Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2', 6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq-and phospholipase C beta-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.1142Ysciescopu

Desipramine Inhibits Histamine H1 Receptor-Induced Ca2+ Signaling in Rat Hypothalamic Cells

The hypothalamus in the brain is the main center for appetite control and integrates signals from adipose tissue and the gastrointestinal tract. Antidepressants are known to modulate the activities of hypothalamic neurons and affect food intake, but the cellular and molecular mechanisms by which antidepressants modulate hypothalamic function remain unclear. Here we have investigated how hypothalamic neurons respond to treatment with antidepressants, including desipramine and sibutramine. In primary cultured rat hypothalamic cells, desipramine markedly suppressed the elevation of intracellular Ca2+ evoked by histamine H1 receptor activation. Desipramine also inhibited the histamine-induced Ca2+ increase and the expression of corticotrophin-releasing hormone in hypothalamic GT1-1 cells. The effect of desipramine was not affected by pretreatment with prazosin or propranolol, excluding catecholamine reuptake activity of desipramine as an underlying mechanism. Sibutramine which is also an antidepressant but decreases food intake, had little effect on the histamine-induced Ca2+ increase or AMP-activated protein kinase activity. Our results reveal that desipramine and sibutramine have different effects on histamine H1 receptor signaling in hypothalamic cells and suggest that distinct regulation of hypothalamic histamine signaling might underlie the differential regulation of food intake between antidepressants

Muscarinic Receptors and BK Channels Are Affected by Lipid Raft Disruption of Salivary Gland Cells

Activity-dependent fluid secretion is the most important physiological function of salivary glands and is regulated via muscarinic receptor signaling. Lipid rafts are important for G-protein coupled receptor (GPCR) signaling and ion channels in plasma membranes. However, it is not well understood whether lipid raft disruption affects all membrane events or only specific functions in muscarinic receptor-mediated water secretion in salivary gland cells. We investigated the effects of lipid raft disruption on the major membrane events of muscarinic transcellular water movement in human salivary gland (HSG) cells. We found that incubation with methyl-β-cyclodextrin (MβCD), which depletes lipid rafts, inhibited muscarinic receptor-mediated Ca2+ signaling in HSG cells and isolated mouse submandibular acinar cells. However, MβCD did not inhibit a Ca2+ increase induced by thapsigargin, which activates store-operated Ca2+ entry (SOCE). Interestingly, MβCD increased the activity of the large-conductance Ca2+-activated K+ channel (BK channel). Finally, we found that MβCD did not directly affect the translocation of aquaporin-5 (AQP5) into the plasma membrane. Our results suggest that lipid rafts maintain muscarinic Ca2+ signaling at the receptor level without directly affecting the activation of SOCE induced by intracellular Ca2+ pool depletion or the translocation of AQP5 into the plasma membrane

Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry.

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2',6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling

Structures of PCBs.

<p>PCB4 (2,2’-dichlorinated biphenyl), PCB19 (2,2’,6-trichlorinated biphenyl), PCB50 (2,2’,4,6-tetrachlorinated biphenyl), and PCB100 (2,2’,4,4’,6-pentachlorinated biphenyl) have chlorine atoms at the <i>ortho</i> position of the phenyl ring, whereas PCB36 (3,3’,5-trichlorinated biphenyl) contains chlorine substitution at the <i>meta</i> position of the phenyl ring.</p

PCB19 blunts thapsigargin-induced increases in sustained [Ca²⁺]_i levels.

<p><b>A</b>, Fura-2-loaded PC12 cells were treated with 1 μM thapsigargin; 5 minutes later (at the sustained phase), cells were challenged with 50 μM of either PCB4, PCB19, or PCB100. Data presented include typical Ca²⁺ traces from more than five independent experiments. <b>B</b>, Concentration-dependent effects of PCBs on thapsigargin-induced SOCE. Decreases in Ca²⁺ levels were monitored upon stimulation with various concentrations of PCB4 (filled triangles), PCB19 (filled squares), PCB50 (blank circles), and PCB100 (blank triangles). Net decreases in [Ca²⁺]_i are expressed as % of controls (thapsigargin-induced Ca²⁺ levels without PCB19 treatment). Each point shown was obtained from triplicate experiments and represents the mean ± SEM. TG, thapsigargin.</p

PCB19 inhibits ionomycin and thapsigargin-induced Ca²⁺ influxes.

<p><b>(A, C, E)</b> Fura-2-loaded PC12 cells were incubated in Ca²⁺-free Locke’s solution, challenged with 50 μM PCB19, 1 μM thapsigargin or 300 nM ionomycin, and treated with 2.2 mM CaCl₂ at the indicated time (arrow). <b>(B, D, F)</b> The [Ca²⁺]i level at point <b>a</b> (Ca²⁺ release) and <b>b</b> (Ca²⁺ influx) were quantitatively analyzed using calcium traces and expressed as % of controls. Number of experiments are depicted in bar graph and each point represents mean ± SEM. TG, thapsigargin. **<i>P</i> < 0.01.</p

PCB19 inhibits thapsigargin-induced SOCE in a manner similar to other SOCE antagonists.

<p><b>A,</b> Fura-2-loaded PC12 cells were treated with 1 μM thapsigargin (TG), then sequentially challenged with 100 μM PCB19 and 30 μM 2-aminoethylphenyl borate (2APB). <b>B,</b> Cells were treated with 1 μM thapsigargin, challenged with 20 μM 2APB, and then treated with 50 μM PCB19. <b>C,</b> Cells were treated with 1 μM thapsigargin, then challenged sequentially with 100 μM PCB19 and 20 μM SK&F96365 (SKF). <b>D,</b> Cells were treated with 1 μM thapsigargin, challenged with 20 μM SK&F96365, and then treated with 50 μM PCB19. The [Ca²⁺]i level at point <b>a</b>, <b>b</b>, and <b>c</b> were quantitatively analyzed using calcium traces. Number of experiments are depicted in bar graph and each point represents mean ± SEM.</p

Ca²⁺ influxes stimulated by PCB19 are relatively small compared to those stimulated by intracellular Ca²⁺-mobilizing chemicals.

<p><b>A</b>, Fura-2-loaded PC12 cells were challenged with 50 μM PCB19 in the presence (left) or absence (right) of 2.2 mM extracellular free Ca²⁺. Ca²⁺ increases were also monitored upon reintroduction of 2.2 mM CaCl₂ in the condition lacking extracellular Ca²⁺. <b>B and C,</b> Experiments were performed as in (<b>A</b>), but with the addition of 1 μM thapsigargin <b>(B)</b> or 300 nM ionomycin <b>(C). D,</b> Peak height of [Ca²⁺]i increase was monitored and represented as mean ± SEM. <b>E and F,</b> Cells were treated with 300 nM ionomycin in the absence of extracellular free Ca²⁺ with (gray trance) or without (black trace) the pretreatment of 50 μM PCB19 for 100 sec. Ca²⁺ influx was then measured upon reintroduction of 2.2 mM CaCl₂ (Ca²⁺) into the extracellular space to monitor the ionomycin-induced Ca²⁺ influx. Number of experiments are depicted in bar graph and each point represents mean ± SEM. TG, thapsigargin.</p

PCB19 does not affect InsP₃ production.

<p><b>A,</b> PC12 cells were treated with 50 μM PCB4, PCB19, PCB50, or PCB100, or 300 nM bradykinin, for 15 seconds. Subsequently, InsP₃ production was measured. <b>B,</b> Cells were preincubated either with (black bar) or without (blank bar) 50 μM PCB19 for 3 min and then treated with 300 nM bradykinin for 15 seconds. Number of experiments are depicted in bar graph and each point represents mean ± SEM. BK, bradykinin.</p