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G. Duncan, L. Wang, D.J. Collison, A.R. Prescott; Sigma Receptor Signalling in the Human Lens–Role of the ER . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4696.
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Purpose: Sigma receptor antagonists have recently been shown to inhibit lens cell growth in human capsular bag preparations and also, surprisingly, to induce melanin granule formation. As Sigma receptors appear to be located in the Endoplasmic Reticulum (ER), this study investigated the impact of Sigma antagonists on calcium signalling in lens cells and also the effect of inhibitors of ER protein trafficking on pigment formation. Methods: Human capsular bags were prepared from donor eyes. They were maintained in Eagle’s minimum essential medium (EMEM) or EMEM supplemented with the Sigma receptor antagonists rimcazole or BD1047. Pigmentation was assessed by digital imaging and electron microscope (EM) techniques. FHL 124 cells were routinely cultured on glass coverslips. Total RNA was extracted from native lens epithelia and cultured FHL 124 cells and expression of the Sigma–1 receptor was assayed by RT–PCR. Following Fura–2 incorporation into FHL 124 cells, ratiometric calcium imaging was performed using epifluorescent microscopy techniques. Results: Message for the Sigma–1 receptor was detected in human lens epithelia and FHL 124 cells. Rimcazole and the specific Sigma–1 antagonist BD1047 (both 3µM) initiated pigment granule formation after 4–6 days exposure. Pigmentation was unaffected by the signalling inhibitors thapsigargin (1µM) and suramin (200µM) but was suppressed by the ER/Golgi protein trafficking inhibitor brefeldin A (10µM). Rimcazole induced a slow increase in intracellular calcium in FHL 124 cells perifused with artificial aqueous humour and also reduced the calcium released from the ER store by subsequent application of G–protein agonists (e.g. ATP). Emptying the store by thapsigargin activated the capacitative calcium entry pathway and entry was blocked by both antagonists. Paradoxically, when the antagonists were applied with the store in the normal full state, calcium entry was stimulated through a non–capacitative pathway. Conclusions:All human lens cells studied expressed the Sigma–1 receptor. Receptor antagonists induced melanin granule synthesis and a crucial step appears to be in ER/Golgi protein trafficking. Although the sigma receptor is located on the ER, agonist binding is signalled to the plasma membrane resulting in the novel reciprocal activation of non–capacitative and capacitative calcium entry pathways. We suggest that Sigma receptors are part of a lens stress response and it is relevant that pigment granules have been detected in human cataract (eg Cataracta nigra).
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