ryanodine and 2-2--3-5--6-pentachlorobiphenyl

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.

Topics: Animals; Arginine; Calcium; Cysteine; Disease Models, Animal; Dose-Response Relationship, Drug; In Vitro Techniques; Inhibitory Concentration 50; Malignant Hyperthermia; Muscle, Skeletal; Mutation; Polychlorinated Biphenyls; Protein Binding; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Swine; Tritium

The mechanisms by which non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and rapamycin interact with ryanodine receptor (RyR) complexes to alter Ca2+ signaling, were explored in intact cerebellar granule neurons. PCB 95 (10 microM, 20 min) significantly increased the number of neurons responding to caffeine. PCB 95 sensitization of RyR-mediated responses was further supported by the observations that ryanodine pretreatment blocked response to caffeine and coplanar 2,4,4',5-tetrachlorobiphenyl (PCB 66), which lacks RyR activity, failed to sensitize neurons. PCB 95 did not significantly alter levels of resting cytosolic Ca2+ nor thapsigargin-sensitive Ca2+ stores, suggesting a more complex mechanism than sensitization from increased cytosolic Ca2+ or an increased endoplasmic reticulum/cytosolic Ca2+ gradient. The immunosuppressant, rapamycin, sensitized neurons to caffeine in a manner similar to PCB 95, suggesting a common mechanism. PCB 95 or rapamycin significantly enhanced Ca2+ responses following N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl4-isoxasolepropiate (AMPA) receptor activation. Store depletion or direct block of RyR with ryanodine enhanced responses to NMDA. PCB 95 further enhanced these responses to NMDA. These results suggest that PCB 95 and rapamycin enhance NMDA- and AMPA-mediated Ca2+ signals by modifying a functional association of the FKBP12/RyR complex that results in amplification of glutamate signaling in cultured cerebellar granule neurons in culture.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Caffeine; Calcium; Calcium Signaling; Cerebellum; Cytosol; Drug Interactions; Immunosuppressive Agents; Neurons; Nitroso Compounds; Organ Culture Techniques; Polychlorinated Biphenyls; Receptors, AMPA; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sirolimus

ortho-Substituted PCBs mobilize Ca2+ from isolated brain microsomes by interaction with FKBP12/RyR complexes. Investigation into the cellular importance of this mechanism was undertaken using PC12 cells by fluoroimaging the actions of specific PCB congeners on [Ca2+]i and pH. RyR and IP3R share a common intracellular Ca2+ store in PC12 cells. Perfusion of nM to low microM PCB95 caused a transient rise of [Ca2+]i that was not completely dependent on extracellular Ca2+. Pre-incubation of the cells with ryanodine or FK506 completely eliminated PCB95 responses, suggesting a primary action on the FKPP12/RyR-sensitive store. PCB95, but not PCB126, induced a gradual decrease in cytosolic pH that could be completely eliminated by FK506 pre-incubation of the cells. Direct respiration measurement using isolated brain mitochondria demonstrated that neither of the PCBs directly altered any stage of mitochondrial respiration. These results revealed that PCB95 disrupts intracellular Ca2+ signaling in PC12 cells by interaction with the FKBP12/RyR complex that in turn accelerated cellular metabolism, possibly affecting signaling between ER and mitochondria. Since ortho-substituted PCBs have been shown to be neurotoxic and may affect neurodevelopment, studies on the molecular mechanism by which they alter cellular signaling may provide valuable information on the physiological roles of FKPB12 and RyR on neuronal functions.

Topics: Acidosis; Animals; Bradykinin; Brain Chemistry; Calcium; Calcium Signaling; Cell Respiration; Enzyme Inhibitors; Estrogen Antagonists; Hydrogen-Ion Concentration; Immunophilins; Inositol Phosphates; Intracellular Fluid; Male; Mitochondria; PC12 Cells; Pheochromocytoma; Polychlorinated Biphenyls; Rats; Rats, Sprague-Dawley; Ryanodine; Structure-Activity Relationship; Tacrolimus

Ortho-substituted polychlorinated biphenyls (PCBs) have been shown to alter microsomal Ca2+ transport by selective interaction with ryanodine receptors (RyRs) of muscle sarcoplasmic reticulum (SR) and brain endoplasmic reticulum. The mechanism underlying the actions of PCBs on Ca2+ transport is further elucidated with skeletal SR enriched in Ry1R. Disruption of the association between immunophilin FKBP12 and Ry1R with FK 506 or rapamycin completely eliminates PCB 95-enhanced binding of [3H]ryanodine (IC50 approximately 35 microM) to Ry1R and PCB 95-induced release of Ca2+ from actively loaded SR vesicles (IC50 approximately 11 microM), demonstrating a FKBP12-dependent mechanism. FK 506 selectively eliminates PCB 95-induced Ca2+ release from SR because Ry1R maintains responsiveness to caffeine and Ca2+. PCB 95 and FK 506 are used to examine the relationship between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive Ca2+ leak pathways present in SR vesicles. Micromolar ryanodine completely blocks ryanodine-sensitive Ca2+ efflux but neither eliminates the ryanodine-insensitive Ca2+ leak unmasked by thapsigargin nor enhances the loading capacity of SR vesicles. PCB 95 alone enhances thapsigargin evoked Ca2+ release and therefore diminishes the loading capacity of SR vesicles. However, in the presence of micromolar ryanodine, PCB 95 dose-dependently eliminates the Ca2+ leak unmasked by thapsigargin and significantly enhances the loading capacity of SR vesicles. The actions of PCB 95 on SR-loading capacity are additive with those of FK 506. Structural specificity for these novel actions are further demonstrated with coplanar PCB 126, which is inactive toward Ry1R and lacks the ability to alter the Ca2+ leak pathway. The results reveal that FKBP12 relates ryanodine-insensitive Ca2+ "leak" and ryanodine-sensitive Ca2+ channel efflux pathways of SR by modulating distinct conformations Ry1R complexes. Noncoplanar PCBs, like PCB 95, alter SR Ca2+ buffering by an FKBP12-mediated mechanism. An immunophilin-based mechanism could account for the toxic actions attributed to certain noncoplanar PCB congeners.

Topics: Animals; Caffeine; Calcium; Calcium Channels; Carrier Proteins; DNA-Binding Proteins; Drug Interactions; Heat-Shock Proteins; Ion Transport; Microsomes; Polychlorinated Biphenyls; Polyenes; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins

We investigated a novel molecular mechanism by which polychlorinated biphenyls (PCBs) alter microsomal Ca2+ transport with sarcoplasmic reticulum (SR) membranes isolated from skeletal and cardiac muscles. Aroclors with an intermediate weight percent of chlorine enhance by >6-fold the binding of 1 nM[3H]ryanodine to its conformationally sensitive site on the SR Ca2+ -release channel [i.e., ryanodine receptor (RyR)] with high potency (EC50=1.4 microM), whereas Aroclors with either high or low chlorine composition show little activity. Structure-activity studies with selected pentachlorobiphenyl congeners reveal a stringent structural requirement for chlorine substitution at the ortho-positions, with 2,2',3,5',6-pentachlorobiphenyl having the highest potency toward skeletal and cardiac isoforms of RyR (EC50=330 nM and 2 microM, respectively). In contrast, 3,3',4,4',5-pentachlorobiphenyl does not enhance ryanodine binding, suggesting that noncoplanarity of the biphenyl rings is required for channel activation. However, 2,2',4,6,6'-pentachlorobiphenyl is significantly less active toward RyR, suggesting that some degree of rotation about the biphenyl bond is required. 2,2',3,5',6-Pentachlorobiphenyl induces a dose-dependent release of Ca2+ from actively loaded SR vesicles with a maximum rate of 1.2 micromol mg-1 min-1 (EC50=1 microM), whereas 3,3',4,4',5-pentachlorobiphenyl (< / = microM) does not alter Ca2+ transport. The mechanism of PCB-induced channel activation involves a significant decrease in the inhibitory potency of Ca2+ and Mg2+ (20-fold and 100-fold, respectively). Neither 2,2',3,5',6- nor 3,3',4,4',5-pentachlorobiphenyl (< / = 10 microM) alters the activity of the skeletal isoform of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase or the cardiac isoform of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase, and PCB-induced Ca2+ release can be fully blocked by either microM ryanodine or ruthenium red. These results are the first to demonstrate a selective ryanodine receptor-mediated mechanism by which ortho-substituted PCBs alter microsomal Ca2+ transport and may have toxicological relevance.

Topics: Animals; Aroclors; Calcium; Calcium Channels; Male; Muscle Proteins; Muscle, Skeletal; Myocardium; Polychlorinated Biphenyls; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Structure-Activity Relationship