The endoplasmic reticulum (ER) is an intracellular organelle of fundamental importance present in all types of eukaryotic cells. The ER lumen is densely packed with numerous enzymatic systems that allow protein synthesis in the rough endoplasmic reticulum and correct post-translational ‘folding’ of these proteins. Any malfunctions in the latter process result in accumulation of unfolded proteins, which in turn activates several signalling systems aimed at appropriate compensatory responses. At the same time the ER is recognised as an important component of a different signalling system, i.e. the cytosolic calcium signalling cascade. Within a framework of this cascade the endoplasmic reticulum serves as a rapidly exchanging Ca²⁺ store, able to release Ca²⁺ ions upon appropriate physiological stimulation. In order for the endoplasmic reticulum to work as a dynamic Ca²⁺ store, a high concentration of free Ca²⁺ has to be maintained within its lumen, where [Ca²⁺] varies between 0.2 and 1 mM. Simultaneously, high intraluminal free Ca²⁺ concentration appears to be a key factor determining the activity of synthesis and processing of proteins within the endoplasmic reticulum, and disruption of endoplasmic reticulum Ca²⁺ homeostasis triggers endoplasmic reticulum stress response.

The importance of the endoplasmic reticulum as a dynamic calcium pool in nerve cells was first appreciated in the late 1980s and early 1990s when several groups reported stimulation-induced cytosolic Ca²⁺ signals recorded from cultured neurones, which were not affected by Ca²⁺ removal from the extracellular media. These [Ca²⁺]_i signals were triggered by either caffeine (potent activator of RyRs) or by neurotransmitters (glutamate or ATP) stimulating metabotropic (i.e. InsP₃ producing) receptors (Verkhratsky & Shmigol, 1996; Verkhratsky & Petersen, 1998).

By imaging free Ca²⁺ concentration within the ER lumen ([Ca²⁺]_L) of cultured peripheral (dorsal rot ganglia) and central (hippocampus) neurones employing low-affinity Ca²⁺ dyes we succeeded in real-time visualisation of [Ca²⁺]_L dynamics in response to physiological and pharmacological stimulation.

In a study of cultured rat DRG neurones we deployed the patch-clamp whole-cell technique to record membrane currents in parallel with simultaneous video-imaging of both [Ca²⁺]_L and calcium concentration in cytosol ([Ca²⁺]_i) using low (Mag-Fura-2) and high (Fluo-3) affinity Ca²⁺-sensitive fluorescent probes (Solovyova et al. 2002). Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca²⁺]_L levels to 40Ð50 % of the resting [Ca²⁺]_L value. Ca²⁺ entry through voltage-gated calcium channels also induced a transient decrease in [Ca²⁺]_L. This [Ca²⁺]_L response was inhibited by 50 mM ryanodine and potentiated by 1 mM of caffeine, indicating that it was directly associated with calcium-induced calcium release (CICR) triggered. Surprisingly the amplitude of CICR was linearly dependent on the amount of Ca²⁺ ions entering the cell with the Ca²⁺ current.

The InsP₃-induced Ca²⁺ release was investigated in saponin-permeabilised DRG neurones pre-loaded with Mag-Fura-2. The permeabilisation effectively removed the sytosolic portion of the dye allowing direct [Ca²⁺]_L monitoring in response to InsP₃ application. We found that both Ca²⁺ release mechanisms, the RyR-mediated and the InsP₃R-mediated, share the same Ca²⁺ pool, further substantiating the idea of one continuous ER Ca²⁺ store (Park et al. 2000).

In central neurones the visualisation of Ca²⁺ was hampered by the prolonged process of removal of the excess of low-affinity Ca²⁺ indicator from the cytosol. By using non-invasive staining with single-wavelength low-affinity Ca²⁺ indicators Mag-Fluo-4 (K_d ~20 µM) and Fluo-3FF (K_d ~40 µM) in combination with confocal imaging we found high levels of resting fluorescence within the cytosol, whereas virtually no signal was detected in the nuclear region. Cell depolarisation triggered a rapid transient increase in fluorescence in the nucleus. This ‘nuclear’ response was greatly attenuated by cell incubation with 100-500 nM thapsigargin. Thus inhibition of Ca²⁺ accumulation into the ER prevented the spread of Ca²⁺ elevation into the central portions of the cell. We suggest that the ER lumen may represent a specific Ca²⁺ ‘tunnel’ (similar to that found in pancreatic acinar cells Ð Petersen et al. 2001) which provides a pathway for rapid Ca²⁺ transport within the cell, thus connecting plasma membrane with cell interior.

This research was supported by a BBSRC research grant (ref. 34/C12751).

All procedures accord with current local guidelines.