Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+ signaling in human retinal pigment epithelial cells.

Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+ signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks.

Bimodal modulation of store-operated Ca2+ channels by clozapine in astrocytes.

Clozapine (Clz) and olanzapine (Olz) are second generation (atypical) antipsychotics, used widely for treating schizophrenia and bipolar disorder. These drugs share multiple sites of actions, however their mechanisms remain incompletely understood. Here, we analyzed the effects of these drugs on primary cultures of rat cortical astrocytes and C6 glioma cells using fura-2-based Ca2+ imaging. C6 cells, but not cortical astrocytes, express the serotonin 2A receptor subtype, which couples to phospholipase C. Clz (1µM) significantly blocked serotonin-induced Ca2+ transients in C6 cells, consistent with known antagonistic actions of Clz. Interestingly, at higher concentrations (>10µM), Clz but not Olz increased intracellular Ca2+ concentrations in both cortical astrocytes and C6 cells. This Clz-induced Ca2+ increase was concentration-dependent and completely blocked by removal of extracellular Ca2+ using ethylene glycol tetraacetic acid (EGTA). Furthermore, 2-aminoethyl diphenylborinate or SKF-96365, blockers for store-operated Ca2+ channels, significantly inhibited the Clz-induced Ca2+ increase. Therefore, we analyzed the effects of Clz and Olz during Ca2+ re-entry through store-operated Ca2+ channels, which was maximized following depletion of internal Ca2+ stores by thapsigargin and EGTA. The results demonstrated that Clz decreased Ca2+ re-entry through store-operated Ca2+ channels in cortical astrocytes and C6 cells whereas Olz failed to modulate the Ca2+ re-entry. These results suggest Clz-specific bimodal actions via store-operated Ca2+ channels in astrocytic cells. Since intracellular Ca2+ homeostasis in astrocytes is an important determinant for neighboring synaptic signal transmission, our results may explain Clz-specific adverse effects or differential actions between Clz and Olz reported in the treatment of psychiatric disorders.