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We recorded Ca2+ sparks and spontaneous transient outward currents (STOCs) simultaneously in smooth muscle cells using whole-cell patch recording and a unique, high-speed widefield digital imaging system to monitor fluo-3 fluorescence in both two and three dimensions (2D and 3D). In 2D imaging, the correlation between the amplitude of a spark and its corresponding STOC was a weak one, and 27 % of the sparks failed to cause STOCs. The STOCless sparks were not significantly different in amplitude from those that caused STOCs. Three-dimensional imaging disclosed that STOCless sparks were located close to the cell surface, and on average their apparent distance from the cell surface was not significantly different from the sparks that cause STOCs. Statistical evaluation of spark clusters disclosed that there were regions of the cell where the probability of spark occurrence was high and others where it was quite low.

1. A digital imaging microscope with fura-2 as the Ca2+ indicator was used to determine the sources for the rise in intracellular calcium concentration ([Ca2+]i) that occurs when the membrane in a cell-attached patch is stretched. Unitary ionic currents from stretch-activated channels and [Ca2+]i images were recorded simultaneously. 2. When suction was applied to the patch pipette to stretch a patch of membrane, Ca2+-permeable cation channels (stretch-activated channels) opened and a global increase in [Ca2+]i occurred, as well as a greater focal increase in the vicinity of the patch pipette. The global changes in [Ca2+]i occurred only when stretch-activated currents were sufficient to cause membrane depolarization, as indicated by the reduction in amplitude of the unitary currents. 3. When Ca2+ was present only in the pipette solution, just the focal change in [Ca2+]i was obtained. This focal change was not seen when the contribution from Ca2+ stores was eliminated using caffeine and ryanodine. 4. These results suggest that the opening of stretch-activated channels allows ions, including Ca2+, to enter the cell. The entry of positive charge triggers the influx of Ca2+ into the cell by causing membrane depolarization, which presumably activates voltage-gated Ca2+ channels. The entry of Ca2+ through stretch-activated channels is also amplified by Ca2+ release from internal stores. This amplification appears to be greater than that obtained by activation of whole-cell Ca2+ currents. These multiple pathways whereby membrane stretch causes a rise in [Ca2+]i may play a role in stretch-induced contraction, which is a characteristic of many smooth muscle tissues.

1. The Ca(2+)-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. 2. 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 (t1/2) to peak for the increase in [Ca2+]m was considerably longer than the t1/2 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. 3. 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 t1/2 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. 4. 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. 5. 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. 6. 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.

1. [Ca2+] was recorded in voltage-clamped gastric myocytes from Bufo marinus. Repolarization to -110 mV following a 300 ms depolarization to +10 mV led to triphasic [Ca2+]i decay, with a fast-slow-fast pattern. After a conditioning train of repetitive depolarizations the duration of the second, slow phase of decay was shortened, while the rate of decay during the third, faster phase was increased by 34 +/- 6% (mean +/- S.E.M., n = 21) when compared with unconditioned transients. 2. [Ca2+]i decay was biphasic in cells injected with the calmodulin-binding peptide RS20, with a prolonged period of fast decay followed by a slow phase. There was no subsequent increase in decay rate during individual transients and no acceleration of decay following the conditioning train (n = 8). Decline of [Ca2+]i in cells injected with the control peptide NRS20 was triphasic and the decay rate during the third phase was increased by 50 +/- 19% in conditioned transients (n = 6). 3. Cell injection with CK3AA, a pseudo-substrate inhibitor of calmodulin-dependent protein kinase II, prevented the increase in the final rate of decay following the conditioning train (n = 6). In cells injected with an inactive peptide similar to CK3AA, however, there was a 45 +/- 17% increase after the train (n = 5). 4. Inhibition of Ca2+ uptake by the sarcoplasmic reticulum with cyclopiazonic acid or thapsigargin did not prevent acceleration of decay. 5. These results demonstrate that [Ca2+]i decay is accelerated by Ca(2+)-calmodulin and calmodulin-dependent protein kinase II. This does not depend on Ca2+ uptake by the sarcoplasmic reticulum but may reflect upregulation of mitochondrial Ca2+ removal.

1. Decay in intracellular calcium concentration ([Ca2+]i) was recorded following step depolarizations in voltage clamped gastric myocytes from Bufo marinus. 2. Depolarizations (300 ms) to +10 mV were followed by three phases of [Ca2+]i decay with repolarization to both -110 and -50 mV. The decline was initially rapid (mean fractional decay rate = 81 +/- 11%s-1 at -110 mV), then slowed (decay rate = 14 +/- 2%s-1) and finally accelerated again (decay rate = 24 +/- 3%s-1; n = 19). 3. The initial phase of rapid decay became shorter as the length of the depolarizing pulse increased but was unaffected by changes in pulse voltage. 4. The delayed acceleration in [Ca2+]i decay was no longer seen when the duration of the depolarizing pulses was reduced to 100 ms, but was clearly evident following 500 ms pulses. This phase was abolished when the depolarizing voltage was altered to minimize the rise in [Ca2+]i. 5. Ryanodine and caffeine had no effect on the temporal profile of [Ca2+]i decay. 6. Removal of extracellular Na+ decreased the decay rate during all three phases at -110 mV, but this effect was particularly marked for the initial rapid phase of decay, the rate of which was reduced by 75%. A delayed increase in decay rate was still seen. 7. Inhibition of mitochondrial Ca2+ uptake with cyanide, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or Ruthenium Red had no effect on the initial rate of [Ca2+]i decay but blocked the delayed acceleration. 8. These results are discussed in terms of a model in which rapid influx of Ca2+ produces a high subsarcolemmal [Ca2+], favouring rapid Ca2+ removal by near-membrane mechanisms, particularly Na(+)-Ca2+ exchange. Mitochondrial Ca2+ removal produces a delayed increase in [Ca2+]i decay if the global [Ca2+]i is raised high enough for long enough.

1. Single smooth muscle cells were isolated intact from the stomach of the toad Bufo marinus. The relaxation of cells following cessation of electrical stimulation was compared with those relaxed by pressure microinjection of either metal ion chelators or cyclic nucleotides. 2. Injection of either a Ca2+ chelator or 3',5'-cyclic AMP slowed or halted shortening and promoted re-extension of a cell or collapse of membrane evaginations (blebs) in a manner similar to that following cessation of electrical stimulation. Collapse of blebs occurred first and was then followed smoothly by the next stage with cells re-extending at maximum rates in one of three ranges at 22 degrees C. These rates, in order of increasing speed, were 0.005, 0.009 and 0.03 cell lengths s-1 after electrical stimulation, 3',5'-cyclic AMP and EDTA injection, respectively. On the other hand, shortening began at a maximum rate of about 0.1 cell lengths s-1 unless a Ca2+ chelator or 3',5'-cyclic AMP was injected about 30 s or less before electrical stimulation. Injection of these agents reduced the speed of shortening by about half. 3. Injection of a liquid per se (e.g. 140 mM-KCl) neither altered action potentials nor duplicated the changes produced by the aforementioned relaxing agents. Large, sustained injections of substances that were not relaxing agents (e.g. dilute KCl) ruptured the membrane without producing any bleb collapse or re-extension of a contracted cell. Blebs not only collapsed rapidly when a relaxing agent was injected but bleb collapse was a much more sensitive indication of relaxation than cell re-extension; small injections of relaxing agents could clearly collapse blebs with no associated measurable change in cell length. This supports the idea previously inferred from fixed or permeabilized cells, that filaments in smooth muscle are organized to produce force over short distances at points along the cell membrane, in addition to shortening along the long axis. 4. Physiological relaxation of smooth muscle can evidently be mimicked by 3',5'-cyclic AMP elevation. Restoring forces may develop during shortening of isolated smooth muscle cells in elements of their cytoskeleton, surface membrane, or contractile filaments. However, these putative forces may not be able to produce physiological re-extension in the absence of a rise in cyclic AMP and/or a fall in [Ca2+].