Mitochondrial Ca²⁺ homeostasis during Ca²⁺ influx and Ca²⁺ release in gastric myocytes from Bufo marinus

Abstract

The Ca2+‐sensitive fluorescent indicator rhod‐2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura‐2 was used in combination with rhod‐2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half‐time (t½) to peak for the increase in [Ca2+]m was considerably longer than the t½ to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t½ to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. Using a wide‐field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR.

The mitochondrial membrane potential and Ca2+ oscillations in smooth muscle

“…However, whether or not the local [Ca 2+ ] c experienced by mitochondria in situ, in intact cells, is sufficient to cause ⌬⌿ m depolarizations comparable to those that occur in isolated mitochondria is unresolved. ⌬⌿ m depolarization has been observed in response to the [Ca 2+ ] c rise produced by trains of plasmalemmal depolarization in single toad gastric myocytes (Drummond et al, 2000). No alterations in ⌬⌿ m were observed in the present study to trains of depolarization (Fig.…”

“…(Brustovetsky and Dubinsky, 2000;Huser et al, 1998), it is less clear whether or not this occurs during [Ca 2+ ] c signaling in intact cells. Transient, or 'flickering', ⌬⌿ m changes have been observed in some (Buckman and Reynolds, 2001;Drummond et al, 2000;Duchen et al, 1998;Haak et al, 2002;Jacobson and Duchen, 2002;Kaftan et al, 2000;O'Reilly et al, 2004;Petronilli et al, 2001) but not all Csordas and Hajnoczky, 2003) (Jacobson and Duchen, 2002) but not in others (O'Reilly et al, 2004).…”

Mitochondrial Ca2+ Uptake Requires Sustained Ca2+ Release from the Endoplasmic Reticulum

“…The first observation is that, upon cell stimulation, [Ca 2ϩ ] m peaks well after [Ca 2ϩ ] c . As previously observed by various groups, there is a short delay in the upstroke, possibly due to the time needed for the diffusion of Ca 2ϩ released by IP 3 receptors through the outer mitochondrial membrane, thus reaching the transport systems (uniporter) of the inner membrane (14,18,19). Then [Ca 2ϩ ] m rises and reaches its maximal value after ϳ10 s (i.e.…”

“…The apparent discrepancy was reconciled by the concept that high [Ca 2ϩ ] microdomains generated at mouth of IP 3 Rs during its activation are sensed by neighboring mitochondria (17), which are thus exposed to [Ca 2ϩ ], that allow efficient Ca 2ϩ uptake. Finally, the diffusion of Ca 2ϩ through the outer mitochondrial membrane creates a lag time between the initial [Ca 2ϩ ] c and [Ca 2ϩ ] m rises into mitochondria (14,18,19). Thus, the properties of mitochondrial Ca 2ϩ accumulation suggest that these organelles may represent the prototype of a cytosolic effector, which requires sustained release of Ca 2ϩ from the ER (and thus maintenance of a high [Ca 2ϩ ] microdomain at ER/mitochondria contacts) to be actively recruited in the calcium signaling pathway.…”

Ca2+Syntillas, Miniature Ca2+Release Events in Terminals of Hypothalamic Neurons, Are Increased in Frequency by Depolarization in the Absence of Ca2+Influx

“…Finally, mitochondria do not appear to be necessary for syntilla generation, because syntillas were still present after 10 min exposure to 2 mM cyanide and 5 mM azide. This treatment was observed to collapse the mitochondrial membrane potential, as measured with a fluorescent probe in parallel experiments (Drummond et al, 2000).…”