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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
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
* Author for correspondence (e-mail: alice_villalobos{at}urmc.rochester.edu)
Accepted 5 December 2006
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Summary |
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70 mmol l–1). Active transepithelial absorption of the
organic anion, 2,4-dichlorophenoxyacetic acid (2,4-D), by IVth CP mounted in
Ussing chambers was measured after in vitro stress, and a marker for
the cellular stress response, inducible heat shock protein 70 (Hsp70), was
assayed by immunoblot analysis. Transient heat stress (a shift from the normal
13.5°C to 23.5°C for 1 h) decreased 2,4-D transport by 66%;
however, the same stress minus TMAO (isosmotic replacement with urea) had no
effect on transport rate. In the absence of TMAO, stress-induced Hsp70
accumulation was more than double that seen in the presence of TMAO. Likewise,
exposure to 50 µmol l–1 Zn for 6 h induced a twofold
greater Hsp70 accumulation in the absence of TMAO than in its presence, and
the higher Hsp70 level was associated with a higher 2,4-D transport rate. Heat
stress and 50 µmol l–1 Zn also induced more pronounced
increases in Hsp70 mRNA in the absence of TMAO. Thus, the cellular stress
response can significantly alter CP organic anion transport capacity, and an
endogenous osmolyte can suppress that response.
Key words: choroid plexus, heat shock response, Hsp70, 2, 4-dichlorophenoxyacetic acid transport, dogfish shark, elasmobranch
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Introduction |
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; Johanson et al.,
2000; Palm et al.,
1995). However, this brain barrier mounts a cellular stress
response (Blake et al., 1990;
Brown, 1991;
Krueger-Naug et al., 2000),
which in other tissues can be generally protective against physicochemical
stressors such as ischemia, hyperthermia, oxidants or heavy metals and may
ameliorate loss of function in CP.
An increased production of heat shock proteins (hsps) results from a
variety of cellular stresses such as hyperthermia and exposure to heavy
metals. Hsps are both chaperones that prevent premature folding of nascent
proteins and cytoprotectants that preserve and restore native conformation of
damaged proteins (Agashe and Hartl,
2000). Denatured or abnormal cellular proteins (misfolded) can
signal an increased activation of the heat shock genes
(Hightower, 1980;
Ananthan et al., 1986;
Hightower, 1991). The
resultant increase in hsp accumulation can improve tolerance of subsequent
stress of equal or greater severity, and such conditioning may predispose the
cell or organism to survive an otherwise lethal stress. In many cases, the
conditioning stress may preserve or enhance certain cell and tissue functions
(Calabrese, 2005). This effect
is associated with elevated cellular levels of hsps, especially
Hsp70/72 (Brown et al.,
1992; Renfro et al.,
1993; Sussman-Turner and
Renfro, 1995; Sussman and
Renfro, 1997; Hightower et
al., 2000), and in several cases increased hsp levels have been
shown to be necessary (Renfro et al.,
1993; Riabowol et al.,
1988) and sufficient (Heads et
al., 1995) for this effect.
Like hsps, many organic osmolytes are also cytoprotectants and act to
stabilize tertiary protein structure. Certain animal groups, such as marine
elasmobranchs, maintain high systemic levels of osmolytes. Sharks naturally
maintain plasma urea concentrations of 350–400 mmol l–1
and trimethylamine oxide (TMAO) concentrations of 70–80 mmol
l–1 (Goldstein et al.,
1967). The intracellular ratio of urea:TMAO + glycine betaine is
near 2:1, and these high levels of TMAO and glycine betaine counteract the
protein destabilizing effects of urea in sharks
(Somero, 1986;
Yancey and Somero, 1979;
Yancey and Somero, 1980).
Examples of protein stabilizing effects of methylamines are also found in the
mammalian renal medulla. Here, as in marine elasmobranchs, urea concentrations
and ionic strength are high enough to destabilize macromolecules
(400–600 mmol l–1), yet the renal tubule cells survive
and efficiently transport inorganic and organic solutes. Urea decreases the
thermal transition temperatures of several critical proteins in the kidney,
whereas the methylamines glycine betaine and glycerylphosphorylcholine
increase them, effectively counteracting urea and, consequently,
temperature-induced protein denaturation
(Burg and Peters, 1998). TMAO
also effectively restores the activity of certain enzymes lost by exposure to
high urea (Palmer et al.,
2000). Other relevant examples include the effective rescuing and
restoration by TMAO of the dysfunctional cystic fibrosis transmembrane
conductance regulator (CFTR) mutant F508 to functional capacity similar to
that of the native protein (Brown et al.,
1996). Proteins exhibiting temperature-sensitive folding defects,
such as the tumor suppressor p53, viral oncogene protein pp60src and ubiquitin
activating enzyme E1, are also restored to near-native phenotype by TMAO
(Brown et al., 1997).
Through their protein stabilizing effects, the osmolytes can attenuate
Hsp70 induction by hyperosmotic stress as well as temperature shock
(Sheikh-Hamad et al., 1994).
Thus, given that osmolytes may protect and salvage protein function in other
transporting epithelia, we asked whether in CP high levels of osmolytes
supplant the role of hsps and modify stress-induced changes. We had previously
established the CP (IVth and lateral) of the dogfish as a model for direct
assessment of transepithelial OA transport, demonstrating active removal or
absorption of two model substrates [2,4-dichlorophenoxyacetic acid (2,4-D) and
fluorescein] from the CSF compartment via a specific and
Na+-dependent pathway similar to the mechanism described for
mammalian CP (Villalobos et al.,
2002). The objective of the present study was to determine how
physicochemical stress in the presence and absence of the endogenous
cytoprotectant, TMAO, may alter OA transport across the vertebrate
blood–CSF barrier. The data indicate that heat shock or zinc exposure
can alter CP transepithelial OA transport and that TMAO diminishes both the
induction of Hsp70 synthesis and the capacity to actively transport OAs under
conditions of physicochemical stress.
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Materials and methods |
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2 kg body mass) were collected from the coastal waters of Mount Desert
Island, Maine, USA and held in large (11 360 liters) tanks of flowing sea
water 1–4 days before use. Experiments were conducted from the period
June through July during which tank temperature ranged from 13 to 19°C.
Animals were decapitated, and the cranial compartment immediately removed,
flooded with ice-cold elasmobranch Ringer (ER; in mmol l–1,
280 NaCl, 6 KCl, 4 CaCl2, 3 MgCl2, 1
NaH2PO4, 0.5 Na2SO4, 350 urea, 72
TMAO, 2.5 glucose and 8 NaHCO3, pH 7.8) and placed on ice. With the
cerebrum and medulla submerged in ice-cold sterile ER, the IVth and lateral
plexuses were excised and cleared of extraneous tissue. Tissues were then
either cultured for 48–72 h with or without TMAO for subsequent heat
stress treatment or incubated without or with TMAO during treatment with zinc.
The present study adheres to the newest Guiding Principles for Research as
outlined by the American Physiological Society
(American Physiological Society,
2002). All investigations involving animals reported in this study
were conducted in conformity with these principles, and the animal protocol
was approved by the Mount Desert Island Biological Laboratory IACUC (protocol
#0606; MDIBL Institutional Assurance #A3562-01).
Explant tissue culture and heat stress treatment
The effects of TMAO on heat-induced modulation of transport and Hsp70
accumulation in IVth and lateral CP, respectively, were investigated in
vitro using isolated tissues that were maintained in culture prior to
initiation of heat stress. This provided a consistent thermal history prior to
further treatment. Immediately following dissection, the IVth CP was bisected
longitudinally, and each half-plexus was rinsed four times in sterile ER.
Subsequently, one half-IVth CP was rinsed twice in sterile Leibovitz's (L-15)
medium (Gibco, Invitrogen, Carlsbad, CA, USA) modified for use with
elasmobranch tissues (L-15E) by supplementation with the following (in mmol
l–1): 142 NaCl2, 2.75 CaCl2, 2
MgCl2, 350 urea and 72 TMAO. The contralateral half-IVth plexus was
rinsed in TMAO-free L-15E medium in which TMAO was replaced isosmotically with
urea. Each half-IVth plexus was placed in a 35 mm Petri dish with 3 ml of the
respective L-15E medium at 13.5°C (humidified air) for a minimum of 48 h
before initiation of stress treatments. Lateral plexuses were prepared in a
similar manner. IVth and lateral CPs were heat-stressed in the same L15E
medium in which they were cultured. Tissues were heated to 23.5°C for 1 h
and then returned to 13.5°C for an additional 1.5 h. Corresponding
non-heated tissues were similarly handled but held at 13.5°C continuously.
At the end of treatment, IVth plexus tissues were mounted in Ussing chambers
for measurement of [14C]2,4-dichlorophenoxyacetic acid
([14C]2,4-D) transport, and segments of lateral plexus were
processed for immunoblot analysis of Hsp70 protein or semi-quantitative RT-PCR
analysis of Hsp70 mRNA.
Zinc treatment
The influences of TMAO on zinc-induced alteration of organic anion
transport and Hsp70 expression were examined in IVth and lateral CP in
vitro. Following harvest, tissues were incubated for 6 h in either L15E
or TMAO-free L-15E with or without 50 µmol l–1
ZnSO4 (13.5°C, humidified air). L-15E medium does not contain
zinc. Following exposure to zinc, all tissues (regardless of the presence of
TMAO during zinc exposure) were rinsed twice with L-15E containing TMAO and
allowed to recover in this complete medium for 1.5 h (13.5°C, humidified
air). Tissues were then mounted in Ussing chambers for measurement of
bioelectrical properties and transepithelial transport of
[14C]2,4-D. Lateral CPs were treated as described for IVth CP and
processed for analysis of Hsp70 accumulation or Hsp70 mRNA.
Transepithelial transport
Ussing flux chambers were used to determine transepithelial transport and
electrophysiological properties of IVth CP. In the dogfish, the IVth CP is a
flat sheet of epithelial tissue overlying the dorsal and lateral surfaces of
the medullary auricles. In a 2–3 kg animal, it is 3–4
cm2 of choroidal epithelium that contains the tight junctional
barriers. After treatment, each half-IVth plexus was transferred onto a piece
of nylon mesh (Nitex®, 150 µm mesh size) and mounted in an Ussing
chamber. Aperture size was 0.2 cm2. Each hemichamber was filled
with ER containing 10 µmol l–1 2,4-D, continually gassed
with humidified 99% O2–1% CO2, and magnetically
stirred. Chamber temperature was maintained at 13.5°C.
Each flux chamber was attached to an automatic voltage clamp (Model
EC4000-2; WPI, Sarasota, FL, USA) interfaced with a computer-controlled data
acquisition board (MacLab, ADI Instruments, Grand Junction, CO, USA). Methods
for determination of electrophysiological properties were described previously
(Villalobos et al., 2002).
Transepithelial electrical resistance (Rt) was used as an
indicator of structural integrity. Transepithelial fluxes of 2,4-D were
determined under short-circuited conditions, i.e. ER on both sides and
transepithelial electrical potential clamped at 0 mV. Under control
conditions, transepithelial potential difference, Rt and
short-circuit current (Isc) remained stable for at least
four hours and were similar to those previously reported
(Villalobos et al., 2002).
Unidirectional OA transport rates were determined by adding
3.7x104 Bq [14C]2,4-D to the CSF-side or
blood-side of each half-plexus as previously described
(Villalobos et al., 2002). The
net transepithelial flux of 2,4-D by a single IVth plexus was calculated by
subtracting the blood-to-CSF (secretory) flux from the CSF-to-blood
(absorptive) flux measured in its respective paired half
(Fig. 1A). In some cases,
non-mediated flux (`leak' flux) was determined after addition of 10 mmol
l–1 para-aminohippuric acid (PAH) together with 100
µmol l–1 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The
remaining uninhibited flux was considered non-mediated, and previous work
showed that it was equivalent to unidirectional blood-to-CSF flux
(Villalobos et al., 2002)
(Fig. 1B). Net active fluxes of
2,4-D (CSF to blood) in control 48-h explants (0.3±0.11 nmol
cm–2 h–1) and freshly isolated CP tissue
(0.4±0.05 nmol cm–2 h–1) were nearly
identical; however, transport rates by control 7.5-h explants averaged
considerably higher than in either of the above (see controls in Figs
2,
4).
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Immunoblot analysis
Hsp70 and actin in control and stressed lateral CP were compared by
immunoblot analysis. After treatment, tissues were rinsed in chilled ER/0.5%
Triton X-100 supplemented with phosphatase and protease inhibitors and then
lysed with sample buffer (65 mmol l–1 Tris-HCl, pH 6.8, 5%
v/v ß-mercaptoethanol, 10% v/v glycerol, 2.3% w/v SDS, 0.5% bromophenol
blue). Proteins in heat-denatured tissue lysates were separated by
electrophoresis (SDS/10% polyacrylamide gel) then electroblotted onto
polyvinylidene difluoride membranes. Membranes were then blocked with 10% milk
protein in Tris-buffered saline containing 0.1% Tween-20 (TBS-T, 1.5 h,
24°C) followed by overnight incubation (4°C) in fresh blocking buffer
with a mouse monoclonal primary antibody against either Hsp70/heat shock
cognate 70 (Hsp70/Hsc70, 1:1000; Stressgen, Victoria, BC, Canada) or
ß-actin (1:500; Sigma-Aldrich, St Louis, MO, USA). For heat shock protein
analysis, membranes were subsequently incubated in 5% milk/TBS-T with
horseradish peroxidase-conjugated secondary antibody against mouse IgG
(1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 24°C, 1.5 h);
immunoreactivity was detected using a luminol-based chemiluminescent substrate
(LumiGLO; Kirkegaard & Perry Labs, Inc., Gaithersburg, MD, USA) and
visualized on X-ray film. For actin analysis, membranes were incubated with
alkaline phosphatase-conjugated secondary antibody against mouse IgG (1:3000;
Stressgen; 24°C, 1.5 h), and immunoreactivity was detected with
chromogenic substrate, BCIP/NBT (Promega, Madison, WI, USA). For each tissue
lysate sample, the relative area and intensity of individual Hsp70 and actin
bands were quantitated by densitometry (NIH Image), and Hsp70 accumulation was
normalized to that of actin.
The antibody used for analysis of Hsp70 protein is immunoreactive with both the inducible and constitutive forms of Hsp70 in mammals, i.e. two bands are detected in mammalian tissues. For shark tissues, only a single band was detected using this antibody; its mass most closely coincides with the constitutive or cognate form of mammalian Hsp70 (70 kDa).
Isolation of mRNA and RT-PCR
Relative levels of Hsp70 mRNA in non-treated and stressed tissue segments
of lateral CP were analyzed by semi-quantitative RT-PCR. Total RNA was
isolated from experimental tissues (RNeasy® Mini kit; Qiagen, Valencia,
CA, USA). Briefly, tissue segments were triturated with guanidine
isothiocyanate-containing buffer with 1% ß-mercaptoethanol and
homogenized using a QiaShredder® spin column (Qiagen). Ethanol was added
to the homogenate, which was then loaded onto a silica-gel membrane to which
total RNA binds. Residual DNA was digested by an on-column procedure
(RNase-Free DNase; Qiagen); DNA fragments and other contaminants were removed
from the column before total RNA was eluted from the column with 30 µl
RNase-free water. For each sample, first-strand cDNA was synthesized from 2.5
µg total mRNA using Superscript III First-Strand Synthesis System SuperMix
(Invitrogen); the mixture was incubated at 65°C for 15 min, chilled on ice
for 1 min, incubated at 50°C for 50 min and finally incubated at 70°C
for 15 min to terminate the reaction. A 5-µl aliquot of each cDNA sample
was amplified by RT-PCR using the forward and reverse primers against known
sequences of Hsp70 genes in other shark species (see below). The PCR reaction
protocol was initiated with a 5-min denaturation at 95°C and followed by
35 cycles of 95°C for 40 s, 56°C for 40 s, 72°C for 40 s; the
reaction was terminated with a 7-min incubation at 72°C. A 14-µl
aliquot of each PCR product from a given experiment was electrophoresed on a
1.5%-agarose gel, along with a DNA ladder (0.5 µg, 100–1000 bp,
GeneRuler; Fermentas, Hanover, MD, USA). The gel was stained with ethidium
bromide to visualize bands of nucleic acids separated from each experimental
sample and the DNA ladder. Intensity and area of each band were analyzed and
compared using NIH Image.
Primers against Hsp70 were designed using the PrimerPremier Program (Premier Biosoft, Palo Alto, CA, USA). Primer design was based on the known Hsp70 gene sequences from 12 other shark species (species and GenBank accession numbers: Alopias superciliosus, AF502451; Alopias vulpinus, AF502457; Cetorhinus maximus, AF502489; Mitsukurina owstoni, AF502477; Isurus oxyrinchus, AF502522; Lamna ditropis, AF502462; Megachasma pelagios, AF502470; Odontaspis ferox, AF502442; Pseudocarcharias kamoharai, AF502484; Carcharias taurus, AF502436; Carcharodon carcharias, AF502529; Alopias pelagicus, AF502445). The forward and reverse primers were 5'-AGCCCAAGGTGAAGGTC-3' and 5'-TGGTGATGGAGGTGTAAAA-3' and the anticipated product size was 597 bp. The predicted range for the ideal annealing temperature was 50–56°C. To test the efficacy of these primers at various annealing temperatures, cDNA was generated from total RNA extracted from representative heat-stressed isolated lateral CP (1-h incubation at 23.5°C followed by 1.5-h incubation at 13.5°C) and amplified by RT-PCR protocols incorporating annealing temperatures of 50°C, 52°C, 54°C and 56°C. Aliquots of the PCR products generated at these respective annealing temperatures were electrophoresed on a single 1.5% agarose gel, and the area, intensity and clarity of each nucleic acid band were integrated and compared (NIH Image). The annealing temperature of 56°C yielded the greatest amount of product with minimal contamination and was utilized in the PCR protocol for comparison of relative levels of Hsp70 mRNA under various experimental conditions. The PCR product was sequenced by the Mount Desert Island Biological Laboratory DNA Sequencing Center. The sequence was CAGGTNNGANAACCTTCTCCCCGAGGAATCTCTTCCATGGTGCTGACCAAGATGAAGGAAACGGCCGAGGCTTACCTGGGCCACACCGTCACCAACGCTGTTATCACTGTGCCCGCTTACTTCAATGACTCCCAGCGCCAGGCAACCAAAGACGCTGGTGTGATCGCTGGTCTCACTGTCCTGCGTGTCATCAATGAGCCGACGGCNGCTGCCATTGCCTACGGNCTATACAAGAAGGGCANAGGTGAGCACAATGTTCTCATCTTTGACTTGNGTGGTGGTACCTTCNACGTCTCTATTCTCACCATTGACNACGGTATCTTTNANGTGAAATCNACNGNTGGTGACACCCACTTGGGANGAGAGGACTTTGATAATCNCNTGGNCANTCNCTTCATTGAGGANTTCAAGCGTAAATACAAGAAGGACATCAGTCATAACAANANGGCNGNCAGGANGCTGAGGACAGCCTGCGAGAGANCAAANAGAACCNTGTCTTCCAGCACCCNAGNNNNTATNGAGATTGACTNTCTGNTTGAAGGCATAGACTTTTACCCCTCCATCNCCAAA (GenBank accession number DQ913093). This segment of the S. acanthias Hsp70 gene was 86–88% identical to Hsp70 gene sequences previously submitted for 12 other shark species. Homology to rat and human Hsp70 genes was 84% and 79%, respectively.
Electron microscopy
After experimental treatments, segments of IVth CP (60 mg wet mass)
were fixed with 1.5% glutaraldehyde and 1.5% formaldehyde in a high
sucrose-cacodylate buffer (150 mmol l–1 sodium cacodylate, 3
mmol l–1 MgCl2 and 20% sucrose, pH 7.4) on ice for
2 h as described previously (Villalobos et
al., 2002). Tissues were rinsed in chilled buffer and stored
overnight at 4°C, post-fixed with 1% glutaraldehyde and 2% osmium
tetroxide and processed for scanning or transmission electron microscopy.
Details were reported previously
(Villalobos et al., 2002).
Chemicals
TMAO, urea, zinc sulfate, unlabeled 2,4-D, probenecid and 2,4,5-T were
purchased from Sigma-Aldrich. KNK437 (Heat Shock Protein Inhibitor I,
N-formyl-3,4-methylenedioxy-benzylidene-
-butrolactam) was
purchased from Calbiochem (La Jolla, CA, USA). [14C]2,4-D
(1.85x109 Bq mmol l–1) was purchased from
American Radiolabeled Chemical, Inc. (St Louis, MO, USA) or Moravek (Brea, CA,
USA). All other chemicals were of the highest grade and obtained from
commercial vendors.
Statistics
Data are presented as means ± standard error (s.e.m.). For 2,4-D
transepithelial flux experiments, control and experimental 60-min fluxes or
treatment-sensitive components were compared by one-tailed Student's paired
t-test. Differences were deemed significant at
P
0.05.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Mosser et al., 1986). Our
stress treatments included time for adequate synthetic activity, and, thus,
the earliest time for tissue assessment was 7.5 h post-treatment in culture
medium (13.5°C, referred to as 7.5-h-explants), at which time the
Hsp70:actin pixel density ratio had stabilized at 1.31±0.17 and was no
different in 48-h-explants (1.42±0.05). Freshly isolated tissues had an
Rt of 87±17 
cm2, which was not
significantly different from that of 7.5-h-explants
(Table 1). However, there was a
3-fold decrease in mean Rt of IVth CP to 25
cm2 in 48-h-explants (Table
1). The presence or absence of TMAO in the culture medium had no
effect on this change. The Rt in either medium was also
unaffected by zinc or heat stress treatments. Similar incubation in the
presence of 0.2% DMSO, however, significantly lowered the
Rt of 7.5-h-explants
(Table 1).
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Exposure of 48-h-explants to an acute temperature elevation of 5°C for 6 h followed by a 1.5 h recovery had no significant effect on transport with or without TMAO (Fig. 2). Acutely increasing the incubation temperature by 10°C for 1 h (henceforth referred to as heat stress) with a 1.5-h recovery at 13.5°C caused a significant decrease in net active transport of 2,4-D from the CSF to blood in the presence of TMAO. However, in the absence of TMAO (isosmotic replacement with urea), active flux was unchanged by heat stress (Fig. 2). The tabulated unidirectional fluxes shown in Fig. 2 indicate that heat stress in the presence of TMAO caused the tissues to become `leaky', i.e. transepithelial 2,4-D flux increased in both directions.
The preservation of transport with heat stress in the absence of the osmolyte was associated with greater accumulation of Hsp70. Experimental treatments to test for the effects of TMAO on stress-induced modulation of Hsp70 paralleled those implemented to investigate the effects of TMAO on stress-induced modulation of 2,4-D transport. In non-heated tissues held continuously for 48–72 h at 13.5°C, accumulation of Hsp70 in the absence of TMAO was comparable to that in the presence of TMAO (Fig. 3). In heat stressed tissues, Hsp70:actin ratios in TMAO-free conditions were, on average, 2-fold greater than the ratios in tissue heat-stressed in the presence of TMAO. Thus, the increased accumulation of Hsp70 was coincident with the tissue-level resistance of the organic anion transport machinery to heat stress.
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Zinc-induced modulation of transport of 2,4-D and Hsp70 accumulation
IVth CP tissues were incubated for 6 h (13.5°C) with or without 50
µmol l–1 ZnCl2 with TMAO or without TMAO; all
tissues were then rinsed and incubated for an additional 1.5-h recovery in
zinc-free medium containing TMAO. Irrespective of the presence of TMAO,
Rt was not altered by zinc exposure
(Table 1). However, zinc
exposure in the absence of TMAO markedly increased net 2,4-D transport from
the CSF side to the blood side (Fig.
4); active transport increased by an average of 40%. By
contrast, zinc exposure in the presence of TMAO had no effect on transport.
The zinc-induced increase in net active transport appeared to be due mostly to
an increase in the unidirectional CSF-to-blood flux; there was little change
in blood-to-CSF flux.
Induction of Hsp70 in CP exposed in vitro to zinc in the presence and absence of TMAO is shown in Fig. 5. In both the presence and absence of TMAO, zinc exposure induced increased accumulation of Hsp70. In tissues incubated with TMAO, zinc increased the accumulation of Hsp70 by 1.36-fold as compared to levels of the protein in tissues not exposed to zinc. However, in tissues incubated without TMAO, zinc exposure increased accumulation of Hsp70 by 2.65-fold as compared to levels of stress protein in TMAO-free tissues not treated with zinc. Furthermore, the pixel density ratio of Hsp70 to that of actin in tissues exposed to zinc in the absence of TMAO was roughly 2-fold greater than the ratio in tissues exposed to the metal in the presence of the osmolyte. Thus, although zinc induced accumulation of Hsp70 in the presence of TMAO, induction of Hsp70 was far more pronounced in the absence of TMAO. The increased level of Hsp70 accumulation was, thus, associated with increased OA transport rate.
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The apparent correlation of increased active OA transport by CP and the
cellular stress response was further examined with the novel inhibitor of
acquisition of thermotolerance and heat shock protein induction, KNK437
(Yokota et al., 2000). Because
zinc was a strong inducer in the absence of TMAO, we repeated the flux
experiments above with and without 100 µmol l–1 KNK437.
Tissues were preincubated with inhibitor for 30 min prior to exposure to 50
µmol l–1 zinc + KNK437 and no TMAO for 6 h followed by 1.5
h recovery in normal L-15E with KNK437. Zinc-exposed controls were treated
identically except with vehicle only (0.2% DMSO). As shown in
Fig. 6, the stimulatory effect
of zinc in the absence of TMAO was completely blocked by KNK437. Net active
flux was reduced about 7-fold. Some non-specific damage may have occurred with
combined zinc and KNK437 exposure, as indicated by the significantly lowered
Rt in the latter (Table
1). In a separate set of tissues, referred to as controls in
Fig. 6, the hsp inhibitor also
strongly inhibited transport in the absence of zinc stimulation
(Fig. 6), although not to the
same extent. Fig. 7 shows that
KNK437 treatment was effective in reducing hsp production in tissues exposed
to zinc in the absence of TMAO. As compared to time-matched control tissues
(no zinc), zinc exposure with recovery increased Hsp70 accumulation by
4.09-fold; in the presence of KNK437, the hsp was induced by 1.45-fold
(N=4). As displayed in the immunoblot shown in
Fig. 7, KNK437 also reduced
Hsp70 expression in non-zinc exposed tissues about 3-fold. In addition, as
compared to Hsp70 accumulation in CP tissue collected directly from the shark,
7.5-h-explant culture at 13.5°C without TMAO increased Hsp70 accumulation
by 2.02-fold. Thus, alterations of OA transport in both zinc-exposed and
control 7.5 h-explanted cultures was associated with relative Hsp70
accumulation.
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Stress-induced transcription of Hsp70 mRNA
Transcription of the Hsp70 gene was evaluated in CP subjected in
vitro to +10°C heat stress or zinc exposure in the presence or
absence of TMAO; expression levels of Hsp70 mRNA were analyzed by
semi-quantitative RT-PCR (Fig.
8). For each individual heat stress or zinc exposure experiment,
paired, equal-sized fragments of CP from a single shark were used. To
investigate the influence of TMAO on heat-induced changes in Hsp70 mRNA
expression, levels of Hsp70 mRNA were compared among tissues held at
13.5°C incubated in both the presence and absence of TMAO and tissues heat
stressed in the presence and absence of the osmolyte (23.5°C for 1 h
followed by 1.5 h at 13.5°C). In the non-heat stressed condition, the
relative level of Hsp70 mRNA in the absence of TMAO was nearly twice that in
the presence of TMAO (1.98±0.13; N=3). Heat stress increased
expression of Hsp70 mRNA irrespective of the presence of TMAO. However, the
relative levels of Hsp70 mRNA in the absence of TMAO were approximately 2-fold
greater than levels in the presence of TMAO (2.24±0.98;
N=3).
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CP ventricular surface morphology and ultrastructure following thermal stress and zinc exposure
The CSF-facing surface topology and ultrastructural morphology of isolated
CP are shown in Fig. 9. As
viewed by transmission electron microscopy, CP is comprised of an epithelial
monolayer rich in mitochondria with densely packed apical microvilli that
interface with the CSF in vivo and an interdigitated basolateral
membrane that interfaces with the interstitial compartment and a discontinuous
layer of adipocytes (the large fat globules can be seen in
Fig. 9F,H,I). As the
capillaries within the stroma are fenestrated, the `blood' compartment is
functionally continuous with the interstitial compartment. The tight junctions
between the epithelial cells form the blood–CSF barrier and appeared to
remain intact under all conditions tested. As viewed by scanning electron
microscopy, the apical microvilli are clavate or club-shaped rather than
filiform (Fig. 9A). The
differences in the ultrastructure and ventricular surface morphology of IVth
CP fixed immediately upon removal from the animal (referred to as in
vivo) compared with non-stressed CP at 7.5 h in L-15E (13.5°C) were
slight (Fig. 9A,F and
Fig. 9B,G). Although
microvillus length was greater in 7.5-h-explants (1.77±0.12 µm
vs 1.17±0.03 µm; P<0.05), there was no
difference in size and shape of the clavate tip of the microvilli (maximum
traverse width: in vivo, 0.30±0.03 µm; non-stressed
explanted tissue, 0.33±0.05 µm; minimum transverse width:
non-stressed, 0.09±0.005 µm; in vivo, 0.07±0.008
µm). Replacement of TMAO with equimolar mannitol in the L-15E had no effect
on morphology (data not shown).
|
Morphological comparisons were made after a 5°C heat stress (18.5°C
for 6 h plus recovery at 13.5°C for 1.5 h), a 10°C heat stress
(23.5°C for 1 h plus recovery at 13.5°C for 1.5 h) and zinc exposure
at 13.5°C (50 µmol l–1 ZnSO4 for 6 h plus
recovery in zinc-free medium for 1.5 h). Transmission electron micrographs
indicated that heat stress and zinc treatment caused no detachment of
epithelium from the basement membrane or any remarkable changes in vascular
elements; tight junctions also remained intact under each stress condition.
The mean length of the microvilli in stressed explanted tissue was comparable
to that in non-stressed explanted tissue (
+5°C, 1.12±0.04
µm; +10°C, 1.62±0.08 µm; Zn, 1.82±0.11 µm).
However, a profound morphological change was the expansion of the tips of the
microvilli in both thermally stressed and zinc-treated tissues.
+5°C with recovery resulted in expansion of the microvilli tips in
the maximum and minimum transverse dimensions to 0.88±0.096 µm and
0.22±0.03 µm, respectively; +10°C induced even greater
and more irregular expansion to 1.28±0.08 µm and 0.20±0.3
µm. Zinc treatment also expanded microvilli tips to a degree comparable to
heat stress; maximum and minimum transverse dimensions were 0.96±0.095
µm and 0.25±0.04 µm, respectively. Ultrastructurally, these
expanded microvilli appeared to be multiple layers and myeloid-like whorls of
plasma membrane; this was particularly noteworthy with +10°C.
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
;
Hightower et al., 2000;
Ikari et al., 2002;
Renfro et al., 1993;
Kultz, 2005). Hsp70 is among
the battery of stress responsive proteins that comprise what Kultz has called
the minimal stress proteome (Kultz,
2005). Thus, inducible Hsp70 is a good indicator of a generalized
cell stress response that includes protein stabilization, DNA repair and free
radical scavenging. The present study of isolated dogfish shark CP showed that
non-lethal physicochemical stressors, such as excessive heat and heavy metal
exposure, not only induced Hsp70 production by shark CP but significantly
impacted the capacity of CP to actively remove OAs from CSF. Notably, however,
this brain barrier's functional response to cellular stress was significantly
impeded by a physiological level of TMAO.
Without application of the aforementioned stresses, the presence or absence
of TMAO during explant culture of shark IVth CP had no effect on net 2,4-D
absorption. However, a +10°C heat stress in the presence of TMAO
was clearly more damaging to transepithelial transport than in the
absence of TMAO. Not only was net active transport decreased, but
passive leak flux was increased. The unchanged Rt
indicated that the latter was not due to a loss of tight junction integrity, a
phenomenon not without precedent that may be due to redistribution of
transporters caused by transient changes in cytoskeletal elements
(Brown et al., 1992).
Transmission electron microscopy revealed pronounced and irregular expansion
of microvilli and melding of plasma membrane at the apical pole of the
epithelium in +10°C stressed tissues; however, there was no
sloughing of cells, detachment from the basement membrane or opening of
junctional complexes. Nevertheless, in the absence of broad destructive
changes in morphology, +10°C heat stress in the presence of TMAO
markedly decreased OA transport. By comparison, transepithelial transport
seemed unaffected by the substantial temperature shift when TMAO was not
present in the incubation medium. Similarly, net active OA transport was
greater in CP exposed to 50 µmol l–1 zinc without TMAO,
and the lower transport capacity in TMAO-supplemented tissues could not be
attributed to differences in either Rt or leak flux. This
lack of change in, or apparent protection of, transport in TMAO-deprived CP
tissues was associated with considerably higher levels of Hsp70. The
suppression of Hsp70 induction by TMAO was consistent with work on the
Madin-Darby canine kidney cell line (MDCK) that showed the stabilizing
osmolyte betaine strongly attenuated the stimulation of increased Hsp70 mRNA
by a denaturing level of hypertonicity and by elevated temperature
(Sheikh-Hamad et al., 1994).
The effect was attributed to greater stabilization of proteins in the presence
of a high concentration (250 mmol l–1) of osmolyte and, as a
consequence, attenuated protein denaturation, thereby decreasing the signal
for increased synthesis of stress proteins
(Hightower, 1980;
Ananthan et al., 1986;
Hightower, 1991). In shark CP,
the putative protein stabilizing effect of TMAO was manifested in the
suppression of Hsp70 production. Support for the TMAO effect was seen in the
effects of the heat shock protein inhibitor KNK437. The latter coincidentally
blocked Hsp70 accumulation and drastically lowered active OA transport.
Interestingly, this effect was also seen in control, explanted tissues,
revealing stress-induced changes brought about by the in vitro
environment.
Proposed mechanisms of cytoprotection usually indicate that it is the
prevention of damage to macromolecules or the facilitation of their repair by
chaperone proteins or chemical cytoprotectants that promotes cell survival.
However, in functional transporting epithelia, an associated stress-induced
increase in transepithelial transport capacity has been observed that may
either compensate for lost capacity due to damage or actually increase total
capacity (Hightower et al.,
2000). Preconditioning of both flounder and porcine cultured renal
proximal tubule with mild heat-stress (i.e. +5°C above baseline with
recovery), which produced pronounced induction of Hsp70 protein in those
tissues, prevented attenuation of transepithelial transport of glucose and
sulfate by severe heat shock (+10°C above baseline with recovery) that was
otherwise observed in naïve tissues
(Renfro et al., 1993;
Sussman and Renfro, 1997). In
flounder proximal tubule, a preconditioning zinc exposure sufficient to induce
synthesis and accumulation of Hsp70 also elicited an increase in the tissue's
capacity to transport sulfate, an effect that may contribute to the apparent
protection of sulfate transport against both severe heat shock and chemical
intoxication (Renfro et al.,
1993). Inhibition of hsp synthesis or gene transcription prevented
the effectiveness of stress preconditioning in protection of solute transport.
In the present study, exposure of shark CP to 50 µmol l–1
zinc elicited a very strong cellular stress response as judged by Hsp70
accumulation. Although zinc is a vital nutrient, essential for growth,
development and reproduction in all organisms and present in at least 300
enzymes including all six IUBMB classes
(Laity et al., 2001), it also
adversely influences the quaternary structure of many proteins. Thus, cells
very efficiently regulate total and free concentrations of intracellular zinc
via coordinated regulation of the influx and efflux of the metal with
its binding to metallothionein (Cousins et
al., 2006; Liuzzi and Cousins,
2004; Tapiero and Tew,
2003). The stress response noted in the present study indicated
significant overload of those mechanisms in the CP. The stimulation of
cellular or integrative tissue function to levels higher than those in
unexposed controls by a chemical denaturant has been frequently reported and
is a manifestation of hormesis (Bukowski
and Lewis, 2000; Calabrese,
2005; Rattan,
2004). Thus, the stimulation by zinc exposure of herbicide
transport from CSF to blood compartments in isolated CP to levels above that
of unexposed tissues is consistent with hormesis associated with the cellular
stress response. This phenomenon has been previously observed in renal
proximal tubule (Renfro et al.,
1993).
As Somero has pointed out (Somero,
1995), both protein turnover and protein function benefit from a
balance between stability and lability. Constitutive as well as stress-induced
hsps prevent nonspecific protein aggregates; however, maintenance of high
levels of hsps greatly increases the cellular energy requirements. Expression
of inducible hsps during nonstressful periods may be detrimental to growth and
lead to accumulation of useless and difficult to remove aggregates of hsps
themselves (Feder et al.,
1992). The suppression of hsp production during stress by TMAO, as
observed in mammalian cell lines and renal medullary cells
(Neuhofer et al., 2005;
Neuhofer et al., 2001) and now
here in a stenohaline marine elasmobranch, may be beneficial to tissues in
environments with only minimal or slow physicochemical fluctuations. Thus, the
necessity to counteract the protein destabilizing effects of high urea and
NaCl may be better served by chemical cytoprotectants rather than by
energetically expensive and continuous synthesis of new hsps. Evolutionarily,
ectotherms living in widely fluctuating environments appear to have a stronger
cellular stress response than those in more stable conditions
(Shabtay and Arad, 2005).
Thus, the comparatively stable oceanic environment and ureosmotic strategy of
osmoregulation may have made chemical cytoprotectants an economical
alternative stress defense for spiny dogfish; however, when extreme stresses
are encountered, these animals may be less able to mount an appropriate
cellular stress response and therefore may be at greater risk of excessive
protein injury.
In summary, active transepithelial absorption of OAs by isolated shark CP was altered in response to thermal stress and heavy metal exposure in a manner contingent on the tissue's ability to mount a cellular stress response. Impairment of Hsp70 upregulation, by inclusion of TMAO or the HSF1 inhibitor KNK437, diminished transepithelial transport under stress conditions. Collectively, these data indicate that induction of heat shock proteins in CP may be critical for sustaining the active and selective exchange of OAs between CSF and blood compartments under conditions of physicochemical stress.
List of symbols and abbreviations
|
Acknowledgments |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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), 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.
