Abstract

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP(3))-mediated increases in cytosolic Ca2+ concentration ([Ca2+](c)). Local Ca2+ release from an InsP(3) receptor (InsP(3)R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP(3)R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP(3)-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP(3)R. Mitochondria may regulate InsP(3)-mediated Ca2+ signals by operating between or within InsP(3)R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP(3)-mediated Ca2+ release by operating within (local) or between (global) InsP(3)R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP(3) in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5-4.0 F/F(0), durations of approximately 112 ms at half-maximum amplitude, and were abolished by the InsP(3)R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized DeltaPsi(M) to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by approximately 66 or approximately 60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by approximately 62%. The "fast" Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acted like mitochondria to prolong InsP(3)-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP(3)R communication at the intra-cluster level and that mitochondria regulate both local and global InsP(3)-mediated Ca2+ signals.

Highlights

  • The mitochondrial uniporter inhibitor, RU360, attenuated Ca2؉ puffs by ϳ62%

  • The “fast” Ca2؉ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N؅,N؅-tetraacetic acid acted like mitochondria to prolong InsP3-mediated Ca2؉ release suggesting that mitochondrial influence is via their Ca2؉ uptake facility

  • Ca2ϩ puffs have been observed in several cell types including Xenopus oocytes, rat basophilic leukemia, glial, PC12, and smooth muscle cells where the localized release forms microdomains of [Ca2ϩ]c, which exceed that of the bulk cytoplasm (6 – 8, 10 –12)

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Summary

EXPERIMENTAL PROCEDURES

Cell Isolation—Male guinea pigs (350 –500 g) were humanely killed by cervical dislocation followed by immediate exsanguination in accordance with the guidelines of the Animal (Scientific Procedures) Act UK 1986. In the majority of experiments, as specified under “Results,” EGTA (250 ␮M to 1 mM) was added to the pipette solution to buffer the [Ca2ϩ]c. As specified under “Results,” BAPTA (250 ␮M) replaced EGTA in the pipette solution to buffer the [Ca2ϩ]c. In some experiments the software program Metafluor (Molecular Devices, Wokingham, UK) was used to obtain longer periods of data acquisition. In these experiments sampling rates of ϳ10 frames/s were used during and 1 frame/s between Ca2ϩ transients. The output intensity of the flash lamp was altered in the Ca2ϩ puff experiments to between 20 and 100% (0.025– 0.19 milliwatts) of the maximum output to control the amount of InsP3 that was uncaged and determined empirically in each cell.

RESULTS
57 Ϯ 8 1486 Ϯ 156
DISCUSSION
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