Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca2+ 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 Ca2+ influx and determining the bulk average cytoplasmic Ca2+ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca2+ 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 Ca2+ current (ICa) and evoked a rise in [Ca2+] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca2+] increase ([Ca2+]c) was approximately uniform, whereas that of the subplasma membrane space ([Ca2+]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 Ca2+ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca2+ channels were not normally constitutively active. The repetitive localized [Ca2+]PM rises (“persistent Ca2+ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca2+ channels regulate the bulk average [Ca2+]c. A dihydropyridine blocker of Ca2+ channels, nimodipine, which blocked ICa and accompanying [Ca2+]c rise, reduced neither the resting bulk average [Ca2+]c (at −70 mV) nor the rise in [Ca2+]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 Ca2+ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca2+]c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca2+]

Microdomains of muscarinic acetylcholine and InsP3 receptors create InsP3 junctions and sites of Ca2+ wave initiation in smooth muscle

Inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c) regulate activities which include division, contraction and cell death. InsP3-evoked Ca2+ release often begins in 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. We addressed 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 initiation site. However, examination of the overlap of mAChR3 and InsP3R1 by centre of mass analysis revealed a small percentage (~10%) of sites which showed colocalisation. Indeed, the extent of colocalisation was greatest at Ca2+ wave initiation site. The initiation site may arise from a selective delivery of InsP3 from mAChR3 activity to particular InsP3R to generate faster local [Ca2+]c increases at sites of co-localization. In support, a localized subthreshold ‘priming’ InsP3 concentration applied rapidly but at regions distant from the initiation site shifted the wave to the site of priming InsP3 release. 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 localized signal by being rapidly and selectively delivered to InsP3

Microdomains of muscarinic acetylcholine and InsP3 receptors create InsP3 junctions and sites of Ca2+ wave initiation in smooth muscle

Inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c) regulate activities which include division, contraction and cell death. InsP3-evoked Ca2+ release often begins in 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. We addressed 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 initiation site. However, examination of the overlap of mAChR3 and InsP3R1 by centre of mass analysis revealed a small percentage (~10%) of sites which showed colocalisation. Indeed, the extent of colocalisation was greatest at Ca2+ wave initiation site. The initiation site may arise from a selective delivery of InsP3 from mAChR3 activity to particular InsP3R to generate faster local [Ca2+]c increases at sites of co-localization. In support, a localized subthreshold ‘priming’ InsP3 concentration applied rapidly but at regions distant from the initiation site shifted the wave to the site of priming InsP3 release. 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 localized signal by being rapidly and selectively delivered to InsP3

Examining the role of mitochondria in Ca2+ Signaling in native vascular 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

From Structure to Function: Mitochondrial Morphology, Motion and Shaping in Vascular Smooth Muscle

The diversity of mitochondrial arrangements, which arise from the organelle being static or moving, or fusing and dividing in a dynamically reshaping network, is only beginning to be appreciated. While significant progress has been made in understanding the proteins that reorganise mitochondria, the physiological significance of the various arrangements is poorly understood. The lack of understanding may occur partly because mitochondrial morphology is studied most often in cultured cells. The simple anatomy of cultured cells presents an attractive model for visualizing mitochondrial behaviour but contrasts with the complexity of native cells in which elaborate mitochondrial movements and morphologies may not occur. Mitochondrial changes may take place in native cells (in response to stress and proliferation), but over a slow time-course and the cellular function contributed is unclear. To determine the role mitochondrial arrangements play in cell function, a crucial first step is characterisation of the interactions among mitochondrial components. Three aspects of mitochondrial behaviour are described in this review: (1) morphology, (2) motion and (3) rapid shape changes. The proposed physiological roles to which various mitochondrial arrangements contribute and difficulties in interpreting some of the physiological conclusions are also outlined