First published online January 17, 2007
Journal of Experimental Biology 210, 541-552 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02681
Trimethylamine oxide suppresses stress-induced alteration of organic anion transport in choroid plexus
Alice R. A. Villalobos1,2,* and
J. Larry Renfro1,3
1 Center for Membrane Toxicological Studies, Mount Desert Island Biological
Laboratory, Salisbury Cove, ME 04672, USA
2 Environmental Medicine, University of Rochester, Rochester, NY 14642,
USA
3 Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269,
USA
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Fig. 1. Two examples of unidirectional and net fluxes of 10 µmol
l–1 [14C]2,4-dichlorophenoxyacetic acid (2,4-D) in
freshly isolated shark IVth choroid plexus (CP) under short-circuited
conditions. (A) Unidirectional absorptive [cerebrospinal fluid-to-blood
(CSF-to-Bl)] and secretory [Bl-to-CSF] fluxes of 2,4-D in paired halves of
IVth CP mounted in Ussing chambers under short-circuited conditions in
elasmobranch Ringer solution. Fluxes were initiated at t=0 by
addition of isotopically labeled 2,4-D. Steady-state flux was apparent by
t=0.5 h. (B) Alternate method of determining unidirectional and net
absorptive flux of 2,4-D in a single one-half IVth CP under short-circuited
conditions. Flux was initiated at t=0 by addition of isotopically
labeled 2,4-D to the CSF compartment without inhibitor. At t=1 h, 100
µmol l–1 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was
added to both blood and CSF compartments, and 10 mmol l–1
para-aminohippuric acid (PAH) was added only to the CSF compartment
(arrow); unidirectional flux was measured for an additional hour. Net flux was
calculated as the difference between the initial 1-h flux (total flux) and
second 1-h flux (inhibitor-insensitive flux).
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Fig. 2. Trimethylamine oxide (TMAO)-sensitivity of heat-induced change in net
active cerebrospinal fluid-to-blood (CSF-to-blood)
[14C]2,4-dichlorophenoxyacetic acid (2,4-D) fluxes across paired
halves of shark IVth choroid plexus following 48 h in explant culture. Fluxes
of 2,4-D were determined after tissues were exposed, ± 72 mmol
l–1 TMAO, to treatment as follows. Control, 7.5 h at
13.5°C;  +5°C heat stress, 18.5°C for 6 h plus recovery at
13.5°C for 1.5 h; +10°C heat stress, 5 h at 13.5°C then 1 h
at 23.5°C followed by recovery at 13.5°C for 1.5 h. Tabulated data
show the mean unidirectional fluxes from which net data were derived. B to C,
blood-to-CSF flux; C to B, CSF-to-blood flux. Data are means ± s.e.m.
(N=6). *Significant effect of TMAO at
P<0.05.
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Fig. 3. Heat shock protein (hsp) accumulation in explanted shark lateral choroid
plexus (CP) after thermal stress in the presence and absence of trimethylamine
oxide (TMAO). Lateral CPs from individual sharks were each divided into three
equal-sized segments, totaling six pieces; tissues were separated into three
pairs and incubated, ± 72 mmol l–1 TMAO, at 13.5°C
for 7.5 h ( ), at 18.5°C for 6 h plus recovery at 13.5°C for 1.5 h
(+5°C), or 23.5°C for 1 h plus recovery at 13.5°C for 1.5 h
(+10°C). (A) Representative immunoblot for Hsp70 and actin accumulation in
lysates of non-heated and heat-stressed lateral CP. (B) Graphical summation of
Hsp70 accumulation (normalized to actin accumulation) in non-heated and
heat-stressed lateral CP (means ± s.e.m., N=4). For each
condition, fold-induction of heat shock protein was calculated by dividing the
normalized Hsp70 accumulation in the absence of TMAO by the normalized Hsp70
accumulation in the presence of TMAO. *Significantly different from
paired tissue treated in the presence of TMAO at P<0.05.
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Fig. 4. Trimethylamine oxide (TMAO)-sensitivity of zinc-induced change in active
cerebrospinal fluid-to-blood (CSF-to-blood)
[14C]2,4-dichlorophenoxyacetic acid (2,4-D) fluxes across paired
halves of shark IVth choroid plexus. Fluxes of 2,4-D were determined after
tissues were exposed, ± 72 mmol l–1 TMAO (13.5°C),
to treatment as follows. Control, 6 h in zinc-free medium + 1.5 h recovery in
L-15E; Zinc-treated, 6 h in 50 µmol l–1 ZnSO4 +
1.5 h recovery in L-15E. Tabulated data show the mean unidirectional fluxes
from which net data were derived; B to C, blood-to-CSF flux; C to B,
CSF-to-blood flux. Data are means ± s.e.m. (N=6).
*Significant effect of zinc at P<0.05.
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Fig. 5. Heat shock protein accumulation in isolated shark lateral choroid plexus
(CP) after zinc exposure in the presence and absence of TMAO. Each lateral CP
from an individual shark was divided in half yielding four segments. Paired
tissue segments were incubated, ± 72 mmol l–1 TMAO
(13.5°C), with 0 or 50 µmol l–1 ZnSO4 for 6
h; all tissues were then incubated in zinc-free medium with TMAO for 1.5 h.
(A) Representative immunoblot for Hsp70 and actin accumulation in lysates of
zinc-free and zinc-exposed lateral CP. (B) Graphical summation of Hsp70
induction by zinc ± TMAO in lateral CP. Hsp70 accumulation was
normalized to that of actin. For zinc exposure with TMAO, fold-induction of
Hsp70 was calculated by dividing the normalized Hsp70 accumulation in
zinc-treated tissue by that in non-exposed timed-control tissue incubated with
TMAO. Likewise, for zinc exposure without TMAO, fold-induction of Hsp70 was
calculated by dividing Hsp70 accumulation in zinc-treated tissue by that in
non-exposed timed-control tissue incubated without TMAO (means ±
s.e.m., N=4; *P<0.05 vs
fold-induction with TMAO).
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Fig. 6. KNK437-sensitivity of zinc-induced change in active cerebrospinal
fluid-to-blood (CSF-to-blood) [14C]2,4-dichlorophenoxyacetic acid
(2,4-D) fluxes across paired halves of shark IVth choroid plexus in the
absence of trimethylamine oxide (TMAO). Fluxes of 2,4-D were determined after
tissues were exposed at 13.5°C, ± 100 µmol l–1
KNK437 (vehicle control was 0.2% DMSO), to treatment as follows. Zinc-treated,
6 h in 50 µmol l–1 ZnSO4 + 1.5 h recovery in
L-15E (N=8); Control, 6 h in zinc-free medium + 1.5 h recovery in
L-15E (N=7). Tabulated data show the mean unidirectional fluxes from
which net data were derived. B to C, blood-to-CSF flux; C to B, CSF-to-blood
flux. Data are means ± s.e.m.; *P<0.05
vs net flux in paired tissue treated without KNK437.
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Fig. 7. Heat shock protein accumulation in isolated shark lateral choroid plexus
(CP) after zinc exposure with KNK437 in absence of trimethylamine oxide
(TMAO). Lateral CPs from an individual shark were divided, yielding four
equal-sized segments; each segment was incubated at 13.5°C in TMAO-free
L15E medium. Tissues were incubated without zinc ± 100 µmol
l–1 KNK437 or with 50 µmol l–1
ZnSO4 ± 100 µmol l–1 KNK437 for 6 h; all
tissues were then incubated for 1.5 h in zinc-free medium containing TMAO
± 100 µmol l–1 KNK437. For tissues not treated with
KNK437, medium contained 0.2% DMSO (vehicle). (A) Representative immunoblot
analysis of Hsp70 and actin accumulation in lysates of lateral CP treated in
the absence of TMAO without zinc or with zinc ± KNK437. (B) Graphical
comparison of Hsp70 induction by zinc exposure ± KNK437 in the absence
of TMAO (mean ± s.e.m.; N=3). Fold-induction of Hsp70 was
calculated by dividing Hsp70 accumulation (normalized to actin) in
zinc-treated tissue without or with KNK437 by the normalized Hsp70
accumulation in timed-control tissue incubated without KNK437.
*P<0.05 vs fold-induction in control and
**P<0.05 vs fold-induction in zinc only,
respectively.
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Fig. 8. Expression of Hsp70 mRNA in isolated lateral choroid plexus (CP) following
+10°C heat shock or zinc exposure in the presence and absence of
trimethylamine oxide (TMAO). Levels of Hsp70 mRNA were compared in non-treated
and stressed lateral CP tissues obtained from the same shark. +10°C heat
stress: tissue segments were incubated in medium with and without TMAO at
13.5°C for 7.5 h () or at 23.5°C for 1 h then 13.5°C for 1.5
h (+10°C). Zinc exposure/recovery: tissue segments were incubated at
13.5°C in medium with and without TMAO for 7.5 h without zinc (No Zn) or
with 50 µmol l–1 ZnSO4 for 6 h then without
zinc for an additional 1.5 h (Zn/Rec). Relative levels of hsp70 mRNA in
non-treated and stressed tissues were analyzed by semi-quantitative RT-PCR.
Shown here is an EtBr-stained 1.5% agarose gel on which aliquots of PCR
products from representative sets of heat-stressed and zinc-exposed tissue,
along with a DNA ladder, were electrophoresed; N=2–3 for each
experiment.
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Fig. 9. Electron micrographs of isolated shark choroid plexus (CP) subjected in
vitro to heat stress or zinc exposure in complete culture medium (L-15E).
(A–E) Scanning electron micrographs of dogfish shark IVth CP ventricular
surface (CSF-side); (F–J) transmission electron micrographs of shark
IVth CP. (A,F) Freshly harvested tissue immediately processed for fixation.
(B,G) Tissues held at 13.5°C for 7.5 h in L-15E. (C,H) Tissues incubated
at 18.5°C for 6 h followed by incubation at 13.5°C for 1.5 h. (D,I)
Tissues incubated initially at 13.5°C for 5 h, then at 23.5°C for 1 h
followed by 13.5°C for 1.5 h. (E,J) Tissues incubated with 50 µmol
l–1 ZnSO4 at 13.5°C for 6 h followed by
recovery in zinc-free medium at 13.5°C for 1.5 h. mv, microvilli; v,
ventricular or cerebrospinal fluid-side; g, fat globule. Scale bars, 5
µm.
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© The Company of Biologists Ltd 2007
