Transformation of local Ca2+ spikes to global Ca2+ transients: the combinatorial roles of multiple Ca2+ releasing messengers

Abstract

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca2+ spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca2+ spikes to global Ca2+ transients, focusing on the interactions of multiple intracellular messengers. ACh-elicited local Ca2+ spikes were transformed into a global sustained Ca2+ response by cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5-trisphosphate (IP3) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP3, whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP3 and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly amplified the local Ca2+ release evoked by a cADPR/IP3 mixture eliciting a vigorous global Ca2+ response. Different combinations of Ca2+ releasing messengers can shape the spatio-temporal patterns of cytosolic Ca2+ signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca2+ signals.

Fig. 1. Globalization of the local ACh response by cADPR and NAADP, but not IP₃. (A) Repetitive spikes of Ca²⁺-sensitive current were evoked by low, just suprathreshold concentrations of ACh. When IP₃ at 15 µM was included in the intracellular pipette solution, the ACh-evoked response was normal (B), but when the intracellular pipette solution contained either cADPR (10 µM) (C) or NAADP (50 nM) (D) ACh elicited larger sustained responses.

Fig. 2. Globalization of the CCK response by IP₃, but not cADPR or NAADP. (A) Repetitive spikes of Ca²⁺-sensitive current were evoked by low, just suprathreshold concentrations of CCK. (B) When IP₃ (15 µM) was included in the intracellular pipette solution, the CCK-evoked response was strongly potentiated (sustained). When the intracellular pipette solution contained either cADPR (10 µM) (C) or NAADP (50 nM) (D), CCK evoked normal non-potentiated (spiking) responses.

Fig. 3. NAADP evokes Ca²⁺ spiking in the secretory pole. Confocal fluorescence microscopy reveals that NAADP, at 50 nM in the intracellular pipette solution, evokes repetitive Ca²⁺ spikes localized in the apical pole. In the picture panel below the current trace is shown (left) the transmitted light picture of the cell investigated (tip of attached patch pipette is seen), (middle) the fluorescence image between spikes, and finally (right) the fluorescence image demonstrating the position of one of the Ca²⁺ spikes evoked by NAADP. By comparison with the transmitted light image, it can be seen that the spike occurs in the apical granular pole of the lower right cell. Calibration of the colour coding of the cytosolic Ca²⁺ concentration is shown at the right-hand side.

Fig. 4. A mixture of IP₃ and cADPR only evokes local Ca²⁺ release. (A) Confocal fluorescence microscopy reveals that cADPR, at 10 µM in the intracellular pipette solution, evokes repetitive Ca²⁺ spikes localized in the apical pole. The repetitive Ca²⁺ spikes evoked by IP₃ (10 µM) are localized in the apical pole (B) and the repetitive short-lasting Ca²⁺ spikes evoked by a mixture of cADPR (10 µM) and IP₃ (15 µM) (C) are also localized in the apical pole (no mutual potentiation).

Fig. 5. A mixture of NAADP and IP₃ evokes locally amplified Ca²⁺ release. (A) IP₃ (15 µM in the intracellular pipette solution) evokes repetitive spikes of Ca²⁺-sensitive current. The underlying Ca²⁺ spikes are localized in the apical pole (see Figure 4B). (B) A mixture of IP₃ (15 µM) and NAADP (50 nM) evokes somewhat amplified repetitive spikes of Ca²⁺-sensitive current. (C) The Ca²⁺ spikes elicited by the mixture of IP₃ and NAADP are localized in the apical pole. Calibration of the colour coding of the cytosolic Ca²⁺ concentration is shown at the bottom.

Fig. 6. NAADP transforms the short-lasting Ca²⁺ spikes evoked by cADPR into long-lasting Ca²⁺ spikes. (A) A mixture of NAADP (50 nM) and cADPR (10 µM) in the intracellular pipette solution evokes repetitive, long-lasting spikes of Ca²⁺-sensitive current with relatively long intervals between the spikes. (B) The result of an experiment simultaneously recording the Ca²⁺-sensitive Cl^– current and the cytosolic Ca²⁺ concentration measured by microfluorimetry in a single pancreatic acinar cell. The short-lasting spikes are local and the long-lasting spikes are global. Calibration of the colour coding of the cytosolic Ca²⁺ concentration is shown at the bottom.

Fig. 7. NAADP transforms the local Ca²⁺ spikes evoked by a cADPR + IP₃ mixture into a sustained global Ca²⁺ elevation. (A) A mixture of short-lasting spikes and sustained activation of Ca²⁺-sensitive current evoked by a triple mixture of NAADP (50 nM), cADPR (10 µM) and IP₃ (15 µM). External application of 20 mM caffeine, used as an IP₃ antagonist, abolished the sustained activation of the Ca²⁺-sensitive current, indicating that functional IP₃ receptors are required to generate the sustained Ca²⁺ signal. (B) The result of an experiment simultaneously recording the Ca²⁺-sensitive current and the cytosolic Ca²⁺ concentration measured by microfluorimetry in a single pancreatic acinar cell. Confocal fluorescence microscopy reveals that the triple mixture of NAADP (50 nM), cADPR (10 µM) and IP₃ (15 µM) in the intracellular pipette solution evokes a sustained global Ca²⁺ elevation. Calibration of the colour coding of the cytosolic Ca²⁺ concentration is shown at the bottom.

Fig. 8. (A) Schematic model showing that every messenger on its own can initiate local Ca²⁺ spikes. (B) There is little interaction between IP₃ and cADPR. (C) NAADP does potentiate the action of cADPR producing long-lasting global spikes at long intervals. (D) In contrast, NAADP only has a locally potentiating effect on the local IP₃-evoked Ca²⁺ spikes. (E) When all three messengers act together a large, sustained, global Ca²⁺ elevation is observed. The apical pole is the most sensitive part of the cell. In the models shown to the right, the basolateral part of the cell contains poorly sensitive Ca²⁺ release units that cannot trigger a wave in the presence of either IP₃, cADPR or NAADP alone. To generate a Ca²⁺ wave across the cell, a combination of potentiated Ca²⁺ release in the apical pole, helping to overcome the mitochondrial barrier, and sensitization of Ca²⁺ release channels by coincident activation of ryanodine, IP₃ and NAADP receptors by their respective messengers in the basal pole is necessary.