Ca2+ homeostasis in the agonist-sensitive internal store: functional interactions between mitochondria and the ER measured In situ in intact cells - PubMed (original) (raw)

Ca2+ homeostasis in the agonist-sensitive internal store: functional interactions between mitochondria and the ER measured In situ in intact cells

B Landolfi et al. J Cell Biol. 1998.

Abstract

Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals.

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Figures

Figure 1

Figure 1

FCCP inhibits ER Ca2+ uptake. Mag-fura-2 measurements of [Ca2+] in agonist-sensitive internal stores in intact cells. (A) Resting intact cell treated with FCCP (1 μM) followed by FCCP 1 BK (100 nM). (B) FCCP administered during store refilling subsequent to BK-induced release inhibited Ca2+ sequestration, but had little effect on the mag-fura-2 ratio when given after treatment with a SERCA blocker, tBHQ (10 μM).

Figure 3

Figure 3

FCCP and oligomycin impede store refilling after BK-induced Ca2+ release in intact mag-fura-2-loaded cells. (A) Control recovery and response in the same cell to hormone after brief pretreatment with FCCP. (Inset) Entire experimental record. (Bottom) Enlarged overlay emphasizing the recovery phases with and without FCCP. (B) As above, profile of store reloading before and after treatment with 1 μM oligomycin. (C) Comparison of store refilling after three separate stimulations with 100 nM BK: first in the presence of 1 μM FCCP in Ca2+-containing medium; second after a 15-min incubation with 40 μM BAPTA-AM (added at arrow); cells were washed with normal Ringer's for 5 min, and were then stimulated with BK in Ca2+-free medium containing 1 μM FCCP. Finally, cells were stimulated in the absence of FCCP, still in Ca2+-free external medium. (D) Cells were loaded with 40 μM BAPTA-AM for 15 min before the start of experiment. (Inset) Responses to 100 nM BK in Ca2+-free medium before and after treatment with 1 μM oligomycin. (Bottom) Enlarged overlay of intraluminal [Ca2+] changes, oligomycin vs. control.

Figure 3

Figure 3

FCCP and oligomycin impede store refilling after BK-induced Ca2+ release in intact mag-fura-2-loaded cells. (A) Control recovery and response in the same cell to hormone after brief pretreatment with FCCP. (Inset) Entire experimental record. (Bottom) Enlarged overlay emphasizing the recovery phases with and without FCCP. (B) As above, profile of store reloading before and after treatment with 1 μM oligomycin. (C) Comparison of store refilling after three separate stimulations with 100 nM BK: first in the presence of 1 μM FCCP in Ca2+-containing medium; second after a 15-min incubation with 40 μM BAPTA-AM (added at arrow); cells were washed with normal Ringer's for 5 min, and were then stimulated with BK in Ca2+-free medium containing 1 μM FCCP. Finally, cells were stimulated in the absence of FCCP, still in Ca2+-free external medium. (D) Cells were loaded with 40 μM BAPTA-AM for 15 min before the start of experiment. (Inset) Responses to 100 nM BK in Ca2+-free medium before and after treatment with 1 μM oligomycin. (Bottom) Enlarged overlay of intraluminal [Ca2+] changes, oligomycin vs. control.

Figure 3

Figure 3

FCCP and oligomycin impede store refilling after BK-induced Ca2+ release in intact mag-fura-2-loaded cells. (A) Control recovery and response in the same cell to hormone after brief pretreatment with FCCP. (Inset) Entire experimental record. (Bottom) Enlarged overlay emphasizing the recovery phases with and without FCCP. (B) As above, profile of store reloading before and after treatment with 1 μM oligomycin. (C) Comparison of store refilling after three separate stimulations with 100 nM BK: first in the presence of 1 μM FCCP in Ca2+-containing medium; second after a 15-min incubation with 40 μM BAPTA-AM (added at arrow); cells were washed with normal Ringer's for 5 min, and were then stimulated with BK in Ca2+-free medium containing 1 μM FCCP. Finally, cells were stimulated in the absence of FCCP, still in Ca2+-free external medium. (D) Cells were loaded with 40 μM BAPTA-AM for 15 min before the start of experiment. (Inset) Responses to 100 nM BK in Ca2+-free medium before and after treatment with 1 μM oligomycin. (Bottom) Enlarged overlay of intraluminal [Ca2+] changes, oligomycin vs. control.

Figure 3

Figure 3

FCCP and oligomycin impede store refilling after BK-induced Ca2+ release in intact mag-fura-2-loaded cells. (A) Control recovery and response in the same cell to hormone after brief pretreatment with FCCP. (Inset) Entire experimental record. (Bottom) Enlarged overlay emphasizing the recovery phases with and without FCCP. (B) As above, profile of store reloading before and after treatment with 1 μM oligomycin. (C) Comparison of store refilling after three separate stimulations with 100 nM BK: first in the presence of 1 μM FCCP in Ca2+-containing medium; second after a 15-min incubation with 40 μM BAPTA-AM (added at arrow); cells were washed with normal Ringer's for 5 min, and were then stimulated with BK in Ca2+-free medium containing 1 μM FCCP. Finally, cells were stimulated in the absence of FCCP, still in Ca2+-free external medium. (D) Cells were loaded with 40 μM BAPTA-AM for 15 min before the start of experiment. (Inset) Responses to 100 nM BK in Ca2+-free medium before and after treatment with 1 μM oligomycin. (Bottom) Enlarged overlay of intraluminal [Ca2+] changes, oligomycin vs. control.

Figure 2

Figure 2

Effects of FCCP on Ca2+ homeostasis in internal stores of digitonin-permeabilized mag-fura-2-loaded BHK-21 cells. Cells were continuously superfused with intracellular buffer. [Ca2+] was clamped with EGTA buffers to 170 nM, and a constant [ATP] of 0.5 mM was maintained. FCCP had no effect on the mag-fura-2 ratio or InsP3-induced release and reloading of internal stores under these conditions.

Figure 4

Figure 4

(A) Effects of changing [ATP] on rates of refilling in digitonin-permeabilized BHK-21 cells loaded with mag-fura-2. Rates of refilling after stimulation with 6 μM InsP3 in the presence of 50 μM ATP is compared with that in 0.5 mM. Maximal recovery velocity was obtained when [ATP] was 200 μM (not shown). (B) Rates of recovery after release with 6 μM InsP3 were barely affected by adding 0.5 mM ADP during the recovery phase. (C) 4 mM ADP (in the presence of 0.5 mM ATP) blocked refilling.

Figure 5

Figure 5

Effects of mitochondrial inhibitors on rates of release in intact mag-fura-2–loaded cells. Data are taken from the records shown in Fig. 3, and are represented on an expanded time scale to allow better appreciation of the release kinetics. (A) FCCP vs. control, (B) oligomycin vs. control, (C) as in Fig. 3_C_, comparison in the same cell between rates of release: (a) in the presence of 1 μM FCCP (FCCP); (b) after a 15-min incubation with 40 μM BAPTA-AM, after which cells were washed with normal Ringer's for 5 min and then stimulated with BK in Ca2+-free medium containing 1 μM FCCP (BAPTA/ FCCP); (c) cells stimulated in the absence of FCCP, still in Ca2+-free external medium (BAPTA control). (D) Data from Fig. 3_D_: rates of release after BAPTA-AM pretreatment in the presence and absence of oligomycin.

Figure 6

Figure 6

ATP modulates InsP3-induced Ca2+ release in digitonin-permeablized cells. (A) Comparison of two sequential stimulations with 6 μM InsP3 in the absence of ATP and in the presence of 5 mM ATP. (B) Increasing [ATP] to 5 mM during the release phase enhances the rate of InsP3-induced Ca2+ discharge.

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