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In smooth muscle, the gating of dihydropyridine-sensitive [Ca.sup.2+] channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for [Ca.sup.2+] influx and determining the bulk average cytoplasmic Ca concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to [Ca.sup.2+] signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (-70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent [Ca.sup.2+] current ([I.sub.Ca]) and evoked a rise in [[Ca.sup.2+]] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [[Ca.sup.2+]] increase ([[[Ca.sup.2+]].sub.c]) was approximately uniform, whereas that of the subplasma membrane space ([[[Ca.sup.2+]].sub.PM]) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active [Ca.sup.2+] channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive [Ca.sup.2+] channels were not normally constitutively active. The repetitive localized [[[Ca.sup.2+]].sub.PM] rises ('persistent [Ca.sup.2+] sparklets') that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active [Ca.sup.2+] channels regulate the bulk average [[[Ca.sup.2+]].sub.c], A dihydropyridine blocker of [Ca.sup.2+] channels, nimodipine, which blocked [I.sub.Ca] and accompanying [[[Ca.sup.2+]].sub.c] rise, reduced neither the resting bulk average [[[Ca.sup.2+]].sub.c] (at -70 mV) nor the rise in [[[Ca.sup.2+]].sub.c], which accompanied an increased electrochemical driving force on the ion by hyperpolarization (-130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent [Ca.sup.2+] channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [[[Ca.sup.2+]].sub.c] regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [[Ca.sup.2+]].

Smooth muscle is activated by plasma-membraneacting agonists that induce inositol (1,4,5)-trisphosphate [Ins(1,4,5)[P.sub.3]] to release [Ca.sup.2+]. from the intracellular sarcoplasmic reticulum (SR) [Ca.sup.2+] store. Increased concentrations of agonist evoke a concentration-dependent graded release of [Ca.sup.2+] in a process called 'quantal' [Ca.sup.2+] release. Such a graded release seems to be incompatible with both the finite capacity of the SR store and the positive-feedback [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR)-like process that is operative at Ins(1,4,5)[P.sub.3] receptors, which--once activated--might be expected to deplete the entire store. Proposed explanations of quantal release include the existence of multiple stores, each with different sensitivities to Ins(1,4,5)[P.sub.3], or Ins(1,4,5)[P.sub.3] receptor opening being controlled by the [Ca.sup.2+] concentration within the SR. Here, we suggest that the regulation of Ins(1,4,5)[P.sub.3] receptors by the [Ca.sup.2+] concentration within the SR explains the quantal [Ca.sup.2+]-release process and the apparent existence of multiple [Ca.sup.2+] stores in smooth muscle.

Mitochondrial Ca2+ uptake contributes important feedback controls to limit the time course of Ca2+signals. Mitochondria regulate cytosolic [Ca2+] over an exceptional breath of concentrations (~200 nM to >10 μM) to provide a wide dynamic range in the control of Ca2+ signals. Ca2+ uptake is achieved by passing the ion down the electrochemical gradient, across the inner mitochondria membrane, which itself arises from the export of protons. The proton export process is efficient and on average there are less than three protons free within the mitochondrial matrix. To study mitochondrial function, the most common approaches are to alter the proton gradient and to measure the electrochemical gradient. However, drugs which alter the mitochondrial proton gradient may have substantial off target effects that necessitate careful consideration when interpreting their effect on Ca2+ signals. Measurement of the mitochondrial electrochemical gradient is most often performed using membrane potential sensitive fluorophores. However, the signals arising from these fluorophores have a complex relationship with the electrochemical gradient and are altered by changes in plasma membrane potential. Care is again needed in interpreting results. This review provides a brief description of some of the methods commonly used to alter and measure mitochondrial contribution to Ca2+ signaling in native smooth muscle. [ABSTRACT FROM AUTHOR]

Increases in cytosolic Ca2+ concentration ([Ca2+]c)mediated by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3, hereafter InsP3] regulate activities that include division, contraction and cell death. InsP3-evoked Ca2+ release often begins at a single site, then regeneratively propagates through the cell as a Ca2+ wave. The Ca2+ wave consistently begins at the same site on successive activations. Here, we address the mechanisms that determine the Ca2+ wave initiation site in intestinal smooth muscle cells. Neither an increased sensitivity of InsP3 receptors (InsP3R) to InsP3 nor regional clustering of muscarinic receptors (mAChR3) or InsP3R1 explained the selection of an initiation site. However, examination of the overlap of mAChR3 and InsP3R1 localisation, by centre of mass analysis, revealed that there was a small percentage (,10%) of sites that showed colocalisation. Indeed, the extent of colocalisation was greatest at the Ca2+ wave initiation site. The initiation site might arise from a selective delivery of InsP3 from mAChR3 activity to particular InsP3Rs to generate faster local [Ca2+]c increases at sites of colocalisation. In support of this hypothesis, a localised subthreshold 'priming' InsP3 concentration applied rapidly, but at regions distant from the initiation site, shifted the wave to the site of the priming. Conversely, when the Ca2+ rise at the initiation site was rapidly and selectively attenuated, the Ca2+ wave again shifted and initiated at a new site. These results indicate that Ca2+ waves initiate where there is a structural and functional coupling of mAChR3 and InsP3R1, which generates junctions in which InsP3 acts as a highly localised signal by being rapidly and selectively delivered to InsP3R1. [ABSTRACT FROM AUTHOR]

Ca2+ may selectively activate various processes in part by the cell's ability to localize changes in the concentration of the ion to specific subcellular sites. Interestingly, these Ca2+ signals begin most often at the plasma membrane space so that understanding subplasma membrane signals is central to an appreciation of local signaling. Several experimental procedures have been developed to study Ca2+ signals near the plasma membrane, but probably the most prevalent involve the use of fluorescent Ca2+ indicators and fall into two general approaches. In the first, the Ca2+ indicators themselves are specifically targeted to the subplasma membrane space to measure Ca2+ only there. Alternatively, the indicators are allowed to be dispersed throughout the cytoplasm, but the fluorescence emanating from the Ca2+ signals at the subplasma membrane space is selectively measured using high resolution imaging procedures. Although the targeted indicators offer an immediate appeal because of selectivity and ease of use, their limited dynamic range and slow response to changes in Ca2+ are a shortcoming. Use of targeted indicators is also largely restricted to cultured cells. High resolution imaging applied with rapidly responding small molecule Ca2+ indicators can be used in all cells and offers significant improvements in dynamic range and speed of response of the indicator. The approach is technically difficult, however, and realistic calibration of signals is not possible. In this review, a brief overview of local subplasma membrane Ca2+ signals and methods for their measurement is provided. © 2012 IUBMB IUBMB Life, 64(7): 573-585, 2012 [ABSTRACT FROM AUTHOR]

Smooth muscle responds to IP3-generating agonists by producing Ca2+ waves. Here, the mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells using localized photolysis of caged IP3 and the caged Ca2+ buffer diazo-2. Waves, evoked by the IP3-generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca2+ concentration ([Ca2+]c) over a single though substantial length (∼30 µm) of the cell. During regenerative propagation, the wave-front was about 1/3 the length (∼9 µm) of the initiation site. The wave-front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP3 may explain the amplitude changes. Ca2+ was required for IP3-mediated wave progression to occur. Increasing the Ca2+ buffer capacity in a small (2 µm) region immediately in front of a CCh-evoked Ca2+ wave halted progression at the site. However, the wave front does not progress by Ca2+-dependent positive feedback alone. In support, colliding [Ca2+]c increases from locally released IP3 did not annihilate but approximately doubled in amplitude. This result suggests that local IP3-evoked [Ca2+]c increases diffused passively. Failure of local increases in IP3 to evoke waves appears to arise from the restricted nature of the IP3 increase. When IP3 was elevated throughout the cell, a localized increase in Ca2+ now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP3 and Ca2+ are required for active propagation of the wave front to occur. J. Cell. Physiol. 224: 334–344, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Smooth muscle responds to IP3-generating agonists by producing Ca2+ waves. Here, the mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells using localized photolysis of caged IP3 and the caged Ca2+ buffer diazo-2. Waves, evoked by the IP3-generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca2+ concentration ([Ca2+]c) over a single though substantial length (∼30 µm) of the cell. During regenerative propagation, the wave-front was about 1/3 the length (∼9 µm) of the initiation site. The wave-front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP3 may explain the amplitude changes. Ca2+ was required for IP3-mediated wave progression to occur. Increasing the Ca2+ buffer capacity in a small (2 µm) region immediately in front of a CCh-evoked Ca2+ wave halted progression at the site. However, the wave front does not progress by Ca2+-dependent positive feedback alone. In support, colliding [Ca2+]c increases from locally released IP3 did not annihilate but approximately doubled in amplitude. This result suggests that local IP3-evoked [Ca2+]c increases diffused passively. Failure of local increases in IP3 to evoke waves appears to arise from the restricted nature of the IP3 increase. When IP3 was elevated throughout the cell, a localized increase in Ca2+ now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP3 and Ca2+ are required for active propagation of the wave front to occur. J. Cell. Physiol. 224: 334–344, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Increases in bulk average cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) are derived from the combined activities of many Ca(2+) channels. Near (<100 nm) the mouth of each of these channels the local [Ca(2+)](c) rises and falls more quickly and reaches much greater values than occurs in the bulk cytoplasm. Even during apparently uniform, steady-state [Ca(2+)] increases large local inhomogeneities exist near channels. These local increases modulate processes that are sensitive to rapid and large changes in [Ca(2+)] but they cannot easily be visualized with conventional imaging approaches. The [Ca(2+)] changes near channels can be examined using total internal reflection fluorescence microscopy (TIRF) to excite fluorophores that lie within 100 nm of the plasma membrane. TIRF is particularly powerful when combined with electrophysiology so that ion channel activity can be related simultaneously to the local subplasma membrane and bulk average [Ca(2+)](c). Together these techniques provide a better understanding of the local modulation and control of Ca(2+) signals.