Non-dioxin-like polychlorinated biphenyl neurotoxic equivalents found in environmental and human samples.

Non-dioxin like polychlorinated biphenyls (NDL PCB) are recognized neurotoxicants with implications on altered neurodevelopment and neurodegeneration in exposed organisms. NDL PCB neurotoxic relative potency schemes have been developed for a single mechanism, namely activity toward the ryanodine receptor (RyR), or combined mechanisms including, but not limited to, alterations of RyR and dopaminergic pathways. We compared the applicability of the two neurotoxic equivalency (NEQ) schemes and applied each scheme to PCB mixtures found in environmental and human serum samples. A multiple mechanistic NEQ predicts higher neurotoxic exposure concentrations as compared to a scheme based on the RyR alone. Predictions based on PCB ortho categorization, versus homologue categorization, lead to a higher prediction of neurotoxic exposure concentrations, especially for the mMOA. The application of the NEQ schemes to PCB concentration data suggests that PCBs found in fish from US lakes represent a considerable NEQ exposure to fish consuming individuals, that indoor air of schools contained high NEQ concentrations representing an exposure concern when inhaled by children, and that levels already detected in the serum of adults and children may contribute to neurotoxicity. With further validation and in vivo exposure data the NEQ scheme would help provide a more inclusive measure of risk presented by PCB mixtures.

A transgenic myogenic cell line lacking ryanodine receptor protein for homologous expression studies: reconstitution of Ry1R protein and function.

CCS embryonic stem (ES) cells possessing two mutant alleles (ry1r-/ry1r-) for the skeletal muscle ryanodine receptor (RyR) have been produced and injected subcutaneously into severely compromised immunodeficient mice to produce teratocarcinomas in which Ry1R expression is absent. Several primary fibroblast cell lines were isolated and subcloned from one of these tumors that contain the knockout mutation in both alleles and exhibit a doubling time of 18-24 h, are not contact growth inhibited, do not exhibit drastic morphological change upon serum reduction, and possess the normal complement of chromosomes. Four of these fibroblast clones were infected with a retrovirus containing the cDNA encoding myoD and a puromycin selection marker. Several (1-2 microg/ml) puromycin-resistant subclones from each initial cell line were expanded and examined for their ability to express myoD and to form multinucleated myotubes that express desmin and myosin upon removal of mitogens. One of these clones (1B5 cells) was selected on this basis for further study. These cells, upon withdrawal of mitogens for 5-7 d, were shown by Western blot analysis to express key triadic proteins, including skeletal triadin, calsequestrin, FK506-binding protein, 12 kD, sarco(endo)plasmic reticulum calcium-ATPase1, and dihydropyridine receptors. Neither RyR isoform protein, Ry1R (skeletal), Ry2R (cardiac), nor Ry3R (brain), were detected in differentiated 1B5 cells. Measurements of intracellular Ca2+ by ratio fluorescence imaging of fura-2-loaded cells revealed that differentiated 1B5 cells exhibited no responses to K+ (40 mM) depolarization, ryanodine (50-500 microM), or caffeine (20-100 mM). Transient transfection of the 1B5 cells with the full-length rabbit Ry1R cDNA restored the expected responses to K+ depolarization, caffeine, and ryanodine. Depolarization-induced Ca2+ release was independent of extracellular Ca2+, consistent with skeletal-type excitation-contraction coupling. Wild-type Ry1R expressed in 1B5 cells were reconstituted into bilayer lipid membranes and found to be indistinguishable from channels reconstituted from rabbit sarcoplasmic reticulum with respect to unitary conductance, open dwell times, and responses to ryanodine and ruthenium red. The 1B5 cell line provides a powerful and easily managed homologous expression system in which to study how Ry1R structure relates to function.

Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor.

Xestospongins (Xe's) A, C, D, araguspongine B, and demethylxestospongin B, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are shown to be potent blockers of IP3-mediated Ca2+ release from endoplasmic reticulum vesicles of rabbit cerebellum. XeC blocks IP3-induced Ca2+ release (IC50 = 358 nM) without interacting with the IP3-binding site, suggesting a mechanism that is independent of the IP3 effector site. Analysis of Pheochromocytoma cells and primary astrocytes loaded with Ca2+-sensitive dye reveals that XeC selectively blocks bradykinin- and carbamylcholine-induced Ca2+ efflux from endoplasmic reticulum stores. Xe's represent a new class of potent, membrane permeable IP3 receptor blockers exhibiting a high selectivity over ryanodine receptors. Xe's are a valuable tool for investigating the structure and function of IP3 receptors and Ca2+ signaling in neuronal and nonneuronal cells.

Sparks and puffs in oligodendrocyte progenitors: cross talk between ryanodine receptors and inositol trisphosphate receptors.

Investigating how calcium release from the endoplasmic reticulum (ER) is triggered and coordinated is crucial to our understanding of how oligodendrocyte progenitor cells (OPs) develop into myelinating cells. Sparks and puffs represent highly localized Ca(2+) release from the ER through ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP(3)Rs), respectively. To study whether sparks or puffs trigger Ca(2+) waves in OPs, we performed rapid high-resolution line scan recordings in fluo-4-loaded OP processes. We found spontaneous and evoked sparks and puffs, and we have identified functional cross talk between IP(3)Rs and RyRs. Local events evoked using the IP(3)-linked agonist methacholine (MeCh) showed significantly different morphology compared with events evoked using the caffeine analog 3,7-dimethyl-1-propargylxanthine (DMPX). Pretreatment with MeCh potentiated DMPX-evoked events, whereas inhibition of RyRs potentiated events evoked by low concentrations of MeCh. Furthermore, activation of IP(3)Rs but not RyRs was critical for Ca(2+) wave initiation. Using immunocytochemistry, we show OPs express the specific Ca(2+) release channel subtypes RyR3 and IP(3)R2 in patches along OP processes. RyRs are coexpressed with IP(3)Rs in some patches, but IP(3)Rs are also found alone. This differential distribution pattern may underlie the differences in local and global Ca(2+) signals mediated by these two receptors. Thus, in OPs, interactions between IP(3)Rs and RyRs determine the spatial and temporal characteristics of calcium signaling, from microdomains to intracellular waves.

Early over-expression of low-affinity [3H]ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major.

Heavy sarcoplasmic reticulum (SR) membranes enriched in [3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17% lower protein recovery in the heavy SR subfractions compared to preparations from paired dystrophic PM (0.80 vs. 0.96 mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7-9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4, and 5.5 weeks ex ovo. [3H]Ryanodine binding to normal SR from PM 2 days ex ovo exhibits nonlinear Scatchard plots which resolve into high-(Kd,app = 18 nM; Bmax = 1.7 pmol/mg protein) and low-(Kd,app = 532 nM; Bmax = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [3H]ryanodine binding sites (Kd,app = 31 nM; Bmax = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respectively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from dystrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [3H]ryanodine and exhibit high densities of low-affinity binding sites (Kd,app = 725-4500 nM; Bmax = 15.4-25.1 pmol/mg protein). Early developmental over-expression of [3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of Mr 340,000 and 320,000 stain on SDS-PAGE. Low-affinity [3H]ryanodine binding sites of dystrophic SR exhibit 4-6-fold higher sensitivity to activation by Ca2+ and altered sensitivity to activation by caffeine and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of inositol 1,4,5-trisphosphate receptors in rat basophilic leukemia cells. I. Multiple conformational states of the receptor in a microsomal preparation.

A detailed characterization of the inositol 1,4,5-trisphosphate (IP3) receptor in rat basophilic leukemia (RBL) cells, a neoplastic mast cell line, has been possible through the growth of solid RBL cell tumors which provide a rich source of IP3 receptor. Equilibrium binding studies show a 1.6 +/- 0.1 pmol/mg of protein maximal binding capacity for [3H]IP3 at optimal Ca2+ (10 microM). The specificity of the RBL cell IP3 receptor towards phosphoinositides, ATP and heparin parallels those previously described with excitable and nonexcitable tissues. [3H]IP3 binding is slightly enhanced from < 1 nM to 10 microM Ca2+ and inhibited by > 10 microM Ca2+. Kinetic and equilibrium studies provide evidence for at least two classes or conformational states of binding sites with pico- and nanomolar affinities. At nM concentrations of IP3, neither binding to the IP3 receptor nor IP3-induced Ca2+ efflux from permeabilized cells demonstrates cooperativity. In contrast, at pM concentrations, IP3 binding kinetics deviate from simple mass action suggesting a complex interaction among binding sites for IP3 on the receptor-channel oligomer. The mechanisms that regulate [3H]IP3 binding in RBL cells are unique when compared to what has been reported in other cells.

Regulation of inositol 1,4,5-trisphosphate receptors in rat basophilic leukemia cells. II. Modulation of the receptor in permeabilized cells by the cytosolic compartment.

Engagement of the IP3 receptor by its ligand releases Ca2+ from intracellular stores of the rat basophilic leukemia (RBL) cell. The IP3 receptor in washed permeabilized cells has high affinity (Kd = 1.2 +/- 0.3 nM) for [3H]IP3 and is not sensitive to physiological concentrations of Ca2+. Moreover, washed permeabilized cells only release small amounts of Ca2+ when stimulated with IP3. When [3H]IP3 binding to permeabilized cells is performed in the presence of cytosolic constituents (unwashed cells), the IP3 receptor has a lower affinity for [3H]IP3 (Kd from 20 to 100 nM) and has enhanced Ca2+ release. Cytosolic supernatant, prepared by centrifugation of permeabilized cells and added back to washed permeabilized cells, decreases [3H]IP3 binding in a dose-dependent manner and increases the amount of Ca2+ released by IP3. Depletion of either MgATP or IP3 in the cytosolic supernatant does not affect the supernatant's ability to decrease [3H]IP3 binding. Though MgATP competitively inhibits [3H]IP3 binding, it cannot fully account for the shift in Kd or the modulation of IP3-stimulated Ca2+ release in the presence of cytosol. These findings suggest that components present in the cytosolic supernatant modulate the function of the IP3 receptor by maintaining it in a low affinity state capable of promoting Ca2+ release.

Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca(2+) release channel/ryanodine receptors.

Maurocalcine is a novel toxin isolated from the venom of the chactid scorpion Scorpio maurus palmatus. It is a 33-mer basic peptide cross-linked by three disulfide bridges, which shares 82% sequence identity with imperatoxin A, a scorpion toxin from the venom of Pandinus imperator. Maurocalcine is peculiar in terms of structural properties since it does not possess any consensus motif reported so far in other scorpion toxins. Due to its low concentration in venom (0.5% of the proteins), maurocalcine was chemically synthesized by means of an optimized solid-phase method, and purified after folding/oxidation by using both C18 reversed-phase and ion exchange high-pressure liquid chromatographies. The synthetic product (sMCa) was characterized. The half-cystine pairing pattern of sMCa was identified by enzyme-based cleavage and Edman sequencing. The pairings were Cys3-Cys17, Cys10-Cys21, and Cys16-Cys32. In vivo, the sMCa was lethal to mice following intracerebroventricular inoculation (LD(50), 20 microg/mouse). In vitro, electrophysiological experiments based on recordings of single channels incorporated into planar lipid bilayers showed that sMCa potently and reversibly modifies channel gating behavior of the type 1 ryanodine receptor by inducing prominent subconductance behavior.

Functional role of hyperreactive sulfhydryl moieties within the ryanodine receptor complex.

Several laboratories using chemically heterogeneous sulfhydryl modifying agents have shown that sarcoplasmic reticulum (SR) Ca2+ channels known as ryanodine receptors (RyRs) are especially sensitive to modification of functionally important cysteine residues. The functional consequence of sulfhydryl modification of RyRs can include phases of activation and inhibition that are very much dependent on the concentration of the reagent used, the length of exposure, and the nature of the chemical reaction the reagent undertakes with sulfhydryl groups. Most challenging is understanding the relationship for how specific sulfhydryl moieties ascribe specific aspects of RyR function. Considering the structural complexity of the RyR complex with its associated proteins, this task is likely to be a formidable one. A small number of hyperreactive thiols have been shown to exist within the RyR complex. Their functional role does not appear to impact directly on channel gating. Rather hyperreactive cysteine (Cys) moieties may represent biochemical components of a redox sensor that conveys information about localized changes in redox potential produced by physiologic (e.g., glutathione, nitric oxide) and pathophysiologic (quinones, reactive oxygen species) channel modulators to the Ca2+ release process. The molecular and functional details of such a redox sensor remains to be elucidated.

Structural aspects of ryanodine action and selectivity.

The topography and toxicological relevance of the Ca2+-ryanodine receptor complex are evaluated with ryanodine and two natural analogues (9,21-didehydro and the new 18-hydroxy), 13 ryanoid derivatives (prepared from ryanodine and didehydroryanodine by functionalizing the available pyrrole, olefin, and hydroxyl substituents), and four degradation products. The potency of ryanoids at the skeletal muscle sarcoplasmic reticulum specific binding site generally parallels their toxicity to mice, supporting the toxicological relevance of the Ca2+-ryanodine receptor. The optimal receptor potency of ryanodine and didehydroryanodine is reduced 3-14-fold by hydroxylation at an isopropyl methyl substituent, epimerization at C9, oxidation or acetylation of the C10-hydroxyl, or epoxidation at the 9,21-position; other ryanoids are less active. Ryanodol and didehydroryanodol, in contrast to ryanodine and didehydroryanodine, have low toxicity to mice and little activity at the mammalian receptor, yet they are potent knockdown agents for injected houseflies or cockroaches, suggesting a possible difference in the target sites of mammals and insects.

Symposium overview: toxicity of non-coplanar PCBs.

Research into the mechanism of toxicity of PCBs has focused on the Ah receptor. However, it is becoming increasingly clear that certain ortho-chlorine-substituted, non-coplanar PCB congeners having low affinity for the Ah receptor exhibit important biological activities. Actions of non-coplanar PCB congeners in a variety of biological systems have been discovered and the mechanisms for these effects are being elucidated. The objectives of this symposium are to examine the state of knowledge concerning the mechanisms of toxic action of non-coplanar PCBs and to identify similarities and differences using a variety of biological systems. Effects to be considered will include: neurotoxicity, estrogenicity, insulin release, neutrophil function, calcium regulation, and relevant signal transduction systems. Finally, the symposium addresses the need to consider non-coplanar congeners within the context of risk assessment. The use of Ah-receptor binding and its associated biological effects to assess the total toxicity of PCBs may no longer be defensible because of the actions produced by non-coplanar congeners. This symposium provides documentation for that conclusion and focuses attention on emerging mechanisms of PCB action that have received relatively little attention to date. The topics presented should be of interest to toxicologists interested in mechanisms of action, in PCB risk assessment, and in regulatory toxicology.

Pharmacological characterization of the specific binding of [3H]ryanodine to rat brain microsomal membranes.

High-affinity binding of [3H]ryanodine has been characterized in rat brain microsomal fractions. Membrane fractions from 4 brain regions (cerebral cortex, cerebellum, hippocampus and brainstem) have been isolated using sucrose density gradient purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the presence of a high-molecular weight protein (Mr approximately 320 kDa), similar to that of ryanodine receptor from muscle sarcoplasmic reticulum (SR). In the presence of high salt (1 M KCl), [3H]ryanodine binds to low density (0.8 M sucrose) cortical microsomal fraction with high affinity (Kd 1.5 nM), and with the highest capacity (Bmax 330 fmol/mg protein). Kinetic analysis of the binding suggests multiple available binding sites for ryanodine. Binding of ryanodine is Ca2+ dependent (ED50 1 microM) and inhibited by Mg2+ and Ruthenium red. Adenine nucleotides have a biphasic effect on the binding of [3H]ryanodine. At low Ca2+ concentration caffeine and daunorubicin enhance the binding of [3H]ryanodine. The inositol 1,4,5-trisphosphate (IP3) binding inhibitor, heparin, has no effect on ryanodine binding, and ryanodine and caffeine do not influence the binding of [3H]IP3, which is enriched in the cerebellar fractions. These data demonstrate significant quantitative differences in the pharmacology of brain and muscle receptors and raise the question as to the physiological role of ryanodine binding proteins in the central nervous system and whether it is coupled to an endoplasmatic reticulum (ER) Ca2+ release channel.

Etiology of sarcoplasmic reticulum calcium release channel lesions in doxorubicin-induced cardiomyopathy.

Alterations in the native function of the ryanodine-sensitive Ca2+ release channel complex of sarcoplasmic reticulum (SR) isolated from rat cardiac ventricles during acute and chronic exposure to doxorubicin are examined. Compared to control SR, actively loaded SR from animals exposed to a single intravenous dose of doxorubicin exhibit faster rates of doxorubicin-induced Ca2+ release and the occupancy of [3H]ryanodine is significantly enhanced with subsequent exposure of SR membranes to doxorubicin in vitro. One week after acute exposure to doxorubicin in vitro, the EC50 for activation of the binding of [3H]ryanodine by Ca2+ is not significantly different from control SR. However, the persistence of doxorubicin-sensitized SR channels appears to be latent since repeated exposure to doxorubicin in vitro significantly enhances receptor occupancy in SR obtained from the treated rats compared to control SR. Ryanodine receptors from rats chronically exposed to doxorubicin consistently exhibit a higher sensitivity to activation Ca2+ which persists at least 4 weeks following the last injection of drug. Chronic exposure produces a concomitant reduction in the capacity of [3H]ryanodine binding sites. The marked decrease in receptor density observed with SR from doxorubicin-treated rats coincides with significant reduction in body weight, suggesting a possible influence of nutrition. However, sodium dodecyl sulfate polyacrylamide electrophoresis indicates no significant loss of the high molecular weight subunit of the ryanodine receptor, suggesting that loss of [3H]ryanodine-binding capacity may be the result of progressive and permanent channel desensitization. Consistent with desensitized receptors, membrane vesicles prepared from rats chronically exposed to doxorubicin take up significantly more Ca2+ and exhibit significantly reduced rates of doxorubicin or Ca2+/ryanodine induced Ca2+ release. The data demonstrates (i) doxorubicin inflicts cumulative SR channel lesions in vivo, (ii) a persistent sensitization of the SR channel to activation by Ca2+ and (iii) a significant and apparently irreversible reduction in the number of functional channel complexes.

Ryanodine induces maturation of embryonic acetylcholinesterase forms in cultured quail myotubes.

[3H]Ryanodine is shown to specifically bind to cultured myotubes from 10 day quail embryo pectoralis. The binding of [3H]ryanodine increases in a time-dependent manner reaching 38 +/- 3 fmol/mg protein at 4 h. A level of theophylline (THEO; 5mM) that induces propagated wave-like contractures, doubles the capacity of the myotubes to bind [3H]ryanodine (78 +/- 7 fmol/mg protein at 4 h). Polycationic ruthenium red (100 microM) only partially inhibits (56%) [3H]ryanodine-binding, whereas the membrane permeable channel antagonist [2,6-dichloro-4-dimethyl-amino-phenyl]-isopropylamine (20 microM) inhibits occupancy > 80%. Ryanodine (10 microM) interferes with THEO-induced contractures. Pretreatment with micromolar ryanodine for 48 h, followed by washout for 48 h, causes a persistent decrease in [3H]ryanodine-binding sites. Persistent [3H]ryanodine receptor blockade coincides with a dramatic shift in AChE forms found in the myotubes. A transition from the embryonic 4S and 7S globular forms to the 20S collagen-tailed (adult) form is evident within 12 hr exposure to ryanodine and progresses after removal of the alkaloid from the culture medium, mimicking the transition that normally occurs during myocyte maturation in vivo. These results suggest that SR Ca++ movements and excitation-contraction coupling may, at least in part, contribute to AChE maturation.

Ortho-substituted 2,2',3,5',6-pentachlorobiphenyl (PCB 95) alters rat hippocampal ryanodine receptors and neuroplasticity in vitro: evidence for altered hippocampal function.

The effects of PCBs on hippocampal function were studied in vitro, by radioligand-receptor binding analysis and electrophysiological measurements of the hippocampal slice preparation. [3H]Ryanodine, a conformation-sensitive probe for ryanodine receptors, was employed to determine how PCBs influence specific high-affinity occupancy to receptors found in microsomes isolated from rat hippocampus. PCB 95 (2,2',3,5'6-pentachlorobiphenyl) exhibited a dose-dependent enhancement of [3H]ryanodine receptor occupancy with an EC50 of 12 microM. In contrast, PCB 66 (2,3'4,4'-tetrachlorobiphenyl) showed no activity toward ryanodine receptors, up to its solubility limit (> or = 200 microM. Population spike (PS) and excitatory postsynaptic potential (EPSP) responses were recorded from striatum pyramidale of the CA1 region, which were generated from single pulse orthodromic stimulation of Schaffer collateral/commissural (SC/C) fibers at striatum radiatum of the hippocampal slice preparation. After the introduction of PCB 95 to the perfusion medium, PCB 95 depressed PS amplitude, especially at high stimulus intensities. Significant reductions in PS and EPSP maxima were seen, even after induction of long term potentiation, a model of neuroplasticity. However, these actions were not observed with PCB 66 which lacks ryanodine receptor activity, implicating a ryanodine receptor-mediated mechanism in the general depression of pyramidal cell excitability seen with PCB 95. Taken together, these results reveal a novel, arylhydrocarbon (Ah) receptor-independent, mechanism by which PCB 95 alters neuronal Ca2+ signaling and neuroplasticity in adult brain.

Long-term effects of developmental exposure to 2,2',3,5',6-pentachlorobiphenyl (PCB 95) on locomotor activity, spatial learning and memory and brain ryanodine binding.

There is mounting evidence that perinatal exposure to ortho-substituted PCB congeners causes neurobehavioral and neurochemical alterations. The molecular mechanism for these effects is not understood, but certain ortho-substituted PCBs have been found to interact specifically with ryanodine-sensitive Ca2+ channels in vitro. These channels are widely expressed in brain and are thought to be responsible for Ca(2+)-induced Ca2+ release. Thus, the ryanodine receptor may represent a selective molecular target through which ortho-substituted PCBs disrupt calcium signaling in neurons, and produce neurochemical and neurobehavioral alterations. Of the PCBs evaluated, 2,2',3,5',6-pentachlorobiphenyl (PCB 95) exhibits the highest potency and efficacy towards the ryanodine receptor in vitro. Therefore, we conducted an in vivo study to investigate the effects of developmental exposure to PCB 95 on neurobehavioral functional and regional brain ryanodine binding. Time-mated Sprague-Dawley rats were dosed with PCB 95 (8 or 32 mg/kg/day) or corn oil vehicle via gavage on gestation days 10-16. One male and one female from each litter were evaluated for neurobehavioral effects. Locomotor activity was evaluated in an automated open field at 35 and 100 days of age. Spatial learning and memory was assessed using an eight arm radial maze working memory task at 60 days of age and a T-maze delayed spatial alternation task at 140 days of age. The animals were then euthanized and [3H] ryanodine binding was assayed in homogenates of cerebral cortex, hippocampus and cerebellum. Rats exposed to PCB 95 showed normal levels of activity as juveniles, but were hypoactive in adulthood. They also showed a faster acquisition of the working memory task on the radial arm maze, but did not differ from controls on the T-maze delayed spatial alteration task. Region-specific changes in ryanodine binding to Ca2+ channels were also observed, with decreased binding in the hippocampus, increased binding in the cerebral cortex and a biphasic effect in the cerebellum. How these changes in ryanodine receptor function are related to the alterations in behavior will be a challenging problem to elucidate.

Ryanodine receptor acts as a sensor for redox stress.

Ryanoids have not attained importance as insecticides, but the increasing number of xenobiotic effectors known to influence Ca2+ signalling by interaction with ryanodine receptors (RyRs) may serve to identify new targets for insect control. A historical review of redox control of microsomal Ca2+ transport is given here, followed by recent evidence indicating that hyperactive Cys residues are an essential component of a transmembrane redox sensor. Focus is on the role of sulfhydryl chemistry in RyR regulation; metabolic quinonoid intermediates from pesticides and other environmental contaminants are of interest in this context.

Autism-specific maternal autoantibodies recognize critical proteins in developing brain.

Autism spectrum disorders (ASDs) are neurodevelopmental in origin, affecting an estimated 1 in 88 children in the United States. We previously described ASD-specific maternal autoantibodies that recognize fetal brain antigens. Herein, we demonstrate that lactate dehydrogenase A and B (LDH), cypin, stress-induced phosphoprotein 1 (STIP1), collapsin response mediator proteins 1 and 2 (CRMP1, CRMP2) and Y-box-binding protein to comprise the seven primary antigens of maternal autoantibody-related (MAR) autism. Exclusive reactivity to specific antigen combinations was noted in 23% of mothers of ASD children and only 1% of controls. ASD children from mothers with specific reactivity to LDH, STIP1 and CRMP1 and/or cypin (7% vs 0% in controls; P<0.0002; odds ratios of 24.2 (95% confidence interval: 1.45-405)) had elevated stereotypical behaviors compared with ASD children from mothers lacking these antibodies. We describe the first panel of clinically significant biomarkers with over 99% specificity for autism risk thereby advancing our understanding of the etiologic mechanisms and therapeutic possibilities for MAR autism.

Perinatal exposure to a noncoplanar polychlorinated biphenyl alters tonotopy, receptive fields, and plasticity in rat primary auditory cortex.

Noncoplanar polychlorinated biphenyls (PCBs) are widely dispersed in human environment and tissues. Here, an exemplar noncoplanar PCB was fed to rat dams during gestation and throughout three subsequent nursing weeks. Although the hearing sensitivity and brainstem auditory responses of pups were normal, exposure resulted in the abnormal development of the primary auditory cortex (A1). A1 was irregularly shaped and marked by internal nonresponsive zones, its topographic organization was grossly abnormal or reversed in about half of the exposed pups, the balance of neuronal inhibition to excitation for A1 neurons was disturbed, and the critical period plasticity that underlies normal postnatal auditory system development was significantly altered. These findings demonstrate that developmental exposure to this class of environmental contaminant alters cortical development. It is proposed that exposure to noncoplanar PCBs may contribute to common developmental disorders, especially in populations with heritable imbalances in neurotransmitter systems that regulate the ratio of inhibition and excitation in the brain. We conclude that the health implications associated with exposure to noncoplanar PCBs in human populations merit a more careful examination.