Biomedical subjects

Isaac N Pessah

Publications and source records attributed to Isaac N Pessah.

At least 19 recordsLinked to original sources

PubMed2006

Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca(2+) microelectrodes in myotubes expressing wild-type RyR1 ((WT)RyR1) or RyR1 with one of four common MH mutations ((MH)RyR1), we measured resting intracellular Ca(2+) concentration ([Ca(2+)](i)). Changes in resting [Ca(2+)](i) produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the (MH)RyR1s had a 2.0- to 3.7-fold higher resting [Ca(2+)](i) than those expressing (WT)RyR1. Exposure of myotubes expressing (MH)RyR1s to ryanodine (500 microM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 microM) had no effects on their resting [Ca(2+)](i). However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca(2+)](i) was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca(2+)](i) in myotubes expressing (MH)RyR1s was significantly greater than that for (WT)RyR1. From these data, we propose that the high resting myoplasmic [Ca(2+)](i) in (MH)RyR1 expressing myotubes is due in part to a related structural conformation of (MH)RyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum.

Allosteric Regulation↗

PubMed2006

Ryanodine receptor type 1 (RyR1) possessing malignant hyperthermia mutation R615C exhibits heightened sensitivity to dysregulation by non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95).

Malignant hyperthermia (MH) susceptibility is conferred by inheriting one of >60 missense mutations within the highly regulated microsomal Ca(2+) channel known as ryanodine receptor type 1 (RyR1). Although MH susceptible patients lack overt clinical signs, a potentially lethal MH syndrome can be triggered by exposure to halogenated alkane anesthetics. This study compares how non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95), a congener identified in environmental and human samples, alters the binding properties of [(3)H]ryanodine to RyR1 in vitro. Junctional sarcoplasmic reticulum (SR) was isolated from skeletal muscle dissected from wild type pigs ((Wt)RyR1) and pigs homozygous for MH mutation R615C ((MH)RyR1), a mutation also found in humans. Although the level of (Wt)RyR1 and (MH)RyR1 expression is the same, (MH)RyR1 shows heightened sensitivity to activation and altered regulation by physiological cations. We report here that (MH)RyR1 shows more pronounced activation by Ca(2+), and is less sensitive to channel inhibition by Ca(2+) and Mg(2+), compared to (Wt)RyR1. In a buffer containing 100nM free Ca(2+), conditions typically found in resting cells, PCB 95 (50-1000nM) enhances the activity of (MH)RyR1 whereas it has no detectable effect on (Wt)RyR1. PCB 95 (2microM) decreases channel inhibition by Mg(2+) to a greater extent in (MH)RyR1 (IC(50) increased nine-fold) compared to (Wt)RyR1 (IC(50) increased by 2.5-fold). PCB95 reduces inhibition by Ca(2+) two-fold more with (MH)RyR1 than (Wt)RyR1. Our data suggest that non-coplanar PCBs are more potent and efficacious toward (MH)RyR1 than (Wt)RyR1, and have more profound effects on its cation regulation. Considering the important roles of Ca(2+) and Mg(2+) in regulating Ca(2+) signals involving RyR channels, these data provide the first mechanistic evidence that a genetic mutation known to confer susceptibility to pharmacological agents also enhances sensitivity to an environmental contaminant.

Animals↗

PubMed2006

Simplified cyclic analogues of bastadin-5. Structure-activity relationships for modulation of the RyR1/FKBP12 Ca2+ channel complex.

Bastadin-5, a brominated macro-dilactam from the marine sponge Ianthella basta, enhances release of Ca2+ from stores within the sarcoplasmic reticulum (SR) of muscle and nonmuscle cells by modulating RyR1/FKBP12 complex. Analogues of bastadin-5 present desirable targets for SAR studies to shed light on the gating mechanism and locus of bastadin-5 binding on these heteromeric channels that mediate essential steps in early coupling of membrane excitation to Ca2+ signaling cascades. Simple, ring-constrained analogues of bastadin-5 were synthesized from substituted benzaldehydes in a convergent manner, featuring an efficient S(N)Ar macroetherification, and evaluated in an assay that measures [3H]-ryanodine that is known to correlate with the functional open state of the Ca2+ channel. The simplified 14-membered ring, atropisomeric analogue (+/-)-7, like bastadin-5, enhanced ryanodine binding to the RyR1/FKBP12 complex (EC50 11 microM), however, unexpectedly, the corresponding achiral 18-membered ring analogue 14 potently inhibited binding (IC50 6 microM) under the same conditions. Structure-activity relationships of both families of cyclic analogues showed activity in a ryanodine binding assay that varied with substitutions of the Br atom on the trisubstituted aryl ring by various functional groups. The most active analogues were those that conserved the dibromocatechol ether moiety that corresponds to the 'western edge' of the bastadin-5 structure. These data suggest that cyclic analogues of bastadin-5 interact with the channel complex in a complex manner that can either enhance or inhibit channel activity.

Animals↗

PubMed2006

Amino acid residues Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol.

The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.

Allosteric Site↗

PubMed2006

Functional coupling between TRPC3 and RyR1 regulates the expressions of key triadic proteins.

We have shown that TRPC3 (transient receptor potential channel canonical type 3) is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was "knocked down" in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts (97.6 +/- 1.9% reduction in mRNA) were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, we found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated (1.46 +/- 1.91-, 1.42 +/- 0.08-, 2.99 +/- 0.32-, and 1.91 +/- 0.26-fold, respectively) in TRPC3 KD. Based on these data, we conclude that expression of TRPC3 is tightly regulated during muscle cell differentiation and propose that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load.

Acetylcysteine↗

PubMed2006

Heptachlor epoxide induces a non-capacitative type of Ca2+ entry and immediate early gene expression in mouse hepatoma cells.

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE) and a related non-tumor promoting OC, delta-hexachlorocyclohexane (delta-HCH), on the dynamics of intracellular calcium (Ca2+) were investigated in mouse 1c1c7 hepatoma cells. HE induced a non-capacitative, Ca2+ entry-like phenomenon, which was transient and concentration-dependent with 10 and 50 microM HE. The plasma membrane Ca2+ channel blocker SKF-96365 antagonized this HE-induced Ca2+ entry. delta-HCH failed to induce Ca2+ entry, rather it antagonized the HE-induced Ca2+ entry. Both HE and delta-HCH induced Ca2+ release from endoplasmic reticulum (ER) at treatment concentrations as low as 10 microM; at 50 microM, the former induced 5x as much Ca2+ release as the latter. The HE-induced Ca2+ release from the ER was antagonized using the IP3 receptor/channel blocker xestospongin C, suggesting that HE induces ER Ca2+ release through the IP3 receptor/channel pore. These results show that the effect of HE on cellular Ca2+ mimics that of mitogens such as epidermal and hepatocyte growth factors. They also provide insight into the similarities and differences between tumorigenic and non-tumorigenic OCs, in terms of the mechanisms and the extent of the [Ca2+]i increased by these agents.

Animals↗

PubMed2006

Structure-activity relationship for noncoplanar polychlorinated biphenyl congeners toward the ryanodine receptor-Ca2+ channel complex type 1 (RyR1).

Ryanodine receptor isoforms are expressed in both excitable and nonexcitable tissues where they form microsomal Ca2+ release channels broadly involved in shaping cellular signaling. In this report, we provide a detailed structure-activity relationship (SAR) for polychlorinated biphenyl (PCB) congeners and metabolites necessary for enhancing ryanodine receptor type 1 (RyR1) activity using [3H]ryanodine ([3H]Ry) binding analysis. The 2,3,6-Cl PCB configuration is most important for optimal recognition by the RyR1 complex and/or critical for sensitizing its activation. Para substitution(s) diminishes the activity with para-chloro having a higher potency than the corresponding para-hydroxy derivative. The addition of a more bulky para-methyl-sulfonyl group eliminates the activity toward RyR1, supporting the importance of the para positions in binding RyR1. The requirement for an intact major T cell immunophilin FKBP12-RyR1 complex was observed with each of 12 active PCB congeners indicating a common mechanism requiring an immunophilin-regulated Ca2+ release channel. An excellent correlation between the relative potencies for doubling [3H]Ry binding and the corresponding initial rates of PCB-induced Ca2+ efflux indicates that [3H]Ry binding analysis provides a measure of dysregulation of microsomal Ca2+ transport. The SAR for activating RyR1 is consistent with those previously reported in several in vivo and in vitro studies, suggesting that a common mechanism may contribute to the toxicity of noncoplanar PCBs. A practical application of the receptor-based screen developed here with RyR1 is that it provides a quantitative SAR that may be useful in predicting biological activity and risk of mixtures containing noncoplanar PCB congeners that have low or a lack of aryl hydrocarbon receptor activity.

Animals↗

PubMed2006

Pharmacologic and functional characterization of malignant hyperthermia in the R163C RyR1 knock-in mouse.

BACKGROUND: Malignant hyperthermia is a pharmacogenetic disorder affecting humans, dogs, pigs, and horses. In the majority of human cases and all cases in animals, malignant hyperthermia has been associated with missense mutations in the skeletal ryanodine receptor (RyR1). METHODS: The authors used a "knock-in" targeting vector to create mice carrying the RyR1 R163C malignant hyperthermia mutation. RESULTS: Validation of this new mouse model of human malignant hyperthermia susceptibility includes (1) proof of transcription of the R163C allele and expression of ryanodine receptor protein in R163C heterozygous and R163C homozygous animals; (2) fulminant malignant hyperthermia episodes in R163C heterozygous mice after exposure to 1.25-1.75% halothane or an ambient temperature of 42 degrees C characterized by increased rectal temperature, respiratory rate, and inspiratory effort, with significant blood biochemical changes indicating metabolic acidosis, ending in death and hyperacute rigor mortis; (3) intraperitoneal pretreatment with dantrolene provided 100% protection from the halothane-triggered fulminant malignant hyperthermia episode; (4) significantly increased sensitivity (decreased effective concentration causing 50% of the maximal response) of R163C heterozygous and homozygous myotubes to caffeine, 4-chloro-m-cresol, and K-induced depolarization; (5) R163C heterozygous and homozygous myotubes have a significantly increased resting intracellular Ca concentration compared with wild type; (6) R163C heterozygous sarcoplasmic reticulum membranes have a twofold higher affinity (Kd = 35.4 nm) for [H]ryanodine binding compared with wild type (Kd = 80.1 nm) and a diminished inhibitory regulation by Mg. CONCLUSIONS: Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome.

Anesthetics, Inhalation↗

PubMed2006

The CHARGE study: an epidemiologic investigation of genetic and environmental factors contributing to autism.

Causes and contributing factors for autism are poorly understood. Evidence suggests that prevalence is rising, but the extent to which diagnostic changes and improvements in ascertainment contribute to this increase is unclear. Both genetic and environmental factors are likely to contribute etiologically. Evidence from twin, family, and genetic studies supports a role for an inherited predisposition to the development of autism. Nonetheless, clinical, neuroanatomic, neurophysiologic, and epidemiologic studies suggest that gene penetrance and expression may be influenced, in some cases strongly, by the prenatal and early postnatal environmental milieu. Sporadic studies link autism to xenobiotic chemicals and/or viruses, but few methodologically rigorous investigations have been undertaken. In light of major gaps in understanding of autism, a large case-control investigation of underlying environmental and genetic causes for autism and triggers of regression has been launched. The CHARGE (Childhood Autism Risks from Genetics and Environment) study will address a wide spectrum of chemical and biologic exposures, susceptibility factors, and their interactions. Phenotypic variation among children with autism will be explored, as will similarities and differences with developmental delay. The CHARGE study infrastructure includes detailed developmental assessments, medical information, questionnaire data, and biologic specimens. The CHARGE study is linked to University of California-Davis Center for Children's Environmental Health laboratories in immunology, xenobiotic measurement, cell signaling, genomics, and proteomics. The goals, study design, and data collection protocols are described, as well as preliminary demographic data on study participants and on diagnoses of those recruited through the California Department of Developmental Services Regional Center System.

Adult↗

PubMed2006

Uncoupling of ATP-mediated calcium signaling and dysregulated interleukin-6 secretion in dendritic cells by nanomolar thimerosal.

Dendritic cells (DCs) , a rare cell type widely distributed in the soma, are potent antigen-presenting cells that initiate primary immune responses. DCs rely on intracellular redox state and calcium (Ca2+) signals for proper development and function, but the relationship between these two signaling systems is unclear. Thimerosal (THI) is a mercurial used to preserve vaccines and consumer products, and is used experimentally to induce Ca2+ release from microsomal stores. We tested adenosine triphosphate (ATP) -mediated Ca2+ responses of DCs transiently exposed to nanomolar THI. Transcriptional and immunocytochemical analyses show that murine myeloid immature DCs (IDCs) and mature DCs (MDCs) express inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) Ca2+ channels, known targets of THI. IDCs express the RyR1 isoform in a punctate distribution that is densest near plasma membranes and within dendritic processes, whereas IP3Rs are more generally distributed. RyR1 positively and negatively regulates purinergic signaling because ryanodine (Ry) blockade a) recruited 80% more ATP responders, b) shortened ATP-mediated Ca2+ transients > 2-fold, and c) produced a delayed and persistent rise (>/= 2-fold) in baseline Ca2+. THI (100 nM, 5 min) recruited more ATP responders, shortened the ATP-mediated Ca2+ transient (>/= 1.4-fold) , and produced a delayed rise (>/= 3-fold) in the Ca2+ baseline, mimicking Ry. THI and Ry, in combination, produced additive effects leading to uncoupling of IP3R and RyR1 signals. THI altered ATP-mediated interleukin-6 secretion, initially enhancing the rate of cytokine secretion but suppressing cytokine secretion overall in DCs.DCs are exquisitely sensitive to THI, with one mechanism involving the uncoupling of positive and negative regulation of Ca2+ signals contributed by RyR1.

Adenosine Triphosphate↗

PubMed2005

Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone marrow dendritic cells by modulating class II MHC expression.

Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O2 atmospheres and omitting 2-Me from the medium were tested upon the generation of GM-CSF expanded BMDCs. DC yield, phenotype and function were compared to BMDCs grown using conventional conditions. All cultures yielded DC subsets with CD11c+ MHC II(NEG), CD11c+ MHC II(INT), CD11c+ MHC II(HI) expression phenotypes, classed as precursor, immature, and mature DCs (IDC, MDC). Low O2 tensions generated significantly fewer precursor DCs, and more IDCs and MDCs. Cytometer sorted precursor DCs expressed surface class II MHC after transfer to low, but not high O2 atmospheres. Expression of myeloid markers was similar between BMDC cultures generated in 5% O2 or conventional conditions, and MDCs from low O2 cultures had the morphology typical of mature myeloid DCs. IDCs and MDCs from low O2 and conventional culture conditions were similarly potent allostimulatory APCs. The O2 tension (but not 2-Me addition) in vitro significantly influences overall DC subset frequencies and yield, and governs DC maturation by regulating the surface class II MHC expression of GM-CSF expanded BMDC cultures.

Animals↗

PubMed2005

Purkinje cell and cerebellar effects following developmental exposure to PCBs and/or MeHg.

We recently reported that rats exposed to PCBs and MeHg during development were impaired on the rotating rod, a test of balance and coordination that is often indicative of cerebellar damage. In addition, developmental PCB exposure is known to dramatically reduce circulating thyroid hormone concentrations, which may have a negative impact on cerebellar development. Therefore, we investigated the effects of combined PCB and MeHg exposure on Purkinje cells and the cerebellum. The serum and brains from littermates of the animals tested on the rotating rod were collected at weaning, and we also collected brains from the adult animals at the end of motor testing. Four groups were studied: 1) vehicle controls, 2) PCBs only (Aroclor 1254, 6 mg/kg/d, oral), 3) MeHg only (0.5 ppm, in dams' drinking water), and 4) PCB+MeHg (at the same doses as in individual toxicant exposures). Female Long-Evans rats were exposed beginning 4 weeks prior to breeding with an unexposed male and continuing until postnatal day (PND) 16. There was a significant reduction in serum T4 and T3 concentrations in the PCB and PCB+MeHg pups on PND21. Golgi-impregnated Purkinje cells were examined in PND21 brains, but there were no significant exposure-related effects on primary dendrite length, branching area, or structural abnormalities. However, all three male exposure groups had a marginally significant increase in Purkinje cell height, which may suggest a subtle thyromimetic effect in the cerebellum. Cresyl-violet stained sections from the adult brains showed no exposure-related effects within paramedian lobule in Purkinje cell number, total lobule volume or layer volumes (molecular, granule cell and white matter layers). Evidence is provided for the dysregulation of expression of cerebellar ryanodine receptor (RyR) isoforms in PCB-exposed brains, and this could contribute to the rotating rod deficit by changing critical aspects of intracellular calcium signaling within the cerebellum.

Administration, Oral↗

PubMed2005

Heat- and anesthesia-induced malignant hyperthermia in an RyR1 knock-in mouse.

Malignant hyperthermia (MH) is a life-threatening disorder characterized by skeletal muscle rigidity and elevated body temperature in response to halogenated anesthetics such as isoflurane or halothane. Mutation of tyrosine 522 of RyR1 (the predominant skeletal muscle calcium release channel) to serine has been associated with human malignant hyperthermia. In the present study, mice created harboring this mutation were found to represent the first murine model of human malignant hyperthermia. Mice homozygous for the Y522S mutation exhibit skeletal defects and die during embryonic development or soon after birth. Heterozygous mice, which correspond to the human occurrence of this mutation, are MH susceptible, experiencing whole body contractions and elevated core temperatures in response to isoflurane exposure or heat stress. Skeletal muscles from heterozygous mice exhibit increased susceptibility to caffeine- and heat-induced contractures in vitro. In addition, the heterozygous expression of the mutation results in enhanced RyR1 sensitivity to activation by temperature, caffeine, and voltage but not uncompensated sarcoplasmic reticulum calcium leak or store depletion. We conclude that the heterozygous expression of the Y522S mutation confers susceptibility to both heat- and anesthetic-induced MH responses.

Anesthesia↗

PubMed2005

Hydroxylated xestospongins block inositol-1,4,5-trisphosphate-induced Ca2+ release and sensitize Ca2+-induced Ca2+ release mediated by ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) often coexist within the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane and coordinate precise spatial and temporal coding of Ca(2+) signals in most animal cells. Xestospongin C (XeC) was shown to selectively block IP(3)-induced Ca(2+) release and IP(3)R-mediated signaling (Gafni et al., 1997). We have further studied the specificity of xestospongin structures possessing ring hydroxyl (-OH) substituents toward IP(3)R, RyR, and ER/SR Ca(2+)-ATPase (SERCA) activities. XeC potently inhibits IP(3)R, weakly inhibits RyR1, and lacks activity toward SERCA1 and SERCA2. XeD (9-OH XeC), 7-OH-XeA, and araguspongin C isolated from the marine sponge Xestospongia species also inhibit IP(3)-mediated Ca(2+) release and lack activity toward SERCA. However, these hydroxylated derivatives possess a unique activity in that they enhance Ca(2+)-induced Ca(2+) release from SR vesicles by a mechanism involving the sensitization of RyR1 channels within the same concentration range needed to block IP(3)-induced Ca(2+) release. These results show that xestospongins and related structures lack direct SERCA inhibitory activity, as suggested by some previous studies. A new finding is that XeD and related structures possessing a hydroxylated oxaquinolizidine ring are IP(3)R blockers that also enhance Ca(2+)-induced Ca(2+) release mediated by RyRs. In intact cells, the actions of XeD are blocked by ryanodine pretreatment and do not interfere with thapsigargin-mediated Ca(2+) mobilization stemming from SERCA block. Hydroxylated bis-oxaquinolizadine derivatives isolated from Xestospongia species are novel bifunctional reagents that may be useful in ascertaining how IP(3)Rs and RyRs contribute to cell signaling.

Alkaloids↗

PubMed2005

Formulation and characterization of an experimental PCB mixture designed to mimic human exposure from contaminated fish.

Each environmental exposure matrix contains a unique mixture of PCB congeners. Since several congener types have multiple and distinct biological actions, it is important to characterize congener profiles in exposure sources. The Fox River Environment and Diet Study (FRIENDS) is assessing the human health effects of consumption of PCB-contaminated fish from the Fox River in northeastern Wisconsin. Concurrent laboratory studies required the formulation of a dosing solution which closely mimicked the human PCB exposure from fish. PCB congener profiles from Fox River walleye were compared to profiles for various theoretical mixtures having different relative percentages of Aroclors by weight. The theoretical mixture which provided the best approximation of the Fox River fish PCB profile contained 35% 1242, 35% 1248, 15% 1254, and 15% 1260. A PCB mixture was formulated to match this theoretical construct, and the congener profile for the mixture of Aroclors was determined by capillary column gas chromatography with electron capture detection (GC/ECD). The relative percent of each congener was compared to the PCB congener profile of the theoretical Aroclor mixture and that for Fox River walleye. The specific congeners differed on average by 17% from the theoretical Aroclor mixture predicted values, and the specific congeners measured in the mixture were on average within 71% of those reported for Fox River fish. The mixture was found to have relatively low AhR activity but high RyR activity. Indirect comparisons suggest that in vivo toxicity was slightly greater than that for Aroclor 1254. This illustrates that Aroclor mixtures are useful for formulating dosing solutions which closely approximate actual environmental exposures.

Animals↗

PubMed2005

Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion.

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

Animals↗

PubMed2005

Ryanodine receptor-mediated rapid increase in intracellular calcium induced by 7,8-benzo(a)pyrene quinone in human and murine leukocytes.

Benzo(a)pyrene (BaP) is an environmentally prevalent polycyclic aromatic hydrocarbon (PAH) known to produce immunotoxicity in murine and human lymphocytes. Previous studies by our lab have shown that certain BaP metabolites increase intracellular Ca(2+) in human and murine lymphocytes. The mechanism by which these BaP metabolites increase Ca(2+) may involve src kinase activation and mitochondrial oxidative stress. We have implicated a new pathway of Ca(2+) elevation in lymphocytes produced by a novel BaP metabolite, BaP-7,8-dione (7,8-BPQ). This ortho quinone is produced from BaP-7,8-dihydrodiol by aldoketoreductase 1C1 (AKR1C) isoforms in human cells. We have previously shown that 7,8-BPQ increases Ca(2+) levels in an in vitro rabbit skeletal muscle sarcoplasmic reticulum (SR) vesicle model via interaction with ryanodine receptors (RyR). In the present study, we found that 7,8-BPQ produced a RyR-dependent rapid increase in intracellular Ca(2+) in the Daudi human B cell line. However, other BP-diones including 1,6-, 3,6-, and 6,12-BPQs failed to produce a rapid increase in Ca(2+). Instead they produced a late increase in intracellular Ca(2+), presumably via a redox-cycling-dependent loss of Ca(2+) buffering capacity by mitochondria. Functional RyR were detected in Daudi using a (3)H-ryanodine binding assay. The studies were extended to normal human peripheral blood and murine spleen cells, where it was found that 7,8-BPQ rapidly elevated intracellular Ca(2+) in B cells and T cells in both species. The Ca(2+)-elevating effect of 7,8-BPQ was prevented by pretreatment with a high concentration of ryanodine (500 muM). Collectively, these results demonstrate a novel mechanism of Ca(2+) elevation by an environmentally relevant metabolite of BaP in murine and human lymphocytes.

Animals↗

PubMed2005

Three structurally related, highly potent, peptides from the venom of Parabuthus transvaalicus possess divergent biological activity.

The venom of South African scorpion Parabuthus transvaalicus contains a novel group of peptide toxins. These peptides resemble the long chain neurotoxins (LCN) of 60-70 residues with four disulfide bridges; however they are 58 residues long and have only three disulfide bridges constituting a new family of peptide toxins. Here we report the isolation and characterization of three new members of this mammal specific group of toxins. Dortoxin is a lethal peptide, bestoxin causes writhing in mice and altitoxin is a highly depressant peptide. Binding ability of these peptides to rat brain synaptosomes is tested. While the crude venom of P. transvaalicus enhances the binding of [(3)H] BTX to rat brain synaptosomes none of these individual toxins had a positive effect on binding. Although the primary structures of these toxins are very similar to birtoxin, their 3D models indicate significant differences. Dortoxin, bestoxin and altitoxin cumulatively constitute at least 20% of the peptide contained in the venom of P. transvaalicus and contribute very significantly to the toxicity of the venom of this medically important scorpion species. Therefore the amino acid sequences presented here can be used to make more specific and effective antivenins. Possible approaches to a systematic nomenclature of toxins are suggested.

Amino Acid Sequence↗