocytes is resistant to anti-

depressants.

Discussion

High-affinity uptake of serotonin, noradrenaline and

dopamine in neurons plays a key role in regulation of

monoaminergic neurotransmission and represents an

important target for pharmacotherapy of neuropsychiatric

disorders [3]. In contrast, histamine is the only monoamine

neurotransmitter lacking high-affinity neuronal uptake [4,

5]. Astrocytes may therefore represent a major site of

histamine uptake in the brain [9, 44, 45]. However, most

previous studies have focused on histamine uptake in

cortical or hypothalamic astrocytes [10–14], although his-

taminergic system spreads throughout the entire central

nervous system with pronounced regional variation in the

anatomical and functional characteristics [1, 4, 46]. In

addition, while dysfunction of histamine transport may be

associated with pathological alterations of local histamine

concentration in specific brain regions such as striatum [1,

47], whether and how astrocytes from different brain

regions participate in histamine uptake has not been thor-

oughly explored. Here we provide evidence that carrier-

mediated histamine transport exists in cerebellar and

striatal astrocytes. In addition, our data suggest regional

differences in characteristics of astroglial histamine uptake.

Neurons lack high-affinity histamine uptake [4, 5].

Furthermore, some synaptosomal studies even failed to

demonstrate saturable histamine transport [8]. In direct

contrast, we detected high-affinity, concentration-depen-

dent histamine uptake in cultured cerebellar and striatal

astrocytes, implicating a role for astrocytes in regulation of

extracellular histamine concentration and histaminergic

neurotransmission in these regions. Since high-affinity

histamine carrier has not been identified, low-affinity, high-

capacity organic cation transporters OCT2 and especially

OCT3 are often considered a major histamine uptake sys-

tem in the brain [17, 23, 31]. However, not only OCT

inhibitors failed to depress histamine uptake in cerebellar

and striatal astrocytes, also its kinetic properties are

markedly different from those reported for OCT2 and

OCT3. In particular, Km values (0.54–0.89 mM) for OCT-

mediated histamine transport [27, 28] are 10- to 100-fold

higher compared to what we observed in cerebellar

(18.5–43.1 lM) and striatal (6.4–8.2 lM) astrocytes.

These findings are consistent with the existence of high-

affinity histamine uptake in astrocyte-enriched brain frac-

tions as well as in cultured cortical or hypothalamic astro-

cytes, where even lower Km values (\1 lM) were observed

[9, 10]. Although transport efficiencies (0.05–0.46 ll/mg

protein/min), especially in astrocytes derived from stria-

tum, were lower than previously reported for rat astro-

cytes (2.25–16.42 ll/mg/min) [10, 12], they were still

significantly higher compared to efficiency of histamine

uptake in rat brain synaptosomes (0.015 and 0.0028 ll/

mg/min for the high and low affinity uptake process,

respectively) [7]. Taken together our results suggest

astrocytes in cerebellum and striatum possess high-

affinity histamine uptake system.

Table 1 Kinetic parameters of specific histamine uptake in neonatal cerebellar, striatal and cortical astrocytes

Cell type Km (CI) (lM) Vmax (CI) (pmol/mg/min) Vmax/Km (CI) (ll/mg protein/min)

Cerebellar astrocytes 43.1 (20.0–89.1)1

25.8 (9.14–42.5)2

18.5 (11.9–34.6)3

13.5 (10.0–20.8)1

10.5 (6.76–14.2)2

8.42 (5.90–14.7)3

0.313 (0.224–0.520)1

0.406 (0.310–0.798)2

0.456 (0.407–0.518)3

Striatal astrocytes 8.23 (6.16–10.6)1

7.21 (5.82–8.59)2

6.35 (5.69–7.18)3

0.48 (0.45–0.52)1

0.46 (0.40–0.51)2

0.43 (0.39–0.48)3

0.059 (0.049–0.074)1

0.063 (0.058–0.071)2

0.068 (0.067–0.069)3

Cortical astrocytes 12.3 (5.84–20.9)1

12.6 (5.65–19.6)2

9.65 (5.11–70.6)3

4.11 (3.51–4.96)1

4.19 (2.84–5.54)2

3.11 (1.67–22.5)3

0.334 (0.228–0.623)1

0.331 (0.260–0.546)2

0.322 (0.313–0.332)3

Vmax: maximal specific uptake rate, CI: 95 % confidence interval1 Woolf plot2 Eadie-Hofstee plot3 Lineweaver-Burk plot, Km: Michaelis–Menten constant

Neurochem Res

123

A corollary to the absence of neuronal high-affinity

histamine uptake system [4, 5], is that histamine is not an

important physiological substrate for the specific, Na?/Cl--

dependent monoamine transporters DAT, SERT and NET

(uptake1), which mediate neuronal monoamine reuptake

[3]. Also, histamine does not inhibit or has only a marginal

effect on Na?-dependent serotonin and dopamine uptake by

SERT and DAT, respectively [20–22]. Nevertheless, oua-

bain-sensitive, Na?-dependent histamine uptake was dem-

onstrated in neonatal as well as adult rat astrocytes [10, 13,

14], implicating a role for a Na?-coupled histamine carrier.

In agreement with these studies, we observed markedly

reduced histamine uptake in cortical astrocytes exposed to

Na?-free medium or ouabain, a Na?/K?-ATPase inhibitor.

Inhibition of Na?/K?-ATPase quickly and markedly

increases intracellular Na? concentration in astrocytes [36],

which subsequently reduces the driving force for Na?-

coupled transport. However, ouabain failed to exert any

effect on histamine transport in cerebellar and striatal

astrocytes, although its concentration was sufficient to

completely inhibit all a-isoforms of Na?/K?-ATPase,

including the ouabain-resistant a2-isoform [37]. Reduction

of histamine uptake in astrocytes incubated in the presence

Fig. 3 Effect of choline and ouabain treatment on histamine uptake

in cultured astrocytes. Histamine uptake in cerebellar (a), striatal

(b) and cortical (c) astrocytes was determined after exposure to

125 mM choline chloride in Na?-free medium or 1 mM ouabain.

Data are mean ± SEM (n = 4–8). *P \ 0.05 versus Basal

Fig. 4 Cultured astrocytes express OCT1, 2 and 3 mRNA. To

validate OCT gene expression assays OCT mRNA expression profile

was determined in rat cerebral cortex (a) and neonatal kidney (b).

OCT1-3 mRNA was then measured in cerebellar (c), striatal (d) and

cortical (e) astrocytes. ACTB was used as the endogenous control.

Data are mean ± SEM (n = 6–9, except kidney, where n = 2) and

are reported as gene expression ratios

Neurochem Res

123

of choline chloride therefore more likely represents choline-

sensitive transport as opposed to Na?-dependent transport.

Consistent with this notion, histamine uptake in hypotha-

lamic minces was inhibited by choline (IC50 = 10.5 mM)

even in the presence of NaCl [23]. Collectively, our data

suggest histamine uptake in cerebellar and striatal astro-

cytes is predominately Na?-independent, whereas hista-

mine transport in cortical astrocytes seems to involve Na?-

coupled carrier.

Histamine is a substrate for vesicular transporter

VMAT2 [6], which is responsible for packaging mono-

amines into synaptic vesicles. Moreover, histamine uptake

in hypothalamic tissue minces is inhibited by serotonin

[23]. Although serotonin is a substrate for OCTs [27, 28],

maximal inhibition caused by OCT inhibitors D22 and

corticosterone in hypothalamic minces was markedly lower

compared to serotonin-sensitive fraction of specific hista-

mine uptake [23], indicating serotonin does not block only

OCT-mediated histamine uptake. A putative histamine

transporter might therefore share the structural and phar-

macological characteristics with other biogenic amine

transporters. Also, it may be sensitive to uptake1 inhibitors.

Consistent with this notion, we have previously observed

reduced histamine accumulation in neonatal and adult

cortical astrocytes upon exposure to non-selective uptake1

inhibitor amitriptyline as well as selective NET inhibitor

desipramine [12, 13]. In contrast, excepting a minor effect

of desipramine, histamine uptake in cerebellar and striatal

astrocytes was resistant to uptake1 inhibitors. Interestingly,

amitriptyline is also inhibitor of Na?/K? ATPase [48, 49],

indicating inhibition of Na?/K? ATPase rather than

blocking histamine transport directly could be the main

mechanism behind reduction of histamine uptake in corti-

cal astrocytes. Indeed, amitriptyline reduced histamine

uptake in ouabain-sensitive cortical rat astrocytes [13], but

not in ouabain-resistant cerebellar and striatal astrocytes.

Similarly, amitriptyline diminished histamine uptake into

chick glial cells [11] and rat brain synaptosomes [7], which

both displayed Na?-dependent and/or ouabain-sensitive

histamine transport. Taken together our findings demon-

strate that unlike astrocytes derived from brain cortex,

cultured cerebellar and striatal astrocytes possess anti-

depressant-insensitive histamine uptake, again suggesting

different regional properties of histamine uptake.

Histamine uptake in cerebellar and striatal astrocytes

was partially choline-sensitive and, as assessed by ouabain,

not dependent on Na?-gradient. In conjunction with OCT1,

2 and 3 mRNA expression, this suggested a role for OCT2

which transports both choline and histamine [26, 41, 50]. In

contrast, OCT1 mediates choline, but not histamine uptake

Fig. 5 Effect of OCT (a) and uptake1 (b) inhibitors on histamine uptake in cultured astrocytes. Total histamine uptake was determined after

exposure to 125 nM [3H]-histamine in the absence or presence of uptake inhibitors. Results are mean ± SEM (n = 6–9). *P \ 0.05 versus Basal

Neurochem Res

123

[25–27, 51]. On the other hand, while OCT3 could con-

tribute to histamine uptake [27, 28], its lowly expression

did not indicate a major role for OCT3-mediated transport.

However, selective OCT inhibitors D22 and corticosterone

did not alter histamine uptake in cerebellar and striatal

astrocytes. Furthermore, OCTs are electrogenic, potential-

sensitive transporters [26]. Depolarization due to inhibition

of Na?/K?-ATPase by ouabain [37] could therefore be

expected to affect uptake of histamine, which is positively

charged under physiological conditions [14]. These results

essentially precluded major involvement of OCTs at least

in histamine concentration range tested in this study, which

was well below Km value for OCT2 and OCT3 [27, 28].

Consistent with our findings, histamine concentration in

cerebellum remained similar between OCT3 knock-out

mice and controls [32]. Similarly, histamine concentration

in striatum of OCT3-deficient mice was unaltered com-

pared with wild type controls under basal conditions as

well as during post-ischemic reperfusion [31]. Collectively,

these data indicate OCT2 and OCT3 do not mediate high-

affinity histamine uptake observed in cultured cerebellar

and striatal astrocytes.

Another interesting candidate for histamine transporter

in the brain is plasma membrane monoamine transporter

(PMAT), a Na?/Cl--independent, potential-sensitive non-

specific organic cation transporter [28, 52]. PMAT medi-

ates low-affinity histamine uptake and is sensitive to D22

and choline, but is not inhibited by corticosterone [28, 52].

Our pharmacological data in cortical astrocytes are there-

fore consistent with possible involvement of PMAT.

However, whereas PMAT is expressed in human astrocy-

toma cells [53], its expression in rodent astroglia has not

been demonstrated [38, 54], indicating PMAT does not

underlie histamine uptake in cultured rat astrocytes.

Although other non-specific organic cation carriers might

be involved in histamine uptake, it is difficult to speculate

on their identity since PMAT and OCTs are the only

known histamine carriers aside from the vesicular trans-

porter VMAT2. Nevertheless, our data suggest that hista-

mine and choline might share transporters other than

OCT2. This idea is tangible since a substantial fraction of

choline-sensitive histamine uptake in hypothalamic minces

appears to be resistant to OCT inhibitors [23]. Interest-

ingly, astrocytes express carnitine/organic cation trans-

porter (OCTN) 2 and choline transporter-like protein 1

(CTL1) [55, 56], which can mediate Na?-independent

uptake of a range of organic cations, including choline and

several OCT substrates. Also, transport of some organic

cations by OCTN2 is potential-insensitive, which resem-

bles our observations in ouabain-treated striatal and cere-

bellar astrocytes. Furthermore, CTL1-mediated transport is

sensitive to D22 and desipramine, but is not inhibited by

corticosterone [56], which mimics properties of histamine

uptake in cortical astrocytes. Despite these similarities it

remains to be seen if OCTN2 and/or CTL1 could play a

role in histamine uptake, not least because histamine does

not depress OCTN2-mediated uptake of carnitine, its pri-

mary substrate [57].

Cell culture represents an artificial milieu, where cul-

tured astrocytes are deprived of normal environmental

cues. Moreover, differences in media compositions or sera

used may directly affect phenotypic properties of cultured

astrocytes [58], including expression of neurotransmitter

carriers [59]. For example, cell culture factors provide one

possible reason why OCT mRNA expression pattern was

different between brain cortex and cultured cortical astro-

cytes. On the other hand, observed differences in OCT

mRNA expression may simply reflect the fact that cortical

homogenates contain heterogeneous cell population. As

well as astrocytes, brain tissue homogenates contain neu-

rons, microglia, oligodendroglia and endothelial cells,

while we investigated OCT expression and histamine

uptake in primary astrocyte cultures. Importantly, pro-

nounced regional differences in OCT distribution between

neurons and astrocytes have been described [29]. Further-

more, primary astrocytes in cell culture seem to retain

biological characteristics related to their area of origin.

Indeed, variations in uptake of serotonin and glutamate in

cultured astrocytes derived from different brain regions

corresponded to regional differences under in vivo condi-

tions [60]. Nevertheless, only limited extrapolations are

possible in our experimental model owing to limited

knowledge of regional variation in histamine uptake in

neonatal rat brain. Also, since ontogenetic development

affects organization of histaminergic system and brain

histamine levels in brain [61], cultured neonatal astrocyte

may not reflect the situation in adult rats. Notwithstanding

these caveats, its tempting to speculate that high-capacity,

intermediate-affinity histamine uptake in cerebellar astro-

cytes may reflect low histamine turnover rate, an indication

of low activity of histaminergic neurons, in cerebellum

[62–64]. In contrast, markedly higher histamine turnover in

striatum [62–64] may necessitate high-affinity uptake for

timely termination of histaminergic neurotransmission.

To summarize, we have demonstrated that astrocytes

derived from cerebellum and striatum possess intermediate-

or high-affinity, saturable histamine uptake. Furthermore, at

least two high-affinity transporters appear to be involved in

astrocytes derived from different brain regions. Na?-inde-

pendent/choline-sensitive carrier predominates in cerebellar

and striatal astrocytes, whereas Na?-dependent transporter

underlies histamine uptake in cortical astrocytes. Collec-

tively, our data therefore indicate existence of regional

variations in characteristics of astrocyte histamine uptake.

These observations allow us to predict subtle differences

in the fine tuning of histaminergic neurotransmission in

Neurochem Res

123

different neonatal rat brain regions. Moreover, our

findings implicate a role for histamine transporters in

regulation of extracellular histamine concentration in

cerebellum and striatum. Inhibition of histamine uptake

might therefore represent a viable option to modulate

histaminergic neurotransmission.

Acknowledgments This work was supported by the research grant

P3-067, J1-2014 of Ministry of Higher Education, Science and

Technology, Republic of Slovenia. We greatly appreciate technical

assistance of Mrs Jozica Kosir.

Conflict of interest The authors declare that they have no conflict

of interest.

References

1. Haas HL, Sergeeva OA, Selbach O (2008) Histamine in the

nervous system. Physiol Rev 88(3):1183–1241. doi:10.1152/

physrev.00043.2007

2. Passani MB, Blandina P (2011) Histamine receptors in the CNS

as targets for therapeutic intervention. Trends Pharmacol Sci

32(4):242–249. doi:10.1016/j.tips.2011.01.003

3. Iversen L (2006) Neurotransmitter transporters and their impact

on the development of psychopharmacology. Br J Pharmacol

147(Suppl 1):S82–S88. doi:10.1038/sj.bjp.0706428

4. Schwartz JC, Arrang JM, Garbarg M, Pollard H, Ruat M (1991)

Histaminergic transmission in the mammalian brain. Physiol Rev

71(1):1–51

5. Haas H, Panula P (2003) The role of histamine and the tubero-

mamillary nucleus in the nervous system. Nat Rev Neurosci

4(2):121–130. doi:10.1038/nrn1034

6. Merickel A, Edwards RH (1995) Transport of histamine by

vesicular monoamine transporter-2. Neuropharmacology 34(11):

1543–1547

7. Sakurai E, Oreland L, Nishiyama S, Kato M, Watanabe T, Yanai

K (2006) Evidence for the presence of histamine uptake into the

synaptosomes of rat brain. Pharmacology 78(2):72–80. doi:

10.1159/000095637

8. Barnes WG, Hough LB (2002) Membrane-bound histamine N-

methyltransferase in mouse brain: possible role in the synaptic

inactivation of neuronal histamine. J Neurochem 82(5):1262–1271

9. Rafalowska U, Waskiewicz J, Albrecht J (1987) Is neurotrans-

mitter histamine predominantly inactivated in astrocytes? Neu-

rosci Lett 80(1):106–110

10. Huszti Z, Imrik P, Madarasz E (1994) [3H]histamine uptake and

release by astrocytes from rat brain: effects of sodium depriva-

tion, high potassium, and potassium channel blockers. Neuro-

chem Res 19(10):1249–1256

11. Huszti Z, Rimanoczy A, Juhasz A, Magyar K (1990) Uptake,

metabolism, and release of [3H]-histamine by glial cells in pri-

mary cultures of chicken cerebral hemispheres. Glia 3(3):

159–168. doi:10.1002/glia.440030303

12. Osredkar D, Burnik-Papler T, Pecavar B, Kralj-Iglic V, Krzan M

(2009) Kinetic and pharmacological properties of [(3)H]-hista-

mine transport into cultured type 1 astrocytes from neonatal rats.

Inflamm Res 58(2):94–102. doi:10.1007/s00011-009-8103-4

13. Perdan-Pirkmajer K, Pirkmajer S, Cerne K, Krzan M (2012)

Molecular and kinetic characterization of histamine transport into

adult rat cultured astrocytes. Neurochem Int 61(3):415–422. doi:

10.1016/j.neuint.2012.05.002

14. Perdan-Pirkmajer K, Mavri J, Krzan M (2010) Histamine

(re)uptake by astrocytes: an experimental and computational

study. J Mol Model 16(6):1151–1158. doi:10.1007/s00894-

009-0624-9

15. Huszti Z, Magyar K, Kalman M (1990) Contribution of glial cells

to histamine inactivation. Agents Actions 30(1–2):237–239

16. Huszti Z, Prast H, Tran MH, Fischer H, Philippu A (1998) Glial

cells participate in histamine inactivation in vivo. Naunyn

Schmiedebergs Arch Pharmacol 357(1):49–53

17. Baganz NL, Horton RE, Calderon AS, Owens WA, Munn JL,

Watts LT, Koldzic-Zivanovic N, Jeske NA, Koek W, Toney GM,

Daws LC (2008) Organic cation transporter 3: keeping the brake

on extracellular serotonin in serotonin-transporter-deficient mice.

Proc Natl Acad Sci USA 105(48):18976–18981. doi:10.1073/

pnas.0800466105

18. Pickel VM, Chan J (1999) Ultrastructural localization of the

serotonin transporter in limbic and motor compartments of the

nucleus accumbens. J Neurosci 19(17):7356–7366

19. Takeda H, Inazu M, Matsumiya T (2002) Astroglial dopamine

transport is mediated by norepinephrine transporter. Naunyn

Schmiedebergs Arch Pharmacol 366(6):620–623. doi:10.1007/

s00210-002-0640-0

20. Giros B, el Mestikawy S, Bertrand L, Caron MG (1991) Cloning

and functional characterization of a cocaine-sensitive dopamine

transporter. FEBS Lett 295(1–3):149–154

21. Ramamoorthy S, Bauman AL, Moore KR, Han H, Yang-Feng T,

Chang AS, Ganapathy V, Blakely RD (1993) Antidepressant- and

cocaine-sensitive human serotonin transporter: molecular clon-

ing, expression, and chromosomal localization. Proc Natl Acad

Sci USA 90(6):2542–2546

22. Demchyshyn LL, Pristupa ZB, Sugamori KS, Barker EL, Blakely

RD, Wolfgang WJ, Forte MA, Niznik HB (1994) Cloning,

expression, and localization of a chloride-facilitated, cocaine-

sensitive serotonin transporter from Drosophila melanogaster.

Proc Natl Acad Sci USA 91(11):5158–5162

23. Gasser PJ, Lowry CA, Orchinik M (2006) Corticosterone-sensi-

tive monoamine transport in the rat dorsomedial hypothalamus:

potential role for organic cation transporter 3 in stress-induced

modulation of monoaminergic neurotransmission. J Neurosci

26(34):8758–8766. doi:10.1523/JNEUROSCI.0570-06.2006

24. Baganz N, Horton R, Martin K, Holmes A, Daws LC (2010)

Repeated swim impairs serotonin clearance via a corticosterone-

sensitive mechanism: organic cation transporter 3, the smoking

gun. J Neurosci 30(45):15185–15195. doi:10.1523/JNEURO

SCI.2740-10.2010

25. Grundemann D, Liebich G, Kiefer N, Koster S, Schomig E

(1999) Selective substrates for non-neuronal monoamine trans-

porters. Mol Pharmacol 56(1):1–10

26. Schomig E, Lazar A, Grundemann D (2006) Extraneuronal mono-

amine transporter and organic cation transporters 1 and 2: a review of

transport efficiency. Handb Exp Pharmacol 175:151–180

27. Amphoux A, Vialou V, Drescher E, Bruss M, Mannoury La Cour

C, Rochat C, Millan MJ, Giros B, Bonisch H, Gautron S (2006)

Differential pharmacological in vitro properties of organic cation

transporters and regional distribution in rat brain. Neurophar-

macology 50(8):941–952. doi:10.1016/j.neuropharm.2006.01.005

28. Duan H, Wang J (2010) Selective transport of monoamine neu-

rotransmitters by human plasma membrane monoamine trans-

porter and organic cation transporter 3. J Pharmacol Exp Ther

335(3):743–753. doi:10.1124/jpet.110.170142

29. Cui M, Aras R, Christian WV, Rappold PM, Hatwar M, Panza J,

Jackson-Lewis V, Javitch JA, Ballatori N, Przedborski S, Tieu K

(2009) The organic cation transporter-3 is a pivotal modulator of

neurodegeneration in the nigrostriatal dopaminergic pathway.

Proc Natl Acad Sci USA 106(19):8043–8048. doi:10.1073/

pnas.0900358106

Neurochem Res

123

30. Gasser PJ, Orchinik M, Raju I, Lowry CA (2009) Distribution of

organic cation transporter 3, a corticosterone-sensitive mono-

amine transporter, in the rat brain. J Comp Neurol 512(4):

529–555. doi:10.1002/cne.21921

31. Zhu P, Hata R, Ogasawara M, Cao F, Kameda K, Yamauchi K,

Schinkel AH, Maeyama K, Sakanaka M (2012) Targeted dis-

ruption of organic cation transporter 3 (Oct3) ameliorates ische-

mic brain damage through modulating histamine and regulatory T

cells. J Cereb Blood Flow Metab 32(10):1897–1908. doi:10.1038/

jcbfm.2012.92

32. Vialou V, Balasse L, Callebert J, Launay JM, Giros B, Gautron S

(2008) Altered aminergic neurotransmission in the brain of

organic cation transporter 3-deficient mice. J Neurochem 106(3):

1471–1482. doi:10.1111/j.1471-4159.2008.05506.x

33. Krzan M, Schwartz JP (2006) Histamine transport in neonatal and

adult astrocytes. Inflamm Res 55(Suppl 1):S36–S37. doi:10.1007/

s00011-005-0031-3

34. Schwartz JP, Wilson DJ (1992) Preparation and characterization

of type 1 astrocytes cultured from adult rat cortex, cerebellum,

and striatum. Glia 5(1):75–80. doi:10.1002/glia.440050111

35. Tuomi JM, Voorbraak F, Jones DL, Ruijter JM (2010) Bias in the

Cq value observed with hydrolysis probe based quantitative PCR

can be corrected with the estimated PCR efficiency value.

Methods 50(4):313–322. doi:10.1016/j.ymeth.2010.02.003

36. Rose CR, Waxman SG, Ransom BR (1998) Effects of glucose

deprivation, chemical hypoxia, and simulated ischemia on Na?

homeostasis in rat spinal cord astrocytes. J Neurosci 18(10):3554–3562

37. Chibalin AV, Heiny JA, Benziane B, Prokofiev AV, Vasiliev AV,

Kravtsova VV, Krivoi II (2012) Chronic nicotine modifies skel-

etal muscle Na, K-ATPase activity through its interaction with

the nicotinic acetylcholine receptor and phospholemman. PLoS

ONE 7(3):e33719. doi:10.1371/journal.pone.0033719

38. Dahlin A, Xia L, Kong W, Hevner R, Wang J (2007) Expression

and immunolocalization of the plasma membrane monoamine

transporter in the brain. Neuroscience 146(3):1193–1211. doi:

10.1016/j.neuroscience.2007.01.072

39. Daws LC (2009) Unfaithful neurotransmitter transporters: focus

on serotonin uptake and implications for antidepressant efficacy.

Pharmacol Ther 121(1):89–99. doi:10.1016/j.pharmthera.2008.

10.004

40. Wu X, Kekuda R, Huang W, Fei YJ, Leibach FH, Chen J,

Conway SJ, Ganapathy V (1998) Identity of the organic cation

transporter OCT3 as the extraneuronal monoamine transporter

(uptake2) and evidence for the expression of the transporter in the

brain. J Biol Chem 273(49):32776–32786

41. Arndt P, Volk C, Gorboulev V, Budiman T, Popp C, Ulzheimer-

Teuber I, Akhoundova A, Koppatz S, Bamberg E, Nagel G,

Koepsell H (2001) Interaction of cations, anions, and weak base

quinine with rat renal cation transporter rOCT2 compared with

rOCT1. Am J Physiol Renal Physiol 281(3):F454–F468

42. Hayer-Zillgen M, Bruss M, Bonisch H (2002) Expression and

pharmacological profile of the human organic cation transporters

hOCT1, hOCT2 and hOCT3. Br J Pharmacol 136(6):829–836.

doi:10.1038/sj.bjp.0704785

43. Perdan K, Kobe Z, Krzan M (2009) Nature of histamine transport

in neonatal rat cultured type 1 astrocytes–organic cation trans-

porters are not involved. Inflamm Res 58(Suppl 1):32–33. doi:

10.1007/s00011-009-0655-9

44. Huszti Z (1998) Carrier-mediated high affinity uptake system for

histamine in astroglial and cerebral endothelial cells. J Neurosci Res

51(5):551–558. doi:10.1002/(SICI)1097-4547(19980301)51:5\551:

AID-JNR1[3.0.CO;2-E

45. Huszti Z (2003) Histamine uptake into non-neuronal brain cells.

Inflamm Res 52(Suppl 1):S03–S06

46. Giannoni P, Passani MB, Nosi D, Chazot PL, Shenton FC, Medhurst

AD, Munari L, Blandina P (2009) Heterogeneity of histaminergic

neurons in the tuberomammillary nucleus of the rat. Eur J Neurosci

29(12):2363–2374. doi:10.1111/j.1460-9568.2009.06765.x

47. Rinne JO, Anichtchik OV, Eriksson KS, Kaslin J, Tuomisto L,

Kalimo H, Roytta M, Panula P (2002) Increased brain histamine

levels in Parkinson’s disease but not in multiple system atrophy.

J Neurochem 81(5):954–960

48. Carfagna MA, Muhoberac BB (1993) Interaction of tricyclic drug

analogs with synaptic plasma membranes: structure-mechanism

relationships in inhibition of neuronal Na?/K(?)-ATPase activ-

ity. Mol Pharmacol 44(1):129–141

49. Sanganahalli BG, Joshi PG, Joshi NB (2000) Differential effects

of tricyclic antidepressant drugs on membrane dynamics–a fluo-

rescence spectroscopic study. Life Sci 68(1):81–90

50. Sweet DH, Miller DS, Pritchard JB (2001) Ventricular choline trans-

port: a role for organic cation transporter 2 expressed in choroid plexus.

J Biol Chem 276(45):41611–41619. doi:10.1074/jbc.M108472200

51. Koepsell H, Lips K, Volk C (2007) Polyspecific organic cation

transporters: structure, function, physiological roles, and bio-

pharmaceutical implications. Pharm Res 24(7):1227–1251. doi:

10.1007/s11095-007-9254-z

52. Engel K, Wang J (2005) Interaction of organic cations with a

newly identified plasma membrane monoamine transporter. Mol

Pharmacol 68(5):1397–1407. doi:10.1124/mol.105.016832

53. Engel K, Zhou M, Wang J (2004) Identification and characteriza-

tion of a novel monoamine transporter in the human brain. J Biol

Chem 279(48):50042–50049. doi:10.1074/jbc.M407913200

54. Vialou V, Balasse L, Dumas S, Giros B, Gautron S (2007)

Neurochemical characterization of pathways expressing plasma

membrane monoamine transporter in the rat brain. Neuroscience

144(2):616–622. doi:10.1016/j.neuroscience.2006.09.058

55. Inazu M, Takeda H, Maehara K, Miyashita K, Tomoda A,

Matsumiya T (2006) Functional expression of the organic cation/

carnitine transporter 2 in rat astrocytes. J Neurochem

97(2):424–434. doi:10.1111/j.1471-4159.2006.03757.x

56. Inazu M, Takeda H, Matsumiya T (2005) Molecular and func-

tional characterization of an Na ? -independent choline trans-

porter in rat astrocytes. J Neurochem 94(5):1427–1437. doi:

10.1111/j.1471-4159.2005.03299.x

57. Ohashi R, Tamai I, Nezu Ji J, Nikaido H, Hashimoto N, Oku A,

Sai Y, Shimane M, Tsuji A (2001) Molecular and physiological

evidence for multifunctionality of carnitine/organic cation trans-

porter OCTN2. Mol Pharmacol 59(2):358–366

58. Codeluppi S, Gregory EN, Kjell J, Wigerblad G, Olson L,

Svensson CI (2011) Influence of rat substrain and growth con-

ditions on the characteristics of primary cultures of adult rat

spinal cord astrocytes. J Neurosci Methods 197(1):118–127. doi:

10.1016/j.jneumeth.2011.02.011

59. Kimelberg HK, Goderie SK, Conley PA, Higman S, Goldschmidt

R, Amundson RH (1992) Uptake of [3H]serotonin and [3H]glu-

tamate by primary astrocyte cultures. I. Effects of different sera

and time in culture. Glia 6 (1):1–8. doi:10.1002/glia.440060102

60. Amundson RH, Goderie SK, Kimelberg HK (1992) Uptake of

[3H]serotonin and [3H]glutamate by primary astrocyte cultures.

II. Differences in cultures prepared from different brain regions.

Glia 6(1):9–18. doi:10.1002/glia.440060103

61. Molina-Hernandez A, Diaz NF, Arias-Montano JA (2012) His-

tamine in brain development. J Neurochem 122(5):872–882. doi:

10.1111/j.1471-4159.2012.07863.x

62. Nishibori M, Oishi R, Saeki K (1984) Histamine turnover in the

brain of different mammalian species: implications for neuronal

histamine half-life. J Neurochem 43(6):1544–1549

63. Oishi R, Nishibori M, Saeki K (1984) Regional differences in the

turnover of neuronal histamine in the rat brain. Life Sci

34(7):691–699

64. Hough LB, Khandelwal JK, Green JP (1984) Histamine turnover

in regions of rat brain. Brain Res 291(1):103–109

Neurochem Res

123