Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors.

Abstract

Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental composition of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 ± 1.1 mmol/kg (dry weight) (mean ± SEM). During a 3-sec nonsaturating light stimulus, -50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio ofMg uptake to Ca released was -0.7. Our results show unambiguously that the ER is the source of Ca2+ release during cell stimulation and suggest that Mg2+ can nearly balance the charge movement of Ca2+.The mobilization of Ca2" from an intracellular store by a reaction cascade comprising a cell-surface receptor, a guanine nucleotide binding protein, a phospholipase C, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an important mechanism of signal transduction (1). A great deal of evidence indicates that the endoplasmic reticulum (ER) is the target of Ins(1,4,5)P3 action (2-5) and the organelle involved in the physiological regulation of cytoplasmic Ca2" in nonmuscle cells (6-8), although some authors proposed that the Ins(1,4,5)P3-sensitive Ca2" pool is located in a separate organelle ("calciosome") (9,10). Direct measurements ofthe Ca2+ content and of Ca2+ changes in the ER (or the calciosomes) have been difficult to obtain. Consequently, little is known about the ion movements that must occur during Ca2+ release, to maintain charge balance across the ER membrane. The large sacs of ER in bee photoreceptors (11) are uniquely suitable for addressing these questions because the size makes them clearly identifiable in ultrathin freeze-dried cryosections and allows unambiguous experiments in which the analyzed area is completely within the ER cisternae. Therefore, we determined by electron probe x-ray microanalysis (EPMA) the elemental composition of the ER in situ, in the dark and during light stimulation, that, in invertebrates, is known to be followed by Ins(1,4,5)P3-mediated (12, 13) intracellular Ca2' release (14,15). We quantitated the amount of Ca2' released from the ER during photoresponse and demonstrate an associated uptake of Mg2' into the ER, suggesting that Mg2+ movements make a major contribution toward maintaining electroneutrality during Ca2' release from the ER in bee photoreceptors. Preliminary reports of these findings have been published (16,17). MATERIALS AND METHODSHoney bee drones (Apis mellifera) were kept in the dark at 32°C to 36°C for up to 10 days and fed a concentrated sugar solution. Decapitation and all subsequent manipulations were done under red light (A > 600 nm), which elicits no electrical response of the photoreceptors (18). Thick slices (600-1000 Am) ofthe drone head (19) remained in oxygenated physiological saline containing 170 mM NaCl, 10 mM KCI, 10 mM MgCl2, 1.6 mM CaC12, 10 mM Tris-HCl (pH ...

Facilitation of Membrane Fusion During Exocytosis and Exocytosis-Coupled Endocytosis and Acceleration of ``Ghost' Detachment in Paramecium by Extracellular Calcium. A Quenched-Flow/Freeze-Fracture Analysis

“…2 values of ∼50 mM [58,59], assuming 20% dry weight. Similar variation ranges were reported for SR, i.e., between 6 mM [24], 15 [23], 20 [22] and 33 mM [60], or for ER in different cells, i.e., between 1 mM [6], 1.5 to 2.5 [11], 3 [36], and 9.5 mM [7]. In sum, if one disregards excessively high values reported in older references for all of these systems, it appears reasonable to assume a total of ∼5 mM Ca 2+ in alveolar sacs.…”

Calcium as a Crucial Cofactor for Low Density Lipoprotein Receptor Folding in the Endoplasmic Reticulum

“…(iv) Calcium waves in the cell cause frequent transient depletion of calcium from the ER, dropping calcium levels from millimolar to 1-50 M levels (22). The charge change in the ER is compensated by parallel influx of other cations, such as Mg 2ϩ or K ϩ in muscle (69) and monovalent cations in hepatocytes (70). It is unlikely that the ER would be dysfunctional every time the cell needs its calcium for signaling.…”

Calcium release‐activated calcium current in rat mast cells.

“…There is evidence from a number of studies that in some cells there is an adjacent co-localization of the plasma membrane and parts of the endoplasmic reticulum (ER) (Rossier, Bird & Putney, 1991;McPherson, McPherson, Mathews, Campbell & Longo, 1992). The ER appears to act, at least in some cells, as a storage organelle for Ca2` (Baumann, Walz, Somlyo & Somlyo, 1991). Other microsomal storage organelles (calciosomes) have been described (Volpe et al 1988) but their localization needs further characterization.…”