(Received for publication, October 22, 1996, and in revised form, March 31, 1997)
From the Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616
A stringent structure-activity relationship among polychlorinated biphenyls (PCBs) possessing two or more ortho-chlorine substituents is observed for activation of ryanodine receptors in mammalian brain, revealing an arylhydrocarbon receptor-independent mechanism through which non-coplanar PCBs disrupt neuronal Ca2+ signaling. Of the congeners assayed, non-coplanar PCB 95 exhibits the highest potency (EC50 = 12-24 µM) toward activating high affinity [3H]ryanodine-binding in rat hippocampus, cerebellum, and cerebral cortex. Coplanar PCB 66 and PCB 126 have no ryanodine receptor activity in all brain regions examined. PCB 95 enhances [3H]ryanodine-binding affinity and capacity by significantly altering modulation by Ca2+ and Mg2+, thereby stabilizing a high affinity conformation of the ryanodine receptor. Ca2+ transport measurements using cortical microsomes reveal that PCB 95 discriminates between inositol 1,4,5-trisphosphate- and ryanodine-sensitive stores. PCB 95 selectively mobilizes Ca2+ from ryanodine-sensitive stores in a dose-dependent manner (EC50 = 3.5 µM) and is completely inhibited by ryanodine receptor blockers, whereas coplanar PCBs are inactive. These data demonstrate that ortho-substituted PCBs disrupt Ca2+ transport in central neurons by direct interaction with ryanodine receptors, showing high selectivity and specificity. Alteration of Ca2+ signaling mediated by ryanodine receptors in specific regions of the central nervous system may account, at least in part, for the significant impact of these agents toward neurodevelopment and neuroplasticity in mammals.
Along with the genetically related inositol 1,4,5-trisphosphate receptors (IP3Rs),1 ryanodine receptors (RyRs) are Ca2+-selective ion channels which regulate the release of Ca2+ from endoplasmic reticulum (ER) during cell activation. Mobilization of ER Ca2+ is essential for determining the amplitude, spatial and temporal fluctuation of intracellular Ca2+, thereby providing important signaling information to the cell (1). In consonance with their fundamental role in cellular Ca2+ signaling, skeletal (Ry1R) and brain (Ry3R) isoforms of RyRs possess a cytoskeletal binding motif which could confer a structural and functional association with L-type voltage-dependent Ca2+ entry channels in skeletal muscle (2-5). Data supporting direct coupling of neuronal L-type voltage-dependent Ca2+ entry and RyRs has been recently provided (6). Most significant is the finding that expression of Ry1R cDNA in dyspedic muscle cells, which lack constitutive expression of Ry1R, restores not only excitation-contraction coupling but also restores L-type voltage-dependent Ca2+ entry (5). Thus mechanical coupling between RyRs and voltage-gated Ca2+ channels within the surface membrane appears to confer reciprocal regulation, a finding that may have broad significance in neuronal cell functions.
Polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans are commonly known as halogenated aromatic hydrocarbons (HAHs), a family of persistent and widely dispersed environmental contaminants. The unique chemical properties and low cost of producing PCBs have contributed to their extensive industrial use (7, 8). The high lipophilicity and chemical stability of PCBs have further resulted in widespread environmental contamination, and there is significant evidence of PCBs accumulation in biota (9). PCBs are found in extracts of virtually all environmental samples as well as in human tissue and breast milk (10, 11).
Among the HAHs various congeners differentially confer ability to bind to a cytosolic receptor, the arylhydrocarbon receptor (AhR). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to the receptor with the highest affinity and confers the highest potency to induce certain toxic responses, such as wasting syndrome, immunosuppression, and teratogenicity. PCB congeners without ortho-chlorine substitutions are able to confer the coplanar structure similar to TCDD and elicit similar toxicity. PCB congeners with single ortho-chlorine slightly favor the non-coplanarity and behave as weak AhR agonists. PCB congeners with two or more ortho-chlorines highly favor the non-coplanar conformation; thus, this group of congeners do not bind to the AhR and exhibit different toxicity. There is emerging evidence indicating that certain ortho-substituted PCB congeners are responsible for the neurotoxic effects of PCBs, including decreased in catecholamine levels in certain brain regions in mammals (12-14) and reduced dopamine levels in rat pheochromocytoma cells (PC12 cells) (15). Perinatal exposure of monkeys and rodents to PCBs resulted in behavioral abnormalities including delayed reflex development, altered activity patterns, learning deficits, and impaired memory (16). Rats perinatally exposed to certain ortho-substituted PCB congeners were impaired in learning a delayed spatial alternation task similar to that employed in the behavioral test of the PCB-exposed monkeys (17). Epidemiological studies reveal that children exposed either perinatally or prenatally to PCBs and other HAHs developed long-lasting cognitive function deficits (18-21). Although the compounds that are responsible for these deficits are unknown, results from animal studies have revealed that ortho-substituted PCBs are probably responsible for the neurotoxicity observed.
In addition to the subtle neurotoxic effects induced by PCBs, the 209 possible congeners in the PCB family that constitute various industrial PCB mixtures has greatly obstructed progress in understanding the cellular and subcellular mechanisms of PCBs action. Studies have demonstrated that certain ortho-substituted PCB congeners are responsible for the neurotoxic action of PCBs, suggesting a non-AhR-mediated pathway. Shain and co-workers performed an extensive structure-activity relationship study on more than 50 PCB congeners (15). ortho-Substituted, non-coplanar PCB congeners were found to be the most active structures in decreasing dopamine level in PC12 cells, whereas coplanar PCBs were found to be inert. However, the underlying molecular mechanisms by which ortho-substituted PCBs induced neurotoxicity are unknown. We have provided evidence of the direct interaction between certain ortho-substituted PCB congeners and the ryanodine-sensitive Ca2+ channel complex (RyRs) localized on sarcoplasmic and endoplasmic reticulum (SR/ER) (22). The studies reported suggest a selective molecular target by which certain ortho-substituted PCB congeners disrupt Ca2+ homeostasis in muscle and within neurons in certain regions of the brain, as RyRs are differentially expressed in the central nervous system.
The present paper demonstrates for the first time a stringent
structure-activity relationship among PCBs possessing two or more
chlorine substitutions in the ortho positions for activation of RyRs of the mammalian central nervous system, revealing an AhR-independent mechanism through which PCBs disrupt neuronal Ca2+ signaling. The most potent congener at the receptor
yet identified, PCB 95 (2,2,3,5
,6-pentachlorobiphenyl), is found to
alter Ca2+ transport across neuronal microsomal membrane
vesicles by a RyR-mediated pathway without affecting the
IP3R-mediated pathway. These actions of PCB 95 at RyRs may
underlie its ability to alter neuronal excitability in rat hippocampal
slices in vitro (23), as well as both locomotor activity and
spatial learning in an in vivo rat model (24). Taken
together, these results demonstrate a RyR-mediated mechanism by which
certain ortho-substituted PCBs alter neuronal
Ca2+ signaling, through which they may alter
neurodevelopment and neurobehavioral function in mammals.
Chemicals
Aroclor 1254 and PCB congeners (>99% pure) were purchased from
Ultra Scientific (North Kensington, RI). [3H]Ryanodine
(60-90 Ci/mmol; > 99%) and [3H]inositol
1,4,5-trisphosphate (15-30 Ci/mmol; > 95%) were purchased from
DuPont NEN. Fluo-3 (pentammonium salt, 90% pure) was purchased from
Molecular Probes (Eugene, OR). All other reagents were of the highest
purity commercially available.
Membrane Preparations
Membrane fractions from cerebellum, cerebral cortex, and hippocampus of rat were prepared as described previously (25). Briefly, tissue from each brain region was homogenized with 10-fold (w/v) ice-cold homogenization buffer consisting of 320 mM sucrose, 5 mM HEPES, pH 7.4, 100 µM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml leupeptin.
Whole Particulate FractionsThe homogenate from each brain
region was centrifuged at 110,000 × g for 1 h.
The whole particulate fractions were obtained by suspending the pellets
in a buffer consisting of 320 mM sucrose, 5 mM
HEPES, pH 7.4, at a protein concentration of 8-12 mg/ml (26). The
protein preparations were then rapidly frozen in liquid N2, and stored at 80 °C until needed.
The homogenate from each brain region was centrifuged at 1,000 × g for 10 min. The membrane pellets were collected, suspended in homogenization buffer with a glass Dounce homogenizer, and re-centrifuged at 1,000 × g. The two supernatant fractions were combined and centrifuged at 8,000 × g for 10 min. The resulting supernatants were then centrifuged at 110,000 × g for 1 h. Finally, the crude microsomal pellets were suspended and stored as described above.
Radioligand Binding Assay
Binding Capacity of CerebellumTo examine the protein dependence of [3H]ryanodine binding in the presence or absence of selected PCB congeners, the binding capacity of the whole particulate fraction isolated from rat cerebellum was determined as a function of protein concentration. Specific binding of 10 nM [3H]ryanodine to cerebellar whole particulate fraction (50-250 µg) was measured in an assay buffer consisting of 140 mM KCl, 15 mM NaCl, 20 mM PIPES, pH 7.4, 100 µM PMSF, 10% sucrose, 10 µM CaCl2, in the presence or absence of either 40 µM PCB 95 or 40 µM PCB 126, in a final volume of 250 µl, as described previously (25). All samples were equilibrated at 37 °C for 3 h with constant shaking. Nonspecific binding was determined by addition of a thousandfold excess of cold ryanodine and averaged 70% of the total binding. Each experiment was performed in duplicate and repeated at least three times with different membrane preparations.
Structure-Activity Relationship of PCBs toward RyRs in CerebellumDose-response relationship for PCB-activated [3H]ryanodine binding to cerebellar microsomes was determined for selected PCB congeners and Aroclor 1254. Specific binding of 2 nM [3H]ryanodine was measured in the presence or absence of 400 nM to 400 µM PCB with 180 µg of crude cerebellar microsomal protein, in an assay buffer consisting of 140 mM KCl, 15 mM NaCl, 20 mM PIPES, pH 7.4, 100 µM PMSF, 10 µM CaCl2, in a final volume of 250 µl (25). Nonspecific binding was determined by addition of a thousandfold excess of cold ryanodine and averaged 35% of the total binding. The potency (EC50 and Hill coefficient) of each PCB toward RyRs was determined by linear regression analysis of logit-log transformation of the data obtained between 10 and 90% of maximal binding.
Saturation Binding of [3H]Ryanodine in CerebellumThe mechanism by which PCB 95 enhanced occupancy of [3H]ryanodine to cerebellum was measured by saturation binding experiments (22). Specific binding of 0.5-20 nM [3H]ryanodine was determined in the presence of 50 µM PCB 95 in an assay buffer (Low Salt Buffer) consisting of 180 µg of cerebellar whole particulate protein, 140 mM KCl, 15 mM NaCl, 20 mM PIPES, pH 7.4, 10% sucrose, 100 µM PMSF, 10 µM CaCl2, 1 mM MgCl2, in a final volume of 250 µl. Control experiments were performed in the absence of PCB by measuring specific binding of 0.5-70 nM [3H]ryanodine in an assay buffer (High Salt Buffer) consisting of 180 µg of cerebellar whole particulate protein, 200 mM KCl, 15 mM NaCl, 20 mM PIPES, pH 7.4, 10% sucrose, 100 µM PMSF, 10 µM CaCl2, 1 mM MgCl2, in a final volume of 250 µl. Binding constants (KD and Bmax) of high affinity [3H]ryanodine-binding sites were obtained from linear regression analysis of the Scatchard plots using ENZFITTER (Elsevier Biosoft, London, United Kingdom) computer software.
Calcium and Magnesium Modulation of [3H]Ryanodine BindingThe ability of PCB 95 to alter Ca2+ modulation of RyRs was performed by measuring the specific binding of 2 nM [3H]ryanodine to 200 µg of cerebellar microsomal protein in the presence of 50 µM PCB 95 in Low Salt Buffer and 12 nM to 200 mM free Ca2+, in a final volume of 250 µl. Free Ca2+ concentrations below 200 µM in the assay were adjusted by adding calculated amount of EGTA based on the SPECS computer software and published stability constants (27). Control experiments were performed in High Salt Buffer containing 200 µM to 200 mM CaCl2.
The ability of PCB 95 to alter Mg2+ inhibition of RyRs was determined in the presence of 2 nM [3H]ryanodine, 200 µg of cerebellar microsomes, and 2 mM to 1 M Mg2+ in Low Salt Buffer containing 100 µM CaCl2, in a final volume of 250 µl. Control experiments were performed in High Salt Buffer containing 100 µM CaCl2. Values for EC50 of Ca2+ and IC50 of Ca2+ and Mg2+ were calculated from the linear regression analysis of logit-log transformation of the data obtained between 10 and 90% of maximal binding.
[3H]IP3 Binding to Brain MicrosomesThe possible influence of PCB 95 on the binding of
[3H]IP3 to cerebellum was determined by
incubating 0-100 µM PCB 95 or 100 µM PCB
126, in the presence of 0.5 nM
[3H]IP3, 200 µg of cerebellar microsomal
protein, 100 mM KCl, 20 mM NaCl, 1 mM Na-EGTA, 0.1% bovine serum albumin, 25 mM
Na2HPO4, pH 8.3, in a final volume of 1 ml
(25). Nonspecific binding was obtained by adding a thousandfold excess
of cold IP3. The samples were permitted to equilibrate
either in an ice bath (4 °C) for 30 min or at 25 °C for 15 min
with constant shaking. Binding data were analyzed with two-tailed
Student's t test, = 0.05.
Ca2+ Transport Assay
Net Ca2+ flux across brain microsomal vesicles was measured with the long-wavelength Ca2+ indicator fluo-3 using a luminescence spectrometer (Amino Bowman series 2, SLM Aminco, Rochester, NY). Two hundred µg of the rat cortical membrane microsome was added into an assay buffer consisting of 40 mM KCl, 62.5 mM KH2PO4, 1 mM NaN3, 10 µM K-EGTA, 8 mM MOPS, pH 7.0, 0.5 µM fluo-3, 1 mM Mg-ATP, 20 µg/ml creatine phosphokinase, 5 mM phosphocreatine, in a final volume of 1.2 ml (28, 29). The transport assays were performed in temperature-controlled cuvettes at 37 °C with constant stirring. The net Ca2+ flux across the vesicles was recorded by measuring the extravesicular free Ca2+ level as fluorescence intensity with excitation and emission wavelengths of 500 and 530 nm, respectively. The free Ca2+ concentration was calculated from fluorescence intensity using the following equation (30).
![]() |
(Eq. 1) |
Aroclor 1254 and selected PCB congeners were examined for their ability to modulate
the high affinity binding of [3H]ryanodine to microsomes
isolated from rat cerebellum, cerebral cortex, and hippocampus in the
presence of physiologically relevant concentrations of intracellular
K+ and Na+. Fig. 1 demonstrated
that binding of [3H]ryanodine (10 nM)
increased linearly as a function of cerebellar protein concentration
and that non-coplanar PCB 95 significantly enhanced occupancy to 50
µg of protein/assay. In contrast, coplanar PCB 126 did not increase
occupancy above control values at any concentrations of protein
examined (Fig. 1). Similar enhancement in the occupancy of nanomolar
[3H]ryanodine was observed with cortical and hippocampal
membrane preparations (data not shown). The influence of
ortho-chloro substitutions on the ability of PCB congeners
to activate the high affinity binding of [3H]ryanodine
was examined with complete dose-response relationships for selected PCB
congeners. Coplanar PCB 66 and PCB 126 had no activity toward RyRs in
microsomes isolated from rat cerebellum, cerebral cortex, or
hippocampus at concentrations up to their solubility limits (200 µM) (Fig. 2, Table I). In
marked contrast, non-coplanar PCB 95 enhanced binding of
[3H]ryanodine, in a dose-dependent manner, to
microsomes isolated from all the brain regions examined. PCB 95 gave
EC50 values (in µM) of 17, 12, and 25 with
microsomes isolated from rat cerebellum, hippocampus, and cerebral
cortex, respectively (Fig. 2, Table I).
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An extended structure-activity relationship was performed with
cerebellar microsomes. Compared with PCB 95, PCB 4 (2,2-dichlorobiphenyl) and PCB 52 (2,2
5,5
-tetrachlorobiphenyl) were
2- and 3-fold less potent, respectively. The position of the chlorine
substituents about the biphenyl ring structure appeared to be as
important as the degree of chlorination for RyR activity, since
2,2
,4,5
,6- and 2,2
,3,4,6-pentachlorobiphenyl (PCB 103 and PCB 88, respectively) were 3- and 5-fold less potent than PCB 95. The stringent
structural requirement for receptor activity was further demonstrated
by the finding that 2,2
,4,4
,5,5
-hexachlorobiphenyl,
2,2
,4,6,6
-pentachlorobiphenyl, and 2,3
,4
,5-tetrachlorobiphenyl (PCB
153, PCB 104, and PCB 70, respectively) were approximately 10-fold less
potent than PCB 95, whereas 2,3
,4,4
-tetrachlorobiphenyl (PCB 66) was
inactive in all of the brain regions tested (Table I). Interestingly, the PCB mixture Aroclor 1254 showed appreciable activity toward RyR
(EC50 = 35 µM in cerebellum). The values of
EC50, maximal occupancy, and Hill coefficient for selected
PCB congeners and Aroclor 1254 are summarized in Table I.
Net Ca2+ transport across rat cortical
microsomal membrane vesicles was measured with the fluorometric dye
fluo-3 under conditions of active Ca2+ loading. In Fig.
3A, a typical trace of the Ca2+
loading phase is shown, and was invariant for each of the experiments shown below. Ca2+ loading was initiated by addition of ATP
followed by serial addition of two 2.4-nmol additions of
Ca2+. No change in fluorescence intensity, (i.e.
no net Ca2+ transport across the membrane vesicles) was
observed before the addition of ATP, suggesting that Ca2+
uptake by the membrane vesicles was ATP-dependent (data not
shown). Addition of the Ca2+ ionophore 4-bromo A-23187
after completion of the loading phase demonstrated that the accumulated
Ca2+ could be rapidly released from the vesicles. Addition
of Ca2+ (2 × 2.4 nmol) at the end of each experiment
verified the linearity and the calibration of the dye signal. In all
the measurements reported in the present study, none of the drugs at
the concentrations used significantly interfered with either the
sensitivity or calibration of the fluo-3 dye (data not shown). Under
these assay conditions, Fmin and
Fmax were 0.5 ± 0.1 and 8.0 ± 0.3, respectively (mean of three determinations). In Fig. 3B,
addition of coplanar PCB 66 or PCB 126 to cortical microsomes actively
loaded with Ca2+ had no effect on the net Ca2+
flux across the vesicles. In marked contrast, addition of PCB 95 induced a rapid release of Ca2+ from the vesicles. The
initial rate of Ca2+ release from the vesicles induced by
PCB 95 (1-10 µM) was dose-dependent (Fig.
3C) with an apparent EC50 of 3.5 µM (Fig. 3C, inset).
RyR Blockers Inhibit PCB 95-induced Ca2+ Release from Brain Microsomes
The mechanism by which PCB 95 induced
Ca2+ release from brain microsomes was further studied
using ruthenium red and ryanodine, known RyR blockers. Fig.
4 shows the response of actively loaded cortical
microsomes to 5 µM PCB 95 (trace A). Addition
of ruthenium red (1 µM) after loading the vesicles with
Ca2+ largely eliminated the response to PCB 95 (~ 94%
inhibition of the initial Ca2+ release rate) (Fig. 4,
trace B). Prior addition of 500 µM ryanodine resulted in a typical biphasic response of the receptor, whereas subsequent addition of PCB 95 failed to mobilize Ca2+ from
the vesicles (~93% inhibition of the initial Ca2+
release rate) (Fig. 4, trace C). These results suggested
that PCB 95 induced Ca2+ release was inhibited by
ryanodine-sensitive Ca2+ channel blockers.
PCB 95 Selectively Targets Ryanodine-sensitive Ca2+ Stores
To discriminate which of the Ca2+ stores in
the microsomal preparation are sensitive to PCB 95, additional
pharmacological studies were performed with IP3 and
ryanodine. Fig. 5 illustrates the ability of
D-IP3 to stereoselectively activate
IP3Rs in the microsomal preparation, since
L-IP3 is inactive (trace a).
Addition of 500 µM ryanodine to the preparation caused a
biphasic response similar to that seen with junctional SR vesicles
isolated from striated muscle (31): initially activating and
subsequently inactivating RyRs, resulting in net re-uptake of the
Ca2+ into the cortical vesicles (Fig. 5, trace
b). Addition of D-IP3 after treating with
ryanodine demonstrated that the IP3Rs maintained their
sensitivity to agonist, as the rate and amount of
IP3-induced Ca2+ release was similar to that
seen with D-IP3 alone (Fig. 5, compare traces a and b). Addition of ryanodine subsequent
to IP3-induced Ca2+ release mobilized stored
Ca2+ with magnitude and kinetics quantitatively similar to
ryanodine-induced Ca2+ release in the absence of
IP3 (Fig. 5, compare traces a and b). Taken together, the results suggested that the IP3- and
ryanodine-sensitive efflux pathways in the cortical microsomal
preparation were on distinct vesicles.
To further determine whether the actions of PCB 95 were selective
toward the ryanodine-sensitive store, PCB 95 was added prior or
subsequent to addition of D-IP3. With either
experimental protocol, PCB 95-induced Ca2+ release was
quantitatively similar (Fig. 6, compare traces
a and b). Addition of 1 µM ruthenium red
after depleting the IP3-sensitive stores completely blocked
PCB 95-induced Ca2+ release (Fig. 6, trace c).
Similarly, prior treatment of the Ca2+-loaded vesicles with
1 µM ruthenium red completely negated the response to PCB
95 without altering the response to D-IP3 (Fig. 6, trace d). Addition of 60 µM of heparin to
Ca2+-loaded vesicles resulted in an instantaneous jump in
the fluo-3 response due to contamination of the drug with 34 pmol of
Ca2+. However, subsequent addition of
D-IP3 failed to induce the release of
Ca2+ even though the preparation remained completely
responsive to PCB 95 (Fig. 6, compare traces a,
b, and e). These results further demonstrated the
presence of distinct IP3- and ryanodine-sensitive vesicles
in the microsomal preparation and that PCB 95 selectively mobilized
Ca2+ by directly interacting with vesicles possessing RyRs.
PCB 95 (5 µM) did not appear to alter the
Ca2+ transport properties of the fraction of
IP3R-containing vesicles. In consonance with this
observation, PCB 95 (50 µM) did not significantly alter
the binding of [3H]IP3 to cerebellar
microsomes, although higher concentrations did produce a statistically
significant enhancement of occupancy that was not seen with coplanar
PCB 126 (Fig. 7).
PCB 95 Stabilizes a High Affinity Conformation of RyRs
The
mechanism by which PCB 95 enhanced the high affinity binding of
[3H]ryanodine to cerebellar microsomes was further
elucidated by performing saturation binding measurements and examining
changes in modulation by Ca2+ and Mg2+. In the
presence of physiologically relevant concentrations of intracellular
Na+ (15 mM) and K+ (140 mM), negligible specific binding of
[3H]ryanodine (0.5-25 nM) was detected (data
not shown), whereas inclusion of PCB 95 (50 µM) to the
same assay medium produced a significant enhancement in the number of
high affinity binding sites for [3H]ryanodine exhibiting
a Bmax of 68 ± 5 fmol/mg and a
KD of 3.7 ± 0.6 nM (Fig. 10). By
contrast, control measurement in the presence of high salt (200 mM KCl) revealed that [3H]ryanodine bound to
RyRs on cerebellar microsomes with Bmax of 52 ± 1 fmol/mg and KD of 24 ± 3 nM (Fig. 8).
In the presence of 50 µM PCB 95 and physiological
concentrations of Na+ and K+, the
EC50 for activation of [3H]ryanodine-binding
sites by Ca2+ was 61 nM (Fig.
9A, Table II). Interestingly,
no consistent inhibition of [3H]ryanodine binding was
observed at Ca2+ as high as 200 mM. In
contrast, control measurements performed in the presence of 200 mM KCl exhibited an IC50 for
Ca2+-activated binding of 1.6 mM and a Hill
coefficient of 3, with near-complete inhibition at 10 mM
Ca2+. Furthermore, in the presence of 50 µM
PCB 95, the IC50 for Mg2+ was 19 mM
and the Hill coefficient was 1.3, and only ~50% of the binding sites
could be inhibited even with 1 M Mg2+ (Fig.
9B, Table II). Control measurement in the presence of 200 mM KCl revealed an IC50 of 1 mM and
Hill coefficient of 3.7 for Mg2+, with >90% inhibition at
10 mM Mg2+.
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The present
study demonstrates that certain ortho-substituted PCB
congeners alter Ca2+ regulation of central neurons by a
RyR-mediated mechanism. In neuronal tissues, two types of ligand-gated
Ca2+ release channels have been found to be localized to
the ER (32, 33): 1) IP3R channels and 2)
Ca2+-induced Ca2+ release channels
(i.e. RyR). Because of structural homologies shared by
IP3Rs and RyRs, it is necessary to determine if
ortho-substituted PCBs discriminate between these microsomal
Ca2+ release channels. In neurons, the ryanodine-sensitive
stores have thus far been found mainly localized in cell soma, whereas the IP3-sensitive stores appear to be equally distributed
in neuronal soma and processes (34, 35). Using autoradiography, Synder and co-workers (33) have demonstrated the differential localization of
IP3Rs and RyRs within the central nervous system. Direct
Ca2+ transport measurements are conducted in the present
study using microsomal membrane isolated form rat cerebral cortex. The
results demonstrate that IP3Rs and RyRs do not share the
same population of vesicles. Despite the presence of a large fraction
of IP3-sensitive vesicles in the microsomal preparation
used in the present study, the potent actions of PCB 95 are found to be
completely selective toward the ryanodine-sensitive microsomes. In
addition, PCB 95-induced Ca2+ mobilization from brain
microsomes is completely blocked by µM ryanodine or
ruthenium red, but not heparin. The inability of PCB 95 (50
µM) to significantly alter the binding of
[3H]IP3 to cerebellar microsomes offers
further support for the hypothesis that PCB 95 selectively targets
RyRs. However, at the present time we cannot discount the possibility
that other PCB structures possess IP3R activity. In light
of the current findings, PCB congeners exhibiting potent activity
toward RyR proteins in the mammalian brain would be expected to alter
Ca2+ signaling and Ca2+-dependent
processes in affected neurons. Although it is currently unclear what
the exact role of ryanodine-sensitive Ca2+ channels is in
the adult mammalian brain, the distinct heterogeneity in the
distribution of neuronal RyRs suggests distinct
Ca2+-associated brain functions for each isoform. Given the
potency and specificity of PCB congeners, they may represent a new
class of molecular probes to define the function of RyRs in the brain. Because of the wide environmental distribution of PCBs, and our emerging understanding on the role of RyRs in neurodevelopment and
neuroplasticity, this newly identified mechanism by which PCBs alter
Ca2+ signaling in mammalian brain may underlie the
neurotoxicity that has been attributed to non-coplanar PCBs
(14).
Three isoforms of RyRs have been shown to be expressed in the mammalian central nervous system, where they are thought to be responsible for Ca2+-induced Ca2+ release (36). In situ hybridization and immunolocalization studies have revealed a cell type-specific pattern of expression in the different regions of the central nervous system (37-40). Several recent studies have provided evidence that RyRs are under strict developmental control. First, Ry1R and Ry3R expression have been shown to be regulated by cytokines and growth factors (41-43). Second, using polymerase chain reaction analysis, Futatsugi et al. (44) have identified two alternatively spliced regions in mRNA of mouse Ry1R, which are characterized by the presence or absence of amino acid sequences that exist within the region where modulatory sites for phosphorylation and binding of Ca2+, calmodulin, and ATP are postulated to exist. The ratio of Ry1R splice variants changes abruptly in the cerebrum between embryonic days 14 and 18. Third, RyRs have been found to be expressed in neural growth cones, where they are thought to play an important role in buffering and releasing Ca2+ during intracellular Ca2+ oscillations (45). Interestingly, RyRs appear to regulate the amplitude of Ca2+ spiking behavior in the growth cone, suggesting a role in signal amplification. Since the homeostatic mechanisms controlling intracellular Ca2+ dynamics of growth cones are likely to be important determinants of growth cone migration during development, it is worthwhile to speculate how prenatal exposure to PCB 95 might alter RyR function and its relationship to reduced motor activity and radial arm maze performance (24). PCB 95 administered perinatally would be expected to alter the sensitivity of Ry1R and Ry2R (cardiac isoform) to normal stimuli. Indeed, Schantz and co-workers have demonstrated that rats exposed to PCB 95 perinatally exhibit significantly altered locomotor activity and responses to a hippocampal learning task. These behavioral changes are correlated with a significant regiospecific changes in [3H]ryanodine-binding (24). If the temporal pattern with which the various isoforms of RyRs expressed are important for normal brain development, then agents that alter the timing or level of their expression could also alter subtle aspects of neurodevelopment.
Studies from Kodavanti and co-workers (46, 47) using primary cerebellar
granule cell cultures suggest a similar structure-activity relationship
of PCBs activity. Inhibition of microsomal and mitochondrial Ca2+-ATPases by PCBs has been proposed to be the molecular
mechanism that perturbs Ca2+ homeostasis in neuronal cells
in culture. In support of the hypothesis, recently Kodavanti and
co-workers (47) have shown that PCB 4 enhances protein kinase C
translocation in neuronal cell culture. However, the present study
provides direct evidence for an alternate mechanism which involves
mobilization of Ca2+ from neuronal ER. Previously, we have
shown that PCB 95 and Aroclor 1254 at 10 µM exhibited
no effect on the activity SERCA1 and SERCA2 pumps (22).
Ryanodine-sensitive Ca2+ channels of the brain appear to be
a selective molecular target for certain non-coplanar PCBs. Aroclor
1254 is a commercial mixture of PCBs, which is composed of 49%
pentachlorinated biphenyls. The high activity of Aroclor 1254 toward
RyRs of the brain implies that several of the constituent congeners
that have yet to be tested possess significant receptor activity.
Coplanar PCB 66, a mono-ortho-substituted tetrachlorobiphenyl, has been shown to possess similar physicochemical properties to PCB 95 based on gas chromatographic retention coefficients (7). Despite their similar hydrophobicity, coplanar PCB 66 is inactive toward modifying RyRs and Ca2+ transport in all the brain regions studied, demonstrating that the number of chlorines in the ortho positions is a major determinant for activity toward RyR. Coplanar PCB 126 has been widely studied for its ability to bind the cytosolic AhR and induce hepatic microsomal enzymes (10). Like PCB 66, PCB 126 does not alter the occupancy of [3H]ryanodine to its high affinity sites in any of the rat brain regions examined, nor does it alter Ca2+ transport across the microsomal vesicles, suggesting that coplanar PCBs cannot directly interact with any of the RyR isoforms present in rat brain. In consonance with these findings, PCB 95, but not PCB 126, alters Ca2+ transport across SR vesicles isolated from either skeletal muscle (enriched in Ry1R and SERCA1 isoforms) or cardiac muscle (enriched in Ry2R and SERCA2 isoforms) by a RyR-mediated mechanism (22).
The data presented in the present paper demonstrate that PCB congeners
with two or three chlorine substituents in the
ortho-position confer the highest potency and efficacy
toward RyRs of the mammalian brain. Of the congeners assayed, PCB 95 (2,2,3,5
,6-pentachlorobiphenyl) and PCB 4 (2,2
-dichlorobiphenyl)
exhibit the highest potency and efficacy. Therefore, the position of
the chlorine substituents on PCBs is more important toward conferring
RyR activity than the degree of chlorination. The four
ortho-chloro substituents of PCB 104 (2,2
,4,6,6
-pentachlorobiphenyl) contribute significant steric
constraint which severely limit rotation about the biphenyl bond. The
finding that PCB 104 has significantly lower receptor potency than PCB
95 and PCB 4 suggests that although a non-coplanar conformation of the
biphenyl structure appears to be critical for receptor activity, a
certain degree of rotational flexibility about the biphenyl bond seems
to be required to produce maximum activation of RyRs. The high receptor
activity exhibited by PCB 4 can reflect an induced fit of this congener
with its binding domain on the receptor complex. Other than three
ortho-chlorine substituents, substitutions at the
meta- and para-positions are also important for
optimal activity at RyRs. This is exemplified by the stringent
structure-activity relationship among pentachlorobiphenyls, which
reveals a ranked potency of 2,2
,3,5
,6- > 2,2
,4,5
,6- > 2,2
,3,4,6-pentachlorobiphenyl.
Typically, studies of the high affinity binding of [3H]ryanodine to RyRs to mammalian brain microsomes have been performed in the presence of high salt (1 M KCl or NaCl) (25, 48-50). [3H]Ryanodine binding to its high affinity sites was shown to be modulated by Ca2+, Mg2+, caffeine, and adenosine nucleotides (25, 50), in a manner qualitatively similar to those reported for skeletal and cardiac SR preparations. The requirement for high salt may be important to stabilize an open conformation of the receptors which recognizes [3H]ryanodine with high affinity but the underlying mechanism has remained unclear. Certain ortho-substituted PCB congeners (e.g. PCB 95) effectively eliminate the requirement of high salt in the assay medium. Results from saturation binding of [3H]ryanodine to cerebellar microsomes in the presence of a minimal concentration of salt (200 mM K+) to permit measurement of high affinity binding of [3H]ryanodine reveal a 6.5-fold lower affinity and 1.5-fold lower capacity compared with an assay medium containing physiological K+ and Na+, and PCB 95. Moreover, the value of KD in the presence of PCB 95 is similar to that previously reported by Zimanyi and Pessah (KD = 1-3 nM) and Padua et al. (KD = 2.4 nM), in the presence of 1 M KCl (25, 50). Thus PCB 95 appears to significantly stabilize a single high affinity state (Ca2+ conducting state) even in the presence of physiologically relevant K+ and Na+.
The mechanism by which PCB 95 favors the high affinity state of brain RyR appears to be related to its ability to dramatically alter the responses of the channel to two important physiological ligands, Ca2+ and Mg2+. It has been previously demonstrated that micromolar Ca2+ is required to activate high affinity binding of [3H]ryanodine to brain microsomes in the presence of 1 M K+ (50). Under the same conditions, millimolar Ca2+ has been shown to fully inhibit the binding of [3H]ryanodine to its high affinity site (50). In the present paper, we report that PCB 95 significantly alters the sensitivity of RyRs to activation and inhibition by Ca2+, shifting the EC50 value to 61 nM and essentially eliminating inhibition. Zimanyi and Pessah have reported an IC50 for Mg2+ of 10 mM in high salt (1 M K+) (25), and Padua and co-workers (50) have reported IC50 values for Mg2+ of 2 mM and 5 mM assayed in 200 mM K+ and 1 M K+, respectively. In this respect, PCB 95 alters Mg2+ inhibition in two important ways; 1) it shifts the IC50 for susceptible sites nearly 20-fold compared with control lacking PCB, and 2) it completely eliminates inhibition for approximately 50% of the measurable sites. Therefore, altered modulation by Ca2+ and Mg2+ appears to underlie the ability of PCB 95 to stabilize a high affinity [3H]ryanodine-binding conformation of RyRs of the brain.
In conclusion, ortho-substituted PCBs are shown for the first time to directly and selectively activate the RyR/Ca2+ release channel complex in adult rat brain. Structure-activity relationship studies performed with selected PCBs indicate a stringent structural requirement for activation of RyRs in central nervous system. ortho-Substituted PCB 95, the most potent congener studied, mobilizes microsomal Ca2+ selectively and specifically from ryanodine-sensitive microsomes. Disruption of Ca2+ homeostasis in the affected regions of the brain by certain ortho-substituted PCBs may contribute significantly in altering neurodevelopment and neuroplasticity function in mammals.
We gratefully acknowledge Tien H. Lam, Lili Chen, and Holger P. Behrsing for excellent technical assistance.