Reversible interruption of ER Ca²⁺ uptake inversely affects ACh-elicited exocytosis in mouse and bovine chromaffin cells

Adrenal medulla chromaffin cells (CCs) are invaluable cell models for neurosecretion studies [5]. Significant differences in Ca²⁺ dynamics and exocytosis exist in CCs from several mammalian species (bovine, rat, guinea pig, cat, human) [7]. Nonetheless, the interest in mouse CCs (MCCs) increased disproportionally after the advent of transgenic mouse models to explore, from the complexities of Ca²⁺ signaling and exocytosis to alterations of Ca²⁺ homeostasis in neurodegenerative diseases [5].

There is a consensus that bovine chromaffin cells (BCCs) express an efficient mechanism of [Ca²⁺]_i signal amplification by which the initial [Ca²⁺]_i elevation opens ryanodine receptors (RyRs) from the ER through which Ca²⁺ is released into the cytosol [2, 8]. An influential article [10] reported that MCCs lack RyR-mediated Ca²⁺-induced Ca²⁺ release (CICR), but two recent studies in CCs from C57BL/6 mice [4, 11] conclusively support the presence of RyR and CICR. A differential role of mitochondria Ca²⁺ handling has also been reported: They sequester a more significant fraction of Ca²⁺ influx in MCCs than in BCCs [1].

Martínez-Ramírez et al., in this issue [9], compare the effects of acute, reversible SERCA pump inhibition by ciclopiazonic acid (CPA) on exocytotic responses and intracellular Ca²⁺ signals elicited by application to MCCs or BCCs of short ACh pulses at different intervals and extracellular [Ca²⁺]. The results could not be more contrasting: enhancement of exocytosis during CPA treatment and inhibition of exocytosis upon CPA washout in MCCs; inhibition of exocytosis during CPA exposure, and rebound exocytosis recovery upon CPA washout in BCCs. These differences, found under identical recording conditions at 37 °C, appear genuine. Intriguingly, CPA only reduced ACh-induced Ca²⁺ signals slightly in both cell types. Why the reversible pharmacological inhibition of ER Ca²⁺ uptake affects so differently ACh-elicited CA exocytosis in MCCs and BCCs?

The authors suggest that the opposite effects of CPA on ACh-induced exocytosis in MCCs and BCCs result from different [Ca²⁺]_i handling by the ER and mitochondria, affecting vesicle traffic and refilling of the rapid release pool. During the SERCA’s acute blockade, more Ca²⁺ is diverted to and taken up by the mitochondria through their Ca²⁺ uniporter. Conversely, upon restoration of ER Ca²⁺ uptake, mitochondria release Ca²⁺ through the Na⁺/Ca²⁺ exchanger, which the ER eventually takes up. As mentioned above, more Ca²⁺ is sequestered by mitochondria in MCCs than in BCCs [1]. However, mitochondrial Ca²⁺ transport cannot explain interspecies differences because it is not affected by the CPA.

It has been demonstrated that the ER can either attenuate or potentiate depolarization-induced Ca²⁺ signals, depending on its Ca²⁺ content. When depleted, a fraction of Ca²⁺ entering the cell during stimulation is captured by the store, acting as a Ca²⁺ sink. Conversely, when the store is full, it acts as a Ca²⁺ source and releases Ca²⁺ by CICR, therefore amplifying Ca²⁺ entry [3, 6].

Thus, the different behavior could be explained by assuming that mouse and bovine CCs differ in the two opposing ER functions: CICR and Ca²⁺ uptake: MCCs display weak CICR and strong Ca sinking. The reverse is true for BCCs. Significantly, CPA inhibits both mechanisms. In MCCs, secretion is enhanced by CPA because Ca²⁺ sinking is reduced, and a larger fraction of the Ca²⁺ entering the cell becomes available to trigger exocytosis. Upon removal of CPA, Ca²⁺ sinking recuperates, and exocytosis diminishes back to control. In BCCs, by discontinuing Ca²⁺ uptake (and allowing ER Ca²⁺ leakage), Ca²⁺ content drops, and CICR is weakened, thus reducing exocytosis. After CPA wash out, CICR recovers its strength, and exocytosis recuperates. See the summarizing scheme of Figure 9 in Martínez-Ramírez et al. [9]. The discordant Ca²⁺ signaling data is irrelevant in this context because Fura-2 reports “bulk [Ca²⁺]_I^“ and the relevant Ca²⁺ signals for exocytosis occur locally, in the submembrane space.

One may wonder if SERCA inhibition would affect differently exocytosis elicited by other stimuli, namely high-K⁺ depolarization, Ca²⁺ release by InsP₃-dependent mechanisms, or caffeine that directly opens RyRs. Also, given the crucial role of CICR in this context, it would be interesting to know if ryanodine, which blocks RyRs, affects exocytosis differently in MCC and BCC during SERCA inhibition.

The ER store’s filling status depends on the cell’s previous electrical activity, the expression level and activity of SERCA, and other Ca²⁺-mobilizing proteins, the expression, and activity of RyRs, InsP₃ receptors etc. Variations of these parameters determine the cell’s response under physiological and pathological scenarios. By clearly demonstrating differences between MCCs and BCCs in the same context, this study underscores the need to understand better the regulatory mechanisms that underlie these phenomena.